The Interactive Fly

Zygotically transcribed genes

RNAi and Posttranscriptional Gene Silencing (page 2 | page1)



miRNA biogenesis and function
  • Small RNA sorting: matchmaking for Argonautes
  • RNAi and PTGS - functions and processes
  • GW182 proteins directly recruit cytoplasmic deadenylase complexes to miRNA targets
  • GW-bodies and P-bodies constitute two separate pools of sequestered non-translating RNAs
  • Drosophila cells use nanotube-like structures to transfer dsRNA and RNAi machinery between cells
  • The interactions of GW182 proteins with PABP and deadenylases are required for both translational repression and degradation of miRNA targets
  • NOT10 and C2orf29/NOT11 form a conserved module of the CCR4-NOT complex that docks onto the NOT1 N-terminal domain
  • HPat a decapping activator interacting with the miRNA effector complex
  • The CCR4 deadenylase acts with Nanos and Pumilio in the fine-tuning of Mei-P26 expression to promote germline stem cell self-renewal
  • Drosophila miR2 primarily targets the m7GpppN cap structure for translational repression
  • An mRNA m7G cap binding-like motif within human Ago2 represses translation
  • miRISC recruits decapping factors to miRNA targets to enhance their degradation
  • Adenylation of maternally inherited microRNAs by Wispy
  • MicroRNAs block assembly of eIF4F translation initiation complex in Drosophila
  • MicroRNA biogenesis via splicing and exosome-mediated trimming in Drosophila
  • Drosophila exoribonuclease nibbler is a tumor suppressor, acts within the RNA(i) machinery and is not enriched in the nuage during early oogenesis
  • Stable intronic sequence RNAs have possible regulatory roles in Drosophila melanogaster
  • Uridylation of RNA hairpins by Tailor confines the emergence of microRNAs in Drosophila
  • Selective suppression of the splicing-mediated microRNA pathway by the terminal uridyltransferase Tailor
  • Molecular basis for cytoplasmic RNA surveillance by uridylation-triggered decay in Drosophila
  • Diverse modes of evolutionary emergence and flux of conserved microRNA clusters
  • Principles of microRNA-target recognition
  • Direct measurement of pervasive weak repression by microRNAs and their role at the network level

    MicroRNA functions
  • Lin-28 regulates oogenesis and muscle formation in Drosophila melanogaster
  • Antisense-mediated depletion reveals essential and specific functions of microRNAs in Drosophila development
  • Distinct populations of primary and secondary effectors during RNAi in C. elegans; Secondary effectors are derived from RNA-directed RNA polymerase (RdRP) activity
  • Genome-wide identification and developmental expression profiling of long noncoding RNAs during Drosophila metamorphosis
  • Comparative expression dynamics of intergenic long noncoding RNAs (lncRNAs) in the genus Drosophila
  • Maternally inherited stable intronic sequence RNA triggers a self-reinforcing feedback loop during development
  • Functionally diverse microRNA effector complexes are regulated by extracellular signaling
  • microRNAs that promote or inhibit memory formation in Drosophila melanogaster
  • Pervasive behavioural effects of microRNA regulation in Drosophila
  • Temporal reciprocity of miRNAs and their targets during the maternal-to-zygotic transition in Drosophila
  • The Smaug RNA-binding protein is essential for microRNA synthesis during the Drosophila maternal-to-zygotic transition
  • Target repression induced by endogenous microRNAs: Large differences, small effects
  • MicroRNA-277 modulates the neurodegeneration caused by Fragile X premutation rCGG repeats
  • FOXO regulates RNA interference in Drosophila and protects from RNA virus infection

    MicroRNA targets
  • Pervasive regulation of Drosophila Notch target genes by GY-box-, Brd-box-, and K-box-class microRNAs
  • Downregulation of the host gene jigr1 by miR-92 is essential for neuroblast self-renewal in Drosophila
  • Robust intestinal homeostasis relies on cellular plasticity in enteroblasts mediated by miR-8-Escargot switch
  • miR-980 is a memory suppressor microRNA that regulates the autism-susceptibility gene A2bp1
  • A neuroprotective role for microRNA miR-1000 mediated by limiting glutamate excitotoxicity
  • Identification of miR-305, a microRNA that promotes aging, and its target mRNAs in Drosophila
  • EXD2 governs germ stem cell homeostasis and lifespan by promoting mitoribosome integrity and translation
  • Novel Triazole linked 2-phenyl benzoxazole derivatives induce apoptosis by inhibiting miR-2, miR-13 and miR-14 function in Drosophila melanogaster
  • Immediate-early alcohol-responsive miRNA expression in Drosophila
  • Multiple in vivo biological processes are mediated by functionally redundant activities of Drosophila mir-279 and mir-996
  • miR-965 controls cell proliferation and migration during tissue morphogenesis in the abdomen
  • Derepressing muscleblind expression by miRNA sponges ameliorates myotonic dystrophy-like phenotypes in Drosophila
  • Presynaptic CamKII regulates activity-dependent axon terminal growth: miR-289 directly represses the translation of CamKII
  • Drosophila miR-956 suppression modulates Ectoderm-expressed 4 and inhibits viral replication
  • miR-31 mutants reveal continuous glial homeostasis in the adult Drosophila brain

    The Piwi-interacting RNA (piRNA) pathway
  • piRNA pathway is not required for antiviral defense in Drosophila melanogaster
  • The C. elegans CSR-1 argonaute pathway counteracts epigenetic silencing to promote germline gene expression
  • The Tudor domain protein Tapas, a homolog of the vertebrate Tdrd7, functions in piRNA pathway to regulate retros in germline of Drosophila melanogaster
  • Antisense transcription of retrotransposons in Drosophila: The origin of endogenous small interfering RNA precursors
  • The Drosophila fragile X mental retardation protein participates in the piRNA pathway
  • RNA editing regulates transposon-mediated heterochromatic gene silencing
  • Natural variation of piRNA expression affects immunity to transposable elements
  • Roles for the Yb body components Armitage and Yb in primary piRNA biogenesis in Drosophila
  • The Yb body, a major site for Piwi-associated RNA biogenesis and a gateway for Piwi expression and transport to the nucleus in somatic cells
  • Export of piRNA precursors by EJC triggers assembly of cytoplasmic Yb-body in Drosophila
  • Production of small non-coding RNAs from the flamenco locus is regulated by the gypsy retrotransposon of Drosophila melanogaster
  • Abundant expression of somatic transposon-derived piRNAs throughout Tribolium castaneum embryogenesis
  • PIWI slicing and RNA elements in precursors instruct directional primary piRNA biogenesis
  • Redundant and incoherent regulations of multiple phenotypes suggest microRNAs' role in stability control
  • Distribution, evolution, and diversity of retrotransposons at the flamenco locus reflect the regulatory properties of piRNA cluster
  • Unique transposon landscapes are pervasive across Drosophila melanogaster genomes
  • Paramutation in Drosophila linked to emergence of a piRNA-producing locus
  • Transgenerationally inherited piRNAs trigger piRNA biogenesis by changing the chromatin of piRNA clusters and inducing precursor processing
  • Panoramix enforces piRNA-dependent cotranscriptional silencing
  • The HP1 homolog Rhino anchors a nuclear complex that suppresses piRNA precursor splicing
  • A transgenerational process defines piRNA biogenesis in Drosophila virilis
  • piRNAs are associated with diverse transgenerational effects on gene and transposon expression in a hybrid dysgenic syndrome of D. virilis
  • Somatic primary piRNA biogenesis driven by cis-acting RNA elements and trans-acting Yb
  • Yb integrates piRNA intermediates and processing factors into perinuclear bodies to enhance piRISC assembly
  • Recurrent gene duplication diversifies genome defense repertoire in Drosophila
  • Splicing-independent loading of TREX on nascent RNA is required for efficient expression of dual-strand piRNA clusters in Drosophila
  • Temperature-responsive miRNAs in Drosophila orchestrate adaptation to different ambient temperatures
  • A heterochromatin-dependent transcription machinery drives piRNA expression
  • Diversity of P-element piRNA production among M' and Q strains and its association with P-M hybrid dysgenesis in Drosophila melanogaster
  • piRNA-mediated regulation of transposon alternative splicing in the soma and germ line
  • Spontaneous gain of susceptibility suggests a novel mechanism of resistance to hybrid dysgenesis in Drosophila virilis

    The small interfering RNA (siRNA) pathway
  • Polymorphism in the processing body component Ge-1 controls resistance to a naturally occurring ehabdovirus in Drosophila
  • Drosophila oncogene Gas41 is RNAi modulator that intersects heterochromatin and siRNA pathway
  • siRNAs from an X-linked satellite repeat promote X-chromosome recognition in Drosophila melanogaster
  • Dicer-2 processes diverse viral RNA species
  • Association of zygotic piRNAs derived from paternal P elements with hybrid dysgenesis in Drosophila melanogaster

    Lin-28 regulates oogenesis and muscle formation in Drosophila melanogaster

    Understanding the control of stem cell (SC) differentiation is important to comprehend developmental processes as well as to develop clinical applications. Lin28 is a conserved molecule that is involved in SC maintenance and differentiation by regulating let-7 miRNA maturation. However, little is known about the in vivo function of Lin28. This study reports critical roles for lin-28 during oogenesis. let-7 maturation was shown to be increased in lin-28 null mutant fly ovaries. lin-28 null mutant female flies display reduced fecundity, due to defects in egg chamber formation. More specifically, in mutant ovaries, the egg chambers were shown to fuse during early oogenesis resulting in abnormal late egg chambers. This phenotype is the combined result of impaired germline SC differentiation and follicle SC differentiation. A model is suggested in which these multiple oogenesis defects result from a misregulation of the ecdysone signaling network, through the fine-tuning of Abrupt and Fasciclin2 expression. These results give a better understanding of the evolutionarily conserved role of lin-28 on GSC maintenance and differentiation (Stratoulias, 2014).

    The Cold-Shock Domain (CSD) protein Lin28 was initially identified in Caenorhabditis elegans (C. elegans) as a component of the heterochronic pathway that regulates the timing of cell fate specification (Ambros, 1984). Subsequent discovery of gene expression regulation through small non-coding RNAs clarified the role of Lin28 in this pathway. The lin-28 mRNA is a conserved target of the let-7 micro-RNA (miRNA) family both in C. elegans and vertebrates. On the other hand, Lin28 inhibits let-7 processing. At the molecular level, Lin28 protein interacts with the let-7 precursor (pre-let-7), resulting in inhibition of let-7 maturation. The let-7 inhibition occurs through the physical interaction of the pre-let-7 loop and Lin28 protein, preventing further processing of pre-let-7 towards the mature form of let-7. Together, these interactions create a feedback loop between Lin28 and let-7, leading to a strict regulation of let-7 maturation (Stratoulias, 2014 and references therein).

    Lin28 raised further interest when it was used, along with Nanog, to replace the factors c-Myc and Klf4 in somatic cell reprogramming. These experiments, together with data from human embryonic stem cells, underscored the important role of lin-28 in pluripotency regulation and maintenance. Besides acting as a negative regulator of let-7 maturation, Lin28 has also been shown to have a direct effect on translation through the recruitment of the RNA Helicase A. This mode of function, independent of let-7 maturation, has been demonstrated in the case of Insulin-like Growth Factor 2 during mouse myogenesis. Lin28 binding on IGF-2 mRNA increases its translation efficiency and therefore facilitates skeletal myogenesis in mice (Stratoulias, 2014 and references therein).

    The Lin28 protein is composed of four domains: a positively charged linker that binds two Cys-Cys-His-Cys (CCHC)-type zinc-binding motifs to the CSD. In mammalian genomes, two paralogs of lin-28 are found, Lin28A and Lin28B. While Lin28B represses let-7 processing in the nucleus to prevent the formation of the precursor form from the primary let-7, Lin28A also blocks cytoplasmic processing of let-7 (Piskounova, 2011). It has recently been shown in mouse that deletion of the Lin28 linker domain alters the protein’s three-dimensional structure and is sufficient to disrupt sequestration of the precursor form of let-7 (pre-let-7) (Stratoulias, 2014).

    The miRNA let-7 family is conserved across diverse animals, functioning to control late temporal transitions during development. During the last decade, the involvement of let-7 in regulating cell differentiation has been analyzed in various contexts, including neural cell specification, stem cell maintenance and hematopoietic progenitor differentiation. While eight different let-7 miRNA genes are annotated in the human genome, only one is found in Drosophila melanogaster. Like in C. elegans, in Drosophila the loss of let-7 expression leads to the modification of temporal regulation of the metamorphosis process. During fly metamorphosis, the expression of let-7 complex (let-7C), a polycistronic locus encoding the let-7, miR-100 and miR-125 miRNAs, is under direct control by the steroid hormone ecdysone. Ecdysone is the central regulator of insect developmental transitions. Therefore, let-7 has been proposed to be part of a conserved, ecdysone regulated pathway that controls the timing of the larva to adult transition (Stratoulias, 2014).

    In addition to affecting the metamorphosis clock, Sokol and colleagues have shown that the let-7 deletion also affects the neuromuscular remodeling that takes place during the larva to adult transition. During neuromuscular remodeling, and under normal conditions, the dorsal internal oblique muscles (DIOMs) disappear 12 hours after emergence of the adult fly from the pupa. However, the adult let-7 mutants retain the DIOMs through adulthood. Deletion of the let-7 gene is sufficient to induce this phenotype, while deletion of either miR-100 or miR-125 genes is not enough to recapitulate the DIOM phenotype. Furthermore, let-7 has been shown to govern the maturation of neuromuscular junction of adult abdominal muscles, through regulation of Abrupt expression (Stratoulias, 2014 and references therein).

    While previous studies have demonstrated that the let-7 target Abrupt and ecdysone signaling are required for oogenesis in fruit fly ovaries, and that the let-7 miRNA family is abundantly expressed both in newborn mouse ovaries and in fly ovaries, no study has been conducted on the role of Lin-28/let-7 network in Drosophila ovaries. Therefore, a study was undertaken of the effects of lin-28 during Drosophila melanogaster development from the egg to the adult, and more particularly during oogenesis (Stratoulias, 2014).

    A lin-28 mutant was generated, and the consequent increase of let-7 maturation was validated. lin-28 knockout resulted in reduced muscular performance and defects in DIOM morphogenesis. These results were in line with the let-7 knock out muscular phenotype described earlier. Moreover, this study identified multiple defects during oogenesis due to abnormal follicle and germline stem cell (FSCs and GSCs respectively) differentiation. A link is proposed between ovarian defects and ectopic expression of Fasciclin2 (Fas2), a known downstream target of the Ecdysone pathway, and a predicted let-7 target (Stratoulias, 2014).

    Because of their role during stem cell differentiation, members of the let-7 miRNA family have been extensively studied. However, the role of lin-28 is still poorly documented. Deletion of let-7 in Drosophila impairs the musculature remodeling during the larva to adult metamorphosis. For instance the DIOMs, muscles which are required for eclosion and which are lost within 12 hours after eclosion, they are maintained during adulthood upon let-7 deletion. By generating the first lin-28 deletion in flies, this study has successfully confirmed the involvement of Lin-28/let-7 regulatory network in DIOM remodeling. This study has shown that deletion of lin-28 leads to over maturation of let-7, which negatively affects, and sometimes prevents DIOM formation. This drastic phenotype leads to a suboptimal muscular phenotype. However, due to a variable penetrance of the lin-28 deletion phenotype, a proportion of the flies could eclose and live as fertile animals (Stratoulias, 2014).

    In addition, a link was discovered between Lin-28 function and oogenesis. The data indicates a role of let-7 during GSC differentiation and egg chamber formation. Because of the importance of these processes, let-7 maturation has to be strictly regulated by Lin-28 activity. It is suggested that a potential network involving Lin-28/let-7/Ecdysone signaling/Abrupt/Fas2 is needed during GSC differentiation and BC migration. The role of Abrupt in downregulating the steroid hormone Ecdysone has previously been demonstrated. Indeed, the loss of Taiman, a target of the transcription factor Abrupt and co-activator of Ecdysone receptor, leads to an increase of undifferentiated GSCs in the germarium due to disruption of Ecdysone signaling. Therefore, by regulating the expression pattern of Abrupt, Lin28/let-7 may adjust the domain of Ecdysone activity, providing a control over the GSCs differentiation and egg chamber maturation during the oogenesis. Indeed, it has been shown that the Ecdysone titre rises during oogenesis at stage 9. While the precise Ecdysone expression pattern is not known, it is suggested that the uniform EcR expression pattern in follicle cells in lin-28 mutants may break the Ecdysone signaling asymmetry needed during proper oogenesis (Stratoulias, 2014).

    Furthermore, a previous study demonstrated the activation of let-7 expression via Ecdysone activity. This study showed that lin-28 deletion, resulted in the alleviation of Lin28's inhibitory role on let-7 maturation. This led to loss of Abrupt, which in turn inhibited Ecdysone activity and maintained Fas2 expression, resulting in BC migration impairment. To test whether the increase of Ecdysone signaling amplifies let-7 expression through a positive feedback loop, a system was generated in which there is no control of either let-7 expression nor of Ecdysone activity. This situation leads to an early cyst fusion, a loss of proper GSC differentiation and a mitotic defect, as was observed in the homozygous lin-28dF30 ovaries. The accumulation of these defects may be enough to trigger apoptosis at mid-oogenesis, a well-known checkpoint previously described (Stratoulias, 2014).

    Interestingly, the variable penetrance of the phenotype allows proper oogenesis and appearance of subfertile adult flies. This suggests a robust molecular network where feedback loops can rescue the system if one component disturbs the balance (Stratoulias, 2014).

    By combining these results with previously published studies, a conserved link is suggested between hormonal signaling and germline stem cell differentiation, involving the let-7 miRNA family. This suggestion is reinforced in the last couple of years by the discovery of dormant ovarian follicles and mitotically active germ cells in adult mammalian ovaries, which are responsive to gonadotropin hormone. Moreover, it has been demonstrated that Lin-28 is involved in germline stem cell regulation in human ovary and in the ovarian surface epithelium of severe ovarian infertility patients axonal projection is critical for assembly of a functional sensory circuit (Stratoulias, 2014).

    Diverse modes of evolutionary emergence and flux of conserved microRNA clusters

    Many animal miRNA loci reside in genomic clusters that generate multicistronic primary-miRNA transcripts. While clusters that contain copies of the same miRNA hairpin are clearly products of local duplications, the evolutionary provenance of clusters with disparate members is less clear. Recently, it was proposed that essentially all such clusters in Drosophila derived from de novo formation of miRNA-like hairpins within existing miRNA transcripts, and that the maintenance of multiple miRNAs in such clusters was due to evolutionary hitchhiking on a major cluster member. However, this model seems at odds with the fact that many such miRNA clusters are composed of well-conserved miRNAs. In an effort to trace the birth and expansion of miRNA clusters that are presently well-conserved across Drosophilids, a broad swath of metazoan species was analyzed, with particular emphasis on arthropod evolution. Beyond duplication and de novo birth, this study highlighted a diversity of modes that contribute to miRNA evolution, including neofunctionalization of miRNA copies, fissioning of locally duplicated miRNA clusters, miRNA deletion, and miRNA cluster expansion via the acquisition and/or neofunctionalization of miRNA copies from elsewhere in the genome. In particular, it is suggested that miRNA clustering by acquisition represents an expedient strategy to bring cohorts of target genes under coordinate control by miRNAs that had already been individually selected for regulatory impact on the transcriptome (Mohammed, 2014).

    Principles of microRNA-target recognition

    MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression in plants and animals. Although their biological importance has become clear, how they recognize and regulate target genes remains less well understood. This study systematically evaluates the minimal requirements for functional miRNA-target duplexes in vivo and classes of target sites with different functional properties are distinguished. Target sites can be grouped into two broad categories. 5' dominant sites have sufficient complementarity to the miRNA 5' end to function with little or no support from pairing to the miRNA 3' end. Indeed, sites with 3' pairing below the random noise level are functional given a strong 5' end. In contrast, 3' compensatory sites have insufficient 5' pairing and require strong 3' pairing for function. Examples and genome-wide statistical support is presented to show that both classes of sites are used in biologically relevant genes. Evidence is provided that an average miRNA has approximately 100 target sites, indicating that miRNAs regulate a large fraction of protein-coding genes and that miRNA 3' ends are key determinants of target specificity within miRNA families (Brennecke, 2005).

    To improve understanding of the minimal requirements for a functional miRNA target site, use was made of a simple in vivo assay in the Drosophila wing imaginal disc. A miRNA was expressed in a stripe of cells in the central region of the disc and its ability to repress the expression of a ubiquitously transcribed enhanced green fluorescent protein (EGFP) transgene containing a single target site in its 3' UTR was assessed. The degree of repression was evaluated by comparing EGFP levels in miRNA-expressing and adjacent non-expressing cells. Expression of the miRNA strongly reduced EGFP expression from transgenes containing a single functional target site (Brennecke, 2005).

    In a first series of experiments it was asked which part of the RNA duplex is most important for target regulation. A set of transgenic flies was prepared, each of which contained a different target site for miR-7 in the 3' UTR of the EGFP reporter construct. The starting site resembled the strongest bantam miRNA site in its biological target hid and conferred strong regulation when present in a single copy in the 3' UTR of the reporter gene. The effects were tested of introducing single nucleotide changes in the target site to produce mismatches at different positions in the duplex with the miRNA (note that the target site mismatches were the only variable in these experiments). The efficient repression mediated by the starting site was not affected by a mismatch at positions 1, 9, or 10, but any mismatch in positions 2 to 8 strongly reduced the magnitude of target regulation. Two simultaneous mismatches introduced into the 3' region had only a small effect on target repression, increasing reporter activity from 10% to 30%. To exclude the possibility that these findings were specific for the tested miRNA sequence or duplex structure, the experiment was repeated with miR-278 and a different duplex structure. The results were similar, except that pairing of position 8 was not important for regulation in this case. Moreover, some of the mismatches in positions 2-7 still allowed repression of EGFP expression up to 50%. Taken together, these observations support previous suggestions that extensive base-pairing to the 5' end of the miRNA is important for target site function (Brennecke, 2005).

    Next the minimal 5' sequence complementarity necessary to confer target regulation was determined. The core of 5' sequence complementarity essential for target site recognition is referred to as the 'seed'. All possible 6mer, 5mer, and 4mer seeds complementary to the first eight nucleotides of the miRNA were tested in the context of a site that allowed strong base-pairing to the 3' end of the miRNA. The seed was separated from a region of complete 3' end pairing by a constant central bulge. 5mer and 6mer seeds beginning at positions 1 or 2 are functional. Surprisingly, as few as four base-pairs in positions 2-5 confers efficient target regulation under these conditions, whereas bases 1-4 are completely ineffective. 4mer, 5mer, or 6mer seeds beginning at position 3 are less effective. These results suggest that a functional seed requires a continuous helix of at least 4 or 5 nucleotides and that there is some position dependence to the pairing, since sites that produce comparable pairing energies differ in their ability to function. These experiments also indicate that extensive 3' pairing of up to 17 nucleotides in the absence of the minimal 5' element is not sufficient to confer regulation. Consequently, target searches based primarily on optimizing the extent of base-pairing or the total, and ranking miRNA target sites according to overall complementarity or free energy of duplex formation might not reflect their biological activity (Brennecke, 2005).

    To determine the minimal lengths of 5' seed matches that are sufficient to confer regulation alone, single sites were tested that pair with eight, seven, or six consecutive bases to the miRNA's 5' end, but that do not pair to its 3' end. Surprisingly, a single 8mer seed (miRNA positions 1-8) is sufficient to confer strong regulation by the miRNA. A single 7mer seed (positions 2-8) is also functional, although less effective. The magnitude of regulation for 8mer and 7mer seeds is strongly increased when two copies of the site are introduced in the UTR. In contrast, 6mer seeds show no regulation, even when present in two copies. Comparable results have been reported for two copies of an 8mer site with limited 3' pairing capacity in a cell-based assay. These results do not support a requirement for a central bulge (Brennecke, 2005).

    From these experiments it is concluded that (1) complementarity of seven or more bases to the 5' end miRNA is sufficient to confer regulation, even if the target 3' UTR contains only a single site; (2) sites with weaker 5' complementarity require compensatory pairing to the 3' end of the miRNA in order to confer regulation, and (3) extensive pairing to the 3' end of the miRNA is not sufficient to confer regulation on its own without a minimal element of 5' complementarity (Brennecke, 2005).

    While recognizing that there is a continuum of base-pairing quality between miRNAs and target sites, the experiments presented here suggest that sites that depend critically on pairing to the miRNA 5' end (5' dominant sites) can be distinguished from those that cannot function without strong pairing to the miRNA 3' end (3' compensatory sites). The 3' compensatory group includes seed matches of four to six base-pairs and seeds of seven or eight bases that contain G:U base-pairs, single nucleotide bulges, or mismatches (Brennecke, 2005).

    It is useful to distinguish two subgroups of 5' dominant sites: those with good pairing to both 5' and 3' ends of the miRNA (canonical sites) and those with good 5' pairing but with little or no 3' pairing (seed sites). Seed sites are considered to be those where there is no evidence for pairing of the miRNA 3' end to nearby sequences that is better than would be expected at random. The possibility cannot be excluded that some sites identified as seed sites might be supported by additional long-range 3' pairing. Computationally, this is always possible if long enough loops in the UTR sequence are allowed. Whether long loops are functional in vivo remains to be determined (Brennecke, 2005).

    Canonical sites have strong seed matches supported by strong base-pairing to the 3' end of the miRNA. Canonical sites can thus be seen as an extension of the seed type (with enhanced 3' pairing in addition to a sufficient 5' seed) or as an extension of the 3' compensatory type (with improved 5' seed quality in addition to sufficient 3' pairing). Individually, canonical sites are likely to be more effective than other site types because of their higher pairing energy, and may function in one copy. Due to their lower pairing energies, seed sites are expected to be more effective when present in more than one copy (Brennecke, 2005).

    Most currently identified miRNA target sites are canonical. For example, the hairy 3' UTR contains a single site for miR-7, with a 9mer seed and a stretch of 3' complementarity. This site has been shown to be functional in vivo , and it is strikingly conserved in the seed match and in the extent of complementarity to the 3' end of miR-7 in all six orthologous 3' UTRs (Brennecke, 2005).

    Although seed sites have not been previously identified as functional miRNA target sites, there is some evidence that they exist in vivo. For example, the Bearded (Brd) 3' UTR contains three sequence elements, known as Brd boxes, that are complementary to the 5' region of miR-4 and miR-79. Brd boxes have been shown to repress expression of a reporter gene in vivo, presumably via miRNAs; expression of a Brd 3' UTR reporter is elevated in dicer-1 mutant cells, which are unable to produce any miRNAs. All three Brd box target sites consist of 7mer seeds with little or no base-pairing to the 3' end of either miR-4 or miR-79. The alignment of Brd 3' UTRs shows that there is little conservation in the miR-4 or miR-79 target sites outside the seed sequence, nor is there conservation of pairing to either miRNA 3' end. This suggests that the sequences that could pair to the 3' end of the miRNAs are not important for regulation as they do not appear to be under selective pressure. This makes it unlikely that a yet unidentified Brd box miRNA could form a canonical site complex (Brennecke, 2005).

    The 3' UTR of the HOX gene Sex combs reduced (Scr) provides a good example of a 3' compensatory site. Scr contains a single site for miR-10 with a 5mer seed and a continuous 11-base-pair complementarity to the miRNA 3' end. The miR-10 transcript is encoded within the same HOX cluster downstream of Scr, a situation that resembles the relationship between miR-iab-5p and Ultrabithorax in flies and miR-196/HoxB8 in mice. The predicted pairing between miR-10 and Scr is perfectly conserved in all six drosophilid genomes, with the only sequence differences occurring in the unpaired loop region. The site is also conserved in the 3' UTR of the Scr genes in the mosquito, Anopheles gambiae, the flour beetle, Tribolium castaneum, and the silk moth, Bombyx mori. Conservation of such a high degree of 3' complementarity over hundreds of millions of years of evolution suggests that this is likely to be a functional miR-10 target site. Extensive 5' and 3' sequence conservation is also seen for other 3' compensatory sites, e.g., the two let-7 sites in lin-41 or the miR-2 sites in grim and sickle (Brennecke, 2005).

    Several families of miRNAs have been identified whose members have common 5' sequences but differ in their 3' ends. In view of the evidence that 5' ends of miRNA are functionally important, and in some cases sufficient, it can be expected that members of miRNA families may have redundant or partially redundant functions. According to this model, 5' dominant canonical and seed sites should respond to all members of a given miRNA family, whereas 3' compensatory sites should differ in their sensitivity to different miRNA family members depending on the degree of 3' complementarity. This is being tested using the wing disc assay with 3' UTR reporter transgenes and overexpression constructs for various miRNA family members (Brennecke, 2005).

    miR-4 and miR-79 share a common 5' sequence that is complementary to a single 8mer seed site in the bagpipe 3' UTR. The 3' ends of the miRNAs differ. miR-4 is predicted to have 3' pairing at approximately 50% of the maximally possible level (~10.8 kcal/mol), whereas the level of 3' pairing for miR-79 is approximately 25% maximum (~6.1 kcal/mol), which is below the average level expected for random matches. Both miRNAs repressed expression of the bagpipe 3' UTR reporter, regardless of the 3' complementarity. This indicates that both types of site are functional in vivo and suggests that bagpipe is a target for both miRNAs in this family (Brennecke, 2005).

    To test whether miRNA family members can also have non-overlapping targets, 3' UTR reporters were used of the pro-apoptotic genes grim and sickle, two recently identified miRNA targets. Both genes contain K boxes in their 3' UTRs that are complementary to the 5' ends of the miR-2, miR-6, and miR-11 miRNA family. These miRNAs share residues 2-8 but differ considerably in their 3' regions. The site in the grim 3' UTR is predicted to form a 6mer seed match with all three miRNAs, but only miR-2 shows the extensive 3' complementarity that would be needed for a 3' compensatory site with a 6mer seed to function (~19.1 kcal/mol, 63% maximum 3' pairing, versus ~10.9 kcal/mol, 46% maximum, for miR-11 and ~8.7 kcal/mol, 37% maximum, for miR-6). Indeed, only miR-2 is able to regulate the grim 3' UTR reporter, whereas miR-6 and miR-11 are non-functional (Brennecke, 2005).

    The sickle 3' UTR contains two K boxes and provides an opportunity to test whether weak sites can function synergistically. The first site is similar to the grim 3' UTR in that it contains a 6mer seed for all three miRNAs but extensive 3' complementarity only to miR-2. The second site contains a 7mer seed for miR-2 and miR-6 but only a 6mer seed for miR-11. miR-2 strongly downregulates the sickle reporter, miR-6 has moderate activity (presumably via the 7mer seed site), and miR-11 has nearly no activity, even though the miRNAs were overexpressed. The fact that a site is targeted by at least one miRNA argues that it is accessible (e.g., miR-2 is able to regulate both UTR reporters), and that the absence of regulation for other family members is due to the duplex structure. These results are in line with what would be expected based on the predicted functionality of the individual sites, and indicate that the model of target site functionality can be extended to UTRs with multiple sites. Weak sites that do not function alone also do not function when they are combined (Brennecke, 2005).

    To show that endogenous miRNA levels regulate all three 3' UTR reporters, EGFP expression was compared in wild-type cells and dicer-1 mutant cells, which are unable to produce miRNAs. dicer-1 clones did not affect a control reporter lacking miRNA binding sites, but showed elevated expression of a reporter containing the 3' UTR of the previously identified bantam miRNA target hid. Similarly, all 3' UTR reporters above were upregulated in dicer-1 mutant cells, indicating that bagpipe, sickle, and grim are subject to repression by miRNAs expressed in the wing disc. Taken together, these experiments indicate that transcripts with 5' dominant canonical and seed sites are likely to be regulated by all members of a miRNA family. However, transcripts with 3' compensatory sites can discriminate between miRNA family members (Brennecke, 2005).

    Experimental tests such as those presented in this study and the observed evolutionary conservation suggest that all three types of target sites are likely to be used in vivo. To gain additional evidence the occurrence of each site type was examined in all Drosophila 3' UTRs. Use was made of the D. pseudoobscura genome, the second assembled drosophilid genome, to determine the degree of site conservation for the three different site classes in an alignment of orthologous 3' UTRs. From the 78 known Drosophila miRNAs, a set of 49 miRNAs with non-redundant 5' sequences was chosen. Whether sequences complementary to the miRNA 5' ends are better conserved than would be expected for random sequences was tested. For each miRNA, a cohort of ten randomly shuffled variants was constructed. To avoid a bias for the number of possible target matches, the shuffled variants were required to produce a number of sequence matches comparable (±15%) to the original miRNAs for D. melanogaster 3' UTRs. 7mer and 8mer seeds complementary to real miRNA 5' ends were significantly better conserved than those complementary to the shuffled variants. Conserved 8mer seeds for real miRNAs occur on average 2.8 times as often as seeds complementary to the shuffled miRNAs. For 7mer seeds this signal was 2:1, whereas 6mer, 5mer, and 4mer seeds did not show better conservation than expected for random sequences. To assess the validity of these signals and to control for the random shuffling of miRNAs, this procedure was repeated with 'mutant' miRNAs in which two residues in the 5' region were changed. There was no difference between the mutant test miRNAs and their shuffled variants. This indicates that a substantial fraction of the conserved 7mer and 8mer seeds complementary to real miRNAs identify biologically relevant target sites (Brennecke, 2005).

    Direct measurement of pervasive weak repression by microRNAs and their role at the network level

    A gene regulatory network (GRN) comprises many weak links that are often regulated by microRNAs. Since miRNAs rarely repress their target genes by more than 30%, doubts have been expressed about the biological relevance of such weak effects. These doubts raise the possibility of under-estimation as miRNA repression is usually estimated indirectly from equilibrium expression levels. To measure miRNA repression directly, transcript synthesis was inhibited in Drosophila larvae and time-course data collected on mRNA abundance, the decline of which reflects transcript degradation. The rate of target degradation in the absence of miR310s, a moderately expressed miRNA family, was found to decrease by 5 to 15%. A conventional analysis that does not remove transcript synthesis yields an estimate of 6.5%, within the range of the new estimates. These data permit further examinations of the repression mechanisms by miRNAs including seed matching types, APA (alternative polyadenylation) sites, effects of other highly-expressed miRNAs and the length of 3'UTR. The direct measurements suggest the latter two factors have a measurable effect on decay rate. The direct measurement confirms pervasive weak repression by miRNAs, supporting the conclusions based on indirect assays. The confirmation suggests that this weak repression may indeed be miRNAs' main function. In this context, the recent proposal is discussed that weak repression is "cumulatively powerful" in stabilizing GRNs (Ma, 2018).

    Antisense-mediated depletion reveals essential and specific functions of microRNAs in Drosophila development

    MicroRNAs are small noncoding RNAs that control gene function posttranscriptionally through mRNA degradation or translational inhibition. Much has been learned about the processing and mechanism of action of microRNAs, but little is known about their biological function. Injection of 2′O-methyl antisense oligoribonucleotides (2'OM-ORNs) into early Drosophila embryos leads to specific and efficient depletion of microRNAs and thus permits systematic loss-of-function analysis in vivo. Twenty-five of the forty-six embryonically expressed microRNAs show readily discernible defects; pleiotropy is moderate and family members display similar yet distinct phenotypes. Processes under microRNA regulation include cellularization and patterning in the blastoderm, morphogenesis, and cell survival. The largest microRNA family in Drosophila (miR-2/6/11/13/308) is required for suppressing embryonic apoptosis; this is achieved by differential posttranscriptional repression of the proapoptotic factors hid, grim, reaper, and sickle. These findings demonstrate that microRNAs act as specific and essential regulators in a wide range of developmental processes (Leaman, 2005).

    miR-9 affects cellularization: Embryos injected with miR-9 antisense 2′OM-ORNs rarely form any cuticle and show virtually no internal differentiation. Examination of early embryogenesis, using phalloidin and DNA staining as well as DIC, reveal severe defects in nuclear division and migration, pole cell formation, cellularization, and in the basal movement of yolk droplets. To establish that these defects are in fact due to depletion of miR-9, whether they can be rescued by genomic overexpression of mir-9 was tested. Expression of mir-9 with a strong maternal driver (nos-Gal4VP16;UAS-mir-9a) has no effect on its own, but significantly ameliorates the phenotype of miR-9 antisense injection, confirming that a reduction in miR-9 activity is responsible for the defect. Most of the processes affected by miR-9 depletion are complex, but all share an involvement of the microtubule cytoskeleton. Therefore, miR-9 may have a single or a small number of phenocritical targets involved in microtubule function, but a more pleiotropic role cannot be excluded (Leaman, 2005).

    miR-31 affects segmentation: In contrast to miR-9, miR-31 depleted embryos complete development but show severe segmentation defects. Embryos show abnormal cuticle patterns, ranging from partial fusions of denticle belts to a complete loss of alternating segments, suggesting that pattern formation is disrupted at the level of the pair rule genes. Further examination of pair rule gene expression in the blastoderm shows severe pattern abnormalities for even skipped (eve) and fushi tarazu (ftz), as well as hairy, indicating that misregulation must occur above the pair rule gene level in the segmentation gene hierarchy. Since pattern formation is affected throughout the segmented portion of the embryo, the regional gap factors are less likely to be responsible than ubiquitous or widely expressed factors such as components of the JAK/STAT pathway, Dichaete, grainy head, or Grunge (Leaman, 2005).

    The miR-310 family affects dorsal closure: Embryos injected with antisense 2′OM-ORNs for the miR-310/311/312/313/92 family show morphogenetic defects in later development. In cuticle preparations, all family members show head-involution defects; in addition, miR-311 and miR-312 show mild dorsal-closure defects, and miR-313 occasional germ band-retraction defects; miR-310 and miR-313 also show occasional segmentation defects. Germ band retraction, dorsal closure, and head involution are interconnected morphogenetic processes that share the involvement of several cellular structures and pathways, including the cytoskeleton and cell junctions, and JNK and Dpp signaling. Note that despite sequence identity at positions 2–8, the members of the miR-310 family show some differences in their depletion phenotypes, suggesting that the 3′ end of the miRNA contributes to the specificity of the miRNA:mRNA pairing (Leaman, 2005).

    miR-2/13 and miR-6 depletion results in catastrophic apoptosis: Embryos injected with miR-2/13 and miR-6 antisense 2′OM-ORNs fail to differentiate normal internal and external structures. At the end of embryogenesis, the embryos fall apart on touch, and no cuticle is recovered. To determine the onset of these problems, blastoderm embryos were examined, and it was found that cellularization and early pattern formation along the anteroposterior axis occur normally for both miRNAs, indicating that early fating and morphogenesis are intact. Interestingly, in miR-6, but not miR-2/13 depleted embryos, pole cell formation at the posterior end is disrupted (Leaman, 2005).

    One possible cause of the catastrophic defects observed in miR-2/13 and miR-6 depleted embryos is excessive and widespread apoptosis. In both miR-2/13 and miR-6 antisense injected embryos, the number of apoptotic cells is greatly increased compared to wild-type by stage 13. Notably, the overall morphology of miR-6 depleted embryos is much more affected than that of miR-2/13 depleted embryos. miR-6 depleted embryos are generally smaller in size and have fewer and abnormally large (para-) segments, suggesting greater excess or earlier onset of apoptosis (Leaman, 2005).

    To determine the specificity of the effects of miR-6 and miR-2/13 antisense injections, genomic rescue experiments were carried out. Embryos ubiquitously overexpressing mir-6 or mir-2 (Actin-Gal4;UAS-mir6-3/2b-2) show normal cell-death patterns. When injected with miR-6 or miR-2/13 antisense, they show significant rescue of miR-6 antisense by mir-6, with respect to both cell death and morphology, and of miR-2/13 antisense by mir-2. Interestingly, crossrescue of miR-6 antisense by mir-2 overexpression and of miR-2/13 antisense by mir-6 is weak (Leaman, 2005).

    The miRNA sequence family miR-6 and miR-2/13 belong to has two additional members, miR-11 and miR-308. Depletion of miR-11 results in a moderate and of miR-308 in a mild increase in apoptosis in midembryogenesis. Thus, for all members of the miR-2 family, antisense-induced depletion results in excess embryonic cell death, but with marked differences in phenotypic strength. This differential could be due to differences in expression level or to sequence divergence and thus differential interaction with target mRNAs (Leaman, 2005).

    The miR-2 family regulates cell survival by translational repression of proapoptotic factors: In Drosophila, three pathways are known to control caspase activity. The main control is thought to come from the proapoptotic factors Hid, Grim, and Reaper (Rpr), which are transcriptionally activated in response to a range of natural and toxic conditions; they promote caspase activation through inhibition of the caspase inhibitor Diap1. The three factors appear to act independently, with each being sufficient to drive apoptosis. When miR-2/13 and miR-6 antisense 2′OM-ORNs are injected into embryos deficient for the hid, grim, and rpr genes (H99 deficiency), they are unable to trigger apoptosis, indicating that these miRNAs act through hid, grim, and/or rpr (Leaman, 2005).

    To determine whether the regulation of the three proapoptotic factors occurs at the transcriptional or at the posttranscriptional level, their RNA expression was examined in miR-2/13 and miR-6 depleted embryos using in situ hybridization and quantitative PCR. No significant increase was found in the expression level or broadening of the pattern compared to control embryos for any of three transcripts, either at embryonic stage 13 or 1 hr earlier at embryonic stage 12. By contrast, the protein expression of Hid is dramatically increased in miR-6 depleted embryos and modestly in miR-2/13 depleted embryos. These results strongly argue against a transcriptional and in favor of a posttranscriptional regulation of the proapoptotic factors by miR-2/13 and miR-6 (Leaman, 2005).

    To test this directly, two existing translation control assays were adapted to the embryonic paradigm. In the first assay, full-length 3′UTRs are fused to a ubiquitously transcribed sensor (tub-GFP); transgenic embryos are injected with sense or antisense 2′OM-ORNs, and GFP fluorescence is measured. The 3′UTRs of hid, grim, rpr, and sickle (skl, a structurally related but less potent proapoptotic factor display marked differences in sensor expression, with rpr showing no expression, hid and skl low uniform expression, and grim strong and spatially modulated expression, indicating that these proapoptotic factors experience quite different levels of translation control. To gauge the efficacy of the assay, hid GFP sensor embryos were injected with bantam antisense 2′OM-ORNs, and mild but statistically significant derepression of GFP expression was found as compared to control, consistent with the weak cell-death phenotype of bantam depleted embryos. Antisense injection of miR-2 family members reveals strong derepression of the hid GFP sensor by miR-6 antisense, but not by miR-2/13, 11, or 308 antisense. Conversely, the grim GFP sensor shows significant derepression as a result of miR-2/13, 11, and 308, but not miR-6 depletion. Finally, the skl GFP sensor shows significant derepression for all four family members (Leaman, 2005).

    To assess effects on rpr, a second, more sensitive assay was developed that employs transient expression of a dual-luciferase vector in injected embryos. For initial comparison with the GFP assay, a hid luciferase sensor was tested against the entire miR-2 family and the same profile was found. The rpr luciferase sensor shows strong derepression in miR-6 and 2/13, moderate derepression in miR-11, and no significant effect in miR-308 depleted embryos. Thus, the 3′UTRs of all four proapoptotic factors are subject to translational repression by the miR-2 family, but each miRNA displays a distinct interaction profile. The interaction preferences correlate well with the observed differences in phenotype: miR-6 has the most severe death phenotype and is the only family member to regulate hid, the factor with the broadest expression and the strongest proapoptotic effect. mir-2/13 and miR-11 have the same overall profile, but they differ in the strength of their interaction with rpr and show a corresponding differential in phenotypic strength. Finally, miR-308, which has the mildest death phenotype, interacts only with the weakly proapoptotic skl and with grim (Leaman, 2005).

    The differences in target interaction profile between the miR-2 family members are pronounced and do not merely reproduce differences in the strength or onset of miRNA expression. This suggests that differential pairing outside the 5′ core sequence shared by all members has an important role in target selection. Computational predictions indicate that miR-2 family binding sites are present in the 3′UTRs of all four proapoptotic factors: rpr and grim have one, hid and skl two predicted sites. All six miRNA target sites lie in sequence blocks that are conserved between the six sequenced Drosophilid species, spanning an evolutionary distance of 40 Myr. Interestingly, for all sites, absolute conservation extends well beyond the bases complementary to the 5′ core of the miRNA and includes adjacent stretches suitable for pairing with the 3′ end. All but one of the sites show Watson-Crick pairing with miRNA positions 2-7 and variable pairing at the 3′ end. One of the hid sites (hid468) has a mismatch in the core but shows strong pairing with miR-6 at the 3′ end. The rules for 3′ pairing between miRNAs and their targets are not yet well understood, but it is clear that the miR-2 family members differ considerably in their ability to form 3′ matches with the six target sites. Further experimentation will be required to better understand how the observed differences in regulatory effect relate to differences in sequence pairing (Leaman, 2005).

    Distinct populations of primary and secondary effectors during RNAi in C. elegans; Secondary effectors are derived from RNA-directed RNA polymerase (RdRP) activity

    RNA interference (RNAi) is a phylogenetically widespread gene-silencing process triggered by double-stranded RNA. In plants and Caenorhabditis elegans, two distinct populations of small RNAs have been proposed to participate in RNAi: 'Primary siRNAs' (derived from Dicer nuclease-mediated cleavage of the original trigger) and 'secondary siRNAs' [additional small RNAs whose synthesis requires an RNA-directed RNA polymerase (RdRP)]. Analyzing small RNAs associated with ongoing RNAi in C. elegans, it was found that secondary siRNAs constitute the vast majority. The bulk of secondary siRNAs exhibit structure and sequence indicative of a biosynthetic mode whereby each molecule derives from an independent de novo initiation by RdRP. Analysis of endogenous small RNAs indicated that a fraction derive from a biosynthetic mechanism that is similar to that of secondary siRNAs formed during RNAi, suggesting that small antisense transcripts derived from cellular messenger RNAs by RdRP activity may have key roles in cellular regulation (Pak, 2007).

    Double-stranded RNA (dsRNA)–triggered gene silencing in eukaryotes appears universally to involve 21- to 25-nucleotide (nt) siRNA effectors. In Drosophila and mammals, siRNAs derive primarily from processing of longer duplexes by Dicer nuclease, forming 21- to 25-nt duplexes possessing 5'-monophosphates, 3'-hydroxyl groups, and 2-nt 3' overhangs. Along with this 'primary' siRNA response, amplification of the RNA trigger population has been proposed to contribute to potency and persistence of gene silencing in several systems. Amplification mechanisms are accompanied in some cases by 'transitive RNAi' phenomena in which dsRNA matching one mRNA region can silence targets bearing homology to other parts of the mRNA. Unlike the situation in plants where 'spreading' of the effector population occurs bidirectionally relative to the target mRNA, transitive RNAi in C. elegans exhibits a strong bias toward sequences upstream of trigger homology. Transitive RNAi requires function of a putative RdRP (RRF-1 in C. elegans soma, SDE1/SGS2 in Arabidopsis thaliana), suggesting several conceivable means for secondary siRNA production. One possibility is that antisense primary siRNAs could act as primers in the RdRP-mediated synthesis of new dsRNAs on an mRNA template. Alternatively, primary siRNAs may merely guide the RdRP to a target, allowing unprimed synthesis either at the cleaved end of the targeted transcript, at a location close to the trigger-target complex, or at a structure such as a free end that might be revealed as aberrant through consequences of the initial RNA-induced silencing complex (RISC)::target interaction (Pak, 2007).

    To better understand signal amplification in C. elegans, small RNAs were characterized from animals undergoing RNAi against an abundantly expressed endogenous gene, sel-1. After reverse transcription, 245,420 18- to 25-nt RNAs were sequenced by means of single-molecule pyrosequencing. Among these sequences, 534 exhibited either a perfect match (428 instances) or single mismatches (106 instances) to sel-1 mRNA. A similar analysis of ~850,000 clones from animals not exposed to dsRNA yielded just one sel-1 small RNA. Most sel-1 small RNAs induced during interference (483) had an antisense orientation, consistent with previous hybridization-based analyses. Of the 51 sense strand clones, 22 showed complementarity to at least one antisense clone (Pak, 2007).

    An incomplete bias was observed in siRNA positions relative to the trigger; of 138 antisense siRNAs outside the original trigger, 110 (80%) occurred on the 5' side. This bias could certainly account for preferential detection of upstream secondary responses in functional and biochemical assays. Twenty-eight observed instances of small antisense RNAs completely downstream of the trigger homology were of particular interest, since these would not have been expected if the sole mode of amplification involved extension by RdRP of existing siRNA triggers that hybridize to the target transcript (Pak, 2007).

    Exon-exon junctions offer a unique opportunity to unequivocally distinguish de novo synthesis of antisense nucleic acids from an mRNA template. It was found 50 sel-1 small antisense RNA sequences that span exon/exon junctions. Of these, 43 fall within the trigger [458 base pairs (bp) of sel-1 cDNA sequence] and thus could have derived directly from triggering dsRNA. Six antisense exon-exon junction sequences upstream of the trigger were recovered (four matching perfectly and two with single mismatches). These imply de novo copying of the mature mRNA template (Pak, 2007).

    The apparent scarcity of sel-1 siRNAs suggested that the procedure for cloning small RNAs (including ligation of linkers to 3' and 5' ends) might underrepresent the siRNA population. To analyze small RNA termini in detail, a number of structure-specific treatments were used. Treatment of RNA with periodate followed by ß elimination results in a shift on a denaturing acrylamide gel, indicating at least one unmodified (cis-diol) 3' terminus. Ribonuclease T (RNaseT) requires a 3'-hydroxyl to degrade single-stranded RNA. Finally, Terminator exonuclease preferentially degrades substrates with a single 5'-phosphate. Although sel-1 siRNAs are susceptible to both ß elimination and RNaseT reactions, they are resistant to Terminator. Control synthetic 25-nt sel-1 RNA oligonucleotides s with 5'-monophosphate and 3'-OH were sensitive to all three treatments. It is surmised that sel-1 siRNAs are blocked at their 5' ends (Pak, 2007).

    It was next asked if a cloning protocol could be designed that would not be biased by the structure at the 5' end on an siRNA. The resulting protocol avoids both (1) the requirement for ligation of the 5' end of the RNA and (2) the possibility that modified 5' ends on small RNAs could affect enzymatic treatments of the paired cDNA strand. 127 sel-1 antisense sequences were observed and zero sense sequences from 1612 total clones using this protocol. For sel-1 antisense sequences, this represents a 40-fold enrichment compared to the 5'-ligation-dependent cloning method, providing further evidence for a prominent population of 5'-blocked siRNAs (Pak, 2007).

    Secondary siRNAs are still recovered in 5'-ligation-dependent cloning, albeit inefficiently, as indicated by the representation of sequences outside the trigger (presumably most siRNAs within the trigger are also secondary). Notably, it was found that small antisense segments cloned with a 5'-ligation-independent procedure were on average 1 nt longer than those cloned with a 5'-ligation-dependent procedure. The substantial increase in incidence of sel-1 clones that followed 5'-ligation-independent cloning indicates that the vast majority of small sel-1 RNAs are modified on their 5' ends, while at most 2 to 3% have simple 5'-phosphate termini that are exposed in vivo or produced by 5' cleavage during the cloning procedure. An assumption that sense and antisense are roughly equal in the primary siRNA pool leads to primary siRNA estimates of <0.6% of the total sel-1 siRNA population and <0.05% of the total 21- to 25-nt RNAs in the animal (Pak, 2007).

    The two methods of cloning were selective for different classes of endogenous small RNAs. microRNAs (miRNAs) appeared much less frequently with the 5'-ligation-independent cloning method, seemingly replaced by endogenous small RNAs corresponding to antisense sequence from coding regions. This analysis suggests that miRNAs and small antisense RNAs could be comparably abundant in C. elegans, with 5' modification of the small antisense RNAs accounting for the predominance of miRNA clones in libraries derived using ligation-dependent schemes. 612 out of 245,420 clones from the 5'-ligation-dependent method and 9 out of 1612 clones from the 5'-ligation-independent method were observed that were perfect antisense copies of exon/exon junctions, suggesting synthesis by RdRP acting on an mRNA template (Pak, 2007).

    To further characterize the modification of siRNAs in C. elegans, a ligation assay and Terminator 5'-exonuclease treatment (both requiring a 5'-phosphate) were used. Sensitivity of the predominant fraction of the siRNAs could be restored by sequential treatment with alkaline phosphatase (which removes any number of 5'-phosphates) and T4 polynucleotide kinase (which adds a single 5'-phosphate), suggesting that the 5' modification was likely to involve additional 5'-phosphate groups on the siRNA. How many phosphates do these molecules have on their 5' ends? Examining relative gel mobilities of the native and dephosphorylated siRNAs, using a variety of gel porosities (and using a series of synthetic RNA markers with different numbers of phosphates), indicated that the predominant fraction of the untreated siRNAs have triphosphate 5' termini (Pak, 2007).

    The results presented here define an RNA population produced de novo during RNAi in C. elegans as a pool of 5'-triphosphate–terminated small antisense molecules templated by the mature mRNA target and covering sequences both upstream and downstream of the original dsRNA trigger. The current working model for amplified gene silencing in C. elegans is that rare primary siRNAs, formed from a long dsRNA trigger, act as guides (presumably in an Argonaute-dependent manner) to recruit RdRP to targeted transcripts. This recruitment leads to de novo synthesis of short antisense RNAs that must be stripped off the template mRNA and incorporated into complexes that are capable of finding additional silencing targets. This model differs from other examples of RdRP action, such as in the generation of ta-siRNAs in A. thaliana where repeated Dicer activity on long RdRP-generated dsRNAs produces a phased distribution of small RNAs (Pak, 2007).

    Although ongoing RNAi is certainly important for de novo synthesis of antisense siRNAs, this process appears to contribute by providing guidance to the RdRP rather than priming activity. Primary silencing targets may or may not be degraded; whatever their fate, however, they remain intact for a sufficient period to be substrates for RdRP activity upstream and (somewhat less efficiently) downstream of the targeting site. This model is consistent with the biochemical properties of characterized cellular RdRPs in that these enzymes are capable of unprimed (as well as primed) synthesis. Initiation at 3' ends of potential templates has been reported for fungal and plant RdRPs; this might allow initial Argonaute-mediated cleavage of mRNA targets to yield ready RdRP substrates (Pak, 2007).

    Previous investigations of siRNA structure revealing double-stranded character, 3' overhangs, and 5'-monophosphate termini were performed in organisms whose genomes do not encode canonical RdRPs. In addition, crystal structures of Argonaute proteins [key executioners in the RNAi pathway indicate considerable specificity in recognizing specific 5' structures in RNA. It is possible that 5'-triphosphate antisense RNAs are themselves inactive in gene silencing, requiring either removal of two terminal phosphates or of the entire first base for activity. Alternatively, triphosphate-terminated small RNAs may be active directly as silencing triggers, potentially through distinct members of the Argonaute family that might recognize guide RNAs with a 5'-triphosphate (Pak, 2007).

    One feature of the proposed mechanism is the involvement of dsRNA trigger only at the earliest stage of the process (production of primary siRNAs). Following this stage, the double-stranded character of the original trigger plays no role in the reaction. Given diverse structural features that could target an aberrant mRNA for RdRP activity, such a system would permit analogous machineries (each involving RdRP, helicase, and Argonaute activities) to serve in amplified surveillance processes triggered by both aberrant mRNA structure and dsRNA (Pak, 2007).

    Genome-wide identification and developmental expression profiling of long noncoding RNAs during Drosophila metamorphosis

    An increasing number of long noncoding RNAs (lncRNAs) have been discovered with the recent advances in RNA-sequencing technologies. lncRNAs play key roles across diverse biological processes, and are involved in developmental regulation. However, knowledge about how the genome-wide expression of lncRNAs is developmentally regulated is still limited. This study performed a whole-genome identification of lncRNAs followed by a global expression profiling of these lncRNAs during development in Drosophila melanogaster. Bioinformatic prediction of lncRNAs were combined with stringent filtering of protein-coding transcripts and experimental validation to define a high-confidence set of Drosophila lncRNAs. 1,077 lncRNAs were identified in the given transcriptomes that contain 43,967 transcripts; among these, 646 lncRNAs are novel. In vivo expression profiling of these lncRNAs in 27 developmental processes revealed that the expression of lncRNAs is highly temporally restricted relative to that of protein-coding genes. Remarkably, 21% and 42% lncRNAs were significantly upregulated at late embryonic and larval stage, the critical time for developmental transition. The results highlight the developmental specificity of lncRNA expression, and reflect the regulatory significance of a large subclass of lncRNAs for the onset of metamorphosis. The systematic annotation and expression analysis of lncRNAs during Drosophila development form the foundation for future functional exploration (Chen, 2016).

    Comparative expression dynamics of intergenic long noncoding RNAs (lncRNAs) in the genus Drosophila

    This study identified and characterized long noncoding RNAs (lncRNAs) in D. pseudoobscura. Using RNA-Seq and computational filtering of protein-coding potential, 1,589 intergenic lncRNA loci were identified in D. pseudoobscura. Multiple sex-specific developmental stages were surveyed and, like in D. melanogaster, increasingly prolific lncRNA expression was found through male development and an overrepresentation of lncRNAs was found in the testes. Other trends seen in D. melanogaster, like reduced pupal expression, were not observed. Nonrandom distributions of female-biased and non-testis-specific male-biased lncRNAs between the X chromosome and autosomes are consistent with selection-based models of gene trafficking to optimize genomic location of sex-biased genes. The numerous testis-specific lncRNAs, however, are randomly distributed between the X and autosomes, and the hypothesis that many of these are likely to be spurious transcripts cannot be rejected. Finally, using annotated lncRNAs in both species, 134 putative lncRNA homologs were found between D. pseudoobscura and D. melanogaster, and many were found to have conserved developmental expression dynamics, making them ideal candidates for future functional analyses (Nyberg, 2016).

    Maternally inherited stable intronic sequence RNA triggers a self-reinforcing feedback loop during development

    Maternally inherited noncoding RNAs (ncRNAs) can regulate zygotic gene expression across generations. Recently, many stable intronic sequence RNAs (sisRNAs), which are byproducts of pre-mRNA splicing, were found to be maternally deposited and persist till zygotic transcription in Xenopus and Drosophila. In various organisms, sisRNAs can be in linear or circular conformations, and they have been suggested to regulate host gene expression. It is unknown whether maternally deposited sisRNAs can regulate zygotic gene expression in the embryos. This study shows that a maternally inherited sisRNA (sisR-4) from the deadpan locus is important for embryonic development in Drosophila. Mothers, but not fathers, mutant for sisR-4 produce embryos that fail to hatch. During embryogenesis, sisR-4 promotes transcription of its host gene (deadpan), which is essential for development. Interestingly, sisR-4 functions by activating an enhancer present in the intron where sisR-4 is encoded. It is proposed that a maternal sisRNA triggers expression of its host gene via a positive feedback loop during embryogenesis (Tay, 2017).

    An mRNA m7G cap binding-like motif within human Ago2 represses translation

    microRNAs (miRNAs) bind to Argonaute (Ago) proteins and inhibit translation or promote degradation of mRNA targets. Human let-7 miRNA inhibits translation initiation of mRNA targets in an m7G cap-dependent manner and also appears to block protein production, but the molecular mechanism(s) involved is unknown and the role of Ago proteins in translational regulation remains elusive. This study identified a motif (MC) within the Mid domain of Ago proteins, which bears significant similarity to the m7G cap-binding domain of eIF4E, an essential translation initiation factor. Conserved aromatic residues were identified within the MC motif of human Ago2 that are required for binding to the m7G cap and for translational repression but do not affect the assembly of Ago2 with miRNA or its catalytic activity. It is proposed that Ago2 represses the initiation of mRNA translation by binding to the m7G cap of mRNA targets, thus likely precluding the recruitment of eIF4E (Kiriakidou, 2007).

    An important feature of miRNA-directed translational repression is its apparent cooperativity: increasing the number of miRNA recognition elements (MREs) in the 3′-UTR of an mRNA target enhances translational repression. Cooperativity is also seen when multiple MREs for different miRNAs are found in the 3′-UTR of the same mRNA target, arguing that common factors, notably Ago proteins, bound to all miRNAs are responsible for the enhanced translational repression. Indeed, this cooperativity is accurately recapitulated in experiments with tethered Ago2; increasing the number of BoxB sites in the 3′-UTR of the reporter leads to enhancement of the translational repression by λN-HA-Ago2. It is proposed that multiple MREs, within the same mRNA target, increase the number of Ago2 molecules bound to the mRNA, thus increasing the probability that they will interact with the m7G cap and augment translational repression by limiting availability of the m7G cap to eIF4E. In this model, Ago2 binds to m7G cap less efficiently than eIF4E. Therefore, optimal repression by Ago2 and thus optimal eIF4E competition would require multiple Ago2 molecules. Weak Ago2 binding to the m7G cap also makes biological sense, since an Ago2 protein with high affinity to the m7G cap would lead to generalized and strong translational inhibition. This model is also consistent with weak translational repression of mRNA targets that bear single MREs. Indeed, the vast majority of mRNA targets contain a single MRE for any given miRNA and the level of translational repression is typically modest (usually 1.5- to 2-fold repression). Such modest and noncomplete repression may also explain why many miRNAs cosediment with actively translating, endogenous, mRNAs in polysomes. Lastly, these findings do not exclude additional mechanisms of miRNA and Ago regulation, perhaps in the presence of additional factors such as inhibition of protein production on actively translating ribosomes during elongation or degradation of mRNAs (Kiriakidou, 2007).

    An important observation is that the MC motif is not detected in Ago proteins from organisms that do not contain miRNAs, or do not use miRNAs for translational repression. Specifically, all mammalian Ago proteins and certain Ago proteins from nematodes and flies, where translational repression by miRNAs has been demonstrated, contain the MC domain, and thus these Ago proteins may be capable of repressing translation. The MC domain is present in Drosophila AGO1, which is required for miRNA function, but not in Drosophila AGO2, which functions predominantly in siRNA pathways, although more recent studies show overlapping functions of Ago1 and Ago2 pathways in flies. The MC domain is present in C.elegans ALG-1 and ALG-2 Ago proteins but absent from the remaining 25 members of the C.elegans Argonaute protein family, consistent with the finding that there are distinct RNAi-related pathways in nematodes, with ALG-1 and ALG-2 proteins participating in the microRNA pathway and all other nematode Argonaute proteins being associated with exo- or endo-RNAi pathways. Finally, the MC domain is absent from Ago proteins in organisms that do not have miRNAs such as fission yeast and Archaea. Although the MC motif is not found in Archaeal Agos, the structures of the P. furiosus and A. aeolicus Ago proteins show that a major portion of the Mid domain is accessible and thus may be capable of interacting with other factors. The MC domain is also not present in PIWI proteins, which are almost exclusively expressed in the germline. Notably, tethering of HIWI, a human PIWI protein, in the 3'-UTR of RL-5BoxB, is unable to repress RL translation. In contrast, tethering of all human Ago proteins (Ago1-4) in the 3'-UTR of RL-5BoxB results in strong repression of RL translation. These studies along with the finding that translational repression is unaffected in Ago2 null mouse embryonic fibroblasts also show that the endonuclease activity of mammalian Ago proteins is not required for translational repression. In flies, PIWI proteins associate with repeat-associated siRNAs. Mammalian PIWI proteins do not assemble with miRNAs or siRNAs but bind to slightly larger RNAs termed piRNAs. The mouse MIWI protein can associate with m7GTP sepharose, suggesting that MIWI proteins may also function in translation. However, since the MC domain is absent from the MIWI protein, it is possible that MIWI contains another cap-binding motif or associates with the cap-analog resin indirectly, via interactions with another cap-binding protein. However, the biochemical function of MIWI proteins and of piRNAs is unknown, and it is difficult to ascertain the functional consequences of this interaction at this point. Finally, the absence of the MC motif from plant Agos is intriguing and suggests that plant miRNAs may not be capable of repressing translation through interactions with the cap (but other mechanisms cannot be excluded). So far translational repression by miRNAs in plants has only been implicated for the control of very few mRNA targets, while most known plant miRNAs show extensive complementarity with their targets, directing target mRNA cleavage (Kiriakidou, 2007).

    Drosophila miR2 primarily targets the m7GpppN cap structure for translational repression

    Understanding the molecular mechanism(s) of how miRNAs repress mRNA translation is a fundamental challenge in RNA biology. This study used a validated cell-free system from Drosophila embryos to investigate how miR2 inhibits translation initiation. By screening a library of chemical m7GpppN cap structure analogs, defined modifications of the triphosphate backbone were identified that augment miRNA-mediated inhibition of translation initiation but are 'neutral' toward general cap-dependent translation. Interestingly, these caps also augment inhibition by 4E-BP. Kinetic dissection of translational repression and miR2-induced deadenylation shows that both processes proceed largely independently, with establishment of the repressed state involving a slow step. These data demonstrate a primary role for the m7GpppN cap structure in miRNA-mediated translational inhibition, implicate structural determinants outside the core eIF4E-binding region in this process, and suggest that miRNAs may target cap-dependent translation through a mechanism related to the 4E-BP class of translational regulators (Zdanowicz, 2009).

    MicroRNAs (miRNAs) are posttranscriptional regulators of a wide range of biological processes including development, growth control, cellular differentiation, and apoptosis. With few exceptions, miRNAs fulfill their regulatory function by imperfect base pairing with the 3′UTR of target mRNAs inhibiting translation and/or triggering mRNA destabilization. Despite active investigation, the molecular mechanism(s) of how miRNAs and their associated proteins mediate their repressive or destabilizing effects remains controversial (Zdanowicz, 2009).

    Much evidence now suggests that miRNAs can regulate translation initiation, although perhaps not exclusively. Since miRNAs induce mRNA deadenylation in vivo and in vitro, this effect could theoretically suffice for inhibition of translation initiation. However, multiple lines of evidence suggest that neither a poly(A) tail nor its removal by deadenylation is required for miRNA regulation, implying that the primary mechanism of inhibition must be sought elsewhere (Zdanowicz, 2009).

    Both cap-dependent small ribosomal subunit recruitment and 60S subunit joining at the translation initiation codon have been reported to be regulated by miRNAs. Binding of the eIF4F complex to the cap promotes recruitment of the small ribosomal subunit to mRNAs and was implicated as a primary target of miRNA regulation by early reports investigating miRNA-mediated control; subsequent work in cultured cells and in vitro systems further supported this notion. However, the strength of conclusions from all of these studies has been questioned on the grounds that they rely on experimental approaches that alter the mode (internal ribosome entry sequences) and/or rate of nonregulated translation. Kinetic modeling studies frame this concern in quantitative terms and actually favored a late step in translation initiation as the likely target for miRNAs (Zdanowicz, 2009).

    To overcome these inherent limitations, a strategy was devised that probes miRNA-mediated regulation without affecting general (nonregulated) cap-dependent translation. This approach, based on modified cap structural analogs, circumvents by its very design the major caveats limiting other experimental approaches used to date. Advantage was also taken of the properties of an in vitro system to probe more deeply the relationship between translational inhibition and mRNA deadenylation. The data unambiguously demonstrate the importance of the cap structure as a primary target for miRNA-mediated translational control and show that deadenylation is an independent, rapid process that can contribute to repression. Moreover, the approach reveals that miR2's cap targeting mechanism bears similarities to repression by 4E-BP, highlighting interactions between cap-bound eIF4E and eIF4G as potential molecular targets of miR-RISC function (Zdanowicz, 2009).

    The discovery of two cap structure analogs that specifically augment miRNA-mediated repression without affecting overall mRNA translation provides a uniquely powerful argument that the cap structure serves as the primary functional target of miR2 translational inhibition. Both modifications to the cap structure also yield stronger repression of translation by 4E-BP. How do these cap modifications selectively sensitize translation to specific inhibitory pathways, and what does this say about how miRNAs target the cap? Structural details of eIF4E binding to the m7GpppN cap structure readily explain why the modifications of cap analogs cap 16 and cap 21 do not interfere with general translation: the modifications affect the end of the triphosphate linker, outside the 'core' m7G nucleotide recognition region that features critical contacts for high-affinity cap binding. Nevertheless, the observation that these modifications result in sensitivity to both miR2-RISC and 4E-BP suggests that changes to this region of the cap subtly affect the way eIF4F interacts with the cap and perhaps also downstream portions of the 5' UTR via eIF4G. Since the effects of these changes are effectively nonconsequential at the level of general translation initiation, and are only revealed in the presence of specific inhibitors, they appear to introduce an Achilles' heel in the translation initiation pathway. Enhanced sensitivity is observed with both 4E-BP and miR2-RISC, but not with m7GpppG cap analog itself, suggesting that miR2-RISC may use a mechanism similar to 4E-BP to target cap function. Since 4E-BP directly interferes with interaction between eIF4E and eIF4G, the results highlight physical interaction between eIF4E and eIF4G–or a related functional step–as a potential target of miRNA action (Zdanowicz, 2009).

    As seen in multiple experimental in vivo and in vitro settings, specific, miR-dependent deadenylation of the reporter mRNA was observed. The data using cap 18 support earlier work suggesting that a poly(A) tail and mRNA deadenylation are not required for miRNA-mediated translational inhibition but can quantitatively contribute to it. At least in vitro, deadenylation is a kinetically rapid process that occurs even under conditions where the mRNA fails to be translationally repressed (lack of preincubation or A capping). In this sense, deadenylation and repression are separable processes that are both specifically triggered by miR2. The data also challenge the recent conclusion that miR/Ago1-mediated translational repression primarily occurs via deadenylation, as m7GpppN-capped mRNA is fully deadenylated but essentially unrepressed if it has not undergone preincubation. Thus, deadenylation cannot be the primary cause of repression (Zdanowicz, 2009).

    Based on published work and the new data, a 'two-hit model' is proposed; the miR-RISC complex affects both ends of mRNAs to which it is bound. Repression primarily targets the cap structure, preventing recruitment of the small ribosomal subunit. This process is normally facilitated and reinforced by the independent action of miR-RISC removal of the poly(A) tail. Both hits converge on the inhibition of cap-dependent small ribosomal subunit recruitment via the eIF4F complex. While this model can explain much of the published work, it does not exclude the existence of additional mechanisms that could target later steps in translation initiation or postinitiation steps (Zdanowicz, 2009).

    GW182 proteins directly recruit cytoplasmic deadenylase complexes to miRNA targets

    miRNAs are posttranscriptional regulators of gene expression that associate with Argonaute and GW182 (Drosophila Gawky) proteins to repress translation and/or promote mRNA degradation. miRNA-mediated mRNA degradation is initiated by deadenylation, although it is not known whether deadenylases are recruited to the mRNA target directly or by default, as a consequence of a translational block. To answer this question, a screen was performed for potential interactions between the Argonaute and GW182 proteins and subunits of the two cytoplasmic deadenylase complexes. Human GW182 proteins were found to recruit the PAN2-PAN3 and CCR4-CAF1-NOT deadenylase complexes through direct interactions with PAN3 and NOT1, respectively. These interactions are critical for silencing and are conserved in D. melanogaster. These findings reveal that GW182 proteins provide a docking platform through which deadenylase complexes gain access to the poly(A) tail of miRNA targets to promote their deadenylation, and they further indicate that deadenylation is a direct effect of miRNA regulation (Braun, 2011; graphic abstract of article).

    Emerging evidence suggests that mRNA deadenylation is part of the mechanism used by miRNAs to silence gene expression. Indeed, deadenylation of miRNA targets has now been reported in zebrafish and C. elegans embryos, human and D. melanogaster cells, and in various cell-free extracts that recapitulate silencing. However, whether miRISCs directly recruit deadenylases to miRNA targets has remained unclear (Braun, 2011).

    This study provides compelling evidence that the silencing domains (SDs) of TNRC6 proteins (human GW182 paralogs) contain binding sites for PAN3 and NOT1, which are subunits of each of the two major cytoplasmic deadenylase complexes. These findings provide strong support for the hypothesis that GW182 proteins enhance poly(A) tail removal by directly recruiting deadenylases to associated mRNA targets. More broadly, these results have implications for the understanding of miRNA-based regulation, because they show that target deadenylation is not merely a consequence of a translational block (Braun, 2011).

    Previous studies have reported conflicting evidence regarding the interaction of deadenylation factors with the two major components of miRISCs (AGO and GW182). Indeed, several studies failed to detect a significant interaction between human AGO or TNRC6 proteins and components of deadenylase complexes, including POP2, CAF1, CCR4a, CCR4b, and PAN2 (Braun, 2011).

    Using coimunoprecipitation and in vitro pull-down assays, it was determined that PAN3 and NOT1 interact directly with TNRC6-SDs, whereas the interaction with PAN2 and the additional components of the CCR4-CAF1-NOT complex is indirect and bridged by PAN3 and NOT1, respectively. These observations provide one explanation for the negative results reported in previous studies. Indeed, other studies focused on the interaction of AGO and GW182 with subunits of the deadenylase complexes that interact indirectly (e.g., the catalytic subunits and NOT3). These indirect interactions are likely to be affected by the efficiency of the immunoprecipitation and the expression of the tagged proteins relative to the expression of the endogenous bridging factors. In agreement with this interpretation, this study showed that human TNRC6C did not coimmunoprecipitate PAN2; nevertheless, an interaction with PAN2 was observed when PAN3 (the bridging factor) was overexpressed (Braun, 2011).

    Previous studies have shown that the silencing domain of GW182 proteins contains two binding sites for PABPC1: one in the PAM2 motif and one in the M2 and C-terminal regions. The PAM2 motif interacts directly with the C-terminal MLLE domain of PABPC1. The M2 and C-terminal regions mediate indirect binding to PABPC1, which is only observed in cell lysates. This study has shown that the TNRC6 M2 and C-term regions mediate direct binding to PAN3. PAN3, in turn, binds to PABPC1 and PAN2 and may act as a bridging factor. It was also shown that the M1, M2, and C-term regions of the silencing domain confer direct binding to NOT1, which, in turn, mediates interaction with the additional subunits of the CCR4-CAF1-NOT complex (Braun, 2011).

    A model is presented that summarizes the interactions uncovered in this work as well as those from previous studies. TNRC6 proteins are recruited to miRNA targets through their interaction with AGOs, and they contact PABPC1 directly through their PAM2 motifs. TNRC6 proteins also bind PAN3 and NOT1 via their Mid and C-term regions, as shown in this study. These interactions may occur consecutively, simultaneously, or alternatively. PAN3 interacts with the catalytic subunit PAN2 . Additionally, PAN3 contains an N-terminal PAM2 motif that could bind to the MLLE domain of a second PABPC1 molecule. Finally, NOT1 recruits the additional subunits of the CCR4-CAF1-NOT complex. Although the detailed molecular interactions between the deadenylases, PABPC1 and TNRC6s need to be further elucidated, an important conclusion emerging from these studies is that TNRC6 proteins engage in multiple interactions with deadenylases and PABPC1 to promote target mRNA degradation. Moreover, the observation that depletion of PAN3 and NOT1 suppresses silencing of an unadenylated reporter, suggests that deadenylase complexes could also contribute to translational repression in addition to promoting deadenylation and decay. Thus, it is possible that translational repression and deadenylation are two distinct outcomes triggered by the recruitment of deadenylase complexes to the 3′UTR of miRNA targets. Further studies will determine how deadenylase complexes interact with TNRC6 proteins at the molecular level, and the role they may play in translational repression (Braun, 2011).

    GW-bodies and P-bodies constitute two separate pools of sequestered non-translating RNAs

    Non-translating RNAs that have undergone active translational repression are culled from the cytoplasm into P-bodies for decapping-dependent decay or for sequestration. Organisms that use microRNA-mediated RNA silencing have an additional pathway to remove RNAs from active translation. Consequently, proteins that govern microRNA-mediated silencing, such as GW182/Gw and AGO1, are often associated with the P-bodies of higher eukaryotic organisms. Due to the presence of Gw, these structures have been referred to as GW-bodies. However, several reports have indicated that GW-bodies have different dynamics to P-bodies. This study used live imaging to examine GW-body and P-body dynamics in the early Drosophila melanogaster embryo. While P-bodies are present throughout early embryonic development, cytoplasmic GW-bodies only form in significant numbers at the midblastula transition. Unlike P-bodies, which are predominantly cytoplasmic, GW-bodies are present in both nuclei and the cytoplasm. RNA decapping factors such as DCP1, Me31B, and Hpat are not associated with GW-bodies, indicating that P-bodies and GW-bodies are distinct structures. Furthermore, known Gw interactors such as AGO1 and the CCR4-NOT deadenylation complex, which have been shown to be important for Gw function, are also not present in GW-bodies. Use of translational inhibitors puromycin and cycloheximide, which respectively increase or decrease cellular pools of non-translating RNAs, alter GW-body size, underscoring that GW-bodies are composed of non-translating RNAs. Taken together, these data indicate that active translational silencing most likely does not occur in GW-bodies. Instead GW-bodies most likely function as repositories for translationally silenced RNAs. Finally, inhibition of zygotic gene transcription is unable to block the formation of either P-bodies or GW-bodies in the early embryo, suggesting that these structures are composed of maternal RNAs (Patel, 2016).

    Polymorphism in the processing body component Ge-1 controls resistance to a naturally occurring ehabdovirus in Drosophila

    Hosts encounter an ever-changing array of pathogens, so there is continual selection for novel ways to resist infection. A powerful way to understand how hosts evolve resistance is to identify the genes that cause variation in susceptibility to infection. Using high-resolution genetic mapping this study has identified a naturally occurring polymorphism in a gene called Ge-1 that makes Drosophila highly resistant to its natural pathogen Drosophila melanogaster sigma virus (DMelSV). By modifying the sequence of the gene in transgenic flies, 26 amino acid deletion in the serine-rich linker region of Ge-1 was identified that is causing the resistance. Knocking down the expression of the susceptible allele leads to a decrease in viral titre in infected flies, indicating that Ge-1 is an existing restriction factor whose antiviral effects have been increased by the deletion. Ge-1 plays a central role in RNA degradation and the formation of processing bodies (P bodies). A key effector in antiviral immunity, the RNAi induced silencing complex (RISC), localises to P bodies, but this study found that Ge-1-based resistance is not dependent on the small interfering RNA (siRNA) pathway. However, Decapping protein 1 (DCP1) was found to protect flies against sigma virus. This protein interacts with Ge-1 and commits mRNA for degradation by removing the 5' cap, suggesting that resistance may rely on this RNA degradation pathway. The serine-rich linker domain of Ge-1 has experienced strong selection during the evolution of Drosophila, suggesting that this gene may be under long-term selection by viruses. These findings demonstrate that studying naturally occurring polymorphisms that increase resistance to infections enables identification of novel forms of antiviral defence, and support a pattern of major effect polymorphisms controlling resistance to viruses in Drosophila (Cao, 2016).

    piRNA pathway is not required for antiviral defense in Drosophila melanogaster

    Since its discovery, RNA interference has been identified as involved in many different cellular processes, and as a natural antiviral response in plants, nematodes, and insects. In insects, the small interfering RNA (siRNA) pathway is the major antiviral response. In recent years, the Piwi-interacting RNA (piRNA) pathway also has been implicated in antiviral defense in mosquitoes infected with arboviruses. Using Drosophila melanogaster and an array of viruses that infect the fruit fly acutely or persistently or are vertically transmitted through the germ line, this study investigated in detail the extent to which the piRNA pathway contributes to antiviral defense in adult flies. Following virus infection, the survival and viral titers of Piwi, Aubergine, Argonaute-3, and Zucchini mutant flies were similar to those of wild type flies. Using next-generation sequencing of small RNAs from wild type and siRNA mutant flies, it was shown that no viral-derived piRNAs are produced in fruit flies during different types of viral infection. This study provides the first evidence that the piRNA pathway does not play a major role in antiviral defense in adult Drosophila and demonstrates that viral-derived piRNA production depends on the biology of the host-virus combination rather than being part of a general antiviral process in insects (Petit, 2016).

    Drosophila cells use nanotube-like structures to transfer dsRNA and RNAi machinery between cells

    Tunnelling nanotubes and cytonemes function as highways for the transport of organelles, cytosolic and membrane-bound molecules, and pathogens between cells. During viral infection in Drosophila, a systemic RNAi antiviral response is established presumably through the transport of a silencing signal from one cell to another via an unknown mechanism. Because of their role in cell-cell communication, this study investigated whether nanotube-like structures could be a mediator of the silencing signal. In the context of a viral infection, the presence of nanotube-like structures is described in different Drosophila cell types. These tubules, made of actin and tubulin, were associated with components of the RNAi machinery, including Argonaute 2, double-stranded RNA, and CG4572. Moreover, they were more abundant during viral, but not bacterial, infection. Super resolution structured illumination microscopy showed that Argonaute 2 and tubulin reside inside the tubules. It is proposed that nanotube-like structures are one of the mechanisms by which Argonaute 2, as part of the antiviral RNAi machinery, is transported between infected and non-infected cells to trigger systemic antiviral immunity in Drosophila (Karlikow, 2016).

    miRISC recruits decapping factors to miRNA targets to enhance their degradation

    MicroRNA (miRNA)-induced silencing complexes (miRISCs) repress translation and promote degradation of miRNA targets. Target degradation occurs through the 5'-to-3' messenger RNA (mRNA) decay pathway, wherein, after shortening of the mRNA poly(A) tail, the removal of the 5' cap structure by decapping triggers irreversible decay of the mRNA body. This study, carried out in Drosophila S2 cells, demonstrates that miRISC enhances the association of the decapping activators DCP1, Me31B and HPat with deadenylated miRNA targets that accumulate when decapping is blocked. DCP1 and Me31B recruitment by miRISC occurs before the completion of deadenylation. Remarkably, miRISC recruits DCP1, Me31B and HPat to engineered miRNA targets transcribed by RNA polymerase III, which lack a cap structure, a protein-coding region and a poly(A) tail. Furthermore, miRISC can trigger decapping and the subsequent degradation of mRNA targets independently of ongoing deadenylation. Thus, miRISC increases the local concentration of the decapping machinery on miRNA targets to facilitate decapping and irreversibly shut down their translation (Nishihara, 2013).

    This study demonstrates that miRISCs enhance the association of DCP1, Me31B and HPat with miRNA targets in a miRNA-dependent manner. This association occurs even when the miRNA target lacks a 5' cap structure, an ORF and a poly(A) tail. Furthermore, mRNA reporters that are immune to deadenylation are degraded through decapping in the presence of the miRNA, indicating that miRISCs can promote decapping independently of deadenylation (Nishihara, 2013).

    It is known that miRNAs promote the degradation of partially complementary targets through the 5'-to-3' decay pathway. In this pathway, decapping is coupled to deadenylation and does not occur on polyadenylated and fully functional mRNAs. This study investigated whether the decapping of miRNA targets occurs by default, as a consequence of this coupling, or whether miRISCs can also recruit decapping factors independently of deadenylation. miRISCs was shown to enhance the association of DCP1, Me31B and HPat with unadenylated 7SL-derived miRNA targets that have been transcribed by Pol III, indicating that the cap, a poly(A) tail and ongoing deadenylation are not required for the recruitment of decapping factors to miRNA targets. DCP1 association with the Alu-miRNA target reporterers, termed EvAluator reporters, was strictly miRNA dependent and stimulated by GW182. miRNAs and GW182 also stimulated the association of HPat and Me13B with the EvAluator reporters, indicating that these decapping factors interact with miRISC components that are bound to EvAluator RNA. However, DCP1 and Me31B did not interact with isolated AGO1 or GW182 in co-immunoprecipitation assays, suggesting that the interaction of decapping factors with miRISC is indirect or that DCP1 and Me31B recognize AGO1 and GW182 as a complex. Indeed, it is possible that the decapping factors are recruited by the PAN2-PAN3 or CCR4-NOT deadenylase complexes, which interact with GW182 proteins directly. Alternatively, DCP1 and Me31B might recognize AGO1 or GW182 only in a certain conformation that is adopted on target binding. Although HPat did interact with AGO1 and GW182 in co-immunoprecipitation assays, these interactions were apparently not sufficient to enhance the association of HPat and a polyadenylated miRNA target. Nevertheless, it is possible that these interactions contribute to the recruitment of HPat to deadenylated or oligoadenylated targets (Nishihara, 2013).

    A previous study in human cells reported that EDC4 co-localized with a specific miRNA target in a miRNA-dependent manner, whereas DCP1 and RCK (the human ortholog of Dm Me31B) associated with the target, regardless of the presence of the miRNA. In agreement with that study, this study observed that decapping factors associate with miRNA targets in the absence of the miRNA; however, it was found that their binding is enhanced by the cognate miRNA. This enhancement was observed for targets that are not degraded or when degradation of the target was partially inhibited and may have escaped detection in co-localization studies (Nishihara, 2013).

    A functional implication for the association of decapping factors with miRNA-targets is that miRNA targets can be decapped and degraded even in the absence of a poly(A) tail or ongoing deadenylation. In combination with previously published data, the current results suggest that miRISC has multiple and redundant activities to ensure robust gene regulation: it induces translational repression, deadenylation and decapping, the latter in both a deadenylation-dependent and -independent manner (Nishihara, 2013).

    Under which circumstances can deadenylation-independent decapping contribute to silencing? Decapping might play a role in silencing specific miRNA targets when deadenylation is blocked or when decapping is blocked and targets that have undergone deadenylation accumulate. Indeed, deadenylation and decapping can be uncoupled on specific mRNAs, in different cell types and under various cellular conditions, leading to the accumulation of deadenylated repressed mRNAs. These mRNAs can re-enter the translational pool on polyadenylation or might be degraded in a deadenylation-independent manner once decapping resumes. For example, in immature mouse oocytes, DCP2 and DCP1 are not detectable, but their expression increases during oocyte maturation. Consequently, in immature oocytes, many maternal mRNAs (most likely including miRNA targets) accumulate in a deadenylated silenced form. These mRNAs may be polyadenylated and translated at later stages of oogenesis or embryogenesis. However, a fraction of these deadenylated targets may be degraded through decapping when DCP2 and DCP1 are expressed. Additionally, DCP1 and DCP2 are phosphorylated under cellular stress conditions, and DCP1 is hyperphosphorylated during mitosis. Under these conditions, a subset of mRNAs is stabilized, suggesting that DCP1 and DCP2 phosphorylation inhibits decapping. Thus, it is possible that under various stress conditions, miRNA targets accumulate in a deadenylated form because decapping is inhibited and that deadenylation-independent decapping is required for the clearance of these targets on return to normal cellular conditions (Nishihara, 2013).

    Notably, in addition to their role in target degradation, decapping activators act as general repressors of translation even in the absence of decapping. Therefore, these factors could play a more direct role in the translational repression of miRNA targets in the absence of mRNA degradation (Nishihara, 2013).

    In contrast to translational repression and deadenylation, decapping irreversibly shuts down translation initiation and commits mRNA to full degradation. Thus, decapping prevents the reversal of miRNA-mediated silencing. However, some miRNA targets have been shown to be released from miRNA-mediated repression in response to extracellular signals, suggesting that decapping is somehow blocked for these targets to allow for a fast reversal of their repression. How decapping is prevented in a target-specific manner remains unclear, but it can reasonable be expected that proteins associated with these targets block decapping in cis by preventing DCP2 access to the cap structure. These proteins may bind the cap structure directly or may act indirectly, for example, by stabilizing binding of the cap-binding protein eIF4E to the mRNA. Proteins that act as inhibitors of DCP2-mediated decapping have been described and include Variable Charged X chromosome VCX-A protein, YB-1, Y14 and Dm CUP. Thus, it is possible that additional proteins that prevent the decapping of specific mRNAs are present in eukaryotic cells. Such mRNA-specific decapping regulators would be likely to play an important role in controlling the reversibility of silencing. Alternatively, mRNAs can be recapped in the cytoplasm; however, how recapping is regulated remains unknown (Nishihara, 2013).

    In addition to the aforementioned sequence-specific decapping regulators, the cap-binding protein eIF4E acts as a general inhibitor of decapping by limiting DCP2 access to the cap structure. Therefore, for decapping to occur, eIF4E needs to dissociate from the 5' end of the mRNA. This study shows that eIF4E remains bound to at least a fraction of silenced miRNA targets in cells in which decapping is blocked. Furthermore, the DCP2 catalytic mutant did not detectably associate with the mRNA target, even though its overexpression inhibited decapping. These observations suggest that DCP2 does not stably associate with miRNA targets. Similarly, DCP2 did not co-localize with miRNA targets in human cells, although in these cells, EDC4 co-localized with the target in a miRNA-dependent manner. Thus, the process of decapping may involve multiple consecutive steps, including the association of decapping activators with the target mRNA in the absence of DCP2, eIF4E dissociation, DCP2 recruitment and cap hydrolysis. The current results suggest that miRISC facilitates an early decapping step by increasing the local concentration of decapping factors on mRNA targets, promoting decapping independently of deadenylation. Further studies are necessary to determine whether, in addition to recruiting decapping factors, miRISC plays a more direct role in accelerating the chemical catalysis step of decapping (Nishihara, 2013).

    MicroRNAs block assembly of eIF4F translation initiation complex in Drosophila

    miRNAs silence their complementary target mRNAs by translational repression as well as by poly(A) shortening and mRNA decay. In Drosophila, miRNAs are typically incorporated into Argonaute1 (Ago1) to form the effector complex called RNA-induced silencing complex (RISC). Ago1-RISC associates with a scaffold protein GW182, which recruits additional silencing factors. Previously studies have shown that miRNAs repress translation initiation by blocking formation of the 48S and 80S ribosomal complexes. However, it remains unclear how ribosome recruitment is impeded. This study examined the assembly of translation initiation factors on the target mRNA under repression. Ago1-RISC was shown to induce dissociation of eIF4A, a DEAD-box RNA helicase, from the target mRNA without affecting 5' cap recognition by eIF4E in a manner independent of GW182. In contrast, direct tethering of GW182 promotes dissociation of both eIF4E and eIF4A. It is proposed that miRNAs act to block the assembly of the eIF4F complex during translation initiation (Fukaya, 2014).

    MicroRNAs (miRNAs) silence their complementary target mRNAs via formation of the effector ribonucleoprotein complex called RNA-induced silencing complex (RISC). The core component of RISC is a member of the Argonaute (Ago) proteins. In Drosophila, miRNAs are sorted into two functionally distinct Ago proteins, Ago1 and Ago2, according to their structural features and the identity of the 5' end nucleotides. Compared to fly Ago2, fly Ago1 shares more common features with mammalian Ago1-4, making it a suitable model for investigating miRNA-mediated gene silencing in animals. Ago1-RISC mediates translational repression as well as shortening of the poly(A) tail followed by mRNA decay (Behm-Ansmant, 2006). While deadenylation per se disrupts the closed-loop configuration of mRNA and leads to inhibition of translation initiation, Ago1-RISC can repress translation independently of deadenylation (Fukaya and Tomari, 2011). Such a deadenylation-independent 'pure' translational repression mechanism seems to be widely conserved among species (Bazzini, 2012, Bethune, 2012, Mishima, 2012 and Iwakawa and Tomari, 2013; Fukaya, 2014 and references therein).

    Ago is not the only protein involved in the miRNA-mediated gene silencing pathway. In flies, a P-body protein GW182 specifically interacts with Ago1, but not with Ago2, through the N-terminal glycine/tryptophan (GW) repeats and provides a binding platform for PAN2-PAN3 and CCR4-NOT deadenylase complexes (Braun, 2011; Chekulaeva, 2011). This protein interaction network is conserved in animals including zebrafish, nematodes, and humans (Fabian, 2011; Kuzuoglu-Ozturk, 2012; Mishima, 2012). Accordingly, GW182 is essential for shortening of the poly(A) tail by miRNAs. In contrast, recent studies revealed that miRNA-mediated translational repression occurs in both GW182-dependent and -independent manners (Fukaya, 2012; Wu, 2013). Previous sedimentation analysis on sucrose density gradient suggested that both of the two translational repression mechanisms block recruitment of the ribosomal 43S preinitiation complex to the target mRNA independently of deadenylation (Fukaya, 2012; Fukaya, 2014 and references therein).

    In eukaryotes, recruitment of the 43S preinitiation complex is initiated by the formation of eukaryotic translation initiation factor 4F (eIF4F). eIF4F is a multiprotein complex composed of the cap-binding protein eIF4E, which recognizes the 7-methyl guanosine (m7G) structure of the capped mRNA; the scaffold protein eIF4G, which interacts with 40S ribosome-associated eIF3 and bridges the mRNA and the 43S preinitiation complex; and the DEAD-box RNA helicase eIF4A, which plays a pivotal role in translation initiation supposedly through unwinding the secondary structure of the 5' UTR for landing of the 43S complex. In addition, the poly(A)-binding protein PABP stimulates translation initiation through its direct interaction with eIF4G. miRNAs likely block one (or more) of these steps to repress translation initiation. It was recently proposed that, in mammals, preferential recruitment of eIF4AII (one of the two eIF4A paralogs) is required for miRNA-mediated translational repression (Meijer, 2013). This model postulates that eIF4AII acts to inhibit rather than activate translation, unlike its major counterpart eIF4AI. However, the role of eIF4AII in translation remains largely unexplored, as opposed to eIF4AI's well-established function to promote translation. Moreover, invertebrates have only one eIF4A, making this model incompatible in flies. Thus, it still remains unclear how miRNAs repress translation initiation. This is largely due to technical limitations in directly monitoring the assembly of the translation initiation complex specifically on the mRNA targeted by miRNAs (Fukaya, 2014).

    Using site-specific UV crosslinking this study examined the association of translation initiation factors on the target RNA under repression. Fly Ago1-RISC specifically induces dissociation of eIF4A from the target mRNA without affecting the 5' cap recognition by eIF4E in a manner independent of GW182 or PABP. On the other hand, direct tethering of GW182 to the target mRNA promotes dissociation of both eIF4E and eIF4A. It is proposed that miRNAs act to block assembly of the eIF4F complex during translation initiation, in addition to their established role in deadenylation and decay of their target mRNAs (Fukaya, 2014).

    Although eIF4G could not be detected via any of the crosslinking positions spanning from 2 nt to 13 nt downstream of the cap, previous studies have shown that noncanonical translation driven by direct tethering of eIF4G to the 5' UTR was fully susceptible to translational repression by Ago1-RISC (Fukaya, 2012). Therefore, it was reasoned that Ago1-RISC directly targets eIF4A rather than eIF4E or eIF4G. In the accompanying paper, Fukao (2014) revealed that human Ago2-RISC specifically induces dissociation of eIF4A-both eIF4AI and eIF4AII-without affecting eIF4E or eIF4G in a cell-free system deriving from HEK293F cells (Fukao, 2014). Thus, eIF4A is likely a target of miRNA action conserved among species. In agreement with this model, miRNA-mediated gene silencing is cancelled by the eIF4A inhibitors silvestrol (Fukao, 2014), hippuristanol, or pateamine A (Leung, 2011; Meijer, 2013) in human cells (Fukaya, 2014).

    GW182 is a well-known interactor of miRNA-associated Ago proteins and is a prerequisite for miRNA-mediated deadenylation/decay of target mRNAs (Behm-Ansmant, 2006). GW182 directly binds to both NOT1 and CAF40/CNOT9, thereby recruiting the CCR4-NOT deadenylase complex to the target mRNA. It has been suggested that the CCR4-NOT complex not only shortens the poly(A) tail but also plays a role in miRNA-mediated translational repression, because direct tethering of the CCR4-NOT complex was capable of inducing translational repression independently of deadenylation. It was originally proposed that, in humans, the CCR4-NOT complex specifically binds to eIF4AII (but not to eIF4AI) to repress translation. However, this model was challenged by recent studies showing that, although the MIFG4 domain of human CNOT1 structurally resembles the middle domain of eIF4G, it does not bind eIF4AI or II but instead partners with the DEAD-box RNA helicase DDX6, which has been implicated in repression of translation initiation and/or translation elongation as well as activation of decapping. Given that miRNAs mediate gene silencing via multiple different pathways, recruitment of DDX6 by GW182 via the CCR4-NOT complex may well play a role in inhibiting protein synthesis from miRNA targets. Indeed, this study observed strong dissociation of both eIF4E and eIF4A by direct tethering of GW182. However, at the physiological stoichiometry between Ago1 and GW182 in S2 cell lysate, eIF4A was specifically dissociated without apparent effect on eIF4E by canonical miRNA targeting, which is in agreement with the result of the reporter assay in S2 cells depleted of each eIF4F component. It is envisioned that, although GW182 is clearly essential for miRNA-mediated deadenylation, the degree of contribution of GW182 to translational repression can vary in different cell types and conditions, depending on the concentrations of GW182 and Ago proteins, as well as their protein interaction networks that are subject to regulation by extracellular signaling. In this regard, direct tethering of GW182 may potentially overestimate its role in miRNA-mediated translational repression (Fukaya, 2014).

    How could Ago1-RISC specifically dissociate eIF4A from the initiation complex? Previous work has shown that none of GW182, the CCR4-NOT complex, or PABP is required for translational repression by Ago1-RISC (Fukaya, 2012). The current data extend these findings to reveal that Ago1-RISC can induce dissociation of eIF4A independently of GW182 or PABP. It is tempting to speculate that an as-yet-unidentified factor associated with Ago1-RISC, or perhaps Ago1-RISC itself, blocks the interaction between eIF4G and eIF4A (e.g., similarly to Programmed Cell Death 4 [PDCD4] whose tandem MA-3 domains compete with the MA-3 domain of eIF4G to bind the N-terminal domain of eIF4A, thereby displacing eIF4A from the eIF4F initiation complex). Alternatively, Ago1-RISC might directly or indirectly inhibit the ATP-dependent RNA-binding activity of eIF4A, which is tightly regulated by its accessory proteins eIF4B and eIF4H (Abramson, 1988; Richter, 1999). Future studies are warranted to determine how miRNAs block the assembly of the eIF4F translation initiation complex (Fukaya, 2014).

    The interactions of GW182 proteins with PABP and deadenylases are required for both translational repression and degradation of miRNA targets

    Animal miRNAs silence the expression of mRNA targets through translational repression, deadenylation and subsequent mRNA degradation. Silencing requires association of miRNAs with an Argonaute protein and a GW182 family protein. In turn, GW182 proteins interact with poly(A)-binding protein (PABP) and the PAN2-PAN3 and CCR4-NOT deadenylase complexes. These interactions are required for the deadenylation and decay of miRNA targets. Recent studies have indicated that miRNAs repress translation before inducing target deadenylation and decay; however, whether translational repression and deadenylation are coupled or represent independent repressive mechanisms is unclear. Another remaining question is whether translational repression also requires GW182 proteins to interact with both PABP and deadenylases. To address these questions, this study characterized the interaction of Drosophila melanogaster GW182 with deadenylases and defined the minimal requirements for a functional GW182 protein. Functional assays in D. melanogaster and human cells indicate that miRNA-mediated translational repression and degradation are mechanistically linked and are triggered through the interactions of GW182 proteins with PABP and deadenylases (Huntzinger, 2013).

    Recent studies indicate that translational repression of miRNA targets precedes deadenylation and decay. This study shows that these two functional outcomes of miRNA regulation are linked and both require the interaction of GW182 proteins with PABP and deadenylases (Huntzinger, 2013).

    The interaction of GW182 proteins with PABP has been well documented using biochemical and structural studies, and the PAM2 motif is highly conserved among vertebrate and insect GW182 proteins. Despite conservation, the study of the role of PABP in silencing in different systems has led to conflicting conclusions. For example, several studies have reported that the PABP–GW182 interaction is important for silencing in Drosophila and human cells and in cell-free systems that recapitulate silencing. Furthermore, PABP depletion prevented miRNA-mediated deadenylation in cell-free extracts from mouse Krebs-2 ascites cells, and mutations in the PAM2 motif of TNRC6C reduced the rate of deadenylation in tethering assays. In addition, a study in Drosophila cell-free extracts wherein silencing is mediated through endogenous preloaded miRISCs indicated that PABP stimulates silencing by facilitating the association of miRISC complexes with mRNA targets. It was also shown that on miRISC binding, PABP progressively dissociated from the mRNA target, in the absence of deadenylation (Huntzinger, 2013).

    In contrast to the studies mentioned above, studies in zebrafish embryos and in a Drosophila cell-free assay wherein miRISCs are loaded with exogenously supplemented miRNA duplexes indicate that PABP is dispensable for miRNA-mediated silencing. Intriguingly, efficient silencing in zebrafish embryos required the GW182 PAM2 motif. Moreover, the observation that multiple and non-overlapping fragments of Drosophila GW182 (including N-term fragments that do not interact with PABP) silenced mRNA reporters in tethering assays was interpreted as evidence that the interaction of GW182 proteins with PABP is not required for silencing. This study shows that unlike in tethering assays, N-term fragments of GW182 fail to restore the silencing of a majority of the reporters tested in complementation assays. Thus, tethering assays bypass the requirement for PABP binding, and may not faithfully recapitulate silencing. Furthermore, the observation that PABP dissociates from the poly(A) tail of miRNA targets in the absence of deadenylationprovides one explanation for the occurrence of silencing in extracts in which PABP has been depleted or displaced from the poly(A) tail using an excess of Paip2 (Huntzinger, 2013).

    In summary, these results confirm and further extend previous observations that a single amino acid substitution in the PAM2 motif of human TNRC6 proteins abolishes PABP binding and impairs silencing activity, despite the interaction of this mutant with deadenylases. Furthermore, Drosophila GW182 N-term protein fragments that bind deadenylases, but not PABP, failed to complement the silencing of eight of the nine reporters tested, although they are active in tethering assays. These results provide evidence for a role of PABP in silencing in human and Drosophila cells. However, it is possible that PABP becomes dispensable for silencing depending on cellular conditions or the nature of the specific mRNA target, as shown, for example, for the F-Luc-Nerfin-1 reporter when silencing is mediated by miR-9b (Huntzinger, 2013).

    The SDs of human TNRC6 proteins directly interact with CNOT1 through tryptophan-containing motifs in the M1, M2 and C-term regions of the S. This study shows that these motifs contribute additively to CNOT1 binding and silencing activity in human cells. Indeed, when at least two motifs are simultaneously mutated, CNOT1 binding is strongly reduced and silencing activity impaired (Huntzinger, 2013).

    The interaction between GW182 and deadenylases is conserved in Drosophila; however, in contrast to human SDs, the Drosophila SD is not sufficient for NOT1 binding. This study shows that in addition to the SD, the Q-rich region is required for full NOT1 binding activity. Thus, although Drosophila GW182 has lost the CIM-2 motif, this protein has acquired additional motifs that can interact with NOT1. This study also shows that in contrast to the human proteins, Drosophila GW182 can interact with NOT2 and PAN3 via N-term sequences. Consequently, Drosophila GW182 can recruit deadenylases in multiple ways. Considering that (1) NOT1 interacts with NOT2, (2) the PAN2–PAN3 complex interacts with PABP and (3) the CCR4–NOT and PAN2–PAN3 complexes form a larger multiprotein complex in vivo, the current observations indicate a high degree of connectivity and redundancy within the GW182 interaction network, which could explain why mutations in individual motifs do not abolish partner binding or silencing activity, but a combination of two or more mutations is required to abrogate binding and silencing activity (Huntzinger, 2013).

    In addition, the ability of Drosophila GW182 N-term fragments to bind deadenylases also explains why these fragments are potent triggers of translational repression and mRNA degradation in tethering assays, whereas the corresponding fragments of the human proteins exhibit only residual activity. As discussed previously, despite their activity in tethering assays, Drosophila GW182 N-term fragments failed to complement the silencing of several of the reporters tested. The reason for the different activities of these fragments in tethering and complementation assays remains unknown (Huntzinger, 2013).

    This study has demonstrated that silencing (i.e. translational repression and target degradation) requires the interaction between GW182 proteins and both PABP and deadenylases. Several lines of evidence support this conclusion. First, the TNRC6C SD, which is sufficient for PABP and deadenylase binding, rescues silencing when fused to a minimal ABD. Similarly, the minimal fragment of Drosophila GW182 that rescues silencing comprises the Q+SD region, which also binds both deadenylases and PABP. Second, the Drosophila GW182 N-term fragments that bind deadenylases but not PABP are generally inactive in complementation assays. Third, mutations that specifically disrupt TNRC6 binding to PABP or deadenylase impair silencing, and mutations that disrupt deadenylase binding exhibit a stronger deleterious effect. Silencing activity is abolished when these mutations are combined. Finally, silencing is inhibited in human cells overexpressing the CNOT1 Mid domain together with a catalytically inactive CNOT7 mutant. In combination with the previously published data, these results indicate that silencing minimally requires an AGO, a GW182 protein, PABP and deadenylases, thus defining the minimal interaction network required for silencing. The findings do not rule out that additional interactions are potentially required to achieve maximal repression, depending on the cellular context or the mRNA target. For example, the P-GL motif is highly conserved and important for silencing in zebrafish embryos. This motif may mediate interactions with additional partners (Huntzinger, 2013).

    The finding that deadenylase complexes, in particular, are required for miRNA-mediated translational repression has broad implications regarding post-transcriptional mRNA regulation. Indeed, in addition to the GW182 proteins, various sequence-specific mRNA-binding proteins, such as Nanos, Bicaudal-C and Pumilio, recruit the CCR4–NOT complex to their mRNA targets. Furthermore, the direct tethering of the subunits of the CCR4–NOT complex represses the translation of mRNA reporters lacking a poly(A) tail, suggesting that the CCR4–NOT complex promotes translational repression in the absence of deadenylation. Therefore, elucidating the mechanism by which the CCR4–NOT complex regulates the fates of mRNA targets promises to increase understanding of the mechanism underlying repression by miRNAs and diverse sequence-specific RNA-binding proteins (Huntzinger, 2013).

    NOT10 and C2orf29/NOT11 form a conserved module of the CCR4-NOT complex that docks onto the NOT1 N-terminal domain

    The CCR4-NOT complex plays a crucial role in post-transcriptional mRNA regulation in eukaryotes. This complex catalyzes the removal of mRNA poly(A) tails, thereby repressing translation and committing an mRNA to degradation. The conserved core of the complex is assembled by the interaction of at least two modules: the NOT module, which minimally consists of NOT1, NOT2 and NOT3, and a catalytic module comprising two deadenylases, CCR4 and POP2/CAF1. Additional complex subunits include CAF40 and two newly identified human subunits, NOT10 and C2orf29. The role of the NOT10 and C2orf29 subunits and how they are integrated into the complex are unknown. This study shows that the Drosophila melanogaster NOT10 and C2orf29 (Not11) orthologs form a complex that interacts with the N-terminal domain of NOT1 through C2orf29. These interactions are conserved in human cells, indicating that NOT10 and C2orf29 define a conserved module of the CCR4-NOT complex. The assembly of the D. melanogaster CCR4-NOT complex was investigated, and it was demonstrated that the conserved armadillo repeat domain of CAF40 interacts with a region of NOT1, comprising a domain of unknown function, DUF3819. Using tethering assays, it was shown that each subunit of the CCR4-NOT complex causes translational repression of an unadenylated mRNA reporter and deadenylation and degradation of a polyadenylated reporter. Therefore, the recruitment of a single subunit of the complex to an mRNA target induces the assembly of the complete CCR4-NOT complex, resulting in a similar regulatory outcome (Bawankar, 2013).

    HPat a decapping activator interacting with the miRNA effector complex

    Animal miRNAs commonly mediate mRNA degradation and/or translational repression by binding to their target mRNAs. Key factors for miRNA-mediated mRNA degradation are the components of the miRNA effector complex (AGO1 and GW182) and the general mRNA degradation machinery (deadenylation and decapping enzymes). The CCR4-NOT1 complex required for the deadenylation of target mRNAs is directly recruited to the miRNA effector complex. However, it is unclear whether the following decapping step is only a consequence of deadenylation occurring independent of the miRNA effector complex or e.g. decapping activators can get recruited to the miRNA effector complex. In this study split-affinity purifications was performed in Drosophila cells and evidence is provided for the interaction of the decapping activator HPat with the miRNA effector complex. Furthermore, in knockdown analysis of various mRNA degradation factors the importance of NOT1 for this interaction was demonstrated. This suggests that deadenylation and/or the recruitment of NOT1 protein precedes the association of HPat with the miRNA effector complex. Since HPat couples deadenylation and decapping, the recruitment of HPat to the miRNA effector complex provides a mechanism to commit the mRNA target for degradation (Barisic-Jager, 2013).

    The CCR4 Deadenylase acts with Nanos and Pumilio in the fine-tuning of Mei-P26 expression to promote germline stem cell self-renewal

    Translational regulation plays an essential role in Drosophila ovarian germline stem cell (GSC) biology. GSC self-renewal requires two translational repressors, Nanos (Nos) and Pumilio (Pum), which repress the expression of differentiation factors in the stem cells. The molecular mechanisms underlying this translational repression remain unknown. This study shows that the CCR4 deadenylase is required for GSC self-renewal; Nos and Pum act through its recruitment onto specific mRNAs. mei-P26 mRNA was identified as a direct and major target of Nos/Pum/CCR4 translational repression in the GSCs. mei-P26 encodes a protein of the Trim-NHL tumor suppressor family that has conserved functions in stem cell lineages. Fine-tuning Mei-P26 expression by CCR4 plays a key role in GSC self-renewal. These results identify the molecular mechanism of Nos/Pum function in GSC self-renewal and reveal the role of CCR4-NOT-mediated deadenylation in regulating the balance between GSC self-renewal and differentiation (Joly, 2013).

    This study provides evidence that the twin gene that encodes the CCR4 deadenylase is essential for GSC self-renewal. GSCs are rapidly lost in twin mutants because they differentiate and cannot self-renew. Clonal analysis shows that twin is required cell autonomously in the GSCs for their self-renewal. Nos and Pum are major factors of GSC self-renewal and are translational repressors. Genetic and protein interactions among twin, nos, and pum indicate that CCR4 acts together with Nos and Pum to promote GSC self-renewal. This identifies the recruitment of the CCR4-NOT deadenylation complex as the molecular mechanism underlying Nos and Pum translational repression in the GSCs. Two mechanisms of action used by Nos/Pum have previously been described in the embryo. First, Nos/Pum represses hb mRNA translation by forming a complex with Brat, which in turn interacts with 4EHP and blocks initiation of translation. Second, Nos/Pum represses cyclin B mRNA translation in the primordial germ cells by recruiting the CCR4-NOT complex through direct interactions between Pum and CAF1 and between Nos and NOT4 (Kadyrova, 2007). Brat is not expressed in GSCs, thus excluding the first mode of Nos/Pum translational repression in these cells. However, Pum, Nos, and CCR4 were found to be present in a complex in GSC-like cells, consistent with the recruitment of the CCR4-NOT complex by Nos/Pum for GSC self-renewal (Joly, 2013).

    Interestingly, a mutant form of CCR4 that is inactive for deadenylation is able to partially rescue the lack of CCR4 in GSCs. This is consistent with CCR4 not being the only deadenylase in the complex (Temme, 2010). However, CCR4 does participate in the deadenylation activity of the complex, probably via a structural role. Furthermore, the CCR4-NOT complex has been shown recently to be involved in direct translational repression, in addition to its role in deadenylation (Chekulaeva, 2011; Cooke, 2010). This dual mode of action of CCR4-NOT might also be relevant to GSCs (Joly, 2013).

    The miRNA pathway also plays a crucial role in GSC self-renewal. A large body of evidence has shown that an important mechanism of silencing by miRNAs involves deadenylation resulting from the recruitment of CCR4-NOT by GW182 bound to Ago1 (for review, see Braun, 2012). Therefore, the CCR4-NOT complex is also likely to contribute to miRNA-mediated translational repression in the GSCs, thus making this complex a central effector of translational repression in the GSCs (Joly, 2013).

    An important result from this study is that mei-P26 mRNA is a major target of Nos/Pum/CCR4 regulation for GSC self-renewal. Nos and Pum are known to be essential players in GSC self-renewal, and many mRNAs are expected to be regulated by this complex. However, to date only one mRNA target of this complex, brat, has been reported. This study has identified another target, mei-P26 mRNA, and has shown that its repression by the Nos/Pum/CCR4 complex has a key role in GSC self-renewal, because the loss of GSCs in the twin mutant is strongly rescued by decreasing mei-P26 gene dosage (Joly, 2013).

    Both Brat and Mei-P26 belong to the Trim-NHL family of proteins, which have conserved functions in stem cell lineages from C. elegans to mouse (for review, see Wulczyn, 2010). Proteins within this family are potential E3 ubiquitin ligases and can act by either activating or antagonizing the miRNA pathway, through their association with Ago1 and GW182. In particular, Mei-P26 function switches from activation of the miRNA pathway in the GSCs to inhibition of the pathway in differentiating cysts where Mei-P26 levels are higher. As such, Mei-P26 plays a central role in the control of cell fate in the GSC lineage. The rescue of the twin mutant phenotype of GSC loss by decreasing mei-P26 gene dosage suggests that the levels of Mei-P26 themselves might be important for this switch of its function. This might provide an explanation as to why such a precise regulation of its level is crucial for GSC self-renewal and differentiation (Joly, 2013).

    Which molecular mechanisms underlie the fine-tuning of Mei-P26 in the GSC lineage? The translational repression of mei-P26 mRNA is not complete in GSCs. This differs from the complete repression by Nos/Pum of cyclin B mRNA in the primordial germ cells, or brat mRNA in the GSCs, and may result from the concomitant activation of mei-P26 by Vasa. Vasa does activate mei-P26 translation, leading to a peak of expression in 8-cell and 16-cell cysts. However, Vasa is expressed in all germ cells, suggesting that it is not the key regulator governing the timing of Mei-P26 peak of expression. It is proposed that translational activation of Mei-P26 by Vasa would be active already in GSCs but counterbalanced by translational repression by Nos/Pum and the CCR4-NOT complex. In cystoblasts, the presence of Bam overcomes Nos/Pum translational repression by decreasing Nos levels, which would thus switch the balance to translational activation by Vasa. This does not lead to a peak of Mei-P26 expression in cystoblasts, but rather to a progressive increase of Mei-P26 levels in proliferating cysts. This progressive accumulation of Mei-P26 could depend on the necessity to build up Vasa-mediated translational activation. However, another possibility could be that a different factor still partially represses mei-P26 translation in cystoblasts and early cysts. A potential candidate is Bam, which has been defined as a translational repressor and has recently been reported to directly repress mei-P26 mRNA translation in the male GSC lineage (Insco, 2012). The Bam expression profile in female germ cells is consistent with this potential role in mei-P26 translational repression, because Bam protein is present from cystoblasts to 8-cell cysts but absent in 16-cell cysts, where Mei-P26 levels are the highest (Joly, 2013).

    Recent advances have established the generality of a central role for translational regulations in adult stem cell lineages. Translational repression is required to prevent the synthesis of differentiation factors whose mRNAs are already present in stem cells. In the Drosophila female GSC lineage, recent work has demonstrated that changes in cell fate are driven by different translational regulation programs; associations between translational repressors evolve to trigger stage-specific regulation of mRNA targets. For example, while Nos/Pum maintain female GSCs by repressing a specific set of mRNAs, Pum associates with Brat in cystoblasts to repress a different set. The Trim-NHL proteins appear to be of particular importance in the translational regulations essential for stem cell fate as exemplified by Mei-P26. The fine-tuning of Mei-P26 protein levels by translational repression is essential for GSC self-renewal and implicate CCR4 in this regulation (Joly, 2013).

    The functions of Trim-NHL proteins are conserved in many adult stem cell lineages in different organisms, and mutations in the corresponding genes lead to highly proliferative tumors. Elucidating the molecular mechanisms behind their translational control is key to deciphering how these proteins regulate adult stem cell fates (Joly, 2013).

    Functionally diverse microRNA effector complexes are regulated by extracellular signaling

    Because microRNAs (miRNAs) influence the expression of many genes in cells, discovering how the miRNA pathway is regulated is an important area of investigation. This study found that the Drosophila miRNA-induced silencing complex (miRISC) exists in multiple forms. A constitutive form, called G-miRISC, is comprised of Ago1, miRNA, and GW182. Two distinct miRISC complexes that lack GW182 are regulated by mitogenic signaling. Exposure of cells to serum, lipids, or the tumor promoter PMA suppressed formation of these complexes. P-miRISC is comprised of Ago1, miRNA, and Loqs-PB, and it associates with mRNAs assembled into polysomes. The other regulated Ago1 complex associates with membranous organelles and is likely an intermediate in miRISC recycling. The formation of these complexes is correlated with a 5- to 10-fold stronger repression of target gene expression inside cells. Taken together, these results indicate that mitogenic signaling regulates the miRNA effector machinery to attenuate its repressive activities (Wu, 2013).

    This study found that different miRISC complexes are present in S2 cells, depending upon extracellular signals received by the cells. A constitutive G-miRISC complex composed of Ago1, miRNA, and GW182 is present under all signaling conditions tested. Other groups have shown that G-miRISC in S2 cells suppresses target mRNAs via inhibition of translation initiation and enhanced mRNA decay. This study found that lipid signaling does not affect G-miRISC but blocks other miRISC complexes from forming. This signaling is likely mediated by PKC because a phorbol ester mimics the effect of lipids on miRISC formation. Signaling blocks the formation of P-miRISC, which contains Ago1, miRNA, and Loqs-PB, but not GW182. P-miRISC represses translation of target mRNAs, which is manifested in polysome association of the complex. Thus, this work reveals a mechanistic shift in miRISC-executed translation repression under the influence of extracellular lipid signals. In the presence of lipid signaling, initiation is inhibited, and this occurs by G-miRISC. In the absence of lipid signaling, it is proposed that cells generate two levels of translational repression: one mediated by G-miRISC that inhibits initiation, and one mediated by P-miRISC that inhibits elongation. It is proposed that each miRISC complex independently represses the same target, and because they act in series (initiation - elongation), the net result on protein synthesis is the product (not sum) of each inhibitory step. This would provide the strongly synergized repression of reporter protein synthesis that was observed after serum withdrawal (Wu, 2013).

    P-miRISC resembles the miRNA loading complex (miRLC) complex in terms of subunit composition (Ago1, Loqs-PB), but the two differ in one important way. Whereas miRLC contains premiRNA, P-miRISC contains mature miRNA. Thus, P-miRISC has an inherent potential to engage target mRNAs via base pairing interactions. It is suggested that P-miRISC is formed by the processing and loading of mature miRNA into Ago1 within the miRLC. Rather than releasing Loqs-PB/Dcr-1 and recruiting GW182, the loaded Ago1 retains Loqs-PB and never recruits GW182. P-miRISC can then engage target mRNAs, but its subunit composition dictates a different mode of repression upon the target (Wu, 2013).

    Although GW182 and Loqs-PB binding to Ago1 are mutually exclusive, P-miRISC is not simply a default state when GW182 recruitment fails to occur. Knockdown of GW182 was insufficient to induce formation of P-miRISC. Moreover, formation of P-miRISC did not appear to occur at the expense of G-miRISC levels, as measured in sedimentation and immunoprecipitation experiments. This suggests a mechanism in which stable loading of miRNA is limited by the availability of cofactors for Ago1. Under serum-fed conditions, only GW182 is available, whereas both GW182 and Loqs-PB are available under serum-free conditions. This possibly offers a rapid way to modulate miRISC levels without the need for synthesis of more cofactors (Wu, 2013).

    The switch in miRISC formation is regulated by PKC, but how this switch occurs is not clear. A recent study demonstrated that the mammalian homolog of Drosophila Ago1 can be phosphorylated by Akt3, which contributes to increased miRISC-mediated translation repression (Horman, 2013). However, no evidence was found for differential phosphorylation of Ago1 in S2 cells. A study of the mammalian ortholog of Loqs-PB, called TRBP, found it to be phosphorylated by ERK kinase in response to PKC. Phosphorylation stabilized miRLC and increased processing of growth-promoting miRNAs. The same mechanism was not shown for Loqs-PB, and examination of the Loqs-PB sequence failed to find strict conservation of those sites (Wu, 2013).

    A second Ago1 complex also appears when lipid signaling is absent. Membrane-associated Ago1 likely contains miRNA, but not Loqs-PB or GW182. Association of mammalian Ago proteins with late endosomes has been previously observed. Drosophila Ago1 has also been observed to associate with endosomes in vivo. Endosomes have been proposed to serve as sites for miRISC turnover whereby miRISC continuously associates and releases from endosomes, constituting a mechanism that promotes miRISC recycling onto new targets. Thus, membrane-associated Ago1 may represent an intermediate in miRISC turnover. If so, where does the membrane- associated Ago1 originate? Several lines of evidence suggest that it originates from P-miRISC. First, its appearance precisely correlates with P-miRISC. Second, it is sensitive to puromycin treatment, which also disrupts association of P-miRISC with polysomes. However, membrane-associated Ago1 does not sediment in ribosome-containing fractions. Third, insulin specifically inhibits membrane-associated Ago1, arguing that membrane-associated Ago1 is not an obligate precursor of P-miRISC. The simplest interpretation of these data is that membrane- associated Ago1 is formed from a P-miRISC precursor. If so, then Loqs-PB dissociation must be involved in the conversion because Loqs-PB is not found in the membrane-associated complex. A similar manner of cofactor stripping was observed for GW182, which dissociated from Ago-miRNA complexes when they associated with endosomes. Perhaps, cofactor dissociation is a fundamental part of the recycling mechanism (Wu, 2013).

    This model might provide some insights into a long-standing controversy in the miRNA field. Some studies have found evidence for translation initiation as the regulated step, whereas others have found evidence for translation elongation. This work provides a potential explanation for these differences. That is, experimental model systems experiencing diverse extracellular signals might respond accordingly to form distinct types of miRISC complexes, which regulate different steps of translation. Thus, all studies have depicted an accurate picture of miRISC activity because signals that dictate miRISC subunit composition affect its mode of action (Wu, 2013).

    microRNAs that promote or inhibit memory formation in Drosophila melanogaster
    microRNAs (miRNAs) are small noncoding RNAs that regulate gene expression post-transcriptionally. Prior studies have shown that they regulate numerous physiological processes critical for normal development, cellular growth control, and organismal behavior. This study systematically surveyed 134 different miRNAs for roles in olfactory learning and memory formation using "sponge" technology to titrate their activity broadly in the Drosophila melanogaster central nervous system. At least five different miRNAs involved in memory formation or retention were identified from this large screen, including miR-9c, miR-31a, miR-305, miR-974, and miR-980. Surprisingly, the titration of some miRNAs increases memory, while the titration of others decreases memory. More detailed experiments were performed on two miRNAs, miR-974 and miR-31a, by mapping their roles to subpopulations of brain neurons and testing the functional involvement in memory of potential mRNA targets through bioinformatics and a RNA interference knockdown approach. This screen offers an important first step toward the comprehensive identification of all miRNAs and their potential targets that serve in gene regulatory networks important for normal learning and memory (Busto, 2015).

    Pervasive behavioural effects of microRNA regulation in Drosophila

    The effects of microRNA (miRNA) regulation on the genetic programs underlying behaviour remain largely unexplored. Despite this, recent work in Drosophila shows that mutation of a single miRNA locus (miR-iab4/iab8) affects the capacity of the larva to correct its orientation if turned upside-down (self-righting, SR) suggesting that other miRNAs might also be involved in behavioural control. This study explores this possibility studying early larval SR behaviour in a collection of eighty-one Drosophila miRNA mutants covering almost the entire miRNA complement of the late embryo. Unexpectedly, it was observed that more than 40% of all miRNAs tested significantly affect SR time revealing pervasive behavioural effects of miRNA regulation in the early larva. Detailed analyses of those miRNAs affecting SR behaviour (SR-miRNAs) show that individual miRNAs can affect movement in different ways suggesting that the workings of distinct molecular and cellular elements are affected by miRNA ablation. Furthermore, gene expression analysis shows that the Hox gene Abdominal-B (Abd-B) represents one of the targets de-regulated by several SR-miRNAs. This work thus reveals pervasive effects of miRNA regulation on a complex innate behaviour in Drosophila and suggests that miRNAs may be core components of the genetic programs underlying behavioural control in other animals too (Picao-Osorio, 2017).

    Temporal reciprocity of miRNAs and their targets during the maternal-to-zygotic transition in Drosophila

    During oogenesis, female animals load their eggs with messenger RNAs (mRNAs) that will be translated to produce new proteins in the developing embryo. Some of these maternally provided mRNAs are stable and continue to contribute to development long after the onset of transcription of the embryonic (zygotic) genome. However, a subset of maternal mRNAs are degraded during the transition from purely maternal to mixed maternal-zygotic gene expression. In Drosophila, two independent RNA degradation pathways are used to promote turnover of maternal transcripts during the maternal-to-zygotic transition. The first is driven by maternally encoded factors, including SMAUG, whereas the second is activated about 2 hr after fertilization, coinciding with the onset of zygotic transcription. This paper reports that a cluster of zygotically expressed microRNAs (miRNAs) targets maternal mRNAs for turnover, as part of the zygotic degradation pathway. miRNAs are small noncoding RNAs that silence gene expression by repressing translation of their target mRNAs and by promoting mRNA turnover. Intriguingly, use of miRNAs to promote mRNA turnover during the maternal-to-zygotic transition appears to be a conserved phenomenon because a comparable role was reported for miR-430 in zebrafish (Giraldez, 2006). The finding that unrelated miRNAs regulate the maternal to zygotic transition in different animals suggests convergent evolution (Bushati, 2008).

    The Drosophila miR-309 cluster contains eight microRNA (miRNA) genes, which encode six different miRNAs. Nucleotides 2 to 8 at the miRNA 5' end comprise the 'seed' region, which serves as the primary determinant of target specificity. The cluster encodes miRNAs with five distinct seed sequences, and so has the potential to regulate a broad spectrum of target messenger RNAs (mRNAs) (Bushati, 2008).

    By using homologous recombination, a mutant was generated in which the 1.1 kb comprising the miR-309 cluster was deleted and replaced with green fluorescent protein (GFP). Northern-blot analysis was used to verify that the first and last miRNAs in the cluster, miR-309 and miR-6, were not produced in the mutant. Homozygous mutant animals completed embryogenesis with no apparent defects in patterning, but approximately 20% died as larvae at different larval stages. Some individuals stopped growing at the size of L2 larva and arrested at this developmental stage for a few days before dying. Approximately 80% of mutants survived to adulthood and were viable and fertile. Introduction of a transgene containing a 2.6 kb fragment of genomic DNA spanning the miRNA cluster restored survival of the mutants to normal levels. The mutant animals showed a developmental delay during larval stages. This delay was suppressed in simultaneously collected and staged mutant larvae carrying the rescue transgene. The phenotypes that result from complete deletion of the three miR-6 miRNA genes (together with the rest of the cluster mRNAs) contrast with the severe embryonic defects that were reported with the use of antisense 2'-O-methyl oligonucleotide injection to deplete miR-6 or miR-286 (Bushati, 2008).

    RNA samples from precisely staged embryos were used to examine the expression of the miR-309 cluster during early embryogenesis. The levels were compared of mature miR-6 and miR-309 in these samples by quantitative real-time polymerase chain reaction (qPCR). Samples were normalized to two reference miRNAs, miR-310 and miR-184, which were found to be expressed at constant levels when normalized to total RNA. miR-6 and miR-309 were expressed at barely detectable levels in RNA collected from embryos during a 30 min period before the onset of zygotic transcription. The miRNAs were then strongly induced coincident with the onset of zygotic transcription. In situ hybridization analysis at this stage, showed expression of the miR-309 cluster primary transcript throughout the embryo, except in pole cells. This transcript was not detectable in miR-309 cluster mutant embryos (Bushati, 2008).

    Although the mature miRNA products persist for some time, the expression of the primary transcript shows a dynamic spatial pattern by in situ hybridization. At the midpoint of cellularization, expression of the cluster is turned off at the posterior pole and in a stripe in the anterior region of the embryo. During gastrulation, expression is lost ventrally and laterally, resulting in transient stripes in the dorsal ectoderm. By the onset of germ-band elongation, the primary transcript was essentially undetectable, but in Northern blots, the mature miRNAs are detectable until larval stages (Bushati, 2008).

    The miR-309 cluster is predicted to target many mRNAs, including those of several genes implicated in embryo patterning. However, immunolabelling for the detection of these proteins did not reveal alterations in their expression levels or patterns in the miR-309 cluster mutant. For example, the expression of the predicted miR-3-miR-309 target Ftz was compared with Even Skipped (which is not a predicted target). There was no striking difference between mutant and control embryos, consistent with the observation that miR-309 cluster mutant embryos did not show discernable embryonic patterning defects. The significance of the dynamics of spatial expression of the cluster miRNAs and the implied potential to regulate genes involved in embryonic patterning remains unclear (Bushati, 2008).

    Given that the early onset of cluster miRNA expression does not appear to play a role in regulating zygotic mRNAs involved in patterning, attention was turned to their potential to regulate the maternal-to-zygotic-transition. Expression was compared of the miR-309 cluster to a high-resolution temporal gene expression profile of early embryonic development. mRNAs with a temporal expression profile most similar to that of the miR-309 cluster contained significantly fewer 7-mers complementary to miR-309 cluster miRNAs in their 3'untranslated regions (UTRs) than would be expected by chance. This suggests that these mRNAs have been under selection to reduce their regulation by the cluster miRNAs with which they are coexpressed. Reciprocally, 7-mer seed matches complementary to cluster miRNAs were enriched in the 3'UTRs of maternal transcripts that were strongly downregulated as miRNA expression increased. The same trends hold true for 6-mer seed matches to cluster miRNAs. For the 6-mer set, the correlation data are more significant because of overall larger numbers of miRNA targets in each bin (Bushati, 2008).

    To investigate whether early zygotic miR-309 cluster miRNA expression might contribute to this downregulation, microarray analyses were performed of control and mutant embryos at 0-1 hr and 2-3 hr of embryonic development. During the first hour, miR-309 cluster miRNAs are expressed at barely detectable levels, whereas they are strongly induced during the 2-3 hr interval. Messenger RNA levels in control and miRNA mutant embryos were compared. Messenger RNAs whose expression was upregulated in the absence of the cluster miRNAs were examined with reference to two sets of maternal mRNAs that had previously been classified as being moderately or strongly downregulated during the maternal-to-zygotic transition. Forty-two of the 291 mRNAs (14%) that normally decrease by more than 3-fold between 2 and 3 hr of embryonic development were upregulated by over 1.5-fold in mutant embryos at this stage. This represents a 5-fold enrichment among the upregulated mRNAs and is statistically significant. The effect of the removal of the miRNAs was stronger in the group of the 32 maternal transcripts annotated to decrease by more than 10-fold at this stage. Thirty-five percent of these were upregulated in the mutant (12/32), a 12.5-fold enrichment (Bushati, 2008).

    The degree of enrichment of these annotated gene sets among upregulated transcripts is likely to underestimate the true degree of correlation, because only 30% of the genome was included in the original classification of moderately or strongly downregulated maternal gene sets. To get a more complete picture, a similar analysis was performed on the larger set of maternal mRNAs. One thousand sixty-five mRNAs were classified as unstable maternal transcripts on the basis of expression profiling of RNA from unfertilized wild-type eggs and assessment of the degree of their destabilization over time. One hundred thirty-eight of the 1065 unstable maternal mRNAs were among the 410 mRNAs upregulated in cluster mutant embryos at 2-3 hr. This represents more than 4-fold enrichment and is statistically highly significant. There was no significant enrichment in 0-1 hr embryos (before the miRNAs are expressed). Much less enrichment was seen in the stable maternal class, which contains both stable transcripts and transcripts that are stable in unfertilized eggs but likely degraded by the zygotic pathway in fertilized embryos. For example, some of the stable maternal class mRNAs have been classified as 3× down or 10× down. Sixteen of these mRNAs were upregulated in the miRNA mutant and probably contribute to the 1.2-fold enrichment of mRNAs classified as maternal stable in this set. This analysis indicates that downregulation of maternal transcripts is impaired in the miRNA cluster mutant, suggesting that these miRNAs play a role in the zygotic pathway of maternal mRNA turnover (Bushati, 2008).

    The foregoing observations suggest that the miRNA cluster and its targets have largely reciprocal temporal expression patterns, a situation analogous to the spatially reciprocal relationship between many miRNAs and their targets at later stages of embryogenesis and to the temporal relationship between the C. elegans heterochronic miRNAs and their targets. To assess the significance of these observations, the occurrence of miRNA cluster target sites among the regulated mRNAs was compared with what would be expected to occur by chance. Among the 410 transcripts upregulated in the miRNA cluster mutant, 96 contained 7-mers complementary to the seed of one or more cluster miRNAs. This represents a statistically significant enrichment of 1.8-fold (Bushati, 2008).

    Among the mRNAs upregulated in cluster mutant embryos at 2-3 hr, mRNAs from a set of maternal mRNAs, which contained such 7-mer sites, were enriched 3.6-fold. The enrichment was 6.4-fold in the class of maternal mRNAs 3× downregulated containing such 7-mers and 48-fold in 10× downregulated set containing miR-309 cluster 7-mer sites. Importantly, no significant enrichment of 7-mers was observed in 0-1 hr embryos, prior to the onset of miRNA cluster expression. Comparable analysis for the larger set of mRNAs produced similar results. Maternal mRNAs containing target sites were enriched 2.5-fold and the set of unstable maternal mRNAs carrying target sites 6-fold among the mRNAs upregulated in cluster mutant embryos at 2-3 hr. Again, no significant enrichment was seen in the 0-1 hr samples (Bushati, 2008).

    These statistical relationships suggest that the regulation of these mRNAs depends on the presence of the miRNA sites. To confirm that such sites are indeed functional, luciferase reporter constructs containing the 3' UTRs of 32 of the affected maternal mRNAs were prepared from the different functional categories mentioned above and expressed together with the miR-309 cluster in Drosophila S2 cells. Twenty-nine of the 32 reporters were statistically significantly downregulated upon miR-309 cluster expression, indicating that they carry functional miR-309 cluster target sites (Bushati, 2008).

    The cluster encodes miRNAs with five different seed sequences, reflecting the capacity to regulate different sets of target mRNAs. To assess the contribution of individual miRNAs to the effects of the cluster as a whole, 7-mer seed matches complementary to individual miR-309 cluster miRNAs were analyzed. Four of the five unique seeds (miR-3 and 309 have the same seed sequence) were significantly enriched among the upregulated mRNAs at 2-3 hr but not at 0-1 hr. The magnitude of the enrichment and the statistical significance were stronger for miR-6, suggesting that it may contribute disproportionately to the effects of the cluster. This might be in part because miR-6 is present in three copies and so might be expressed at a higher level than the others. These data suggest that, with the possible exception of miR-286, the five distinct miRNAs encoded in the cluster act in concert to regulate a broad spectrum of mRNAs during the maternal-to-zygotic transition (Bushati, 2008).

    SMAUG has been identified as a key component of the maternal system for maternal mRNA turnover in the embryo (Tadros, 2007), whereas the evidence presented above suggests that the miR-309 cluster acts zygotically to promote turnover of maternal mRNAs. A priori, these systems might be functionally related, acting in concert. Alternatively, they might represent independent systems. To explore these possibilities, the degree to which the sets of targets regulated by these two systems overlap was examined (Bushati, 2008).

    Of the 1065 unstable maternal transcripts identified by Tadros (2007), 710 were identified as SMAUG targets by expression profiling of RNA from unfertilized eggs laid by smaug mutant flies (note: SMAUG is deposited maternally and acts on maternally deposited mRNAs). As mentioned before, 138 of the transcripts upregulated in the miR-309 cluster mutant at 2-3 hr were classified as unstable maternal transcripts, which represents more than 4-fold enrichment. Ninety-two of these transcripts were also targeted by SMAUG, which represents more than 4-fold enrichment. Of these, 20 (21.7%) had 7-mer seed matches complementary to cluster miRNAs in their 3' UTRs and so might represent a set of mRNAs potentially coregulated by the maternal and zygotic systems. Other mRNAs among the SMAUG targets were not affected in the miRNA cluster mutants -- for example, Hsp83, whose downregulation depends strongly on the SMAUG system. Of the 355 unstable transcripts that had been reported to be SMAUG independent, 46 were among the 410 mRNAs upregulated in the miR-309 cluster mutant embryos. This represents a more than 4-fold enrichment. Eighteen (39%) of these carry 7-mers complementary to miR-309 cluster miRNAs, an 8-fold enrichment. This set includes mRNAs such as orb, oskar, and exuperantia and may represent the set of mRNAs regulated mainly by the zygotic system. Together, these data suggest that the maternal and zygotic systems regulate distinct but overlapping sets of maternal mRNAs (Bushati, 2008).

    The observation that some SMAUG targets also appear to be targets of the zygotic system raised the question of whether there might be a genetic interaction between the two systems. It can be expected that there might be an additive effect of removing two systems that share some common targets (if it is assumed that the common targets contribute to the mutant phenotype). To address this, it was asked whether removing one copy of maternal SMAUG would enhance the severity of the zygotic miR-309 cluster mutant phenotypes. No difference was observed in embryonic survival rates between miR-309 cluster mutants and those also lacking one copy of maternal SMAUG. However, there appeared to be a small reduction in survival of miR-309 cluster mutant larvae whose mothers lacked one copy of SMAUG, from 85% ± 5% to 69% ± 12%. This difference was, however, not statistically significant (t test = 0.06). The marginal reduction in survival might reflect an additive effect of perturbing both systems on their common targets. It is possible that a further reduction of SMAUG activity might result in a statistically significant effect. At present, though, it is not possible to conclude that there is an interaction that is more than additive between the two systems (Bushati, 2008).

    These findings indicate that the early zygotic onset of miR-309 cluster miRNA expression acts to promote the turnover of many maternally deposited mRNAs. Failure to downregulate maternal mRNAs by this zygotic mechanism has knock-on effects on zygotic gene expression and may result in a late onset phenotype reflected by reduced survival and delayed larval development for many of the surviving animals. Elimination of the early zygotic expression of the miR-430 miRNA gene family also led to substantial misregulation of maternal mRNAs and to a late onset zygotic defect in Zebrafish (Giraldez, 2006). Although miRNAs have been shown to act to ensure a proper transition between maternal and zygotic gene expression programs in flies and fish, the miRNAs involved are not conserved. Perhaps the fact that miRNAs act in part by leading to mRNA deadenylation, and subsequent destabilization, provided a means to promote turnover of a selected set of maternally deposited mRNAs. miRNAs may have been co-opted independently during evolution to fulfill a comparable function in different animals. The mechanistic basis for their action and the biological output are both conserved, but the miRNAs themselves and the identity of their targets are not. This may be an example of convergent evolution (Bushati, 2008).

    The Smaug RNA-binding protein is essential for microRNA synthesis during the Drosophila maternal-to-zygotic transition

    Metazoan embryos undergo a maternal-to-zygotic transition (MZT) during which maternal gene products are eliminated and the zygotic genome becomes transcriptionally active. During this process RNA-binding proteins (RBPs) and the microRNA-induced silencing complex (miRISC) target maternal mRNAs for degradation. In Drosophila, the Smaug (SMG), Brain tumor (BRAT) and Pumilio (PUM) RBPs bind to and direct the degradation of largely distinct subsets of maternal mRNAs. SMG has also been shown to be required for zygotic synthesis of mRNAs and several members of the miR-309 family of microRNAs (miRNAs) during the MZT. This study carried out global analysis of small RNAs both in wild type and in smg mutants. It was found that 85% all miRNA species encoded by the genome are present during the MZT. Whereas loss of SMG has no detectable effect on Piwi-interacting RNAs (piRNAs) or small interfering RNAs (siRNAs), zygotic production of more than 70 species of miRNAs fails or is delayed in smg mutants. SMG is also required for the synthesis and stability of a key miRISC component, Argonaute 1 (AGO1), but plays no role in accumulation of the Argonaute-family proteins associated with piRNAs or siRNAs. In smg mutants, maternal mRNAs that are predicted targets of the SMG-dependent zygotic miRNAs fail to be cleared. BRAT and PUM share target mRNAs with these miRNAs but not with SMG itself. The study hypothesizes that SMG controls the MZT, not only through direct targeting of a subset of maternal mRNAs for degradation but, indirectly, through production and function of miRNAs and miRISC, which act together with BRAT and/or PUM to control clearance of a distinct subset of maternal mRNAs (Luo, 2016).

    To identify small RNA species expressed during the Drosophila MZT and to assess the role of SMG in their regulation 18 small-RNA libraries were produced and sequenced: nine libraries from eggs or embryos produced by wild-type females and nine from smg-mutant females. The 18 libraries comprised three biological replicates each from the two genotypes and three time-points: (1) 0-to-2 hour old unfertilized eggs, in which zygotic transcription does not occur and thus only maternally encoded products are present; (2) 0-to-2 hour old embryos, the stage prior to large-scale zygotic genome activation; and (3) 2-to-4 hour old embryos, the stage after to large-scale zygotic genome activation. After pre-alignment processing, a total of ~144 million high quality small-RNA reads was obtained and 110 million of these perfectly matched the annotated Drosophila genome (Luo, 2016).

    Loss of SMG had no significant effect on piRNAs and siRNAs, or on the Argonaute proteins associated with those small RNAs: Piwi, Aubergine (AUB), AGO3, and AGO2, respectively. In contrast, loss of SMG resulted in a dramatic, global reduction in miRNA populations during the MZT as well as reduced levels of AGO1, the miRISC-associated Argonaute protein in Drosophila (Luo, 2016).

    A pre-miRNA can generate three types of mature miRNA: (1) a canonical miRNA, which has a perfect match to the annotated mature miRNA; (2) a non-canonical miRNA, which shows a perfect match to the annotated mature miRNA but with additional nucleotides at the 5'- or 3'- end that match the adjacent primary miRNA sequence, and (3) a miRNA with non-templated terminal nucleotide additions (an NTA-miRNA), which has nucleotides at its 3'-end that do not match the primary miRNA sequence (Luo, 2016).

    In these libraries a total of 364 distinct miRNA species were identified that mapped to miRBase, comprising 85% (364/426) of all annotated mature miRNA species in Drosophila. Thus, the vast majority of all miRNA species encoded by the Drosophila genome are expressed during the MZT. Overall, in wild type, an average of 75% of all identified miRNAs fell into the canonical category. The remaining miRNAs were either non-canonical (10%) or NTA-miRNAs (15%) (Luo, 2016).

    To validate these sequencing results, those mature miRNA species identified in the data that perfectly matched the Drosophila genome sequence (i.e., canonical and non-canonical) were compared with a previously published miRNA dataset from 0 to 6 hour old embryos. To avoid differences caused by miRBase version, data sets from previous study were remapped to miRBase Version 19 and f99% of their published miRNA species were found to be on the miRNA list (176/178 mature miRNA species comprising 161 canonical miRNA s and 94 non-canonical miRNA s) . There were an additional 181 mature miRNA species in the library that had not been identified as expressed in early embryos in the earlier study (Luo, 2016).

    As a second validation, the list of maternally expressed miRNA species (those present in the 0-to-2 hour wild-type unfertilized egg samples) were compared with the most recently published list of maternal miRNAs, which had been defined in the same manner. 99% of the 86 published maternal miRNA species were on this study's maternal miRNA list (85/86). An additional 144 maternal miRNA species in the library were identified that had not been observed in the previous study. Identification of a large number of additional miRNA species in unfertilized eggs and early embryos can be attributed to the depth of coverage of the current study. The current dataset, therefore, provides the most complete portrait to date of the miRNAs present during the Drosophila MZT (Luo, 2016).

    Next, global changes in miRNA species during the MZT were analyzed in wild-type embryos. A dramatic increase was observed in the proportion of miRNAs relative to other small RNAs that was due to an increase in absolute miRNA amount rather than a decrease in the amount of other types of small RNAs. In wild-type 0-to-2 hour unfertilized eggs, the proportion of the small RNA libraries comprised of canonical and non-canonical miRNAs was 12.8%. These represent maternally loaded miRNAs since unfertilized eggs do not undergo zygotic genome activation. The proportion of small RNAs represented by miRNAs increased dramatically during the MZT, reaching 50.7% in 2-to-4 hour embryos. The other abundant classes of small RNAs underwent either no change or relatively minor changes over the same time course. It is concluded that there is a large amount of zygotic miRNA synthesis during the MZT in wild-type embryos (Luo, 2016).

    For more detailed analysis of the canonical, non-canonical and NTA isoforms focus was placed on 154 miRNA species that possessed an average of > 10 reads per million (RPM) for all three isoform types in one or more of the six sample sets. A focus was placed on changes in wild type. Among all miRNAs, in wild type the proportion of canonical isoforms increased over the time-course from 69% to 83%, the proportion of non-canonical miRNAs remained constant (from 9% to 10%) , and the proportion of the NTA-miRNAs decreased (from 22% to 7%). These results derive from the fact that, during the MZT, the vast majority of newly synthesized miRNAs were canonical, undergoing a more than seven-fold increase from 103,105 to 744,043 RPM; that non-canonical miRNAs underwent a comparable, nearly seven-fold, increase from 13,902 to 92,199; whereas NTA-miRNAs underwent a less than two-fold increase, from 32,840 to 63,847, thus decreasing in relative proportion (Luo, 2016).

    Whereas the proportion of the small-RNA population that was comprised of miRNAs increased fourfold over the wild-type time-course, concomitant with increases in overall miRNA abundance, there was no such increase in the smg mutant embryos: 21.9% of the small RNAs were miRNAs in 0-to-2 hour unfertilized smg mutant eggs (mean RPM = 203,415) and 20.5% (mean RPM = 196,110) were miRNAs in 2-to-4 hour smg mutant embryos (Luo, 2016).

    This difference between wild type and smg mutants could have resulted from the absence of a small number of extremely highly expressed miRNA species in the mutant. Alternatively, it may have been a consequence of a widespread reduction in the levels of all or most zygotically synthesized miRNAs in smg mutants. To assess the cause of this difference, canonical miRNA reads were graphed in scatter plots. These showed that a large number of miRNA species had significantly reduced expression levels in 0-to-2 and in 2-to-4 hour smg-mutant embryos relative to wild type. Most of the down-regulated miRNA species exhibited a more than four-fold reduction in abundance relative to wild type. Furthermore, this reduction occurred for miRNA species expressed over a wide range of abundances in wild type (Luo, 2016).

    Box plots were then used to analyze the canonical, non-canonical and NTA isoforms of the 154 miRNA species identified in the previous section. These showed that, in wild type, the median abundance of canonical, non-canonical and 3' NTA miRNAs increased significantly in 0-to-2 and in 2-to-4 hour embryos relative to 0-to-2 hour unfertilized eggs. In contrast, there was no significant increase in the median abundance of any of the three isoforms of miRNAs in the smg-mutant embryos. Also for all three isoform types, when each time point was compared between wild type and smg mutant, there was no difference between wild type and mutant in 0-to-2 hour unfertilized eggs but there was a highly significant difference between the two genotypes at both of the embryo time-points. Whereas the abundance of miRNAs differed between wild-type and mutant embryos, there was no difference in length or first-nucleotide distribution of canonical miRNAs, nor in the non-templated terminal nucleotides added to NTA-miRNAs (Luo, 2016).

    As described above, during the wild-type MZT canonical miRNAs comprised the major isoform that was present (69% to 83% of miRNAs). It was next asked whether miRNA species could be categorized into different classes based on their expression profiles during the wild-type MZT. 131 canonical miRNA species that had > 10 mean RPM in at least one of the six datasets were analyzed. Hierarchical clustering of their log 2 RPM values identified five distinct categories of canonical miRNA species during the MZT. The effects of smg mutations on each of these classes were analyzed (Luo, 2016).

    The data are consistent with a model in which SMG degrades its direct targets without the assistance of miRNAs whereas a large fraction of the indirectly affected maternal mRNAs in smg mutants fails to be degraded by virtue of being targets of zygotically produced miRNA species that are either absent or present at significantly reduced levels in smg mutants. Thus, SMG is required both for early, maternally encoded decay and for late, zygotically encoded decay. In the former case SMG is a key specificity component that directly binds to maternal mRNAs; in the latter case SMG is required for the production of the miRNAs (and AGO1 protein) that are responsible for the clearance of an additional subset of maternal mRNAs (Luo, 2016).

    In Drosophila, the stability of miRNAs is enhanced by AGO1 and vice versa. Since miRNA levels are dramatically reduced in smg mutants, Ago1 mRNA and AGO1 protein levels were assessed during the MZT both in wild type and in smg mutants. In wild type, AGO1 levels were low in unfertilized eggs and 0-to-2 hour embryos but then increased substantially in 2-to-4 hour embryos. These western blot data are consistent with an earlier, proteomic, study that reported a more than three-fold increase in AGO1 in embryos between 0-to-1.5 hours and 3-to-4.5 hours. In contrast to AGO1 protein, it was found using RT-qPCR that Ago1 mRNA levels remained constant during the MZT. Taken together with a previous report that Ago1 mRNA is maternally loaded, the increase in AGO1 protein levels in the embryo is, therefore, most likely to derive from translation of maternal Ago1 mRNA rather than from newly transcribed Ago1 mRNA (Luo, 2016).

    Next, AGO1, AGO2, AGO3, AUB and Piwi protein levels were analyzed in eggs and embryos from mothers carrying either of two smg mutant alleles: smg1 and smg47. The smg mutations had no effect on the expression profiles of AGO2, AGO3, AUB or Piwi. In contrast, in smg-mutant embryos, the amount of AGO1 protein at both 0-to-2 and 2-to-4 hours was reduced relative to wild type and this defect was rescued in embryos that expressed full-length, wild-type SMG from a transgene driven by endogenous smg regulatory sequences. The reduction of AGO1 protein levels in smg mutants was not a secondary consequence of reduced Ago1 mRNA levels since Ago1 mRNA levels in both the smg-mutant and the rescued-smg-mutant embryos were very similar to wild type (Luo, 2016).

    A plausible explanation for the decrease in AGO1 levels in smg mutants is the reduced levels of miRNAs, which would then result in less incorporation of newly synthesized AGO1 into functional miRISC and consequent failure to stabilize the AGO1 protein. To assess this possibility, a time-course in wild-type unfertilized eggs was analyzed in which zygotic genome activation and, therefore, zygotic miRNA synthesis, does not occur. It was found that AGO 1 levels were reduced in 2-to-4 hour wild-type unfertilized eggs compared with wild-type embryos of the same age. This result is consistent with a requirement for zygotic miRNAs in the stabilization of AGO1 protein (Luo, 2016).

    Next, wild-type unfertilized egg and smg-mutant unfertilized egg time-courses were compared, and AGO1 levels were found to be further reduced in the smg mutant relative to wild type. This suggests that SMG protein has an additional function in the increase in AGO1 protein levels that is independent of SMG's role in zygotic miRNA production (since these are produced in neither wild-type nor smg-mutant unfertilized eggs) (Luo, 2016).

    To assess whether this additional function derives from SMG's role as a post-transcriptional regulator of mRNA, smg1 mutants were rescued either with a wild-type SMG transgene driven by the Gal4:UAS system (SMGWT) or a GAL4:UAS-driven transgene encoding a version of SMG with a single amino-acid change that abrogates RNA-binding (SMGRBD) and, therefore, is unable to carry out post-transcriptional regulation of maternal mRNAs. It was found that, whereas AGO1 was detectable in both unfertilized eggs and embryos from SMGWT-rescued mothers, AGO1 was undetectable in unfertilized eggs from SMGRBD-rescued mothers and was barely detectable in embryos from these mothers. Thus, SMG's RNA-binding ability is essential for its non-miRNA-mediated role in regulation of AGO1 levels during the MZT (Luo, 2016).

    Since the abundance of SMGWT and SMGRBD proteins is very similar, the preceding result excludes the possibility that it is physical interaction between SMG and AGO1 that stabilizes the AGO1 protein. It was previously shown that the Ago1 mRNA is not bound by SMG. Thus, SMG must regulate one or more other mRNAs whose protein products, in turn, affect the synthesis and/or stability of AGO1 protein. It is known that turnover of AGO1 protein requires Ubiquitin-activating enzyme 1 (UBA1) and is carried out by the proteasome . It was previously shown that the Uba1 mRNA is degraded during the MZT in a SMG-dependent manner and that both the stability and translation of mRNAs encoding 19S proteasome regulatory subunits are up-regulated in smg-mutant embryos. It is speculated that increases in UBA1 and proteasome subunit levels in smg mutants contribute to a higher rate of AGO1 turnover and, thus, lower AGO1 abundance than in wild type (Luo, 2016).

    AGO1 physically associates with BRAT. It is not known whether AGO1 interacts with PUM but it has been reported that, in mammals and C. elegans , Argonaute-family proteins interact with PUM/PUF-family proteins. Recent studies identified direct target mRNAs of the BRAT and PUM RBPs in early Drosophila embryos and showed through analysis of brat mutants that, during the MZT, BRAT directs late (i.e., after zygotic genome activation) decay of a subset of maternal mRNAs. These data permitted asking whether the maternal mRNAs that are predicted to be indirectly regulated by SMG via its role in miRISC production might be co-regulated by BRAT and/or PUM (Luo, 2016).

    A highly significant overlap was found between the predicted miRNA-dependent indirect targets of SMG and both BRAT-and PUM-bound mRNAs in early embryos. This suggests that BRAT and PUM might function together with miRISC during the MZT to direct decay of maternal mRNAs (Luo, 2016).

    Given that BRAT and PUM bind to largely non-overlapping sets of mRNAs during the MZT, there are three types of hypothetical BRAT-PUM-miRISC-containing complexes: one with both BRAT and PUM, one with BRAT only, one with PUM only. To assess this possibility for a specific set of zygotically produced miRNAs, the lists of mRNAs stabilized in 2-to-3 hour old embryos from miR-309 deletion mutants were compared to the lists of BRAT and PUM direct-target mRNAs. There was no significant overlap of PUM-bound mRNAs with those up-regulated in miR-309 mutants. However, there was a highly significant overlap of mRNAs up-regulated in miR-309-mutant embryos with BRAT-bound mRNAs. These results lead to the hypothesis that BRAT (but not PUM) co-regulates clearance of miR-309-family miRNA target maternal mRNAs during the MZT (Luo, 2016).

    Target repression induced by endogenous microRNAs: Large differences, small effects

    MicroRNAs are small RNAs that regulate protein levels. It is commonly assumed that the expression level of a microRNA is directly correlated with its repressive activity - that is, highly expressed microRNAs will repress their target mRNAs more. This study investigated the quantitative relationship between endogenous microRNA expression and repression for 32 mature microRNAs in Drosophila melanogaster S2 cells. In general, more abundant microRNAs were found to repress their targets to a greater degree. However, the relationship between expression and repression is nonlinear, such that a 10-fold greater microRNA concentration produces only a 10% increase in target repression. The expression/repression relationship is the same for both dominant guide microRNAs and minor mature products (so-called passenger strands/microRNA* sequences). However, examples were found of microRNAs whose cellular concentrations differ by several orders of magnitude, yet induce similar repression of target mRNAs. Likewise, microRNAs with similar expression can have very different repressive abilities. The association of microRNAs with Argonaute proteins does not explain this variation in repression. The observed relationship is consistent with the limiting step in target repression being the association of the microRNA/RISC complex with the target site. These findings argue that modest changes in cellular microRNA concentration will have minor effects on repression of targets (Kozomara, 2014. PubMed).

    MicroRNA biogenesis via splicing and exosome-mediated trimming in Drosophila

    microRNAs (miRNAs) are approximately 22 nucleotide regulatory RNAs derived from hairpins generated either by Drosha cleavage (canonical substrates) or by splicing and debranching of short introns (mirtrons). The 5' end of the highly conserved Drosophila mirtron-like locus mir-1017 is coincident with the splice donor, but a substantial 'tail' separates its hairpin from the 3'splice acceptor. Genetic and biochemical studies define a biogenesis pathway involving splicing, lariat debranching, and RNA exosome-mediated 'trimming,' followed by conventional dicing and loading into AGO1 to yield a miRNA that can repress seed-matched targets. Analysis of cloned small RNAs yielded six additional candidate 3' tailed mirtrons in D. melanogaster. Altogether, these data reveal an unexpected role for the exosome in the biogenesis of miRNAs from hybrid mirtron substrates (Flynt, 2010).

    Canonical miRNAs derive from primary miRNA (pri-miRNA) transcripts bearing one or more imperfect hairpins typically ~55–70 nt in length. In animals, pri-miRNAs are cleaved by the nuclear Drosha RNase III enzyme to release pre-miRNA hairpins, which are cleaved by the cytoplasmic Dicer RNase III enzyme to generate miRNA/miRNA* duplexes. One strand is preferentially selected for incorporation into an Argonaute complex, which uses the miRNA as a guide to identify mRNA targets for degradation and/or translational inhibition. Animal miRNAs usually target partially complementary mRNAs, often involving 7 nt of Watson-Crick basepairing to positions 2–8 of the miRNA (the 'seed') (Flynt, 2010).

    The repertoire of miRNA-class regulatory RNAs was expanded by the discovery of short hairpin introns known as mirtrons. Mirtrons bypass Drosha cleavage by exploiting the spliceosome to generate their precursor ends. Following lariat debranching, linearized mirtrons adopt hairpin structures that are diced and loaded into Argonaute proteins as functional miRNAs. While best characterized in Drosophila, mirtrons exist in species as diverse as nematodes, and potentially plants (Flynt, 2010).

    In the atypical Drosophila mirtron-like locus (mir-1017), only the 5' hairpin terminus coincides with a splice junction; a substantial 3' tail follows to its 3' splice junction. This study provides genetic and biochemical evidence that mir-1017 generates a miRNA-class regulatory RNA via a multistep process involving intron splicing and debranching, exosome-mediated trimming of the 3' tail, and dicing. Analysis of Drosophila small RNA data revealed additional intronic hairpins bearing 3' tails that are processed into miRNA/miRNA* duplexes, revealing a subfamily of miRNAs that transit an exosome-mediated biogenesis pathway (Flynt, 2010).

    This study shows that a subset of Drosophila mirtrons encode a terminal extension 3' of the pre-miRNA hairpin, which is 'trimmed' by the RNA exosome (see Pathways that generate miRNA-class regulatory RNAs from short hairpins in Drosophila. ). Otherwise, tailed mirtrons are similar to conventional mirtrons in that they bypass the Microprocessor by accessing the splicing and debranching pathway. Most of the current studies focused on the 3' tailed mirtron mir-1017, which is strictly conserved across Drosophilid evolution and regulates conserved target genes including yan. More recently-evolved substrates were also identified that appear to access the 3'tailed mirtron pathway. Currently, 151 canonical miRNAs, 18 conventional mirtrons and 7 tailed mirtrons have been identified in Drosophila melanogaster. The conventional mirtron pathway is envisioned as an 'add-on' to the canonical miRNA pathway, in which splicing has evolved to generate substrates that exploit a pre-existing canonical pathway. Similarly, it is hypothesized that the tailed mirtron pathway represents an 'add-on' to the conventional mirtron pathway, whereby the RNA exosome has been recruited to permit access of an asymmetric mirtron into the canonical miRNA pathway (Flynt, 2010).

    The RNA exosome is well known for its role in the turnover of normal mRNAs and abnormal transcripts. However, this study provides additional evidence for positive roles of the exosome in the biogenesis of non-coding RNAs. In previous studies, the exosome was shown to be required for maturation of rRNA, snRNAs and snoRNAs through 3'–5' trimming of terminal nucleotides. Thus, the consequence of blocking exosome processing of these substrates is the retention of undesired 3' nucleotides. The role of the exosome in biogenesis of 3' tailed mirtrons is distinct in that substrate trimming is prerequisite for subsequent steps in substrate metabolism. In this sense it is reminiscent of processing of yeast 5.8S rRNA, which involves consecutive exonucleolytic processing reactions by the exosome followed by the Rex proteins. The evidence that 3' trimming is mediated by the nuclear Exo11 complex via Rrp6 suggests that the trimming and dicing reactions are compartmentalized in the cell. Removal of the 3' tail may be requisite for efficient export by Exportin-5, which is selective for hairpins with a short 3′ overhang (Flynt, 2010).

    Other non-canonical substrates generate miRNA-class regulatory RNAs, including certain snoRNAs. A viral tRNA/miRNA fusion was found to use tRNaseZ to liberate a pre-miRNA hairpin. In addition, siRNA-class regulatory RNAs derive from other classes of inverted repeat transcripts, such as hairpin RNAs and endo-shRNAs. Finally, a variety of trans-encoded substrates, generating either perfect or imperfect dsRNA, access Dicer pathways to generate endo-siRNAs in Drosophila and mammals. Altogether, a multitude of biogenesis pathways have emanated from the simple building blocks of cis- or trans-encoded dsRNA and a Dicer-class enzyme to generate diverse regulatory RNAs (Flynt, 2010).

    Drosophila exoribonuclease nibbler is a tumor suppressor, acts within the RNA(i) machinery and is not enriched in the nuage during early oogenesis

    micro RNAs (miRNAs) are important regulators of many biological pathways. A plethora of steps are required to form, from a precursor, the mature miRNA that eventually acts on its target RNA to repress its expression or to inhibit translation. Recently, Drosophila nibbler (nbr) has been shown to be an important player in the maturation process of miRNA and piRNA. Nbr is an exoribonuclease which helps to shape the 3' end of miRNAs by trimming the 3' overhang to a final length. In contrast to previous reports on the localization of Nbr, this study reports that 1) Nbr is expressed only during a short time of oogenesis and appears ubiquitously localized within oocytes, and that 2) Nbr was is not enriched in the nuage where it was shown to be involved in piwi-mediated mechanisms. To date, there is little information available on the function of nbr for cellular and developmental processes. Due to the fact that nbr mutants are viable with minor deleterious effects, this study used the GAL4/UAS over-expression system to define novel functions of nbr. Hitherto unknown functions of nbr are revealed; 1) as a tumor suppressor and 2) as a suppressor of RNA(i). Finally, it was confirmed that nbr is a suppressor of transposon activity. These data suggest that nbr exerts much more widespread functions than previously reported from trimming 3' ends of miRNAs only (Castillejo-Lopez, 2018).

    Stable intronic sequence RNAs have possible regulatory roles in Drosophila melanogaster

    Stable intronic sequence RNAs (sisRNAs) have been found in Xenopus tropicalis, human cell lines, and Epstein-Barr virus; however, the biological significance of sisRNAs remains poorly understood. This study identified sisRNAs in Drosophila melanogaster by deep sequencing, reverse transcription polymerase chain reaction, and Northern blotting. A sisRNA (sisR-1) was characterized from the regena (rga) locus and was found to be processed from the precursor messenger RNA (pre-mRNA). A cis-natural antisense transcript (ASTR) from the rga locus was also documented that is highly expressed in early embryos. During embryogenesis, ASTR promotes robust rga pre-mRNA expression. Interestingly, sisR-1 represses ASTR, with consequential effects on rga pre-mRNA expression. These results suggest a model in which sisR-1 modulates its host gene expression by repressing ASTR during embryogenesis. The study proposes that sisR-1 belongs to a class of sisRNAs with probable regulatory activities in Drosophila (Pek 2015).

    Uridylation of RNA hairpins by Tailor confines the emergence of microRNAs in Drosophila

    Uridylation of RNA species represents an emerging theme in post-transcriptional gene regulation. In the microRNA pathway, such modifications regulate small RNA biogenesis and stability in plants, worms, and mammals. This study reports Tailor, an uridylyltransferase that is required for the majority of 3' end modifications of microRNAs in Drosophila and predominantly targets precursor hairpins. Uridylation modulates the characteristic two-nucleotide 3' overhang of microRNA hairpins, which regulates processing by Dicer-1 and destabilizes RNA hairpins. Tailor preferentially uridylates mirtron hairpins, thereby impeding the production of non-canonical microRNAs. Mirtron selectivity is explained by primary sequence specificity of Tailor, selecting substrates ending with a 3' guanosine. In contrast to mirtrons, conserved Drosophila precursor microRNAs are significantly depleted in 3' guanosine, thereby escaping regulatory uridylation. These data support the hypothesis that evolutionary adaptation to Tailor-directed uridylation shapes the nucleotide composition of precursor microRNA 3' ends. Hence, hairpin uridylation may serve as a barrier for the de novo creation of microRNAs in Drosophila (Reimão-Pinto, 2015)

    Selective suppression of the splicing-mediated microRNA pathway by the terminal uridyltransferase Tailor

    Several terminal uridyltransferases (TUTases) are known to modulate small RNA biogenesis and/or function via diverse mechanisms. This study demonstrates that Drosophila splicing-derived pre-miRNAs (mirtrons) are efficiently modified by the previously uncharacterized TUTase, Tailor. Tailor is necessary and sufficient for mirtron hairpin uridylation, and this modification inhibits mirtron biogenesis. Genome-wide analyses demonstrate that mirtrons are dominant Tailor substrates, and three features contribute to substrate specificity. First, reprogramming experiments show Tailor preferentially identifies splicing-derived miRNAs. Second, in vitro tests indicate Tailor prefers substrate hairpins over mature miRNAs. Third, Tailor exhibits sequence preference for 3'-terminal AG, a defining mirtron characteristic. The study supports the notion that Tailor preferentially suppresses biogenesis of mirtrons, an evolutionarily adventitious pre-miRNA substrate class. Moreover, preferential activity of Tailor on 3'-G canonical pre-miRNAs and specific depletion of such loci from the pool of conserved miRNAs are detected. Thus, Tailor activity may have had collateral impact on shaping populations of canonical miRNAs (Bortolamiol-Becet, 2015).

    Molecular basis for cytoplasmic RNA surveillance by uridylation-triggered decay in Drosophila

    The posttranscriptional addition of nucleotides to the 3' end of RNA regulates the maturation, function, and stability of RNA species in all domains of life. This study shows that in flies, 3' terminal RNA uridylation triggers the processive, 3'-to-5' exoribonucleolytic decay via the RNase II/R enzyme CG16940, a homolog of the human Perlman syndrome exoribonuclease Dis3l2. Together with the TUTase Tailor, dmDis3l2 forms the cytoplasmic, terminal RNA uridylation-mediated processing (TRUMP) complex that functionally cooperates in the degradation of structured RNA RNA immunoprecipitation and high-throughput sequencing reveals a variety of TRUMP complex substrates, including abundant non-coding RNA, such as 5S rRNA, tRNA, snRNA, snoRNA, and the essential RNase MRP. Based on genetic and biochemical evidence, a key function is proposed of the TRUMP complex in the cytoplasmic quality control of RNA polymerase III transcripts. Together with high-throughput biochemical characterization of dmDis3l2 and bacterial RNase R, these results imply a conserved molecular function of RNase II/R enzymes as "readers" of destabilizing posttranscriptional marks - uridylation in eukaryotes and adenylation in prokaryotes - that play important roles in RNA surveillance (Reimao-Pinto, 2016).

    MicroRNA-277 modulates the neurodegeneration caused by Fragile X premutation rCGG repeats

    Fragile X-associated tremor/ataxia syndrome (FXTAS), a late-onset neurodegenerative disorder, has been recognized in older male fragile X premutation carriers and is uncoupled from fragile X syndrome. Using a Drosophila model of FXTAS, it has been shown that transcribed premutation repeats alone are sufficient to cause neurodegeneration. miRNAs are sequence-specific regulators of post-transcriptional gene expression. To determine the role of miRNAs in rCGG repeat-mediated neurodegeneration, miRNA expression was profiled, and selective miRNAs were identified, including mir-277 stem loop, that are altered specifically in Drosophila brains expressing rCGG repeats. Their genetic interactions with rCGG repeats were tested, and it was found that miR-277 can modulate rCGG repeat-mediated neurodegeneration. Furthermore, Drep-2 and Vimar were identified as functional targets of miR-277 that could modulate rCGG repeat-mediated neurodegeneration. Finally, it was found that hnRNP A2/B1, an rCGG repeat-binding protein, can directly regulate the expression of miR-277. These results suggest that sequestration of specific rCGG repeat-binding proteins could lead to aberrant expression of selective miRNAs, which may modulate the pathogenesis of FXTAS by post-transcriptionally regulating the expression of specific mRNAs involved in FXTAS (Tan, 2012).

    Fragile X syndrome (FXS), the most common form of inherited mental retardation, is caused by expansion of the rCGG trinucleotide repeat in the 5' untranslated region (5' UTR) of the fragile X mental retardation 1 (FMR1) gene, which leads to silencing of its transcript and the loss of the encoded fragile X mental retardation protein (FMRP). Most affected individuals have more than 200 rCGG repeats, referred to as full mutation alleles. Fragile X syndrome carriers have FMR1 alleles, called premutations, with an intermediate number of rCGG repeats between patients (>200 repeats) and normal individuals (<60 repeats). Recently, the discovery was made that male and, to a lesser degree, female premutation carriers are at greater risk of developing an age-dependent progressive intention tremor and ataxia syndrome, which is uncoupled from fragile X syndrome and known as fragile X-associated tremor/ataxia syndrome (FXTAS). This is combined with cognitive decline associated with the accumulation of ubiquitin-positive intranuclear inclusions broadly distributed throughout the brain in neurons, astrocytes, and in the spinal column (Tan, 2012).

    At the molecular level, the premutation is different from either the normal or full mutation alleles. Based on the observation of significantly elevated levels of rCGG-containing FMR1 mRNA, along with either no detectable change in FMRP or slightly reduced FMRP levels in premutation carriers, an RNA-mediated gain-of-function toxicity model has been proposed for FXTAS. Several lines of evidence in mouse and Drosophila models further support the notion that transcription of the CGG repeats leads to this RNA-mediated neurodegenerative disease. The hypothesis is that specific RNA-binding proteins may be sequestered by overproduced rCGG repeats in FXTAS and become functionally limited, thereby contributing to the pathogenesis of this disorder. There are three RNA-binding proteins found to modulate rCGG-mediated neuronal toxicity: Pur α, hnRNP A2/B1, and CUGBP1, which bind rCGG repeats either directly (Pur α and hnRNP A2/B1) or indirectly (CUGBP1, through the interaction with hnRNP A2/B1) (Tan, 2012).

    MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression at the post-transcriptional level by targeting mRNAs, leading to translational inhibition, cleavage of the target mRNAs or mRNA decapping/deadenylation. Mounting evidence suggests that miRNAs play essential functions in multiple biological pathways and diseases, from developmental timing, fate determination, apoptosis, and metabolism to immune response and tumorigenesis. Recent studies have shown that miRNAs are highly expressed in the central nervous system (CNS), and some miRNAs have been implicated in neurogenesis and brain development (Tan, 2012).

    Interest in the functions of miRNAs in the CNS has recently expanded to encompass their roles in neurodegeneration. Investigators have begun to reveal the influence of miRNAs on both neuronal survival and the accumulation of toxic proteins that are associated with neurodegeneration, and are uncovering clues as to how these toxic proteins can influence miRNA expression. For example, miR-133b is found to regulate the maturation and function of midbrain dopaminergic neurons (DNs) within a negative feedback circuit that includes the homeodomain transcription factor Pitx3 in Parkinson's disease. In addition, reduced miR-29a/b-1-mediated suppression of BACE1 protein expression contributes to Aβ accumulation and Alzheimer's disease pathology. Moreover, the miRNA bantam is found to be a potent modulator of poly-Q- and tau-associated degeneration in Drosophila. Other specific miRNAs have also been linked to other neurodegenerative disorders, such as spinocerebellar ataxia type 1 (SCA1) and Huntington's disease (HD). Therefore, miRNA-mediated gene regulation could be a novel mechanism, adding a new dimension to the pathogenesis of neurodegenerative disorders (Tan, 2012).

    This study shows that fragile X premutation rCGG repeats can alter the expression of specific miRNAs, including miR-277, in a FXTAS Drosophila model. miR-277 modulates rCGG-mediated neurodegeneration. Furthermore, Drep-2, which is associated with the chromatin condensation and DNA fragmentation events of apoptosis, and Vimar, a modulator of mitochondrial function, were identified two of the mRNA targets regulated by miR-277. Functionally, Drep-2 and Vimar could modulate the rCGG-mediated neurodegeneration, as well. Finally, hnRNP A2/B1, an rCGG repeat-binding protein, can directly regulate the expression of miR-277. These data suggest that hnRNP A2/B1 could be involved in the transcriptional regulation of selective miRNAs, and fragile X premutation rCGG repeats could alter the expression of specific miRNAs, potentially contributing to the molecular pathogenesis of FXTAS (Tan, 2012).

    Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder that afflicts fragile X syndrome premutation carriers, with earlier studies pointing to FXTAS as an RNA-mediated neurodegenerative disease. Several lines of evidence suggest that rCGG premutation repeats may sequester specific RNA-binding proteins, namely Pur α, hnRNP A2/B1, and CUGBP1, and reduce their ability to perform their normal cellular functions, thereby contributing significantly to the pathology of this disorder. The miRNA pathway has been implicated in the regulation of neuronal development and neurogenesis. A growing body of evidence has now revealed the role of the miRNA pathway in the molecular pathogenesis of neurodegenerative disorders. This study demonstrates that specific miRNAs can contribute to fragile X rCGG repeat-mediated neurodegeneration by post-transcriptionally regulating target mRNAs that are involved in FXTAS. miR-277 plays a significant role in modulating rCGG repeat-mediated neurodegeneration. Overexpression of miR-277 enhances rCGG repeat-induced neuronal toxicity, whereas blocking miR-277 activity could suppress rCGG repeat-mediated neurodegeneration. Furthermore, Drep-2 and Vimar were identified as the functional miR-277 targets that could modulate rCGG repeat-induced neurodegeneration. Finally, hnRNP A2/B1, an rCGG repeat-binding protein, can directly regulate the expression of miR-277. These biochemical and genetic studies demonstrate a novel miRNA-mediated mechanism involving miR-277, Drep-2, and Vimar in the regulation of neuronal survival in FXTAS (Tan, 2012).

    Several lines of evidence from studies in mouse and Drosophila models strongly support FXTAS as an RNA-mediated neurodegenerative disorder caused by excessive rCGG repeats. The current working model is that specific RNA-binding proteins could be sequestered by overproduced rCGG repeats in FXTAS and become functionally limited, thereby contributing to the pathogenesis of this disorder. Three RNA-binding proteins are known to modulate rCGG-mediated neuronal toxicity: Pur α, hnRNP A2/B1, and CUGBP1, which bind rCGG repeats either directly (Pur α and hnRNP A2/B1) or indirectly (CUGBP1, through the interaction with hnRNP A2/B1); how the depletion of these RNA-binding proteins could alter RNA metabolism and contribute to FXTAS pathogenesis has thus become the focus in the quest to understand the molecular pathogenesis of this disorder. Nevertheless, the data presented in this study suggest that the depletion of hnRNP A2/B1 could also directly impact the transcriptional regulation of specific loci, such as miR-277. It is known that hnRNPs can interact with HP1 to bind to genomic DNA and modulate heterochromatin formation. The results indicate that hnRNP A2/B1 could participate in the transcriptional regulation of miR-277; however, it remains to be determined whether other loci could be directly regulated by hnRNP A2/B1, as well. Identifying those loci will be important to better understand how the depletion of rCGG repeat-binding proteins could lead to neuronal apoptosis (Tan, 2012).

    In recent years, several classes of small regulatory RNAs have been identified in a range of tissues and in many species. In particular, miRNAs have been linked to a host of human diseases. Some evidence suggests the involvement of miRNAs in the emergence or progression of neurodegenerative diseases. For example, accumulation of nuclear aggregates that are toxic to neurons have been linked to many neurodegenerative diseases, and miRNAs are known to modulate the accumulation of the toxic proteins by regulating either their mRNAs or the mRNAs of proteins that affect their expression. Moreover, miRNAs might contribute to the pathogenesis of neurodegenerative disease downstream of the accumulation of toxic proteins by altering the expression of other proteins that promote or inhibit cell survival. The current genetic modifier screen revealed that miR-277 could modulate rCGG repeat-mediated neurodegeneration. By combining genetic screen and reporter assays, Drep-2 and Vimar were identified as the functional targets of miR-277 that could modulate rCGG-mediated neurodegeneration. The closest ortholog of miR-277 in human is miR-597 based on the seed sequence. It would be interesting to further examine the role of miR-597 in FXTAS using mammalian model systems (Tan, 2012).

    Drep-2 is associated with the chromatin condensation and DNA fragmentation events of apoptosis. Drep-2 is one of four Drosophila DFF (DNA fragmentation factor)-related proteins. While Drep-1 is a Drosophila homolog of DFF45 that can inhibit CIDE-A mediated apoptosis. Drep-2 has been shown to interact with Drep-1 and to regulate its anti-apoptotic activity. Vimar is a Ral GTPase-binding protein that has been shown to regulate mitochondrial function via an increase in citrate synthase activity . In the presence of fragile X premutation rCGG repeats, overexpression of miR-277 will suppress the expression of both Drep-2 and Vimar, thereby altering anti-apoptotic activity as well as mitochondrial functions, which have been linked to neuronal cell death associated with neurodegenerative disorders in general. Interestingly, a significant reduction of Drep-2 mRNA was seen in the flies expressing rCGG repeats, while Vimar mRNA levels remained similar to control flies. This observed difference may be due to the fact that miRNA could be involved in different modes of action, including mRNA cleavage, translational inhibition and mRNA decapping/deadenylation its target mRNAs (Tan, 2012).

    In summary, this study provides both biochemical and genetic evidence to support a role for miRNA and its selective mRNA targets in rCGG-mediated neurodegeneration. The results suggest that sequestration of specific rCGG repeat-binding proteins can lead to aberrant expression of selective miRNAs that could modulate the pathogenesis of FXTAS by post-transcriptionally regulating the expression of specific mRNAs involved in this disorder. Identification of these miRNAs and their targets could reveal potential new targets for therapeutic interventions to treat FXTAS, as well as other neurodegenerative disorders (Tan, 2012).

    Dicer-2 processes diverse viral RNA species

    RNA silencing pathways play critical roles in gene regulation, virus infection, and transposon control. RNA interference (RNAi) is mediated by small interfering RNAs (siRNAs), which are liberated from double-stranded (ds)RNA precursors by Dicer and guide the RNA-induced silencing complex (RISC) to targets. Although principles governing small RNA sorting into RISC have been uncovered, the spectrum of RNA species that can be targeted by Dicer proteins, particularly the viral RNAs present during an infection, are poorly understood. Dicer-2 potently restricts viral infection in insects by generating virus-derived siRNAs were studied from viral RNA. To better characterize the substrates of Dicer-2, the virus-derived siRNAs produced during the Drosophila antiviral RNAi response to four different viruses using high-throughput sequencing. It was found that each virus was uniquely targeted by the RNAi pathway; dicing substrates included dsRNA replication intermediates and intramolecular RNA stem loops. For instance, a putative intergenic RNA hairpin encoded by Rift Valley Fever virus generates abundant small RNAs in both Drosophila and mosquito cells, while repetitive sequences within the genomic termini of Vaccinia virus, which give rise to abundant small RNAs in Drosophila, were found to be transcribed in both insect and mammalian cells. Moreover, evidence is provided that the RNA species targeted by Dicer-2 can be modulated by the presence of a viral suppressor of RNAi. This study uncovered several novel, heavily targeted features within viral genomes, offering insight into viral replication, viral immune evasion strategies, and the mechanism of antiviral RNAi (Sabin, 2013).

    Association of zygotic piRNAs derived from paternal P elements with hybrid dysgenesis in Drosophila melanogaster

    P-element transposition in the genome causes P-M hybrid dysgenesis in Drosophila melanogaster. Maternally deposited piRNAs suppress P-element transposition in the progeny, linking them to P-M phenotypes; however, the role of zygotic piRNAs derived from paternal P elements is poorly understood. This study investigated the genomic constitution and P-element piRNA production derived from fathers. As a result, males were characterized of naturally derived Q, M' and P strains, which show different capacities for the P-element mobilizations introduced after hybridizations with M-strain females. The amounts of piRNAs produced in ovaries of F1 hybrids varied among the strains and were influenced by the characteristics of the piRNA clusters that harbored the P elements. Importantly, while both the Q- and M'-strain fathers restrict the P-element mobilization in ovaries of their daughters, the Q-strain fathers supported the production of the highest piRNA expression in the ovaries of their daughters, and the M' strain carries KP elements in transcriptionally active regions directing the highest expression of KP elements in their daughters. Interestingly, the zygotic P-element piRNAs, but not the KP element mRNA, contributed to the variations in P transposition immunity in the granddaughters. It is concluded that the piRNA-cluster-embedded P elements and the transcriptionally active KP elements from the paternal genome are both important suppressors of P element activities that are co-inherited by the progeny (Wakisaka, 2018).

    RNA editing regulates transposon-mediated heterochromatic gene silencing

    Heterochromatin formation drives epigenetic mechanisms associated with silenced gene expression. Repressive heterochromatin is established through the RNA interference pathway, triggered by double-stranded RNAs (dsRNAs) that can be modified via RNA editing. However, the biological consequences of such modifications remain enigmatic. This study shows that RNA editing regulates heterochromatic gene silencing in Drosophila. The binding activity of an RNA-editing enzyme was used to visualize the in vivo production of a long dsRNA trigger mediated by Hoppel transposable elements. Using homologous recombination, this trigger was deleted, dramatically altering heterochromatic gene silencing and chromatin architecture. Furthermore, it was shown that the trigger RNA is edited and that dADAR serves as a key regulator of chromatin state. Additionally, dADAR auto-editing generates a natural suppressor of gene silencing. Lastly, systemic differences in RNA editing activity generates interindividual variation in silencing state within a population. These data reveal a global role for RNA editing in regulating gene expression (Savva, 2013).

    This study pursued an observation of the in vivo localization of the RNA-editing enzyme, dADAR, to the proof of its action on an endogenously expressed inverted repeat of the TE, Hoppel. The results explicitly demonstrate a functional intersection between the processes of RNA editing and RNA silencing. Previous studies in Drosophila implicate Hoppel and the RNAi pathway in determining the global silencing state of chromosome 4, although no dsRNA trigger had been experimentally identified. This study showed that the inverted repeat acts as a genetic element, Hok, and regulates PEV, the global architecture of chromosome 4, and silences the Hoppel transposase. As a general mechanism, ADAR's action on dsRNA should oppose RNAi. It was shown that deficiency for ADAR acts as a global enhancer of silencing state, and dADAR hypomorphism even extends lifespan. In Drosophila, gene silencing decreases with age and has been implicated in the aging process (Wood, 2010). Thus, substantial decreases in ADAR activity may lead to lifespan extension through increased silencing. Interestingly, polymorphisms within a human ADAR gene have been associated with extreme longevity, indicating that interventions involving ADAR activity may be capable of affecting lifespan. Importantly, mutations in human ADAR1 cause Aicardi–Goutières syndrome in which it is hypothesized that ADAR has a role in regulating dsRNA metabolism from repeated elements in the human genome. Thus, the current data are consistent with a conserved role in the regulation of dsRNA levels in animals through RNA editing or RNA binding (Savva, 2013).

    Mechanistically, evidence is provided that dADAR auto-editing has evolved as a natural inhibitor of RNAi, generating dADARG. In dAdar null or dAdarS genetic backgrounds, no dADARG can be produced. Thus, both backgrounds effectively act as enhancers of PEV (E(var)). In the wild-type background, PEV occurs to the extent that each animal expresses dADAR (and the corresponding amount of dADARG). In the extreme, the dAdarG background acts as a strong suppressor of PEV (Su(var)). How can a single amino acid change in dADAR protein affect such a silencing switch? It is speculated that dADARG may interfere indirectly with Dicer activity on dsRNA, simply by blocking access via binding irrespective of editing activity, analogous to the FHV-B2 protein. Alternately, a recent study showed a direct functional interaction between mammalian ADAR and Dicer that is necessary for the processing of small RNAs (Ota, 2013). If dADAR has a similar interaction, it could also mediate all of the effects in the model via dominant-negative interactions of dADARG with Dicer, whereas dADARS (which encodes the conserved amino acid) would function in a similar manner described in mammals to promote small RNA biogenesis. Further biochemical experiments will be necessary to determine whether this phenomenon is conserved across species and the exact molecular mechanisms through which dADARG exerts its effects (Savva, 2013).

    The most engaging aspect of these results lay in their implications for somatic regulation of heterochromatin functioning as a safeguard of transposon activity, especially in the nervous system. The RNA-induced silencing complex isolated from Drosophila tissue-culture cells was shown to be programmed with esiRNAs, largely derived from transposon sequences, a significant portion of which bears the signature of a single dADAR modification (Kawamura, 2008). Likewise, in C. elegans, ADAR activity has a profound effect on the abundance and identity of small RNA profiles. Further experiments in this system using deep sequencing technologies will be necessary to shed light on the effects of ADAR on endo-siRNA abundances and functionality. It is envisioned that such RNA-editing-mediated effects may be quite specific to the nature of individual dsRNA triggers. Studies in both mammals and Drosophila have shown that TEs are mobile in the nervous system, revealing an intriguing mechanism for the generation of somatic mutations potentially conferring adaptive value in individuals (Li, 2013; Muotri, 2005; Perrat, 2013). This study demonstrates a mechanistic link between RNA editing and the regulation of transposon silencing, particularly in the nervous system, which may have domesticated uses as diversifiers of neuronal genomes on a neuron-to-neuron and an individual-to-individual basis. The implications of these results, given the universal prevalence of dsRNAs as a component of transcriptomes, are that ADAR activity has an evolved role in determining the fate of RNAs entering silencing pathways, thus globally influencing somatic genomic integrity, gene expression and downstream organismal phenotypes (Savva, 2013).

    Natural variation of piRNA expression affects immunity to transposable elements

    In the Drosophila germline, transposable elements (TEs) are silenced by PIWI-interacting RNA (piRNA) that originate from distinct genomic regions termed piRNA clusters and are processed by PIWI-subfamily Argonaute proteins. This study explores the variation in the ability to restrain an alien TE in different Drosophila strains. The I-element is a retrotransposon involved in the phenomenon of I-R hybrid dysgenesis in Drosophila melanogaster. Genomes of R strains do not contain active I-elements, but harbour remnants of ancestral I-related elements. The permissivity to I-element activity of R females, called reactivity, varies considerably in natural R populations, indicating the existence of a strong natural polymorphism in defense systems targeting transposons. To reveal the nature of such polymorphisms, ovarian small RNAs between R strains with low and high reactivity were compared. It was shown that reactivity negatively correlates with the ancestral I-element-specific piRNA content. Analysis of piRNA clusters containing remnants of I-elements shows increased expression of the piRNA precursors and enrichment by the Heterochromatin Protein 1 homolog, Rhino, in weak R strains, which is in accordance with stronger piRNA expression by these regions. To explore the nature of the differences in piRNA production, weak and strong strains were analyzed and it was shown that the efficiency of maternal inheritance of piRNAs as well as the I-element copy number are very similar in both strains. At the same time, germline and somatic uni-strand piRNA clusters generate more piRNAs in strains with low reactivity, suggesting the relationship between the efficiency of primary piRNA production and variable response to TE invasions. The strength of adaptive genome defense is likely driven by naturally occurring polymorphisms in the rapidly evolving piRNA pathway proteins. The study hypothesizes that hyper-efficient piRNA production is contributing to elimination of a telomeric retrotransposon HeT-A, which was observed in one particular transposon-resistant R strain (Ryazansky, 2017).

    Antisense transcription of retrotransposons in Drosophila: The origin of endogenous small interfering RNA precursors

    To repress transposons and combat genomic instability, eukaryotes have evolved several small RNA mediated defense mechanisms. Specifically, in Drosophila somatic cells, endogenous small interfering (esi)RNAs suppress retrotransposon mobility. EsiRNAs are produced by Dicer-2 processing of double-stranded RNA precursors, yet the origins of these precursors are unknown. This study shows that most transposon families are transcribed in both the sense and antisense direction. LTR retrotransposons are generated from intra element transcription start sites with canonical RNA polymerase II promoters. Retrotransposon antisense transcripts were shown to be less polyadenylated than sense transcripts, which may promote nuclear retention of antisense transcripts and the double-stranded RNAs they form. Dicer-2 RNAi-depletion causes a decrease in the number of esiRNAs mapping to retrotransposons. These data support a model in which double-stranded RNA precursors are derived from convergent transcription and processed by Dicer-2 into esiRNAs that silence both sense and antisense retrotransposon transcripts. Reduction of sense retrotransposon transcripts potentially lowers element specific protein levels to prevent transposition. This mechanism preserves genomic integrity and is especially important for Drosophila fitness because mobile genetic elements are highly active (Russo, 2015).

    Drosophila oncogene Gas41 is RNAi modulator that intersects heterochromatin and siRNA pathway

    Glioma amplified sequence41 (Gas41) is a highly conserved putative transcription factor that is frequently abundant in human gliomas. Gas41 shows oncogenic activity by promoting cell growth and viability. This study shows Gas41 is required for proper functioning of RNAi machinery in the nuclei, though three basic structural domains of RNAi components PAZ, PIWI and dsRNA binding are absent in the structural sequences. Variations of structural domains are highly conversed among prokaryotes and eukaryotes. Gas41 interacts with cytological RNase III enzyme Dicer1 both biochemically and genetically. However, Drosophila Gas41 functions as chromatin remodeler and interacts with different heterochromatin markers and repeat induced transgene silencing by modulating PEV. This study also shows that transcriptional inactive Gas41 mutant interferes the functional assembly of heterochromatin associated proteins, H3K9me2 and HP1 in developing embryos. A reduction of heterochromatic markers is accompanied with mini-w promoter sequence in Gas41 mutants. These findings suggest that, Drosophila Gas41 guides the repeat associated gene silencing, and Dicer1 interaction thereby depicting a new role of the Gas41. It is a critical RNAi component. In Drosophila, Gas41 plays a dual role. In one hand, it seems to participate with Dicer 1 in the RNAi pathway and alternatively also participate in repeat-induced gene silencing by accumulating heterochromatin proteins at the mw array promoters. Therefore, it proposes an intriguing and seemingly paradoxical new finding in RNA technology in the process of heterochromatin gene silencing (Gandhi, 2014).

    siRNAs from an X-linked satellite repeat promote X-chromosome recognition in Drosophila melanogaster

    Highly differentiated sex chromosomes create a lethal imbalance in gene expression in one sex. To accommodate hemizygosity of the X chromosome in male fruit flies, expression of X-linked genes increases twofold. This is achieved by the male- specific lethal (MSL) complex, which modifies chromatin to increase expression. Mutations that disrupt the X localization of this complex decrease the expression of X-linked genes and reduce male survival. The mechanism that restricts the MSL complex to X chromatin is not understood. The siRNA pathway has been shown to contribute to localization of the MSL complex, raising questions about the source of the siRNAs involved. The X-linked 1.688 g/cm3 satellite related repeats (1.688X repeats; 359-bp repeat unit) are restricted to the X chromosome and produce small RNA, making them an attractive candidate. RNA from these repeats was tested for a role in dosage compensation, and ectopic expression of single-stranded RNAs from 1.688X repeats was found to enhance the male lethality of mutants with defective X recognition. In contrast, expression of double-stranded hairpin RNA from a 1.688X repeat generated abundant siRNA and dramatically increased male survival. Consistent with improved survival, X localization of the MSL complex was largely restored in these males. The striking distribution of 1.688X repeats, which are nearly exclusive to the X chromosome, suggests that these are cis-acting elements contributing to identification of X chromatin (Menon, 2014: PubMed).

    FOXO regulates RNA interference in Drosophila and protects from RNA virus infection

    Small RNA pathways are important players in posttranscriptional regulation of gene expression. These pathways play important roles in all aspects of cellular physiology from development to fertility to innate immunity. However, almost nothing is known about the regulation of the central genes in these pathways. The forkhead box O (FOXO) family of transcription factors is a conserved family of DNA-binding proteins that responds to a diverse set of cellular signals. FOXOs are crucial regulators of cellular homeostasis that have a conserved role in modulating organismal aging and fitness. This study shows that Drosophila FOXO (dFOXO) regulates the expression of core small RNA pathway genes. In addition, increased dFOXO activity results in an increase in RNA interference (RNAi) efficacy, establishing a direct link between cellular physiology and RNAi. Consistent with these findings, dFOXO activity is stimulated by viral infection and is required for effective innate immune response to RNA virus infection. This study reveals an unanticipated connection among dFOXO, stress responses, and the efficacy of small RNA-mediated gene silencing and suggests that organisms can tune their gene silencing in response to environmental and metabolic conditions (Spellberg, 2015).

    Despite its importance, almost nothing is known about how the protein components of the small RNA pathways are transcriptionally regulated in the cell. Currently only studies addressing the transcriptional regulation of germ-line small RNA pathways (piRNA) have been reported. Nothing is reported on the transcriptional regulation of the protein components of the dominant somatic cell small RNA pathways, the miRNA and siRNA pathways. This study found dFOXO at the promoters of many core small RNA pathway genes. Components of the miRNA, siRNA, and piRNA pathways are all bound by dFOXO, suggesting an integrated control of the small RNA pathways (Spellberg, 2015).

    The current work focused on the core small RNA pathway genes dominant in somatic cells. The transcription of the Ago2, Ago1, and Dcr2 genes were found to be increased during dFOXO activation. The effect of this dFOXO activation is augmented RNAi efficacy even with an unchanged limiting level of the dsRNA trigger. This result suggests the RNA-mediated gene silencing response is not constant but it is tunable to cellular physiology. This notion is consistent with previous work showing enhanced RNAi-based phenotypes in a daf-2/INR mutant of C. elegans and greater knockdown of target genes with dsRNA in Drosophila S2 cells after serum starvation. Both of these conditions increase FOXO activity (Spellberg, 2015).

    It is interesting to note that Dcr1, the core miRNA dicer, does not seem to be a dFOXO target. This finding is despite the fact that the core miRNA argonaute, Ago1, is a dFOXO target. There is limited evidence for Ago1 involvement in inhibiting viral replication. However, there is evidence showing changes in the miRNA RISC and enhanced silencing by miRNAs under serum-starved conditions. This effect is achieved through the increased recruitment of GW182 (Gawky) to the miRNA RISC. Based on dFOXO ChIP data, GW182 is also a dFOXO target. Rather than dealing directly with a viral infection, dFOXO's up-regulation of these miRNA factors may be a stress responsive mechanism to repress translation initiation, a previously described role for dFOXO during stress (Spellberg, 2015).

    dFOXO was found to be activated by viral infection to a comparable level as another well-defined physiological signal, serum starvation. Activated dFOXO can decrease viral load in cell culture and is required for effective resistance to RNA virus infection. The FOXO family of transcription factors responds to a multitude of cellular and extracellular signals. The current study shows dFOXO provides a link among cellular physiology, the RNAi pathway, and innate immunity enhancing the effectiveness of silencing and allowing the RNAi pathway to respond dynamically to changes in cellular homeostasis (Spellberg, 2015).

    The importance of the RNAi pathway for viral immunity in invertebrates is well defined. However, the role of RNAi in viral immunity for mammals is still an open question. The mammalian cellular innate immune system differs from lower organisms, relying strongly on the IFN response during a viral infection. However, in cell types that lack a fully developed IFN response, RNAi plays an important role in viral defense. Additionally, several viruses that infect mammalian cells contain genes that suppress the RNAi response. This result suggests an ongoing battle between RNAi-based innate immunity and viruses. There is a growing appreciation for the role of FOXOs in mammalian immune regulation. If conservation of the function of FOXO-small RNA regulation exists in mammals, there are potential therapeutic benefits (Spellberg, 2015).

    Pervasive regulation of Drosophila Notch target genes by GY-box-, Brd-box-, and K-box-class microRNAs

    Although hundreds of distinct animal microRNAs (miRNAs) are known, the specific biological functions of only a handful are understood at present. Three different families of Drosophila miRNAs directly regulate two large families of Notch target genes, including basic helix-loop-helix (bHLH) repressor and Bearded family genes. These miRNAs regulate Notch target gene activity via GY-box (GUCUUCC), Brd-box (AGCUUUA), and K-box (cUGUGAUa) motifs. These are conserved sites in target 3'-untranslated regions (3'-UTRs) that are complementary to the 5'-ends of miRNAs, or 'seed' regions. Collectively, these motifs represent >40 miRNA-binding sites in Notch target genes, and all three classes of motif are shown to be necessary and sufficient for miRNA-mediated regulation in vivo. Importantly, many of the validated miRNA-binding sites have limited pairing to miRNAs outside of the "box:seed" region. Consistent with this, it was found that seed-related miRNAs that are otherwise quite divergent can regulate the same target sequences. Finally, it is demonstrated that ectopic expression of several Notch-regulating miRNAs induces mutant phenotypes that are characteristic of Notch pathway loss of function, including loss of wing margin, thickened wing veins, increased bristle density, and tufted bristles. Collectively, these data establish insights into miRNA target recognition and demonstrate that the Notch signaling pathway is a major target of miRNA-mediated regulation in Drosophila (Lai, 2005).

    The E(spl)-C and Brd-C of Drosophila melanogaster (Dm) contain two large families of direct Notch target genes, including seven bHLH repressor-encoding genes and 10 Bearded family genes. With the exception of E(spl)mbeta and Ocho, all of these genes contain GY-box (GUCUUCC), Brd-box (AGCUUUA), and/or K-box (UGUGAU) motifs in their 3'-UTRs, which are propose to be miRNA-binding sites. Nine of these genes contain three or more box sites, a density that is especially remarkable when one considers how short their 3'-UTRs are (often <350 nt in length). The conservation of these sites were systematically assessed in their orthologs from Drosophila pseudoobscura (Dp) and Drosophila virilis (Dv), species that are ~30 million and 60 million years diverged from Dm, respectively. 33/51 Brd-boxes, GY-boxes, and K-boxes have been perfectly conserved and reside in syntenic locations among all three species; 11 additional sites are identical in two of the three species. This indicates that all three motifs are under strong selective constraint (Lai, 2005).

    Closer examination of nucleotide divergence surrounding these boxes has revealed some unexpected features that are germane to the proposition that these boxes represent miRNA-binding sites. These features are best illustrated by comparing rapidly evolving genes. Notably, Bearded is an unusually rapidly evolving protein, with only 15 residues preserved between Dm and Dv orthologs (out of 81 and 66 amino acids, respectively), and Dv Bearded has a significantly different arrangement of these 3'-UTR motifs. The 3'-UTR of Dv E(spl)m5 is also quite different from its counterparts in Dm/Dp. Alignment of Dm/Dp orthologs of Bearded and E(spl)m5 reveals that sequences upstream of most GY-boxes are well conserved; these regions include most sequences presumed to pair with miR-7. Similar patterns are seen for many other GY-boxes in other Notch target genes. However, the sequence upstream of many Brd- and K-boxes is strongly diverged, so that only 'box'-pairing is often preserved. In fact, many Brd- and K-boxes generally lack extensive pairing to miRNAs outside of the 'box' sequence. These factors likely preclude their identification by various published computational algorithms for miRNA-binding sites. Indeed, Brd- and K-boxes in Notch target genes have been deemed unlikely to represent miRNA-binding sites. In contrast, rapid divergence of the upstream portion of miRNA-binding sites is consistent with the idea that pairing between the miRNA "seed" (positions ~2-8) and the 3'-UTR 'box' (approximately the last one-third of the miRNA-binding site) is most critical for miRNA-mediated regulation (Lai, 2005).

    It is also noted that precise spacing of several motif occurrences that are closely paired is also conserved, even though orthologous 3'-UTRs otherwise display significant insertions and deletions. In these cases, one would presume that simultaneous binding of miRNAs to their respective sites would not be possible unless the 3'-end of the downstream miRNA was unpaired, a configuration that unexpectedly proved functional in vitro. Finally, there are a few nonconserved boxes in these 3'-UTRs (7/51 total sites). In several cases, the nonconserved site is highly related to a neighboring conserved site [i.e., the first and second GY-boxes of Dp E(spl)m4 are equally similar to the first GY-box in Dm E(spl)m4; the third and fourth Brd-boxes in Dp E(spl)m5 are highly related to the third Brd-box in Dm E(spl)m5], implying that these nonconserved sites may be functional, newly evolved miRNA-binding sites (Lai, 2005).

    GY-box-, Brd-box-, and K-box-class miRNAs are highly conserved among diverse insects, and many are, indeed, identical. Therefore Brd-boxes, GY-boxes, and K-boxes were sought in the predicted 3'-UTRs of E(spl)bHLH and Brd genes from mosquitoes, bees, and moths; these species cover ~350 million years of divergence from Drosophila. Impressively, homologs of both E(spl)bHLH and Brd genes in these highly diverged species all contain multiple copies and multiple classes of 'box' motifs in their 3'-UTRs. This strongly suggests that regulation by all three families of miRNAs is an ancient feature of Notch target gene regulation in insects (Lai, 2005).

    To directly test the capacity of miRNAs to regulate the 3'-UTRs of these Notch target genes, an in vivo assay was used. The target in this assay is a ubiquitously expressed reporter (tub>GFP or arm>lacZ) fused to an endogenous 3'-UTR (a 3'-UTR sensor). The reporter transgene is introduced into a genetic background in which a UAS-DsRed-miRNA transgene is activated with dpp-Gal4 or ptc-Gal4. This results in ectopic miRNA production in a stripe of red-fluorescing cells at the anterior–posterior boundary of imaginal discs. Inhibition of the green reporter within the red miRNA-misexpressing domain reflects direct miRNA-mediated negative regulation. Focus was placed on the central wing pouch region of the wing imaginal disc (Lai, 2005).

    The ability of sensor transgenes for most Bearded family genes [Bob, Bearded, Tom, Ocho, E(spl)malpha, and E(spl)m4] and most E(spl)bHLH repressor genes [E(spl)mgamma, E(spl)mdelta, E(spl)m3, E(spl)m5, and E(spl)m8] to be regulated by ectopic GY-box-, Brd-box-, and K-box-class miRNAs was extensively analyzed. Sensor expression is influenced by the level to which it is negatively regulated by endogenous factors, including miRNAs. In this assay, the disc sensor must be expressed at sufficient levels before one can observe its knock-down by ectopic miRNAs. 3'-UTR sensor constructs for different Notch target genes accumulate to different levels in vivo, consistent with variable amounts of endogenous miRNA-mediated regulation. Nevertheless, it was possible to reliably detect expression of all sensors excepting E(spl)m8. As detailed in the following three sections, these sensors were used to unequivocally demonstrate GY-boxes, Brd-boxes, and K-boxes to be sites of miRNA-mediated negative regulation by corresponding families of complementary miRNAs in vivo (Lai, 2005).

    miR-7 is the only known Drosophila miRNA whose 5'-end is complementary to the GY-box (GUCUUCC). miR-7 has been shown to regulate three GY-box targets, including two members of the E(spl)-C, E(spl)m3 and E(spl)m4. While these two genes scored well in a genome-wide prediction of miR-7 targets, many other members of the Brd-C and E(spl)-C also contain between one and three GY-boxes in their 3'-UTRs [Bob, Bearded, Tom, E(spl)mgamma, E(spl)m5]. Of these, only Tom was computationally identified as a compelling candidate for miR-7 (Lai, 2005).

    The specificity of the disc sensor assay was assayed by showing that neither an empty tub-GFP sensor nor an Ocho sensor were affected by miR-7. The previous experiments done with E(spl)m3 and E(spl)m4 were repeated and it was observed that both were, indeed, inhibited by ectopic miR-7. This assay was used to demonstrate that miR-7 negatively regulates all seven GY-box-containing members of the Brd-C and E(spl)-C, including those with single sites [E(spl)m3, E(spl)mgamma, and Bearded], those with two sites [E(spl)m4, Tom, Bob], and those with three sites [E(spl)m5]. These data convincingly support the hypothesis that GY-boxes are general signatures of miR-7-binding sites in Notch target genes, irrespective of the overall amount of pairing between miR-7 and sequences outside of the GY-box. In order to more definitively demonstrate that miR-7-mediated regulation occurs through identified GY-boxes, mutant sensors bearing point mutations in the GY-boxes were tested. A Bearded sensor carrying five point mutations in its single GY-box no longer responded to miR-7. In a more stringent test, an E(spl)m5 sensor carrying 2-nt mutations in each of its three GY-boxes was generated. These targeted changes also abolished the ability of miR-7 to negatively regulate E(spl)m5. Therefore, ~7 continuous base pairs between the 'box' motif and its cognate miRNA seed are critical for in vivo target regulation. It is also noted that when mutant 3'-UTRs are tested, a mild increase in reporter activity in miRNA-misexpressing cells was sometimes observed, the reason for which has not been determined (Lai, 2005).

    Previous work has suggested synergism between miRNA-binding sites on the same transcript. Multiple GY-box 3'-UTRs were generally subject to greater regulation than single-site 3'-UTRs, even though the amount of miR-7 pairing to individual GY-boxes in multiple-site 3'-UTRs is often less than its pairing with single GY-box 3'-UTRs. Indeed, negative regulation of E(spl)m4, Tom, Bob, and E(spl)m5 by miR-7 was qualitatively indistinguishable from an artificial sensor containing two perfectly miR-7-complementary sites, even though many sites in these genes display relaxed pairing with miR-7 outside of GY-boxes. This suggests that as little as 7–8 nt of complementarity may suffice for miRNA target recognition, especially where multiple sites are present. However, since all three single GY-box-containing 3'-UTRs were also regulated by miR-7, synergism is not required for biologically significant regulation by miRNAs (Lai, 2005).

    There are two Drosophila miRNAs, miR-4 and miR-79, whose 5'-ends are complementary to the Brd-box (AGCUUUA). Both miRNAs are resident in miRNA clusters, and miR-4 resides in particularly dense clusters containing several unrelated miRNAs. Use was made of a UAS-DsRed-miR-286, miR-4, miR-5 transgene that is referred to as "UAS-miR-4" and a UAS-DsRed-miR-79 transgene. miR-4 and miR-79 have only limited similarity outside of their Brd-box seed, and there is little indication from pairwise alignments that these miRNAs are specifically "tuned" to different Brd-box sites in Notch target genes. In fact, all of these Brd-boxes lack the extended complementarity to miRNAs that is typical of miR-7:GY-box pairs, and no Notch target genes were previously predicted computationally as targets of miR-4 or miR-79 (Lai, 2005).

    Seven Brd-box-containing Notch target genes were validated as being regulated by Brd-box-family miRNAs, including those with single sites [Tom, E(spl)mdelta, E(spl)mgamma] and those with multiple sites [Bearded, E(spl)malpha, E(spl)m4, and E(spl)m5]. Curiously, the negative regulatory effects of miR-4 on E(spl)mgamma, E(spl)malpha, E(spl)m4, and E(spl)m5 were greater than those of miR-79 on these same 3'-UTRs, even though miR-4 is no more complementary to these sites than is miR-79. Nevertheless, the common ability of miR-4 and miR-79 to down-regulate individual sensors indicates that cross-regulation of individual sites by multiple members of a given miRNA family may occur. Notably, both miRNAs are expressed at high levels during embryonic development (Lai, 2005).

    The specificity of miR-4 and miR-79 was tested using two mutant Bearded sensors, one bearing several point mutations in each of its three Brd-boxes and another containing mutations in the Brd-boxes and the GY-box. In both cases, the mutant transgenes accumulate to higher levels, consistent with relief from negative regulation by endogenous Brd-box-class miRNAs in the wing disc. In addition, they are no longer responsive to ectopic Brd-box-class miRNAs, indicating that the observed regulation occurs directly via Brd-boxes. As well, this experiment demonstrates that regulation by the miR-4 transgene is not attributable to miR-286 and miR-5 carried on this construct. Nevertheless, this miRNA construct efficiently down-regulates a miR-5 sensor containing two miR-5 sites, indicating that the other miRNAs carried on this construct are functional. As a final test of the specificity of this assay, it was observed that this three-miRNA construct fails to inhibit the expression of an empty tub-GFP sensor (Lai, 2005).

    Having demonstrated that Brd-boxes are bona fide miRNA-binding sites, it was asked whether regulation of the Bearded 3'-UTR by miR-7 requires the presence of Brd-boxes. This might be the case, for example, if negative regulation of a given 3'-UTR required synergism between different types of miRNA-binding sites. A Bearded 3'-UTR carrying mutations in each of the three Brd-boxes was observed to be still strongly inhibited by miR-7, indicating that individual types of miRNA-binding sites suffice for regulation in this assay (Lai, 2005).

    The largest family of Drosophila miRNAs includes those whose 5'-ends are complementary to the K-box (cUGUGAUa, where the lowercase nucleotides represent positions of strong bias). The K-box is also the most pervasive motif within these Notch target genes; it is present in almost every member of the Brd-C and E(spl)-C [excepting E(spl)mbeta and Ocho, which lack any box motifs]. The maximum overall site complementarity of any given K-box site to any K-box family miRNAs is generally modest, and less than that seen with other demonstrated targets of the K-box family miRNA miR-2, namely, the proapoptotic genes grim, reaper, and sickle. In fact, the sole Notch target gene that was predicted informatically as a target of a K-box family miRNA in any study was E(spl)m8: miR-11, and this pair ranked only 46th (Lai, 2005).

    The ability was tested of two quite distinct K-box family miRNAs, those of the miR-2 cluster (miR-2a-1, miR-2a-2, and miR-2b-2) and miR-11, to regulate K-box-containing 3'-UTRs. Given the abundance of K-box complementary miRNAs (as a class, they are among the more frequently cloned fly miRNAs), the occupancy of K-box sites by endogenous K-box-class miRNAs may be near-saturating in some cases. In fact, negative regulation of E(spl)m8, whose K-boxes mediate 10-fold negative regulation and nearly eliminate expression of this sensor, could not be convincingly demonstrated. In spite of this, positive evidence was obtained that four other K-box-containing 3'-UTRs, E(spl)m4, Bob, E(spl)malpha, and E(spl)mdelta, are directly regulated by K-box-family miRNAs, although the amount of regulation observed was weaker than that seen with GY-box- or Brd-box-class miRNAs. As was the case with the two Brd-box-class miRNAs, both miR-2 and miR-11 are capable of regulating some of the same K-box-containing targets. This constitutes further evidence for the possibility of cross-regulation of miRNA-binding sites, even where the miRNAs in question display very little similarity outside of their seeds (Lai, 2005).

    In performing pairwise tests of these miRNAs with Notch target gene sensors, two instances were observed of miRNA-mediated regulation of sensors lacking canonical boxes. (1) It was observed that the E(spl)mdelta sensor was inhibited by miR-7. Although E(spl)mdelta lacks a canonical GY-box, it does contain a GY-box-like site that would have a single G:U base pair with the miR-7 seed. The nucleotides that are 5' and 3' to the box are also paired with miR-7, and there is a significant region of pairing to the 3'-end of the miRNA. These factors may allow this site to be recognized by miR-7. The 9-mer AGUUUUCCA is found in both Dp and Dv orthologs of E(spl)mdelta, indicating that this site is under selection and therefore is likely important for regulation of E(spl)mdelta. (2) It was observed that the Bob sensor was negatively regulated by both Brd-box-class miRNAs, miR-4 and miR-79. Although Bob lacks a canonical Brd-box, it does contain two matches to positions 2-7 of the Brd-box, which would pair to positions 2-7 of the miR-4/79. In this regard, this type of site is reminiscent of the 6-mer K-box, which pairs to positions 2-7 of K-box miRNAs. One of these Brd-box-like sites is conserved in Dp, and the syntenic site in Dv is, in fact, a canonical Brd-box, further indicating a functional relationship between Bob and miRNAs of the Brd-box family (Lai, 2005).

    The apparent functionality of these noncanonical sites led to a search for other such sites in Notch target 3'-UTRs. Although one might expect to find many-fold more copies of degenerate sites relative to canonical sites, instead only a few additional examples of relaxed GY-box-like or Brd-box-like sites were found. For comparison, there are 28 canonical sites of these classes in Notch target 3'-UTRs (16 Brd-boxes and 12 GY-boxes), but only three additional examples of a 7-mer box-like site with a G:U base-pair to a miRNA seed [all are GY-box-like sites in E(spl)mdelta, E(spl)m3, and E(spl)m7]. In addition, there are only five additional examples of sites that match only positions 2-7 of the GY-box or the Brd-box [all of which are Brd-box-like sites: the two in Bob, one in E(spl)m7, one in E(spl)malpha, and one in E(spl)mdelta]. These considerations strongly suggest that the much more restricted, canonical sites are actively selected for function in these Notch target 3'-UTRs, a conclusion that is bolstered by the patterns of evolutionary conservation of these sites (Lai, 2005).

    These experiments presented thus far demonstrate that target gene 3'-UTRs harboring sequence elements with Watson-Crick complementarity to the 5'-ends of miRNAs are, indeed, regulated by these miRNAs in vivo, and that such sites are necessary for miRNA-mediated regulation. Are these sites sufficient for regulation by complementary miRNAs? Although a variety of studies of model sites in tissue culture assays indicate site sufficiency, tests in animals suggest that miRNA site context can be less forgiving in vivo. For example, certain reporters containing multimers of six lin-4 or three let-7 sites are not appropriately regulated by lin-4 or let-7 in nematodes. In addition, mutation of sequences outside of the let-7-binding sites in lin-41 abolishes regulation by let-7 in vivo. Therefore, it was of interest to test the functionality of GY-boxes, Brd-boxes, and K-boxes when abstracted from endogenous 3'-UTR context (Lai, 2005).

    To do so, a tandem of isolated GY-box, Brd-box, and K-box elements were cloned from Bob, Bearded, and E(spl)m8, respectively, into tub-GFP transgenes. Also mutant versions were cloned containing single changes in the Brd-box sites or dual changes in the GY-boxes. The ability of these 'box' sensors to respond to exogenously expressed miRNAs was tested. It was found that wild-type GY-box, Brd-box, and K-box sensors are all negatively regulated by corresponding miRNAs. These data directly demonstrate that all three types of box sites are sufficient for miRNA-mediated negative regulation. In contrast, mutant box sensors are nonfunctional in this assay. Since the mutant box sensors contain only one or two changes in each site, these data provide strong in vivo support for the idea that Watson-Crick pairing to the 5'-end of the miRNA (the "seed") is the key essential feature of miRNA target recognition. As a further test of this idea, the ability of the three different K-box miRNAs, miR-6, miR-2, and miR-11, to down-regulate a miR-6 sensor was tested. All three inhibited miR-6 sensor expression, consistent with the ability of seed-pairing to mediate regulation by miRNAs (Lai, 2005).

    With these UAS-miRNA transgenic lines in hand, the consequences of ectopically expressing miRNAs on Drosophila development were tested. It should be noted that Notch target-regulating miRNAs were fully expected to regulate other functionally unrelated targets in vivo. For example, it has been established that K-box-family miRNAs also negatively regulate the proapoptotic genes reaper, sickle, and grim via K-boxes in their 3'-UTRs, while Brd-box-family miRNAs target the mesodermal determinant bagpipe via a Brd-box in its 3'-UTR. Therefore, even if ectopic miRNAs are able to affect normal development, they would not necessarily be expected to affect Notch signaling exclusively. Nevertheless, it has been previously reported that ectopic miR-7 induces loss of molecular markers of wing margin development, resulting in wing notching. This indicates that phenotypic characterization of miRNA misexpression can be informative (Lai, 2005).

    Using an independently derived UAS-miR-7 construct lacking DsRed, it was verified that dpp-Gal4>miR-7 wings display notching and loss of Cut expression at the developing wing margin of wing imaginal discs; the size of the L3-L4 intervein domain was also reduced. It was next observed that ectopic K-box miRNAs of the miR-2a-1, miR-2a-2, miR-2b-2 cluster or miR-6-1, miR-6-2, miR-6-3 cluster had similar effects on wing margin development, although two UAS-transgenes were necessary to produce this effect. Also loss of anterior crossvein and occasional L3 vein breaks was observed, although these are not indicative of loss of N signaling. More generalized expression of miR-7 using bx-Gal4 induced strong thickening of wing veins, which is indicative of compromised Notch signaling during lateral inhibition of wing veins. Expression of K-box miRNAs using bx-Gal4 had severe effects on wing development, resulting in tiny, crumpled wings. It is suspected that this results from misregulation of non-Notch-pathway-related targets. The Brd-box miRNAs miR-4 and miR-79 and the K-box miRNA miR-11 did not affect wing margin development, even when these transgenes were present in two copies, indicating that this phenotype is not generally due to misexpression of miRNAs. However, miR-79 induced strong wing curling at high levels, potentially due to misregulation of non-Notch-pathway-related targets (Lai, 2005).

    Next, focus was placed on development of the adult peripheral nervous system, as exemplified by the bristle sensory organs that decorate the body surface. A classic role for Notch signaling is to restrict the number of sensory organ precursors. It was found that misexpression of miR-6 using bx-Gal4 results in a strong increase in microchaete bristle density and clustered dorsocentral macrochaetes, phenotypes that are consistent with loss of Notch signaling during lateral inhibition of sensory organ precursors. Ectopic miR-2 had a similar, but milder, effect and mostly induced clustered dorsocentral and scutellar macrochaetes. Therefore, divergent members of the K-box miRNA family have similar effects on sensory organ development, consistent with data indicating that seed-related miRNAs can regulate overlapping sets of target genes. Ectopic miR-7 also induces macrochaete tufting, which correlates with the differentiation of supernumerary sensory organ precursors in wing imaginal discs. Finally, occasional duplication of bristles was observed upon misexpression of the Brd-box miRNA mir-79, although this construct also induced occasional bristle loss. Ectopic expression of miRNAs does not in itself induce bristle defects per se, since misexpression of miR-4 or miR-11 does not interfere with bristle development (Lai, 2005).

    Overall, the ability of different classes of Notch-regulating miRNAs to specifically induce phenotypes that are characteristic of Notch pathway loss of function in multiple developmental settings is a strong indication that Notch pathway targets validated in this study are key endogenous targets of these miRNAs (Lai, 2005).

    It appears, therefore, that cells go through a significant amount of trouble to actively inhibit Notch signaling. Core components of the Notch pathway are subject to significant negative regulation at every step in their life cycle, including at the transcriptional, post-transcriptional, and post-translational levels. For example, in the absence of activated nuclear Notch, CSL proteins are transcriptional repressors that actively repress Notch target gene activity. In addition, multiple dedicated ubiquitin ligases promote degradation of Notch pathway components, including the receptor Notch itself. To this list, may be added transcripts of most direct Notch target genes in Drosophila that are negatively regulated by multiple families of miRNAs (Lai, 2005).

    The evidence provided in this study to support this conclusion is that (1) three different classes of miRNA-binding sites (GY-boxes, Brd-boxes, and K-boxes) are pervasive among two major classes of Notch target genes; (2) all three classes of motif are selectively constrained in 3'-UTRs during evolution; (3) transcripts bearing these box sites are negatively regulated by complementary miRNAs in vivo; (4) all three classes of sites are both necessary and sufficient for miRNA-mediated regulation in vivo; and (5) ectopic expression of Notch target-regulating miRNAs phenocopies Notch pathway loss of function during multiple developmental settings. Perhaps most importantly, it has been shown that genomic transgenes specifically mutated for miRNA-binding sites are sufficiently hyperactive so as to perturb normal development of the peripheral nervous system. This places these Drosophila Notch target genes in a relatively select group of miRNA targets for which miRNA-mediated regulation is phenotypically essential for normal development (Lai, 2005).

    While most of the previously characterized in vivo targets of miRNAs are of the 'extensive pairing' variety, many of the validated targets in this study display much more limited 'box:seed'-pairing to miRNAs. In fact, within the context of the set of Notch target gene 3'-UTRs, the presence of conserved GY-boxes, Brd-boxes, and K-boxes allowed for highly effective prediction of miRNA:target relationships. This is the case even without first taking into account the extent of miRNA-pairing outside of box motifs. Rapid divergence of sequences upstream of box motifs, particularly those of the Brd-box and K-box classes, further indicates that extensive pairing is not selected for in these bona fide target sites. Consistent with this, multiple lines of evidence are presented that show that divergent seed-related miRNAs can regulate overlapping sets of target in vivo. Conversely, the importance of pairing between 3'-UTR boxes to miRNA seeds was demonstrated by endogenous 3'-UTR and box sufficiency tests, where even single-nucleotide disruption of seed-pairing abolishes regulation by miRNAs in vivo (Lai, 2005).

    Identification and characterization of miRNA-binding sites in these Notch target 3'-UTRs mesh well with other recent bioinformatics and experimental studies that together help to define the 'look' of miRNA-binding sites. The concept of using conserved 'boxes' with Watson-Crick complementarity to miRNA seeds to identify miRNA targets is at the heart of the TargetScanS approach. A recent study has identify statistically significant signal not only for conserved 3'-UTR sites that match positions 2-8 of the miRNA (as is characteristic of the Brd-box and GY-box), but also for matches to positions 2-7 of the miRNA (as is characteristic of the K-box). In addition, a significant bias was identified for the nucleotide corresponding to position one of the miRNA to be an adenosine in predicted target sites. Interestingly, 27/42 (64%) of GY-boxes, Brd-boxes, and K-boxes in Dm Notch target genes also have an adenosine in this position, consistent with the notion that this feature can help to identify genuine target sites. These results are also consistent with directed tests of model sites using an imaginal disc sensor assay. Together with the recent observation that miRNAs can down-regulate large numbers of transcripts that contain box:seed matches in their 3'-UTRs, a current view emerges that conserved 3'-UTR boxes that are 6-7 nt in length and complementary to the 5'-ends of miRNAs need to be considered seriously as functional regulatory sites. While seed-pairings with G:U base pairs are evidently not generally selected for, evidence is shown that rare sites of this class are functional. This is consistent with other studies that demonstrate that G:U seed-pairing impairs, but does not necessarily abolish target site function (Lai, 2005).

    Finally, the presence of multiple classes of miRNA-binding sites in most Notch target gene 3'-UTRs raises the possibility of combinatorial regulation. Although this has been widely suggested as a formal possibility, extensive evidence has been provided that 3'-UTRs can bear multiple classes of functional sites. Phylogenetic considerations indicate that 10 different Notch target genes are likely regulated by multiple classes of miRNAs, and direct experimental support of this was provided for six Notch target genes. Multiple Brd-box-, K-box-, and GY-box-class miRNAs are present at high levels in the Drosophila embryo, and the Brd-box miRNA miR-4 is co-transcribed with the K-box miRNAs miR-6-1, miR-2, miR-3, suggesting that combinatorial control of Notch target genes actually occurs during normal development. Future studies are aimed at examining how different miRNA-binding sites collectively contribute to overall regulation of an individual gene (Lai, 2005).

    Of the few animal miRNAs whose in vivo functions and targets are well understood, most act as genetic switches that determine binary, on/off states of target gene activity. For example, lin-4 and let-7 are temporal switches that control progression through nematode larval stages by inhibiting their targets at designated times in development. lsy-6 and miR-273 are spatial switches whose extremely restricted cell-type-specific expression patterns control neuronal identity. In these cases, temporally or spatially restricted miRNA expression is central to their control of specific processes, and each of these miRNAs appears to have a small number of key targets (Lai, 2005).

    A different rationale is proposed for Brd-box and K-box miRNAs during Drosophila development. Although endogenous territories of GY-box-mediated regulation are not known, negative regulation by Brd-boxes and K-boxes appears spatially and temporally ubiquitous. Thus, Notch target transcripts of the Brd family and E(spl)bHLH families are subject to modes of miRNA-mediated regulation that operate in all cells, even though the genes themselves display highly restricted patterns of spatial expression. This suggests that these miRNAs are not dedicated to regulating Notch signal transduction, but may 'tune' the expression of many target genes. Indeed, the K-box-family miRNAs miR-2, miR-6, and miR-11 also directly regulate K-box-containing proapoptotic genes, and the Brd-box-family miRNAs miR-4 and miR-79 regulate the mesodermal determinant bagpipe. One prediction is that even though mutation of Brd-boxes and K-boxes in individual Notch target genes results in specific defects in Notch-mediated cell fate decisions, mutation of Brd-box and K-box miRNAs would have more general developmental consequences. This is supported by the observation that many, but not all, of the phenotypes induced by ectopic expression of Notch-regulating miRNAs appear to be obviously related to repression of Notch pathway activity (Lai, 2005).

    An important advance of this study is the in vivo validation of a large number of biologically relevant miRNA targets that are minimally paired to miRNAs outside of the 'box:seed' region. Since modestly complementary sites are both necessary and sufficient for miRNA-mediated regulation, it might be relatively easy for novel miRNA-binding sites to arise in 'tuning' targets. Indeed, a subset of box sites has apparently newly evolved during Drosophilid radiation. In the greater context of insect Notch target genes, it appears to have been important that they be negatively regulated by miRNAs, although the precise numbers and arrangement of different sites is variable. These features of tuning targets seem to allow for highly customized regulation of individual genes (Lai, 2005).

    The experimental validation of many tuning targets may be challenging or impossible to obtain where quantitative regulation is subtle. Nevertheless, minor changes in gene activity, even of a fraction of a percent, could become highly significant when selecting the fitness of individuals at the population level. Deep evolutionary profiling of related species will therefore be key to revealing the full complement of biologically important miRNA-binding sites. The data suggest that multiple classes of miRNA-binding sites can be recognized with confidence as highly conserved 3'-UTR 'boxes' complementary to miRNA seeds, and this approach has been applied to the analysis of mammalian genomes. By mid-2005, 12 Drosophila genomes will be completed, which should enable high-confidence identification of miRNA-binding sites on the genome-wide scale -- even in cases in which only 7 nt of the target are paired to a miRNA (Lai, 2005).

    Recent computational work pointed to regulation of vertebrate Notch and Delta by miR-34; however, no Notch target genes were similarly singled out in various bioinformatics efforts. miR-34 is conserved in flies; however, inspection of fly Notch or its ligands Delta and Serrate failed to reveal 'boxes' that might indicate similar regulation by miR-34. Brd-box-, GY-box-, and K-box-complementary miRNAs are likewise conserved between flies and vertebrates. Are any vertebrate Notch target genes predicted to be targeted by these miRNAs by virtue of 'boxes'? Although Brd proteins have thus far been found only in insects, E(spl)bHLH proteins are conserved in and are primary effectors of Notch signaling in all vertebrates. No enrichment for Brd-boxes, GY-boxes, and K-boxes is observed across the set of vertebrate E(spl)bHLH 3'-UTRs as a whole. However, members of a specific subset of E(spl)-related repressors, named the Hey genes, contain a preponderance of these boxes in their 3'-UTRs. This appears to be the case in a variety of mammals (human, mouse, and rat) and fish (fugu and zebrafish). Therefore, miRNA-mediated regulation may be a conserved feature of Notch target genes, a scenario that is under current experimental investigation (Lai, 2005).

    Robust intestinal homeostasis relies on cellular plasticity in enteroblasts mediated by miR-8-Escargot switch

    This study used tracing methods that allow simultaneously capturing the dynamics of intestinal stem and committed progenitor cells (called enteroblasts) and intestinal cell turnover with spatiotemporal resolution. Intestinal stem cells (ISCs) divide 'ahead' of demand during Drosophila midgut homeostasis. Their newborn enteroblasts, on the other hand, take on a highly polarized shape, acquire invasive properties and motility. They extend long membrane protrusions that make cell-cell contact with mature cells, while exercising a capacity to delay their final differentiation until a local demand materializes. This cellular plasticity is mechanistically linked to the epithelial-mesenchymal transition (EMT) programme mediated by escargot, a snail family gene. Activation of the conserved microRNA miR-8/miR-200 in 'pausing' enteroblasts in response to a local cell loss promotes timely terminal differentiation via a reverse EMT by antagonizing escargot. These findings unveil that robust intestinal renewal relies on hitherto unrecognized plasticity in enteroblasts and reveal their active role in sensing and/or responding to local demand (Antonello, 2015).

    The robustness of intestinal cell renewal relies on cellular plasticity in committed progenitor cells and a rather loose regulation of ISCs proliferation. One key finding is that stem cells divide continually and generate a 'stock' of committed progenitor cells that do not terminally differentiate right away but postpone their final differentiation for long time intervals in the absence of a local epithelial cell loss. Accordingly, one noticeable change in newborn progenitor cells after their (enterocyte) fate commitment is their transformation from rounded cells to spindle-shaped cells that appear to actively monitor their surroundings by extending long membrane actin-rich protrusions that make cell-cell contact with mature epithelial cells and their mother ISCs. Timely terminal differentiation with epithelial cell loss is orchestrated by activation of a conserved pro-epithelial microRNA, in turn, directly repressing the repressors of differentiation. A microRNA-induced repression of the repressors of differentiation provides a faster mechanism than one involving a transcriptional regulator since synthesizing a miRNA likely requires less time than synthesizing a protein. Importantly, mutual antagonism between the microRNA (MiR-8/miR-200) and its targets (Escargot/Snail2 and Zfh1/ZEB) may serve to slow down the mesenchymal-to-epithelial process inside individual mesenchymal/progenitor cells until they are successfully integrated in the epithelium. Consistently, abrupt transition as in mir-8 overexpressing midguts results in erroneous tissue repair (Antonello, 2015).

    Supply and demand in business production involves frequently two alternative solutions called 'make-to-stock' and 'make-to-order'. In 'make-to-stock' or MTS, production is continuous so that response to customers can be supplied immediately. However, as production is not based on actual demand, the MTS solution is not robust against fluctuations in demand and errors in forecasting can result in shortages (if there is insufficient residual stock) or overproduction. In 'make-to-order', or MTO, production only starts upon receiving a customer's order, thereby precisely matching production to demand. However, the MTO generates a delay in the response and can be less efficient and competitive than the MTS paradigm. The dynamics of stem cells and committed progenitor cells in the midgut suggests a hybrid solution between MTS and MTO -- reminiscent to the business solution known as delayed differentiation. Thus, in basal homeostasis, production of new cells to replace cell loss occurs in two stages: (1) a 'make-to-stock' stage where committed progenitor cells are continually generated and 'stocked' in an 'undifferentiated' state; and (2) a 'make-to-order' stage where terminal differentiation takes place only in response to a local demand. In mice and humans, the rapid turnover that occurs in the small intestinal epithelium is thought to be the result of continual shedding of superficial cells balanced by the continual stem cell production. The mechanism described in this study may be more general than expected and could account for how murine cells after fate commitment like the secretory-committed cells defer for long periods their terminal differentiation (Buczacki et al, 2013; Antonello, 2015).

    Escargot/Snail2 sustains the undifferentiated state and self-renewing divisions of midgut intestinal stem cells. However, the committed progenitor cells also express escargot and apparently at higher levels than the stem cells. It is hypothesized that below a certain threshold level, Escargot maintains stemness and a partial EMT that may facilitate regular cell division and a topologically confined position at the base of the intestinal epithelium. Conversely, when Escargot surpasses a certain threshold level, it promotes a full EMT that confers invasive properties and motility for the successful response and integration of the newly differentiated cells in the preexisting epithelium. Intriguingly, the enteroendocrine cells appear to escape from this block in terminal differentiation and differentiate at the normal rate in the absence of escargot. There is as yet no explanation for the behaviour of these progenitor cells (Antonello, 2015).

    Mechanistically, the different levels of escargot could be achieved via Notch signalling pathway, which is prominently activated in enterocyte-committed progenitors. Notch signalling activates directly zfh1 gene and Zfh1, a homolog of the mammalian stemness and EMT-determinant Zeb1,2, and binds to the escargot promoter region, and this study shows that Zfh1 acts genetically upstream of escargot. Thus, progenitor cells receiving Notch signalling might enhance escargot transcriptional levels via Notch-induced zfh1 transcription. Such regulatory mechanism would explain, for example, that loss of Notch results in stem-like/round cells (Antonello, 2015).

    In mammalian cell culture, the EMT process has been linked to the acquisition of stem-like nature via an interplay between the ZEB1,2 and Snail transcription factors and the microRNAs of the miR-200 family. Moreover, EMT determinants often regulate each other to promote EMT. Thus, the interactions between Escargot/Snail2, zfh1/Zeb and miR-8/miR-200 that were identified in this study exemplify the conservation of the regulatory mechanisms involved in EMT/MET and stemness in an in vivo context and a normal physiology of an adult organism. However, this study shows that escargot-zfh1 promotes stemness and full EMT/invasive properties in distinct cell populations and likely at different concentration levels, highlighting the utility of Drosophila midgut as a model to dissect out mechanisms linking physiological EMT to cellular plasticity and stemness as well as provide novel insights linking polyploidy and EMT towards stemness (Antonello, 2015).

    Although midgut mesenchymal/progenitor cells have motility, most of them maintain their own local area as clearly defined by Flybow clonal analysis. This situation is similar to the leading edge mesenchymal cells during collective cell migration. Midgut enteroblasts retain contact via E-cadherin with their mother ISC, a process that might be regulated by escargot as in tracheal cells. Cell-cell contact is crucial to sustain Notch signalling in committed progenitor cells and likely to help to stabilize polarity of enteroblasts and their membrane protrusions that contact mature cells. Through these protrusions, mesenchymal/enteroblasts might actively monitor their surroundings. When a protrusion detects changes in tension and mechanical forces generated during the elimination of a dying cells, a positive input might be created that triggers the activation of expression of the microRNA mir-8 in the particular progenitor cell which, in turn, promotes the epithelial state and integration of the newly differentiated cell in the epithelium. Adhesion via E-cadherin could facilitate communication between an epithelial cells and a mesenchymal/progenitor cell in its vicinity so that a single, newly differentiated cell fills the gap left by the cleared cell (Antonello, 2015).

    Dynamic pseudopodia in migrating cells have been proposed as a mechanism for temporal and spatial sensing during cell migration. Direction sensing is also consistent with time-lapse data showing individual progenitor cells re-adjusting position in the homeostatic midguts. Transduction of mechanical cues via YAP and TAZ (called Yorkie in flies) is functionally involved in differentiation of mesenchymal stem cells. Hence, Drosophila Hippo/Yorkie-YAP in mature enterocytes is a primary candidate pathway for a potential transduction of mechanical cues activating mir-8 in response to cell death (Antonello, 2015).

    In summary, the miR-8-escargot-zfh1 axis and the EMT/MET programme provides a conceptual shift of the current stem cell-centred view of tissue renewal and offers a starting point for investigating how mature cells speak with neighbouring committed progenitor cells to ensure that epithelial cell loss and cell addition are kept in balance (Antonello, 2015).

    miR-980 is a memory suppressor microRNA that regulates the autism-susceptibility gene A2bp1

    MicroRNAs have been associated with many different biological functions, but little is known about their roles in conditioned behavior. This study demonstrates that Drosophila miR-980 is a memory suppressor gene functioning in multiple regions of the adult brain. Memory acquisition and stability were both increased by miR-980 inhibition. Whole cell recordings and functional imaging experiments indicated that miR-980 regulates neuronal excitability. This study identified the autism susceptibility gene, A2bp1, as an mRNA target for miR-980. A2bp1 levels varied inversely with miR-980 expression; memory performance was directly related to A2bp1 levels. In addition, A2bp1 knockdown reversed the memory gains produced by miR-980 inhibition, consistent with A2bp1 being a downstream target of miR-980 responsible for the memory phenotypes. These results indicate that miR-980 represses A2bp1 expression to tune the excitable state of neurons, and the overall state of excitability translates to memory impairment or improvement (Guven-Ozkan, 2016).

    MicroRNAs (miRNAs) are small (21–23 nt), non-coding RNAs that repress gene expression to regulate cellular development and physiology. A short seed sequence (6–8 nt) located at the 5' end of miRNAs binds to complementary sequences in the 3'-UTR of target mRNAs torepress mRNA expression by blocking translation and/or promoting degradation of the mRNA target). Thus, miRNAs offer a relatively rapid, analog, and cell-type-specific control mechanism for the epigenetic expression of genomic information in both time and space (Guven-Ozkan, 2016).

    One aspect of miRNA function that remains understudied concerns the roles for these molecules in learning and memory, a primary adaptive function of the CNS. Prior studies revealed that broad insults to the miRNA processing pathway impairs memory formation in both Drosophila and the mouse. Although eukaryotic genomes encode hundreds of distinct miRNAs and they are generally expressed at high levels in the CNS, only a handful of specific miRNAs have been studied and implicated in memory formation through roles in neuronal maturation, connectivity, and synaptic plasticity (Guven-Ozkan, 2016).

    To identify the miRNAs that participate in the biology of memory formation, a large scale, comprehensive screen was conducted using a transgenic approach to systematically inhibit 134 different miRNAs, using a 'microRNA sponge' technique. The influences of 134 miRNAs were surveyed for effects on intermediate term (ITM, i.e., at 3 hr after conditioning), olfactory aversive memory. From this screen, several new miRNAs were identified that function to inhibit or promote memory formation at this time point. MiR-980, when inhibited, was shown to enhance memory formation. Thus, MiR-980, a member of the miR-22 family of vertebrate miRNAs, was classified as having a memory suppressor function (Guven-Ozkan, 2016).

    This study characterize the memory suppressing function of miR-980. Among the mRNA targets for miR-980, it was demonstrated that the autism-susceptibility gene, Ataxin2 binding protein 1 (A2bp1, also known as Rbfox-1, Fox-1) is a primary target responsible for miR-980-directed memory suppression. A2bp1 is a known RNA binding protein involved in alternative splicing of a network of critical neuronal genes during development and in adults and in addition to autism (ASD), is associated with intellectual disability and epilepsy. Opposite to the role for miR-980, A2bp1 as a memory-promoting gene. Combined data advance understanding of the miR-22 family of miRNAs, showing that in Drosophila the magnitude of memory formation is a direct function of miR-980 abundance and of its primary mRNA target for this function, A2bp1 (Guven-Ozkan, 2016).

    A behaviorally based 'miRNA sponge screen' was conducted to systematically identify the miRNAs involved in Drosophila olfactory aversive learning and memory. The results offer five major advances in knowledge about the function of this class of regulatory molecules: (1) miR-980 functions to suppress memory formation by acting in multiple types of neurons within the olfactory nervous system; (2) miR-980 works as a suppressor of acquisition and memory stability; (3) miR-980 suppresses the excitability of excitatory neurons; (4) the memory suppressor functions of miR-980 are mediated largely by the inhibition of the autism-susceptibility gene, A2bp1; and (5) A2bp1, itself, is a memory-promoting gene (Guven-Ozkan, 2016).

    One surprising observation made in this study was that inhibition of miR-980 in multiple neurons within the olfactory nervous system enhances memory performance, as was anticipated, finding a single cellular focus for its effects. Initially, it was difficult to understand how a single microRNA could modify behavioral memory when altered in one of many different types of neurons. This was reconciled by showing that excitability of projection neurons is enhanced with inhibited miR-980 function, offering the explanation that increased signaling, in general, within the olfactory nervous system enhances behavioral memory. This model provides a general explanation for the effects of miR-980 that function in multiple classes of excitable neurons (Guven-Ozkan, 2016).

    It is proposed that the role of miR-980 in excitability accounts for the increased acquisition when the miRNA is inhibited. An increase in excitable state may simply enhance the signaling through different neuron types within the olfactory nervous system as the organism integrates sensory information into memory. A corollary of this idea is that normal acquisition is a composite effect of multiple neurons within the circuit conveying the sensory information being learned. Although it is possible that increased acquisition also accounts for the increased memory performance observed when immediate performance scores were normalized, an alternative possibility is that miR-980 may have distinct roles in acquisition and memory stability. For instance, although the increased acquisition is attributed to increased neuronal excitability, the increased memory after acquisition may be due to altered regulation of molecules involved in synaptic transmission (Guven-Ozkan, 2016).

    miR-980 belongs to the miR-22 family of miRNAs found in mammals. Within the nervous system, the miR-22 family has been reported to participate in neuroprotection, neurodegeneration, neuroinflammation, neurodevelopment. Thus, although this family appears to have multiple roles in the nervous system and disease, the current studies identify members of this family as specifically involved in the suppression of memory formation. Given the functional association between miR-980 and A2bp1 shown here, it is also tempting to speculate that the miR-980/miR-22 family of miRNAs might be associated with autism spectrum disorders. No evidence for this possibility has yet been reported, but the expression of miR-22 is reduced in attention deficit hyperactivity disorder (ADHD) and is genetically associated with panic disorder and anxiety in humans. Thus, there are neuropsychiatric links to miR-22 , which could potentially be through a role in excitability. Moreover, miR-22 represses the tumor suppressor gene PTEN in transformed human bronchial epithelial cells, and PTEN is known to be involved in Cowden syndrome and ASD in humans (Guven-Ozkan, 2016).

    Behavioral, molecular, cellular, and genetic data together argue that A2bp1 is a primary target of miR-980 for memory suppression. First, A2bp1 is broadly expressed in the fly brain, consistent with a broad nervous system requirement for miR-980. Second, there are three miR-980 binding sites in A2bp1 3' UTR making it a strong candidate mRNA target for miR-980 regulation. Third, an in vitro mRNA binding experiment was performed using biotinylated mature miR-980 as bait, and eight times more A2bp1 mRNA was successfully captured using wild-type miR-980 versus a form mutated for the seed region. Fourth, A2bp1 shows the precise abundance/behavior relationship predicted as a direct target of miR-980. Overexpression of A2bp1 increases memory; miR-980 suppression increases memory. A2bp1 knockdown impairs memory; miR-980 overexpression impairs memory. Fifth, A2bp1 protein abundance varies as expected by manipulation of miR-980 levels. Overexpression of miR-980 decreases A2bp1 protein abundance and miR-980 suppression increases A2bp1 protein abundance. Finally, reducing A2bp1 levels using RNAi in miR-980-inhibited flies reversed the memory improvement. This finding is consistent with the model that A2bp1 is genetically downstream of miR-980 and a major mediator of the phenotype. However, the possibilities cannot be excluded that there may be additional miR-980 targets that participate in memory suppression and miR-980 regulation of A2bp1 could be indirect. A simple model for miR-980/A2bp1 interactions and function seem to be at odds with an observation made about A2bp1 using mammalian models. In the mouse, neuronal-specific knockout of A2bp1 increases excitability in the dentate gyrus, a result opposite of that predicted by the current model. This difference might reflect species or cell type differences, the complexity of the gene with its dozens of isoforms, or the multiple layers of regulation on A2bp1 expression. Bioinformatics analyses predict multiple miRNAs as binding to the A2bp1 3' UTR and regulating its expression. Thus, its basal or regulated expression level due to changes in physiological state could be a composite of (Guven-Ozkan, 2016).

    A2bp1 is associated with autism and epilepsy in human patients functioning presumably by regulating alternative splicing during both development and in adults). It has been proposed that changes in gene-splicing alter the relative abundance of protein isoforms, which remodels protein networks and increases the risk for autism. Consistent with this thought, transcriptome analyses from ASD brains identified A2bp1 as one hub gene that is dysregulated in patients with autism. A2bp1 was originally identified through its interaction with Ataxin-2. Pn-specific knockdown of Ataxin-2 impairs long-term olfactory habituation-associated structural and functional plasticity by regulating the miRNA pathway. Future studies will shed light on whether memory phenotypes of A2bp1 are dependent on Ataxin-2. It is intriguing that the current studies show that adult stage-specific increases in A2bp1 abundance improve aversive olfactory memory, independent of any developmental function for the protein, and human ASD is a spectrum brain disorder that is associated with poor to extraordinarily robust learning and memory capacities. It is speculated that the different protein interaction networks that form due to varying levels of A2bp1 function account for the range of intellectual abilities observed in ASD. Drosophila may prove to be a much speedier and simpler system to dissect the specific effect of A2bp1 abundance on the emergence of protein interaction networks and their influence on cognitive abilities (Guven-Ozkan, 2016).

    A neuroprotective role for microRNA miR-1000 mediated by limiting glutamate excitotoxicity

    Evidence has begun to emerge for microRNAs as regulators of synaptic signaling, specifically acting to control postsynaptic responsiveness during synaptic transmission. This report provides evidence that Drosophila melanogaster miR-1000 acts presynaptically to regulate glutamate release at the synapse by controlling expression of the vesicular glutamate transporter (VGlut). Genetic deletion of miR-1000 led to elevated apoptosis in the brain as a result of glutamatergic excitotoxicity. The seed-similar miR-137 regulates VGluT2 expression in mouse neurons. These conserved miRNAs share a neuroprotective function in the brains of flies and mice. Drosophila miR-1000 showed activity-dependent expression, which might serve as a mechanism to allow neuronal activity to fine-tune the strength of excitatory synaptic transmission (Verma, 2015).

    miRNAs have emerged in recent years as important regulators of homeostatic mechanisms. Changes in miRNA expression and activity have been linked to neurodegenerative disorders. A growing body of evidence suggests that miRNAs are neuroprotective in the aging brain, as well as in the control of synaptic function and plasticity. Mouse miR-134 acts postsynaptically to regulate synapse strength, and miR-181 and miR-223 regulate glutamate receptors, thereby affecting postsynaptic responsiveness to glutamate. miR-1, a muscle-specific miRNA, acts in a retrograde fashion at the neuromuscular junction to regulate the kinetics of synaptic vesicle exocytosis. However, there are few examples of miRNAs acting directly in the presynaptic terminal to control synaptic strength. miR-485, which is found presynaptically, has been shown to control the expression of synaptic vesicle protein SV2A, thereby affecting synapse density and GluR2 receptor levels (Verma, 2015).

    This paper reports that Drosophila miR-1000 regulates neurotransmitter release from presynaptic terminals. miR-1000 regulates expression of the VGlut, which loads glutamate into synaptic vesicles. Genetic ablation of miR-1000 leads to glutamate excitotoxicity, resulting in early-onset neuronal death. Presynaptic regulation of miR-1000 is activity dependent and may serve as a mechanism for tuning synaptic transmission. Evidence is presented that this regulatory relationship is conserved in the mammalian CNS, with a seed-similar miRNA, miR-137, conferring neuroprotection through regulation of VGluT2. The consequences of misregulation of glutamatergic signaling can be severe: excitotoxicity due to excessive glutamate release has been implicated in ischemia and traumatic brain injury, as well as in neurodegenerative conditions such as Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis (Verma, 2015).

    Although postsynaptic regulation of glutamate receptor activity has been well studied, much less is known about presynaptic regulation of glutamatergic signaling. These findings suggest that miR-1000 acts presynaptically to regulate VGlut expression and thereby control synaptic glutamate release. It is tempting to speculate that this could provide a mechanism for tuning synaptic output and locally modulating synaptic strength. Such a mechanism would be most useful if the miRNA itself could be regulated in an activity-dependent manner. Evidence is provided that miR-1000 expression is regulated by light in vivo, presumably reflecting photoreceptor activity in the eye. This in turn leads to light-regulated regulation of VGlut reporter levels. These findings lend support to the notion of activity-dependent regulation of miR-1000 activity. An in depth exploration of these issues awaits the development of methods to monitor changes in presynaptic miRNA levels in real time (Verma, 2015).

    Failure of miR-1000-mediated regulation of VGlut led to excess glutamate release and resulted in excitotoxicity. Consistent with these findings, Gal4-directed overexpression of VGlut has been reported to cause neurodegeneration. Notably, elevated levels of vertebrate VGluTs have been associated with excitotoxicity in animal models of epilepsy and traumatic brain injury. The GAERS rat epilepsy model shows elevated levels of VGluT2 but not of VGluT1. Similarly, in a model of stroke, ischemic injury was found to result in elevated expression of VGluT1 but not of VGluT2. VGluT1 levels are regulated by methamphetamine treatment, likely contributing to excitotoxic consequences of methamphetamine abuse. VGluT1 levels have also been reported to increase in rat brains following antidepressant treatment (Verma, 2015).

    In the mouse, miR-223 acts on postsynaptic glutamate receptors and has a neuroprotective role in vivo. These findings provide evidence that miR-1000 has a neuroprotective role mediated through regulation of presynaptic glutamate release and that this regulatory mechanism is conserved for miR-137 and VGluT2 in the mouse. Together, these studies show that miRNA-mediated regulation of glutamatergic activity acts pre- and post-synaptically to modulate synaptic transmission and to protect against excitotoxicity. In this context, it is noteworthy that miR-137 is reported to be enriched at synapses. miR-137 levels were found to be low in a subset of Alzheimer's patients with elevated serine palmitoyltransferase 1 expression leading to increased ceramide production. Single nucleotide polymorphisms affecting miR-137 target sites could lead to low-level constitutive overexpression of its targets, even when the SNP is present in a single copy. A single nucleotide polymorphism affecting miR-137 has also been identified as a risk factor for schizophrenia. It will be of interest to learn whether misregulation of VGluT2 expression contributes to these neurological conditions. The current findings raise the possibility that miRNA mediated regulation makes VGluT and other miRNA targets possible risk factors in neurodegenerative disease (Verma, 2015).

    Identification of miR-305, a microRNA that promotes aging, and its target mRNAs in Drosophila

    MicroRNAs (miRNAs) are involved in the regulation of important biological processes. This study describes a novel Drosophila miRNAs involved in aging. We selected eight Drosophila miRNAs, displaying high homology with seed sequences of aging-related miRNAs characterized in other species, and investigated whether the over-expression of these miRNAs affected aging in Drosophila adult flies. The lifespan of adults over-expressing miR-305, a miRNA showing high homology with miR-239 in C. elegans, was significantly shorter. Conversely, a reduction in miR-305 expression led to a longer lifespan than that in control flies. miR-305 over-expression accelerated the impairment of locomotor activity and promoted the age-dependent accumulation of poly-ubiquitinated protein aggregates in the muscle, as flies aged. Thus, this study shows that the ectopic expression of miR-305 has a deleterious effect on aging in Drosophila. RNA-Seq was performed o identify the targets of miR-305. Several mRNAs encoding antimicrobial peptides and insulin-like peptides were discovered, whose expression changed in adults upon miR-305 over-expression. We further confirmed, by qRT-PCR, that miR-305 over-expression significantly decreases the mRNA levels of four antimicrobial peptides. As these mRNAs contain multiple sequences matching the seed sequence of miR-305, it is speculated that a reduction in target mRNA levels, caused by ectopic miRNA expression, promotes aging (Ueda, 2018).

    EXD2 governs germ stem cell homeostasis and lifespan by promoting mitoribosome integrity and translation

    Mitochondria are subcellular organelles that are critical for meeting the bioenergetic and biosynthetic needs of the cell. Mitochondrial function relies on genes and RNA species encoded both in the nucleus and mitochondria, and on their coordinated translation, import and respiratory complex assembly. This study characterize EXD2 (exonuclease 3'-5' domain-containing 2), a nuclear-encoded gene, and showed that it is targeted to the mitochondria and prevents the aberrant association of messenger RNAs with the mitochondrial ribosome. Loss of EXD2 results in defective mitochondrial translation, impaired respiration, reduced ATP production, increased reactive oxygen species and widespread metabolic abnormalities. Depletion of the Drosophila melanogaster EXD2 orthologue (CG6744) causes developmental delays and premature female germline stem cell attrition, reduced fecundity and a dramatic extension of lifespan that is reversed with an antioxidant diet. These results define a conserved role for EXD2 in mitochondrial translation that influences development and ageing (Silva, 2018).

    Novel Triazole linked 2-phenyl benzoxazole derivatives induce apoptosis by inhibiting miR-2, miR-13 and miR-14 function in Drosophila melanogaster

    Apoptosis is an important phenomenon in multi cellular organisms for maintaining tissue homeostasis and embryonic development. Defect in apoptosis leads to a number of disorders like- autoimmune disorder, immunodeficiency and cancer. 21-22 nucleotides containing micro RNAs (miRNAs/miRs) function as a crucial regulator of apoptosis alike other cellular pathways. Recently, small molecules have been identified as a potent inducer of apoptosis. This study has identified novel Triazole linked 2-phenyl benzoxazole derivatives (13j and 13h) as a negative regulator of apoptosis inhibiting micro RNAs (miR-2, miR-13 and miR-14) in a well established in vivo model Drosophila melanogaster where the process of apoptosis is very similar to human apoptosis. These compounds inhibit miR-2, miR-13 and miR-14 activity at their target sites, which induce an increased caspase activity, and in turn influence the caspase dependent apoptotic pathway. These two compounds also increase the mitochondrial reactive oxygen species (ROS) level to trigger apoptotic cell death (Mondal, 2017).

    Immediate-early alcohol-responsive miRNA expression in Drosophila

    At the core of the changes characteristic of alcoholism are alterations in gene expression in the brain of the addicted individual. These changes are believed to underlie some of the neuroadaptations that promote compulsive drinking. Unfortunately, the mechanisms by which alcohol consumption produces changes in gene expression remain poorly understood. MicroRNAs (miRNAs) have emerged as important regulators of gene expression because they can coordinately modulate the translation efficiency of large sets of specific mRNAs. This study investigated the early miRNA responses elicited by an acute sedating dose of alcohol in the Drosophila model organism. In this analysis, the power of next-generation sequencing was combined with Drosophila genetics to identify alcohol-sensitive miRNAs and to functionally test them for a role in modulating alcohol sensitivity. Fourteen known Drosophila miRNAs, and 13 putative novel miRNAs were identified that respond to an acute sedative exposure to alcohol. Using the GeneSwitch Gal4/UAS system, a subset of these ethanol-responsive miRNAs was functionally tested to determine their individual contribution in modulating ethanol sensitivity. Two microRNAs were identified that when overexpressed significantly increased ethanol sensitivity: miR-6 and miR-310. MicroRNA target prediction analysis revealed that the different alcohol-responsive miRNAs target-overlapping sets of mRNAs. Alcoholism is the product of accumulated cellular changes produced by chronic ethanol consumption. Although all of the changes described here are extremely rapid responses evoked by a single ethanol exposure, understanding the gene expression changes that occur in the first few minutes after ethanol exposure will help categorization of ethanol responses into those that are near instantaneous and those that are emergent responses produced only by repeated ethanol exposure (Ghezzi, 2016).

    Multiple in vivo biological processes are mediated by functionally redundant activities of Drosophila mir-279 and mir-996

    Drosophila mir-279 has been reported as essential to restrict the emergence of CO2-sensing neurons, to maintain circadian rhythm, and to regulate ovarian border cells. The mir-996 locus is located near mir-279 and bears a similar seed, but they otherwise have distinct, conserved, non-seed sequences, suggesting their evolutionary maintenance for separate functions. Single and double deletion mutants were generated of the mir-279 and mir-996 hairpins, and cursory analysis suggested that mir-996 was dispensable. However, discrepancies in the strength of individual mir-279 deletion alleles led to the the finding that extant mir-279 mutants are deficient for mature mir-996, even though they retain its genomic locus. Therefore a panel of genomic rescue transgenes was engineered into the double deletion background, allowing a pure assessment of mir-279 and mir-996 requirements. Surprisingly, detailed analyses of viability, olfactory neuron specification, and circadian rhythm indicate that mir-279 is completely dispensable. Instead, an endogenous supply of either mir-279 or mir-996 suffices for normal development and behavior. Sensor tests of nine key mir-279/996 targets showed their similar regulatory capacities, although transgenic gain-of-function experiments indicate partially distinct activities of these miRNAs that may underlie that co-maintenance in genomes. Altogether, this study elucidated the unexpected genetics of this critical miRNA operon, and provides a foundation for their further study. More importantly, these studies demonstrate that multiple, vital, loss-of-function phenotypes can be rescued by endogenous expression of divergent seed family members, highlighting the importance of this miRNA region for in vivo function (Sun, 2015).

    miR-965 controls cell proliferation and migration during tissue morphogenesis in the abdomen

    Formation of the Drosophila adult abdomen involves a process of tissue replacement in which larval epidermal cells are replaced by adult cells. The progenitors of the adult epidermis are specified during embryogenesis and, unlike the imaginal discs that make up the thoracic and head segments, they remain quiescent during larval development. During pupal development, the abdominal histoblast cells proliferate and migrate to replace the larval epidermis. This study provides evidence that the microRNA, miR-965, acts via string and wingless to control histoblast proliferation and migration. Ecdysone signaling downregulates miR-965 at the onset of pupariation, linking activation of the histoblast nests to the hormonal control of metamorphosis. Replacement of the larval epidermis by adult epidermal progenitors involves regulation of both cell-intrinsic events and cell communication. By regulating both cell proliferation and cell migration, miR-965 contributes to the robustness of this morphogenetic system (Verma, 2015).

    The findings of this study link regulation of the miR-965 microRNA to the onset of histoblast proliferation at the larval to pupal transition. Previous reports have provided evidence that Ecdysone signaling activates string expression to trigger the onset of histoblast proliferation at the beginning of pupal development (Ninov, 2009). The current findings provide evidence that Ecdysone signaling works though regulation of miR-965, which in turn regulates string. Interestingly, evidence was also found for negative feedback regulation of miR-965 on EcR. Mutual repression circuitry of this type can contribute a switch-like function: EcR activity lowers miR-965 activity, which allows greater EcR expression/activity by alleviating miR-965 mediated repression. In a circuit of this design, there will be a delay between reduced transcription of the miRNA primary transcript and the decay of the mature miRNA product. Hence sustained EcR activity is needed to throw the switch (Verma, 2015).

    EcR shows positive transcriptional autoregulation and this is buffered by miR-14 in a mutual repression circuit (Varghese, 2007). Positive feedback allows for a sharp switch-like response, but also makes the system very sensitive to stochastic fluctuation in EcR activity. Coupling EcR positive auto-feedback to miRNA-mediated repression allows a robust switch function upon Ecdysone stimulation, while protecting the system from the effects of biological noise. This study provides evidence that miR-965 plays an analogous role in regulating EcR response and suggests that miR-965 confers robustness to the EcR response in the histoblasts (Verma, 2015).

    Upregulation of string in the miR-965 mutant contributes to the defects in histoblast proliferation. How misregulation of string might contribute to the migration defects is less immediately obvious. Previous work has shown that cell cycle progression in the histoblast population is required to trigger programmed cell death in the surrounding larval epidermal cells (LECs). Evidence has been provided that cell growth and the expansion of the histoblast nests may be required to elicit LEC apoptosis. Although the mechanism by which expansion of the histoblasts triggers LEC death is not clear, elevated string expression in the miR-965 mutant is likely to be responsible for the cell cycle progression defects during this phase, hindering normal LEC removal and histoblast migration (Verma, 2015).

    Persistence of the LECs might also be a consequence of the increased expression of Wg protein in the mutant histoblast nests. Wg acts in combination with EGFR and Dpp signals to control abdominal segment patterning. These signals are thought to control differential cell adhesion, which may be important for elimination of the LECs as well as for proper segmental fusion of the histoblast nests. Elevated expression of Wg protein may lead to an expanded range of action, perhaps resulting in ectopic Wg activity in the LECs (Verma, 2015).

    Each adult abdominal segment has a well-defined anterior-posterior polarity. Wg is required from 15–20 hr APF for bristle formation and from 18–28 hr APF for tergite differentiation and pigmentation. Overexpression of wg has been shown to cause ectopic bristle formation, and shaggy mutant clones, which constitutively activate wg signaling, can cause polarity reversal in abdominal bristles, while EGFR, FGF, dpp and Notch signaling have no effect on the polarity of bristles in adult epidermis. Wg levels are normally higher in the posterior region of the anterior histoblast nests and lower more anteriorly. The current finding that Wg levels were elevated and that the distribution of Wg was broader than normal suggests ectopic Wg activity throughout the histoblast nest, including cells that normally experience low Wg levels. Ectopic spread of Wg could be responsible for the formation of ectopic bristles and for the occasional instances of polarity reversal observed in the anterior part of tergites in the miR-965 mutants (Verma, 2015).

    Replacement of the larval epidermis during metamorphosis involves regulation of both cell-intrinsic events in the abdominal histoblasts and communication between histoblasts and the larval cells they will replace. miR-965 acts on at least two separate processes required during histoblast morphogenesis. A miRNA with multiple targets can add a layer of regulation, acting across different pathways to integrate their activities. In doing so, the miR-965 miRNA appears to contribute to the robustness of this complex morphogenetic system (Verma, 2015).

    Derepressing muscleblind expression by miRNA sponges ameliorates myotonic dystrophy-like phenotypes in Drosophila

    Myotonic Dystrophy type 1 (DM1) originates from alleles of the DMPK gene with hundreds of extra CTG repeats in the 3' untranslated region (3' UTR). CUG repeat RNAs accumulate in foci that sequester Muscleblind-like (MBNL) proteins away from their functional target transcripts. Endogenous upregulation of MBNL proteins is, thus, a potential therapeutic approach to DM1. This study identifies two miRNAs, dme-miR-277 and dme-miR-304, that differentially regulate muscleblind RNA isoforms in miRNA sensor constructs. It was shown that their sequestration by sponge constructs derepresses endogenous muscleblind not only in a wild type background but also in a DM1 Drosophila model expressing non-coding CUG trinucleotide repeats throughout the musculature. Enhanced muscleblind expression results in significant rescue of pathological phenotypes, including reversal of several mis-splicing events and reduced muscle atrophy in DM1 adult flies. Rescued flies have improved muscle function in climbing and flight assays, and have longer lifespan compared to disease controls. These studies provide proof of concept for a similar potentially therapeutic approach to DM1 in humans (Cerro-Herreros, 2016).

    Presynaptic CamKII regulates activity-dependent axon terminal growth: miR-289 directly represses the translation of CamKII

    Spaced synaptic depolarization induces rapid axon terminal growth and the formation of new synaptic boutons at the Drosophila larval neuromuscular junction (NMJ). This study identified a novel presynaptic function for the Calcium/Calmodulin-dependent Kinase II (CamKII) protein in the control of activity-dependent synaptic growth. Consistent with this function, both total and phosphorylated CamKII (p-CamKII) are were found to be enriched in axon terminals. Interestingly, p-CamKII appears to be enriched at the presynaptic axon terminal membrane. Moreover, levels of total CamKII protein within presynaptic boutons globally increase within one hour following stimulation. These effects correlate with the activity-dependent formation of new presynaptic boutons. The increase in presynaptic CamKII levels is inhibited by treatment with cyclohexamide suggesting a protein-synthesis dependent mechanism. Previous work has found that acute spaced stimulation rapidly downregulates levels of neuronal microRNAs (miRNAs) that are required for the control of activity-dependent axon terminal growth at this synapse. The rapid activity-dependent accumulation of CamKII protein within axon terminals is inhibited by overexpression of activity-regulated miR-289 in motor neurons. Experiments in vitro using a CamKII translational reporter show that miR-289 can directly repress the translation of CamKII via a sequence motif found within the CamKII 3' untranslated region (UTR). Collectively, these studies support the idea that presynaptic CamKII acts downstream of synaptic stimulation and the miRNA pathway to control rapid activity-dependent changes in synapse structure (Nesler, 2016).

    Acute spaced synaptic depolarization rapidly induces the formation of new synaptic boutons at the larval NMJ. These immature presynaptic outgrowths, also known as "ghost boutons", are characterized by the presence of synaptic vesicles but by a lack of active zones and postsynaptic specializations. IA wild-type third instar larval NMJ will typically have about 2 ghost boutons. Using an established synaptic growth protocol, a robust increase in the number of ghost boutons was observed following 5 x K+ spaced stimulation. It has been shown that activity-dependent ghost bouton formation involves both new gene transcription and protein synthesis. Furthermore, new presynaptic expansions can form within 30 min of stimulation even after the axon innervating the NMJ has been severed. These findings suggest that a local mechanism (i.e. local signaling and/or translation) is required for the budding and outgrowth of new axon terminals. As expected, application of the translational inhibitor cyclohexamide during the recovery phase prevented the formation of new ghost boutons (Nesler, 2016).

    It has been shown previously that the outgrowth of new synaptic boutons in response to spaced depolarization requires the function of activity-regulated neuronal miRNAs including miR-8, miR-289, and miR-958 (Nesler, 2013). This implies that mRNAs encoding for synaptic proteins might be targets for regulation by these miRNAs. Focused was placed on CamKII for three reasons. (1) CamKII has been shown to have a conserved role in the control of long-term synaptic plasticity and its expression at synapses requires components of the miRNA pathway. Furthermore, the fly CamKII mRNA contains two predicted binding sites for activity-regulated miR-289. (2) CamKII and PKA both phosphorylate and actives synapsin. At the fly NMJ, a synapsin-dependent mechanism is required for a transient increase in neurotransmitter release in response to tetanic stimulation. Synapsin also redistributes to sites of activity-dependent axon terminal growth and regulates outgrowth via a PKA-dependent pathway. (3) Presynaptic CamKII has been shown to function in axon pathfinding in cultured Xenopus neurons. It seemed likely that activity-dependent ghost bouton formation and axon guidance might share similar molecular machinery (Nesler, 2016).

    It was postulated that presynaptic CamKII was required to control activity-dependent axon terminal growth at the larval NMJ. To address this question, CamKII expression was disrupted in motor neurons using two transgenic RNAi constructs. Depletion of presynaptic CamKII with both transgenes prevented the formation of new ghost boutons in response to spaced stimulation. Thus, presynaptic CamKII is necessary to control the formation of new synaptic boutons (Nesler, 2016).

    To further confirm that presynaptic CamKII function was required for activity-dependent growth, a transgenic line was used that inducibly expressed an inhibitory peptide (UAS-CamKIIAla). As in mammals, the activation of Drosophila CamKII by exposure to calcium leads to the autophosphorylation of a conserved threonine residue within the autoinhibitory domain (T287 in Drosophila). Activation of CamKII then confers an independence to calcium levels that persists until threonine-287 is dephosphorylated. The synthetic Ala peptide mimics the autoinhibitory domain and its transgenic expression is sufficient to substantially inhibit endogenous CamKII activity. Expression of the Ala inhibitory peptide in larval motor neurons disrupted the formation of new ghost boutons following spaced synaptic depolarization. These observations are consistent with results from CamKII RNAi (Nesler, 2016).

    Together, these data suggest that presynaptic CamKII function is required to control new ghost bouton formation in response to acute synaptic activity. Similarly, presynaptic CamKII has been implicated in controlling both bouton number and morphology during development of the larval NMJ. Reducing neuronal CamKII levels by RNAi has recently been shown to significantly reduce the number of type 1b boutons at the larval NMJ suggesting that presynaptic CamKII is required to control normal synapse development. In contrast, presynaptic expression of the Ala inhibitory peptide has no effect on the total number of type 1 synaptic boutons. Given that the Ala peptide does not completely inhibit CamKII autophosphorylation, it is suggested that the activation of CamKII in response to acute spaced synaptic depolarization is likely to be more sensitive to disruption then during NMJ development (Nesler, 2016).

    It was next asked if presynaptic CamKII could induce activity-dependent axon terminal growth at the NMJ. The overexpression of genes that are necessary for the control of ghost bouton formation generally does not cause an increase in the overall number of new synaptic boutons following 5 x high K+ spaced. Instead, overexpression often leads to an increased sensitization of the synapse to subsequent stimuli (for example, significant growth is observed after 3 x instead of 5 x high K+). The overexpression of a wild-type CamKII transgene in motor neurons caused an increase of 71% in ghost bouton numbers in 3 x K+ spaced stimulation larvae compared to 0 x K+ controls. While this is trending towards an increase, it did not reach statistical significance, even though expression levels were substantially higher than endogenous CamKII. Thus, increased CamKII is not sufficient to stimulate activity-dependent axon terminal growth (Nesler, 2016).

    To further investigate the role of presynaptic CamKII in activity-dependent axon terminal growth, the effect of transgenic neuronal overexpression of either an overactive form of CamKII (CamKIIT287D) or a form that is incapable of remaining active in the absence of elevated calcium (CamKIIT287A). Much like C380-Gal4/+ controls, presynaptic expression of either transgene had no significant effect on the number of ghost boutons in 3 x high K+ stimulation. Again, levels of CamKII protein in axon terminals in both transgenic lines were elevated relative to controls. Collectively, these data suggest that constitutive activation of CamKII is not sufficient to sensitize the NMJ to stimulation (Nesler, 2016).

    The results suggest that the temporal and/or spatial regulation of CamKII expression or activation is likely required to control activity-dependent growth. In support, the Drosophila CamKII protein has been shown to phosphorylate and regulate the activity of the Ether-a-go-go (Eag) potassium channel in motor neuron axon terminals. In turn, CamKII is bound and locally activated by phosphorylated Eag. This local activation can persist even after calcium levels have been reduced. CamKII autophosphorylation and Eag localization to synapses requires the activity of the membrane-associated Calcium/Calmodulin-associated Serine Kinase, CASK. The presynaptic coexpression of CASK with CamKIIT287D reverses (to wild-type levels) the increase in type 1b boutons observed when CamKIIT287D is overexpressed alone. Thus, a mechanism exists at the larval NMJ that allows for the persistence of local CamKII activation in the absence of additional stimul (Nesler, 2016).

    After establishing that CamKII has a novel presynaptic function in activity-dependent ghost bouton formation, the distribution of CamKII protein at the larval NMJ was examined closely. It has previously been reported that CamKII strongly colocalizes with postsynaptic Discs large (DLG), the Drosophila ortholog of mammalian PSD-95, around the borders of type 1 synaptic boutons. In support, an anti-CamKII antibody coimmunoprecipitates DLG from larval body wall extracts. Interestingly, while DLG is pre-dominantly postsynaptic at the developing NMJ it is also initially expressed in the presynaptic cell and at least partially overlaps with presynaptic membrane markers in axon terminals. It has been demonstrated that while fly CamKII colocalizes with DLG within dendrites of adult olfactory projection neurons (PNs), it also localizes to presynaptic boutons within those same neurons. Consistent with the latter observations (using a different CamKII antibody), it has been shown that CamKII is substantially enriched in presynaptic terminals of type 1b boutons. To resolve these inconsistent results, both antibodies against CamKII were used to more closely analyze the localization of CamKII at the third instar larval NMJ. First, double labeling of wild-type NMJs with a monoclonal CamKII antibody and anti-horseradish peroxidase (HRP), a marker for Drosophila neurons, confirmed that CamKII was enriched in presynaptic boutons in a pattern very similar to that of HRP. A closer examination of confocal optical sections revealed that almost all CamKII localized to the presynaptic terminal and was not significantly enriched either (1) at sites surrounding presynaptic boutons, or (2) in the axons innervating synaptic arbors (Nesler, 2016).

    Within boutons, CamKII appeared to be predominantly cytoplasmic but was sometimes localized to discrete puncta that were reminiscent of antibody staining for active zones. Prolonged depolarization of hippocampal neurons with K+ leads to mobilization of CamKII from the cytoplasm to sites near active zones. Moreover, using a fluorescent reporter for CamKII activity, high frequency stimulation causes the very rapid (on the order of minutes) activation of presynaptic CamKII and promotes its translocation from the cytoplasm to sites near active zones. To address this possibility, larval NMJs were double labeled with antibodies targeting both CamKII and DVGLUT, the Drosophila vesicular glutamate transporter, in order to visualize active zones. As predicted, it was found that some presynaptic CamKII colocalized with DVGLUT in type 1b and 1s boutons. Thus, in some type 1 synaptic boutons, CamKII protein is enriched in or near active zones (Nesler, 2016).

    To confirm that CamKII was enriched in presynaptic boutons, wild-type NMJs were double labelled with a polyclonal CamKII antibody and anti-DLG. CamKII did partially colocalize with DLG at the border of type 1 synaptic boutons. However, in this study, CamKII was primarily localized to the presynaptic side of the synapse. Collectively, this study provides strong evidence that CamKII is expressed on both the pre- and postsynaptic side of the synapse but that it is clearly enriched within presynaptic boutons at the larval NMJ. This localization is analogous to CamKII distribution in mammalian axons (Nesler, 2016).

    After demonstrating that total CamKII was enriched in presynaptic axon terminals, it was next asked if any of this protein was active by assessing phosphorylation of threonine-287 using a phospho-specific polyclonal antibody. It was found that pT287 CamKII staining intensity was strong and fairly uniform in presynaptic boutons and weakly stained axons innervating synaptic arbor. Closer examination of confocal optical sections revealed that almost all p-CamKII colocalized with HRP in the presynaptic terminal and only sparsely stained the body wall muscle (Fig. 3A′). Presynaptic CamKII RNAi almost completely disrupted p-CamKII in axon terminals leaving some residual staining in the presynaptic bouton and surrounding muscle suggesting that the antibody is specific. To further demonstrate this presynaptic localization, it was found that p-CamKII staining clearly does not overlap with postsynaptic DLG but does colocalize strongly with immunostaining using the monoclonal total CamKII antibody (Nesler, 2016).

    Collectively, three different antibodies were used to show that CamKII enriched in presynaptic axon terminals. Next, it was asked as to how this enrichment was occurring. In Drosophila and mammalian neurons, the CamKII mRNA is transported to dendritic compartments and locally translated in response to synaptic stimulation. This spatial and temporal regulation requires sequence motifs found within the 5' and 3' UTRs of the CamKII transcript. In contrast, the localization of CamKII to axon terminals of Drosophila PNs does not strictly require the CamKII 3'UTR suggesting that enrichment in presynaptic boutons occurs through a mechanism that does not strictly require local translation. In mammalian neurons, CamKII is enriched in axon terminals where it can associate with synaptic vesicles and synapsin I. Recently, it has been shown that mammalian CamKII and the synapsin proteins are both conveyed to distal axons at rates consistent with slow axonal transport, with a small fraction of synapsin cotransported with vesicles via fast transport (Nesler, 2016).

    Because activity-dependent growth at the larval NMJ requires the miRNA pathway and new protein synthesis, it was asked if the localization of CamKII protein to axon terminals might require the CamKII 3'UTR. As expected, when expression was specifically driven in larval motor neurons, a transgenic CamKII:EYFP fusion protein regulated by the CamKII 3'UTR localized strongly to presynaptic boutons at the larval NMJ. However, very similar results were observed using the same CamKII:EYFP fusion protein regulated by a heterologous 3'UTR. Taken together, these data suggest that localization of CamKII protein to presynaptic boutons at the NMJ does not require mRNA transport and local translation. Thus, it is concluded that most of the Drosophila CamKII protein found in motoneuron axon terminals is likely there due to the transport of cytosolic CamKII from the cell body to synapses via a mechanism involving axonal transport. (Nesler, 2016).

    It was of interest to determining how CamKII might be regulating activity-dependent axon terminal growth, and it was speculated that either the levels or distribution of CamKII protein might be altered in response to spaced depolarization. It first asked if high K+ stimulation resulted in an increase in CamKII protein within motoneuron axon terminals. Larval preparations were stimulated, and changes in the levels of CamKII protein in presynaptic boutons was examined by immunohistochemistry and quantitative confocal microscopy. Following spaced stimulation, CamKII staining within boutons rapidly increased (in ~ 1 h) by an average of 26%. This increase in immunofluorescence was global and did not appear to be localized to particular regions of the NMJ (i.e., near obvious presynaptic outgrowths). CamKII has been reported to very rapidly translocate to regions near active zones in response to high frequency stimulation. However, when compared to DVGLUT levels in unstimulated and stimulated larvae, no significant increase in CamKII immunofluorescence was observed indicating that translocation does not occur or does not persist in the current assay. To determine if this increase in CamKII enrichment required new protein synthesis, larval preparations were incubated with the translational inhibitor cyclohexamide during the recovery phase. Surprisingly, this treatment completely blocked the activity-dependent affects on presynaptic CamKII enrichment within axon terminals. Thus, spaced high K+ stimulation results in a rapid increase in CamKII levels in presynaptic boutons via some mechanism that requires activity-dependent protein synthesis (Nesler, 2016).

    Next, it was asked if the levels or distribution of p-CamKII changed in response to spaced stimulation. Larval preparations were stimulated exactly as described above and analyzed by confocal microscopy. Interestingly, p-CamKII staining was enriched at the presynaptic membrane of many axon terminals following spaced depolarization (Nesler, 2016).

    Given the requirement for new protein synthesis, it was speculated that the additional CamKII protein in axon terminals could be derived from a pool of CamKII mRNA that is rapidly transcribed and translated in the soma in response to spaced depolarization. This newly translated CamKII would then be actively transported out to axon terminals via standard mechanisms. If this were true, it would be expected that elevated CamKII levels could be detected in the larval ventral ganglion. To examine this process more closely, global total CamKII expression levels within the larval ventral ganglion were assayed by Western blot analysis. It was found that two distinct isoforms of CamKII are expressed in explanted larval ventral ganglia, Surprisingly, no increase was observed in CamKII protein levels in the ventral ganglion (Nesler, 2016).

    What is the source of this new presynaptic CamKII protein? Three possible explanations are proposed. First, new CamKII protein might be transcribed and translated in the motor neuron cell body. However, this new protein would be rapidly transported away to axon terminals in response to spaced depolarization. Second, some CamKII protein is found in the axons innervating the NMJ (seen using the p-CamKII antibody). It is possible that activity stimulates the rapid transport of an existing pool of CamKII protein from distal axons into axon terminals. This process would be sensitive to translational inhibitors. Finally, a pool of CamKII mRNA might be actively transported into axon terminals and then locally translated in response to spaced depolarization. This would account for the both the dependence on translation and for increased CamKII enrichment in presynaptic boutons (Nesler, 2016).

    Thus far, this study has shown that activity-dependent ghost bouton formation correlates with a protein synthesis-dependent increase in CamKII levels within presynaptic boutons at the larval NMJ. The activity-dependent translation of CamKII in olfactory neuron dendrites in the adult Drosophila brain requires components of the miRNA pathway. Within the CamKII 3'UTR, there are two putative binding sites for activity-regulated miR-289. These two binding sites were of particular interest. It was previously shown that levels of mature miR-289 are rapidly downregulated in the larval brain in response to 5 x high K+ spaced training (Nesler, 2013). Moreover, presynaptic overexpression of miR-289 significantly inhibits activity-dependent ghost bouton formation at the larval NMJ (Nesler, 2013). Based on these data, it was speculated that CamKII might be a target for regulation by miR-289 (Nesler, 2016).

    To determine if CamKII is a target for repression by miR-289 in vivo, a transgenic construct containing the primary miR-289 transcript was overexpressed in motor neurons and CamKII enrichment was examined by anti-CamKII immunostaining and quantitative confocal microscopy. Relative to controls, the presynaptic overexpression of miR-289 completely abolished the observed activity-dependent increase in CamKII immunofluorescence. When analyzing global CamKII levels within axon terminals during NMJ development, presynaptic miR-289 expression led to a slight decrease in CamKII immunofluorescence. This trend is similar to results observed following treatment with cyclohexamide during the recover period. The lack of full repression by miR-289 is not surprising given that one miRNA alone is often not sufficient to completely repress target gene expression (Nesler, 2016).

    To directly test the ability of miR-289 to repress translation of CamKII, a reporter was developed where the coding sequence for firefly luciferase (FLuc) was fused to the regulatory CamKII 3'UTR (FLuc-CamKII 3'UTR). When this wild-type reporter was coexpressed with miR-289 in Drosophila S2 cells, expression of FLuc was significantly reduced. In contrast, when this reporter was coexpressed with miR-279a, a miRNA not predicted to bind to the CamKII 3'UTR, no repression was observed. To confirm that repression of the FLuc-CamKII reporter by miR-289 was via a specific interaction, the second of two predicted miR-289 binding sites was mutagenized. Binding site 2 (BS2) was a stronger candidate for regulation because it is flanked by AU-rich elements (AREs) and miR-289 has been shown to promote ARE-mediated mRNA instability through these sequences. Moreover, it is well established that the stabilization and destabilization of neuronal mRNAs via interactions between AREs and ARE-binding factors plays a significant role in the establishment and maintenance of long-term synaptic plasticity in both vertebrates and invertebrates. Altering three nucleotides within BS2 in the required seed region binding site was sufficient to significantly disrupt repression of the reporter by miR-289. The minimal predicted BS2 sequence was cloned into an unrelated 3'UTR and it was asked if miR-289 could repress translation. Coexpression of the FLuc-SV-mBS2 reporter with miR-289 led to significant repression. Taken together, these results indicate that the BS2 sequence is both necessary and sufficient for miR-289 regulation via the CamKII 3′UTR (Nesler, 2016).

    The most important conclusion of this study is that presynaptic CamKII is required to control activity-dependent axon terminal growth at the Drosophila larval NMJ. First, it was shown that CamKII is necessary to control ghost bouton formation in response to spaced synaptic depolarization. Next, it was demonstrated that spaced stimulation correlates with a rapid protein synthesis dependent increase in CamKII immunofluorescence in presynaptic boutons. This increase is suppressed by presynaptic overexpression of activity-regulated miR-289. Previous work has shown that overexpression of miR-289 in larval motor neurons can suppress activity-dependent axon terminal growth (Nesler, 2013). This study demonstrated that miR-289 can repress the translation of a FLuc-CamKII 3'UTR reporter via a specific interaction with a binding site within the CamKII 3'UTR ( Fig. 6C-E). Collectively, this experimental evidence suggests that CamKII functions downstream of the miRNA pathway to control activity-dependent changes in synapse structure. (Nesler, 2016).

    Thus, CamKII protein is expressed in the right place to regulate rapid events that are occurring within presynaptic boutons. Several questions remain regarding CamKII function in the control of activity-dependent axon terminal growth. First, it is unclear what the significance might be of a rapid increase of total CamKII in presynaptic terminals. Why is the pool of CamKII protein that is already present not sufficient to control these processes? Similar questions have been asked regarding activity-dependent processes occurring within dendrites. It is postulated that the CamKII mRNA might be locally translated in axon terminals. It has been proposed that local mRNA translation might be (1) required for efficient targeting of some synaptic proteins to specific sites, or (2) local translation may in and of itself be required to control activity-dependent processes at the synapse. Second, the impact of spaced depolarization on CamKII function needs to be assessed and downstream targets of CamKII phosphorylation involved in these processes need to be identified. One very strong candidate is synapsin which, at the Drosophila NMJ, has been shown to rapidly redistribute to sites of new ghost bouton outgrowth in response to spaced stimulation. Finally, the idea that CamKII might work through a Eag/CASK-dependent mechanism to control activity-dependent axon terminal growth needs to be examined (Nesler, 2016).

    Drosophila miR-956 suppression modulates Ectoderm-expressed 4 and inhibits viral replication

    Small non-coding microRNAs (miRNAs) can modulate the outcome of virus infection. This study explored the role of miRNAs in insect-virus interactions, in vivo, using the natural Drosophila melanogaster-Drosophila C virus (DCV) model system. Comparison of the miRNA expression profiles in DCV-infected and uninfected flies showed altered miRNA levels due to DCV infection, with the largest change in abundance observed for miR-956-3p. Knockout of miR-956 resulted to delayed DCV-induced mortality and decreased viral accumulation compared to wild-type flies. A screen of 84 putative miR-956-3p target genes identified regulation of Ectoderm-expressed 4 (Ect4), a negative regulator of MYD88- and TRIF-dependent toll-like receptor signaling pathway, in miR-956 knockout flies and, separately, DCV infection. In Ect4 knockdown flies DCV-induced mortality occurred more quickly and virus accumulation was increased. Taken together, results suggest that the host-protective and antiviral consequences of miR-956 suppression during in vivo infection of D. melanogaster with its natural pathogen DCV is conferred through miR-956-3p induction of its target Ect4 (Monsanto-Hearne, 2016).

    The C. elegans CSR-1 argonaute pathway counteracts epigenetic silencing to promote germline gene expression

    Organisms can develop adaptive sequence-specific immunity by reexpressing pathogen-specific small RNAs that guide gene silencing. For example, the C. elegans PIWI-Argonaute/piwi-interacting RNA (piRNA) pathway recruits RNA-dependent RNA polymerase (RdRP) to foreign sequences to amplify a transgenerational small-RNA-induced epigenetic silencing signal (termed RNAe). This study provides evidence that, in addition to an adaptive memory of silenced sequences, C. elegans can also develop an opposing adaptive memory of expressed/self-mRNAs. This mechanism, which can prevent or reverse RNAe, is referred to as RNA-induced epigenetic gene activation (RNAa). CSR-1 (an Argonaute homolog), which engages RdRP-amplified small RNAs complementary to germline-expressed mRNAs, is required for RNAa. A transgene with RNAa activity also exhibits accumulation of cognate CSR-1 small RNAs. These findings suggest that C. elegans adaptively acquires and maintains a transgenerational CSR-1 memory that recognizes and protects self-mRNAs, allowing piRNAs to recognize foreign sequences innately, without the need for prior exposure (Seth, 2013).

    The Tudor domain protein Tapas, a homolog of the vertebrate Tdrd7, functions in piRNA pathway to regulate retrotransposons in germline of Drosophila melanogaster

    Piwi-associated RNAs (piRNAs) are a special class of small RNAs that provide defense against transposable elements (TEs) in animal germline cells. In Drosophila, germline piRNAs are thought to be processed at a unique perinuclear structure, nuage, which houses piRNA pathway proteins including the Piwi clade of Argonaute family proteins, along with several Tudor domain proteins, RNA helicases and nucleases. Tudor domain protein Tejas (Tej), an ortholog of vertebrate Tdrd5, is an important component of the piRNA pathway. The current study identified the paralog of Drosophila tej gene, tapas (tap), which is an ortholog of vertebrate Tdrd7. Like Tej, Tap is localized at the perinuclear structure in germline cells called nuage. The tap loss alone leads to a mild increase in transposon expression and decrease in piRNAs targeting transposons expressed in the germline. tap genetically interacts with other piRNA pathway genes, and Tap physically interacts with piRNA pathway components, such as Piwi family proteins Aubergine (Aub) and Argonaute3 (Ago3) and the RNA helicases Vasa (Vas) and Spindle-E (SpnE). tap together with tej is required for survival of germline cells during early stages and for polarity formation. It was further observed that loss of tej and tap together results in more severe defects in piRNA pathway in germline cells compared to single mutants: the double mutant ovaries exhibit mislocalization of piRNA pathway components and significantly greater reduction of piRNAs against transposons predominantly expressed in germline compared to single mutants. The single or double mutants did not have any reduction in piRNAs mapping to transposons predominantly expressed in gonadal somatic cells and those derived from unidirectional clusters such as flamenco. Consistently, the loss of both tej and tap function results in mislocalization of Piwi in germline cells, while Piwi remains localized to the nucleus in somatic cells. These data suggest that Tej and Tap work together for germline maintenance and piRNA production in germline cells. These observations suggest that tej and tap work together for the germline maintenance. tej and tap also function in a synergistic manner to maintain examined piRNA components at the perinuclear nuage and for piRNA production in Drosophila germline (Patil, 2014).

    The Drosophila fragile X mental retardation protein participates in the piRNA pathway

    RNA metabolism controls multiple biological processes, and a specific class of small RNAs, called piRNAs, act as genome guardians by silencing the expression of transposons and repetitive sequences in the gonads. Defects in the piRNA pathway affect genome integrity and fertility. The possible implications in physiopathological mechanisms of human diseases have made the piRNA pathway the object of intense investigation, and recent work suggests that there is a role for this pathway in somatic processes including synaptic plasticity. The RNA-binding fragile X mental retardation protein (FMRP, also known as FMR1) controls translation and its loss triggers the most frequent syndromic form of mental retardation as well as gonadal defects in humans. This study demonstrates for the first time that germline, as well as somatic expression, of Drosophila Fmr1 (denoted dFmr1), the Drosophila ortholog of FMRP, are necessary in a pathway mediated by piRNAs. Moreover, dFmr1 interacts genetically and biochemically with Aubergine, an Argonaute protein and a key player in this pathway. These data provide novel perspectives for understanding the phenotypes observed in Fragile X patients and support the view that piRNAs might be at work in the nervous system (Bozzetti, 2015).

    dFmr1 is a translational regulator and its role in the miRNA pathway is widely accepted. This study provides several lines of evidence that dFmr1 can be considered as a ‘bona fide’ member of the piRNA pathway that keeps repetitive sequences and transposons silenced. First, dFmr1 mutant testes display crystalline aggregates, as do other mutants of the piRNA pathway. Second, the levels of cry (Suppressor of Stellate)-specific and transposon-specific piRNAs dramatically decrease in dFmr1 mutant testes. Third, as a consequence of this decrease, the Ste RNA is produced and, in addition, transposons are expressed at higher levels than in wt animals. Fourth, dFmr1 mutant animals display fertility defects, a phenotype shown by several mutations affecting the piRNA pathway. The fact that earlier screens did not identify dFmr1 as a member of the somatic piRNA pathway could be due to the heterogeneous phenotypes observed with the somatic transposons (this study) and/or to the material used for those assays. The crySte system thus proves very efficient for identifying new members of this important pathway (Bozzetti, 2015).

    The movement of transposable elements is one of the molecular causes of DNA instability and sterility. Considering that human patients mutant for FMRP also display defects in male and female gonads, it will be interesting to characterize the activity of transposons and repetitive sequences in the gonads of mice or humans that are mutant for the FMRP pathway, although there might be no observable defects in mammals because they express three members of the FMRP family versus the single ortholog in fly. Finally, mutations affecting the piRNA pathway might also induce gonadal defects in humans (Bozzetti, 2015).

    Until now, the members of the piRNA pathway controlling the crySte interaction, including Aub, have been described as being required in the male germline. Surprisingly, the conditional dFmr1 rescue and KD experiments demonstrate that dFmr1 controls the piRNA pathway both in the germline and in the somatic cells of the gonad, which raises questions as to the somatic contribution of other members of the piRNA pathway in the male gonad. The phenotypes induced by somatic Aub expression also suggest that the hub expresses one or more AGO proteins that are involved in the somatic piRNA-mediated Ste silencing and that interact with dFmr1; however, the only other protein of the Piwi clade present in the somatic tissue, Piwi, does not participate in Ste silencing. Based on preliminary data, this study proposes that AGO1 might be one such protein. First, AGO1/+ testes display Ste-made crystals, as do testes expressing UAS-AGO1 RNAi driven by the upd-Gal4 driver. Second, aubsting rescues the AGO1-mediated crystal phenotype. Third, AGO1 and dFmr1 interact biochemically and are known to interact genetically in the ovaries to control germline stem cell maintenance, as well as in the nervous system, where they modulate synaptic plasticity. Taken together, these data suggest that AGO1 contributes to the piRNA pathway that controls the cry–Ste system in the somatic part of the gonad (Bozzetti, 2015).

    The finding that Aub somatic expression affects the NMJ and counteracts the AGO1 loss of function phenotype is also unexpected. Recent work has documented the activation of piRNA pathway in the nervous system in flies, mice, humans and molluscs and it has been proposed that synaptic plasticity, cognitive functions and neurodegeneration might involve the control of genome stability, even though the precise mode of action and impact of this pathway are not completely understood. Because Aub is not required in the larval somatic tissues, its ectopic expression could affect the NMJ by replacing AGO1 in its known role on the miRNA pathway. However, AGO1 might also affect the NMJ through the piRNA pathway, much in the same way as AGO1 loss of function affects a piRNA pathway in the gonad. Even though AGO1 has been previously described as being exclusively involved in the miRNA pathway, some degree of overlapping between different RNAi pathways has been recently described: (1) the double-stranded-RNA-binding protein Loquacious (Loqs) is involved in the miRNA pathway and in the endogenous siRNA pathway, (2) AGO1 and AGO2 can compete for binding with miRNAs, and (3) ectopic expression of Aub in the soma competes for the siRNAs pathway mediated by AGO2. In addition, miRNAs have been demonstrated to have a role on easi-RNA biogenesis in plants. In a similar manner, AGO1 could act on piRNAs through its activity on the miRNA pathway. Although future studies will clarify the connection between AGO1 and the piRNA pathway, the present data provide novel perspectives in the field and could have a broad relevance to diseases affecting cognitive functions (Bozzetti, 2015).

    Expression, genetic and biochemical data indicate that Aub and dFmr1 interact directly. dFmr1 has been proposed to bind specific cargo RNAs and the human FMRP binds small RNA, in addition to mRNAs. Similarly, the Aub–dFmr1 interaction might allow the targeting of piRNAs to the transcripts of repetitive sequences and transposable elements, dFmr1 providing the molecular link between small RNAs and AGO proteins of the RISC (Bozzetti, 2015).

    The Aub and dFmr1 proteins colocalize and likely interact in the piRNA pathway in a specific stage of testis development and also have additional functions that are independent from each other. Typically, dFmr1 accumulates at high levels in more differentiated cells of the testis, where Aub is not detectable, likely accounting for the axoneme phenotype described in dFmr1 testes. In the future, it will be interesting to analyze whether the other genes involved in the piRNA pathway in testis are also required at specific stages, as also recently found in the ovary (Bozzetti, 2015).

    Finally, FMRP proteins work in numerous molecular networks, show complex structural features (TUDOR, KH, NLS, NES RGG domains) and are characterized by widespread expression and subcellular localization (cytoplasm, nucleus, axons, dendrites, P bodies), providing versatile platforms that control mRNA and small RNA metabolism (e.g. translation, degradation and transport). Understanding whether FMRP proteins interact with other members of the piRNA pathway, whether this interaction is modulated physiologically and how does the interaction with this pathway compare with that observed with other AGO proteins will clarify the role and mode of action this family of proteins in small RNA biogenesis and metabolism (Bozzetti, 2015).

    The biogenesis of the piRNAs requires two pathways. The primary pathway involves Piwi and predominantly occurs in the somatic tissues. The ping-pong pathway involves Aub, as well as AGO3, and predominantly occurs in the germline, where Aub is thought to bind an antisense piRNA, to cleave the sense transcript from an active transposon and to produce a sense piRNA that is loaded onto AGO3. The AGO3–piRNA complex binds complementary transcripts from the piRNA cluster, producing the so-called secondary piRNAs by an amplification loop. Although the piRNA pathways have emerged as a very important tool to understand the role of RNA metabolism in physiological and pathological conditions, the relationship and interactions among the involved proteins are not simple to interpret, mostly because not all the players have been characterized. Moreover, recent data support the hypothesis that the somatic and the germline piRNA pathways share components: for example, shutdown (shu), vreteno (vret) and armitage (arm) affect primary as well as ping-pong pathways in ovaries. Results from this study call for a role of dFmr1 in both piRNA pathways at least in testes. Based on the alignment of the human, mouse and fly FMRP family members, dFmr1 might participate in piRNA biogenesis as a Tudor domain (TDRD) containing protein (Bozzetti, 2015).

    TDRDs are regions of about 60 amino acids that were first identified in a Drosophila protein called Tudor. In the recent years, the requirement of TDRD proteins in piRNA biogenesis and metabolism has become evident. Typically, the founding member of the family, Tudor, binds AGO proteins and helps them interact with specific piRNAs. Among the different TDRD proteins, fs(1)Yb works in the primary pathway; Krimper, Tejas, Qin/Kumo, and PAPI work in the ping-pong pathway; and Vret works in both systems. PAPI, the only TDRD protein that has a modular structure closely related to dFmr1 (two KH domains and one TDRD), interacts with the di-methylated arginine residues of AGO3 and controls the ping-pong cycle in the nuage. At least during the early stages of testis development, dFmr1 might interact with Aub in a similar way. Given that TDRDs are involved in the interactions between proteins and in the formation of ribonucleoprotein complexes, future studies will assess whether RNAs mediate the Aub–dFmr1 interaction (Bozzetti, 2015).

    In conclusion, the discovery of dFmr1 as a player in the piRNA pathway highlights the importance of the fly model. Data from this study also adds a new perspective to understanding the role and mode of action of this protein family and the physiopathological mechanisms underlying the Fragile X syndrome (Bozzetti, 2015).

    Roles for the Yb body components Armitage and Yb in primary piRNA biogenesis in Drosophila

    PIWI-interacting RNAs (piRNAs) protect genome integrity from transposons. In Drosophila ovarian somas, primary piRNAs are produced and loaded onto Piwi. This study describes roles for the cytoplasmic Yb body components Armitage and Yb in somatic primary piRNA biogenesis. Armitage binds to Piwi and is required for localizing Piwi into Yb bodies. Without Armitage or Yb, Piwi is freed from the piRNAs and does not enter the nucleus. Thus, piRNA loading is required for Piwi nuclear entry. It is proposed that a functional Piwi-piRNA complex is formed and inspected in Yb bodies before its nuclear entry to exert transposon silencing (Saito, 2010).

    In Drosophila, three sets of endogenous small RNAs have been identified so far: microRNAs (miRNAs), endogenous siRNAs (endo-siRNAs/esiRNAs), and PIWI-interacting RNAs (piRNAs). Of these, piRNAs are considered unique because of their germline-specific expression and specific interaction with germline-specific Argonaute proteins, PIWI proteins. The identification of the piRNAs associated with three PIWI proteins (Aubergine [Aub], Argonaute 3 [AGO3], and Piwi) has revealed distinct features of piRNAs associated with each PIWI and has led to two models for piRNA biogenesis: the primary processing pathway and the amplification loop pathway. In the amplification loop model, the Slicer (endonuclease) activity of Aub and AGO3 determines the formation of the 5' end of piRNAs. Zucchini (Zuc), a putative cytoplasmic nuclease, is involved in the primary processing pathway; however, its precise molecular function remains unclear. Furthermore, the factors other than zuc required for primary piRNA biogenesis are unknown (Saito, 2010).

    The ovarian somatic cell (OSC) line consists of ovarian somas only. The expression of Aub and AGO3 is not detectable in OSCs because both proteins are germ cell-specific. This implies that the amplification loop does not operate in OSCs. However, OSCs express piRNAs and are loaded onto Piwi, indicating that the piRNAs in OSCs are generated specifically through the primary processing pathway. Thus, OSCs are an ideal tool to elucidate the molecular mechanisms of primary piRNA processing and Piwi function. Loss of zuc function drastically reduced the level of primary piRNAs in the ovaries. This was recapitulated in OSCs: Zuc depletion by RNAi caused a severe reduction in the piRNA level in OSCs. This result prompted a screen for other factors necessary for primary piRNA production using RNAi in OSCs (Saito, 2010).

    To identify the genes required for somatic primary piRNA biogenesis, RNAi-based screening was performed in OSCs. The genes screened included armitage (armi), spindle-E (spn-E), and maelstrome (mael), all of which are implicated in piRNA biogenesis. However, their roles in somatic primary piRNA production remain unknown. Depletion of Armi reduced the piRNA levels to an extent very similar to that of Piwi and Zuc depletion, indicating that Armi is necessary for primary piRNA biogenesis in OSCs. Depletion of Mael and Spn-E showed little or no effect on piRNA accumulation in OSCs. Mutations in both genes have been shown to significantly reduce the piRNA levels in ovaries. Thus, spn-E and mael are factors functioning in the amplification loop. Depletion of Dicer1 and Dicer2 had little or no effect on the piRNA levels, confirming that neither protein is necessary for piRNA production (Saito, 2010).

    Armi is the Drosophila ortholog of Arabidopsis Silencing-Defective 3 (SDE3) and mammalian Moloney leukemia virus 10 (MOV10). These orthologs contain a conserved ATP-dependent RNA helicase domain at their C termini and have been implicated in small RNA-mediated gene silencing. However, their precise functions remain unknown. To gain further insight into the function of Armi in somatic primary piRNA processing, a monoclonal antibody was produced against Armi. Western blotting showed a discrete band in both ovary and cultured Schneider2 (S2) cell lysates, indicating that Armi expression is not germline-specific. The ~150-kDa protein immunopurified from S2 cells with the anti-Armi antibody was confirmed to be Armi by mass spectrometry (Saito, 2010).

    Immunostaining of OSCs and ovaries with the anti-Armi antibody confirmed an earlier observation that Armi is a cytoplasmic protein. The Armi signals were detected in both somatic and germ cells of ovaries. The somatic signal was considered a background signal because it did not disappear even in armi homozygous mutant egg chambers. In the present study, the cytoplasmic signal in OSCs mostly disappeared when Armi was depleted by RNAi. Thus, it is concluded that Armi is expressed in both somatic and germ cells in ovaries (Saito, 2010).

    The subcellular localization of Armi in the armi trans-heterozygous mutants appeared very similar to that in the homozygous mutants. In addition, Western blotting revealed a band corresponding to Armi in the armi ovaries. By what mechanisms Armi is expressed in the mutant somas remains unclear. The simplest explanation is that the armi gene uses two distinct genomic elements as promoters in ovarian somas. In fact, armi homozygous mutants weakly express a shorter armi transcript than that expressed in the wild-type strain (Saito, 2010).

    The Armi signal in germ cells was rather weak, and only a small proportion of Armi accumulated at, or near, the nuage, an electron-dense structure associated with nurse cell nuclei. Thus, Armi might not be a component of the nuage per se. This correlates well with the fact that armi mutations barely affected the ability of the ovaries to amplify endogenous piRNAs. In ovarian somas, Armi accumulated strongly at discrete cytoplasmic foci. Each somatic cell contained one or several foci. Interestingly, the Armi-positive foci were often located near the nucleus in both ovaries and OSCs (Saito, 2010)

    Piwi is required for the silencing of transposons in gonads. In fact, Piwi depletion in OSCs caused derepression of transposons, as with Armi, Yb, and Zuc depletion. Under conditions where endogenous Piwi was depleted, expression of myc-Piwi-r, which was designed to be RNAi-insensitive, rescued transposon silencing. However, myc-Piwi-δN, which lacks 72 amino acids at the N terminus of Piwi and thus does not localize to the nucleus, did not rescue transposon silencing, although it does associate with piRNAs to the same extent as does the wild-type Piwi. myc-Piwi-δN13, which lacks 13 amino acids at the N terminus, behaved similarly. On the other hand, myc-Piwi-DDAA-r, a Slicer mutant of Piwi, could bind to mature piRNAs in OSCs, as does the wild-type Piwi, and rescued transposon silencing. These results might suggest that Piwi must be localized in the nucleus to silence the transposable elements, and that Piwi Slicer activity is unnecessary for its function. It is assumed that this system has evolved to prevent nascent Piwi, not loaded with piRNAs, from being imported into the nucleus. In other words, only the functional Piwi-piRNA complex (piRISC) formed at Yb bodies could be transported to the nucleus. At present, the mechanisms of this control system remain unclear. In the nongonadal somatic S2 cell line, where the expression of piRNAs is undetectable, transfected Piwi is localized to the nucleus, indicating that 'empty' Piwi can be transported to the nucleus. It seems that the machineries necessary for the nuclear transport of Piwi might recognize different features of Piwi in different cell types (Saito, 2010).

    How is piRNA-free Piwi restrained in the cytoplasm in OSCs? One possibility is that some unknown protein binds the N-terminal end of Piwi, where its NLS (nuclear localization signal) resides, and interferes with the nuclear import machinery's ability to recognize Piwi as a cargo. The nuclear localization inhibitory factors may be retained on Piwi until a functional Piwi-piRNA complex is formed at Yb bodies. Once the complex is formed, a conformational change in Piwi would be induced, which would release the regulatory factors and reveal the Piwi NLS for recognition by the nuclear import machinery. It would be very interesting to determine the proteins that are associated with Piwi in OSCs under conditions of Armi or Zuc depletion, thus identifying the protein factors that restrain Piwi in the cytoplasm until it is loaded with mature piRNAs at Yb bodies (Saito, 2010).

    The Yb body, a major site for Piwi-associated RNA biogenesis and a gateway for Piwi expression and transport to the nucleus in somatic cells

    Despite exciting progress in understanding the Piwi-interacting RNA (piRNA) pathway in the germ line, less is known about this pathway in somatic cells. Previous work has shown that Piwi, a key component of the piRNA pathway in Drosophila, is regulated in somatic cells by Yb, a novel protein containing an RNA helicase-like motif and a Tudor-like domain. Yb is specifically expressed in gonadal somatic cells and regulates piwi in somatic niche cells to control germ line and somatic stem cell self-renewal. However, the molecular basis of the regulation remains elusive. This study reports that Yb recruits Armitage (Armi), a putative RNA helicase involved in the piRNA pathway, to the Yb body, a cytoplasmic sphere to which Yb is exclusively localized. Moreover, co-immunoprecipitation experiments show that Yb forms a complex with Armi. In Yb mutants, Armi is dispersed throughout the cytoplasm, and Piwi fails to enter the nucleus and is rarely detectable in the cytoplasm. Furthermore, somatic piRNAs are drastically diminished, and soma-expressing transposons are desilenced. These observations indicate a crucial role of Yb and the Yb body in piRNA biogenesis, possibly by regulating the activity of Armi that controls the entry of Piwi into the nucleus for its function. Finally, this study has discovered putative endo-siRNAs in the flamenco locus and the Yb dependence of their expression. These observations further implicate a role for Yb in transposon silencing via both the piRNA and endo-siRNA pathways (Qi, 2011).

    This study reports that Yb is a novel component of the somatic piRNA pathway. Because Yb is localized only in the Yb body in somatic cells, the results further implicate the Yb body as a key site in the cytoplasm for piRNA biogenesis in ovarian somatic cells. How is Yb involved in the piRNA pathway? Previous studys have shown that Yb genetically acts upstream of Piwi to regulate its expression in somatic cells, yet physically, Armi is the only known piRNA pathway component that colocalizes and physically interacts with Yb. While this manuscript was in preparation and under consideration, Olivieri (2010) and Saito (2010) also reported the Armi-Yb interaction and their role in the piRNA pathway (Qi, 2011).

    In addition, Haase (2010) reported the role of Armi in Piwi and piRNA expression. These studies verify the current observations. It is likely that Yb regulates Piwi via Armi, a RNA helicase. It is possible that Yb first recruits Armi to the Yb body, where the Armi-Yb complex, possibly involving other factors, might then serve as a site for the biogenesis and/or loading piRNAs and/or other factors to Piwi. The resulting Piwi-piRNA complex then enters the nucleus to achieve epigenetic regulatory functions. Such epigenetic regulation then leads to transposon silencing in somatic cells and niche cell function. Without Yb, Armi might fail to facilitate the biogenesis and/or loading of piRNA to Piwi. The unloaded Piwi might then fail to enter the nucleus and is subject to degradation. Of course, other possibilities exist, such as Armi-Yb interaction leading to the regulation of transcription or translation of Piwi. In any case, such regulation is specific to Piwi because Aub and Ago3 are not expressed in somatic cells in the ovary, and Yb mutations do not affect the expression or localization of Aub and Ago3 (Qi, 2011).

    Although Yb has long been regarded as a protein required in somatic niche cells to regulate germ line and follicle stem cell division, its underlying molecular mechanism remains elusive. The current study reveals a molecular mechanism through which Yb functions in the niche cells: it regulates Piwi activity, possibly via Armi. The activated Piwi then enters the nucleus to epigenetically regulate the niche cell genome, which ensures the genome integrity and defines the niche signaling function toward stem cells (Qi, 2011).

    In addition to its involvement in the piRNA pathway, Yb appears to be also involved in the endo-siRNA pathway. The gypsy6 endo-siRNA, which is decreased by 8-fold in the Yb mutant, is from the flamenco cluster. The flamenco cluster generates both endo-siRNAs and piRNAs. It is possible that the precursor transcripts of the piRNA pathway also serve as precursors of the endo-siRNA pathway, either in their original forms or as processed intermediates. Alternatively, piRNAs can target the single-stranded precursor of endo-siRNAs to form mature double-stranded precursors to produce endo-siRNAs. In either case, the dual role of Yb is reminiscent of the possible involvement of Piwi in the miRNA pathway. These observations together point to an 'inconvenient' fact, i.e. the specificity of Ago and Piwi proteins with respect to the siRNA/miRNA pathway versus piRNA pathway is only in a relative sense, just like the specificity of Drosophila Ago1 and Ago2 for the siRNA versus miRNA pathway is also a relative sense. In fact, the data suggest that Yb is required for transposon silencing, likely via both the piRNA and endo-siRNA pathways. Further investigation of the Yb-mediated mechanism should reveal a new dimension of the biogenesis and regulatory function of the piRNA pathway (Qi, 2011).

    Export of piRNA precursors by EJC triggers assembly of cytoplasmic Yb-body in Drosophila

    PIWI-interacting RNAs (piRNAs) are effectors of transposable element (TE) silencing in the reproductive apparatus. In Drosophila ovarian somatic cells, piRNAs arise from longer single-stranded RNA precursors that are processed in the cytoplasm presumably within the Yb-bodies. piRNA precursors encoded by the flamenco (flam) piRNA cluster accumulate in a single focus away from their sites of transcription. This study identifies the exportin complex containing Nxf1 and Nxt1 as being required for flam precursor nuclear export. Together with components of the exon junction complex (EJC), it is necessary for the efficient transfer of flam precursors away from their site of transcription. Indeed, depletion of these components greatly affects flam intra-nuclear transit. Moreover, Yb-body assembly is dependent on the nucleo-cytoplasmic export of flam transcripts. These results suggest that somatic piRNA precursors are required for the assembly of the cytoplasmic transposon silencing machinery (Dennis, 2016).

    Production of small non-coding RNAs from the flamenco locus is regulated by the gypsy retrotransposon of Drosophila melanogaster

    Protective mechanisms based on RNA silencing directed against the propagation of transposable elements are highly conserved in eukaryotes. The control of transposable elements is mediated by small non-coding RNAs, which derive from transposon-rich heterochromatic regions that function as small RNA-generating loci. These clusters are transcribed and the precursor transcripts are processed to generate piRNAs and endo-siRNAs, which silence transposable elements in gonads and somatic tissues. The flamenco locus is a Drosophila melanogaster small RNA cluster that controls gypsy and other transposable elements, which has played an important role in understanding how small non-coding RNAs repress transposable elements. This study describe a cosuppression mechanism triggered by new euchromatic gypsy insertions in genetic backgrounds carrying flamenco alleles defective in gypsy suppression. The silencing of gypsy was found to be accompanied by the silencing of other transposons regulated by flamenco, and of specific flamenco sequences from which small RNAs against gypsy originate. This cosuppression mechanism seems to depend on a post-transcriptional regulation that involves both endo-siRNA and piRNA pathways and is associated with the occurrence of developmental defects. In conclusion, it is proposed that new gypsy euchromatic insertions trigger a post-transcriptional silencing of gypsy sense and antisense sequences, which modifies the flamenco activity. This cosuppression mechanism interferes with some developmental processes presumably by influencing the expression of specific genes (Guida, 2016).

    Abundant expression of somatic transposon-derived piRNAs throughout Tribolium castaneum embryogenesis

    Piwi-interacting RNAs (piRNAs) are a class of short (~26-31-nucleotide) non-protein-coding RNAs expressed in the metazoan germline. The piRNA pathway in arthropods is best understood in the ovary of Drosophila melanogaster, where it acts to silence active transposable elements (TEs). Maternal loading of piRNAs in oocytes is further required for the inheritance of piRNA-mediated transposon defence. However, understanding of the diversity, evolution and function of the piRNA complement beyond drosophilids is limited. The red flour beetle, Tribolium castaneum, is an emerging model organism separated from Drosophila by ~ 350 million years of evolution that displays a number of features ancestral to arthropods, including short germ embryogenesis. This study characterized the maternally deposited and zygotically expressed small RNA and mRNA complements throughout T. castaneum embryogenesis. Beetle oocytes and embryos of all stages were found to be abundant in heterogeneous ~ 28-nucleotide RNAs. These small RNAs originate from discrete genomic loci enriched in TE sequences and display the molecular signatures of transposon-derived piRNAs. In addition to the maternally loaded primary piRNAs, Tribolium embryos produce secondary piRNAs by the cleavage of zygotically activated TE transcripts via the ping-pong mechanism. The two Tribolium piRNA pathway effector proteins, Tc-Piwi/Aub and Tc-Ago3, are also expressed throughout the soma of early embryos. These results show that the piRNA pathway in Tribolium is not restricted to the germline, but also operates in the embryo and may act to antagonize zygotically activated transposons. Taken together, these data highlight a functional divergence of the piRNA pathway between insects (Ninova, 2017).

    PIWI slicing and RNA elements in precursors instruct directional primary piRNA biogenesis

    PIWI proteins and PIWI-interacting RNAs (piRNAs) mediate repression of transposons in the animal gonads. Primary processing converts long single-stranded RNAs into approximately 30-nt piRNAs, but their entry into the biogenesis pathway is unknown. This study demonstrates that an RNA element at the 5' end of a piRNA cluster-which has been termed piRNA trigger sequence (PTS)-can induce primary processing of any downstream sequence. It is proposes that such signals are triggers for the generation of the original pool of piRNAs. It was also demonstrated that endonucleolytic cleavage of a transcript by a cytosolic PIWI results in its entry into primary processing, which triggers the generation of non-overlapping, contiguous primary piRNAs in the 3' direction from the target transcript. These piRNAs are loaded into a nuclear PIWI, thereby linking cytoplasmic post-transcriptional silencing to nuclear transcriptional repression (Homolka, 2016).

    Redundant and incoherent regulations of multiple phenotypes suggest microRNAs' role in stability control

    A "simple regulation" model for how microRNAs (miRNAs) function posits "one target-one phenotype" control under which most targeting is nonfunctional. In an alternative "coordinate regulation" model, multiple targets are assumed to control the same phenotypes coherently, and most targeting is functional. Both models have some empirical support but pose different conceptual challenges. This study concurrently analyzes multiple targets and phenotypes associated with the miRNA-310 family (miR310s) of Drosophila. Phenotypic rescue in the mir310s knockout background is achieved by promoter-directed RNA interference that restores wild-type expression. For one phenotype (eggshell morphology), redundant regulation was observed, hence rejecting "simple regulation" in favor of the "coordinate regulation" model. For other phenotypes (egg-hatching and male fertility), however, one gene shows full rescue, but three other rescues aggravate the phenotype. Overall, phenotypic controls by miR310s do not support either model. Like a thermostat that controls both heating and cooling elements to regulate temperature, redundancy and incoherence in regulation generally suggest some capacity in stability control. These results therefore support the published view that miRNAs play a role in the canalization of transcriptome and, hence, phenotypes (Liufu, 2017).

    Distribution, evolution, and diversity of retrotransposons at the flamenco locus reflect the regulatory properties of piRNA cluster

    Most understanding of Drosophila heterochromatin structure and evolution has come from the annotation of heterochromatin from the isogenic y; cn bw sp strain. However, almost nothing is known about the heterochromatin's structural dynamics and evolution. This study has focused on a 180-kb heterochromatic locus producing Piwi-interacting RNAs (piRNA cluster), the flamenco (flam) locus, known to be responsible for the control of at least three transposable elements (TEs). Its detailed structure is reported in three different Drosophila lines chosen according to their capacity to repress or not to repress the expression of two retrotransposons named ZAM and Idefix, and they were shown to display high structural diversity. Numerous rearrangements due to homologous and nonhomologous recombination, deletions and segmental duplications, and loss and gain of TEs are diverse sources of active genomic variation at this locus. Notably, a correlation is evidenced between the presence of ZAM and Idefix in this piRNA cluster and their silencing. They are absent from flam in the strain where they are derepressed. It was shown that, unexpectedly, more than half of the flam locus results from recent TE insertions and that most of the elements concerned are prone to horizontal transfer between species of the melanogaster subgroup. A model is built showing how such high and constant dynamics of a piRNA master locus open the way to continual emergence of new patterns of piRNA biogenesis leading to changes in the level of transposition control (Zanni, 2013).

    The piRNA pathway plays a crucial role in TE silencing and is conserved among species. However, the mechanism by which this system adapts to new mobile elements is still obscure. The current data show a high insertion rate of recent TEs in the flam piRNA cluster far exceeding that previously suspected. A model has been developed in which Rhino protein might interact with the integration machinery of TEs to direct their integration into heterochromatin and, more specifically, into piRNA clusters. The current results concerning ZAM and Idefix highlight how the presence or absence of retrotransposons in piRNA-producing loci makes some Drosophila lines more susceptible to TE invasions than others, and thus how piRNA clusters affect the genomic TE distribution. A strict correlation was observed between the presence of ZAM and Idefix in the flam locus and their silencing. Consistently, their deletion from the flam locus observed in the Rev line is correlated with their activation, characterized by high mobilization, instability, and copy accumulation. A deeper analysis of the flam structure revealed that deletions occur frequently in the locus. Mostly, they affect internal segments of TEs, ranging from several base pairs up to several kilobases, affecting both ancient TEs, remaining as vestiges in the locus, and recently inserted TEs. The longest internal deletions affecting retrotransposons are due to homologous recombination between LTRs leading to the complete elimination of internal sequences. Moreover, large deletions may eliminate several TEs within one mutational event, as seen for ZAM and Idefix in the Rev line. At the same time, insertions also occur within the flam locus, as exemplified by the high proportion of recently inserted TEs, the recent insertions of 412 and Stalker2 in the Iso1A strain, and short and long segmental duplications. Such genetic dynamics of a piRNA master locus open the way to a constant emergence of new patterns of piRNA biogenesis potentially leading to changes in the level of transposition control (Zanni, 2013).

    The present data fit well with a model of TE invasion and its subsequent control by the invaded species as follows. The best genetic background for a TE to invade a genome and have full activity should be a 'virgin' genome devoid of any related copy. The best chance to find a virgin genome is to invade another species by horizontal transfer. In this genome, the incoming TE is not silenced, and is thus able to transpose at high frequency. A period of instability of the newly acquired TE results in its increased copy number. Insertions into piRNA clusters like flam are then highly probable because the current data evidence high content of recent TEs in such loci. These insertions would be associated with production of corresponding piRNAs and silencing of homologous elements. Thus, as soon as one copy of the TE is inserted in a piRNA cluster, a time of stability follows. This suggests that TEs regulated by a certain piRNA cluster should be present only once in this locus, as is seen for most TEs within flam. One or several deletion events can then lead to elimination of TE copies from the locus. A new period of activity of the remaining functional elements in the genome starts. Because deletion events may delete several elements from the locus, transposition bursts may happen involving several different TEs at the same time. This new period of instability for the TEs offers the opportunity to insert into a piRNA cluster again. When this occurs, stability is regained. Thus, transposition bursts, periods of stability, and periods of instability shaping the Drosophila genome would be directly correlated to the mutational events that affect piRNA clusters like flam. This scenario supports the hypothesis proposed by Le Rouzic (2005) that successful invasion of a population by TEs should be possible "thanks to an initial transposition burst followed by a strong limitation of their activity" (Zanni, 2013).

    Rounds of high transposition rate can trigger genetic instabilities and disease-associated mutations, but there is no doubt that they also play an essential role in the evolution of species. Actually, the current Drosophila genome witnesses multiple transposition bursts over time for most of the TE families, resulting in ancient and recent copies being present in the genome (examples from this study are Blood, Stalker2, Stalker4, Gypsy1, and Phidippo). The case of Pifo depicted in this study is different and certainly represents a case of a new invasion of D. melanogaster, because no ancient Pifo elements can be found in the genome. Such high dynamicity of piRNA clusters should also remodel heterochromatic regions in other Drosophila species. In D. erecta and D. yakuba, flam loci have been shown to contain a large amount of TEs that are completely different from the D. melanogaster flam elements. These data illustrate the dynamics of piRNA clusters and their coevolution with the rest of the genome regarding TE content. They also highlight the essential role that piRNA clusters might play in speciation by remodeling via TE control of large genomic regions (Zanni, 2013).

    Unique transposon landscapes are pervasive across Drosophila melanogaster genomes

    To understand how transposon landscapes (TLs) vary across animal genomes, this study describes a new method called the Transposon Insertion and Depletion AnaLyzer (TIDAL) and a database of >300 TLs in Drosophila melanogaster (TIDAL-Fly). This analysis reveals pervasive TL diversity across cell lines and fly strains, even for identically named sub-strains from different laboratories such as the ISO1 strain used for the reference genome sequence. On average, >500 novel insertions exist in every lab strain, inbred strains of the Drosophila Genetic Reference Panel (DGRP), and fly isolates in the Drosophila Genome Nexus (DGN). A minority (<25%) of transposon families comprise the majority (>70%) of TL diversity across fly strains. A sharp contrast between insertion and depletion patterns indicates that many transposons are unique to the ISO1 reference genome sequence. Although TL diversity from fly strains reaches asymptotic limits with increasing sequencing depth, rampant TL diversity causes unsaturated detection of TLs in pools of flies. Finally, novel transposon insertions were shown to negatively correlate with Piwi-interacting RNA (piRNA) levels for most transposon families, except for the highly-abundant roo retrotransposon. This study provides a useful resource for Drosophila geneticists to understand how transposons create extensive genomic diversity in fly cell lines and strains (Rahman, 2015).

    Paramutation in Drosophila linked to emergence of a piRNA-producing locus

    A paramutation is an epigenetic interaction between two alleles of a locus, through which one allele induces a heritable modification in the other allele without modifying the DNA sequence. The paramutated allele itself becomes paramutagenic, that is, capable of epigenetically converting a new paramutable allele. This study describes a case of paramutation in animals showing long-term transmission over generations. Previously a homology-dependent silencing mechanism has been characterized that is referred to as the trans-silencing effect (TSE), involved in P-transposable-element repression in the germ line. This study now shows that clusters of P-element-derived transgenes that induce strong TSE can convert other homologous transgene clusters incapable of TSE into strong silencers, which transmit the acquired silencing capacity through 50 generations. The paramutation occurs without any need for chromosome pairing between the paramutagenic and the paramutated loci, and is mediated by maternal inheritance of cytoplasm carrying Piwi-interacting RNAs (piRNAs) homologous to the transgenes. The repression capacity of the paramutated locus is abolished by a loss-of-function mutation of the aubergine gene involved in piRNA biogenesis, but not by a loss-of-function mutation of the Dicer-2 gene involved in siRNA production. The paramutated cluster, previously producing barely detectable levels of piRNAs, is converted into a stable, strong piRNA-producing locus by the paramutation and becomes fully paramutagenic itself. This work provides a genetic model for the emergence of piRNA loci, as well as for RNA-mediated trans-generational repression of transposable elements (de Vanssay, 2012).

    Transgenerationally inherited piRNAs trigger piRNA biogenesis by changing the chromatin of piRNA clusters and inducing precursor processing

    Small noncoding RNAs that associate with Piwi proteins, called piRNAs, serve as guides for repression of diverse transposable elements in germ cells of metazoa. In Drosophila, the genomic regions that give rise to piRNAs, the so-called piRNA clusters, are transcribed to generate long precursor molecules that are processed into mature piRNAs. How genomic regions that give rise to piRNA precursor transcripts are differentiated from the rest of the genome and how these transcripts are specifically channeled into the piRNA biogenesis pathway are not known. This study found that transgenerationally inherited piRNAs provide the critical trigger for piRNA production from homologous genomic regions in the next generation by two different mechanisms. First, inherited piRNAs enhance processing of homologous transcripts into mature piRNAs by initiating the ping-pong cycle in the cytoplasm. Second, inherited piRNAs induce installment of the histone 3 Lys9 trimethylation (H3K9me3) mark on genomic piRNA cluster sequences. The heterochromatin protein 1 (HP1) homolog Rhino binds to the H3K9me3 mark through its chromodomain and is enriched over piRNA clusters. Rhino recruits the piRNA biogenesis factor Cutoff to piRNA clusters and is required for efficient transcription of piRNA precursors. It is proposed that transgenerationally inherited piRNAs act as an epigenetic memory for identification of substrates for piRNA biogenesis on two levels: by inducing a permissive chromatin environment for piRNA precursor synthesis and by enhancing processing of these precursors (Le Thomas, 2014).

    A previous study and the current results reveal an essential role for a maternally transmitted transgenerationally inherited cytoplasmic factor in the generation of piRNAs. de Vanssay (2012) showed that a maternal factor supplied to the progeny by females expressing piRNAs from the T1 locus activates piRNA generation from the homologous inactive BX2 locus. Furthermore, maintaining the activity of T1 in the subsequent generation also requires the maternal factor. This observation was extended to other systems, and it was shown that generation of piRNAs from a single-copy transgene inserted into a telomeric piRNA cluster also depends on a maternally transmitted cytoplasmic factor. This maternal factor also activates piRNA generation from a single-copy euchromatic sequence, which simultaneously becomes the target of repression and the source of new piRNAs. Finally, the activity of endogenous clusters in D. melanogaster also seems to require a maternally inherited factor: An analysis of previously published piRNA profiles in interspecies hybrids between D. melanogaster females and Drosophila simulans males showed that only D. melanogaster piRNA clusters generate piRNAs, while D. simulans piRNA clusters are inactive (Le Thomas, 2014).

    What is the nature of the maternally supplied epigenetic factor that triggers piRNA generation in the progeny? Multiple lines of evidence point to piRNAs themselves as the carriers of this epigenetic signal. First, as initially shown by de Vanssay (2012), the epigenetic signal produced by the T1 locus does not require inheritance of the locus itself, indicating that the signal has a nonchromosomal nature. This eliminates the possibility that the signal is any kind of chromatin mark linked to the active locus. Second, the process of BX2 activation genetically depends on piRNA pathway genes but is independent of Dicer, which is required for siRNA biogenesis (de Vanssay, 2012). Third, both piRNAs and Piwi proteins are inherited from the maternal germline to the early embryos, while piRNAs are not transmitted through the sperm. Finally, piRNAs can be sequence-specific guides to identify and activate homologous loci. Importantly, recent studies have shown that piRNAs and the nuclear Piwi protein trigger installation of the H3K9me3 mark on homologous targets, providing a possible mechanism by which inherited piRNAs could lead to chromatin changes. Together, these results strongly support the role of inherited piRNAs as a transgenerationally transmitted epigenetic signal that activates piRNA generation from homologous loci in the progeny (Le Thomas, 2014).

    How can transgenerationally inherited piRNAs activate piRNA generation from homologous loci? The results imply two mechanisms that cooperate and work at different steps of piRNA biogenesis. In the cytoplasm, transgenerationally inherited piRNAs activate processing of complementary transcripts by the ping-pong amplification loop, as evidenced by a dramatic increase in piRNAs generated by the ping-pong processing upon MD of cognate piRNAs (Le Thomas, 2014).

    In the ping-pong processing, initial piRNAs guide generation of secondary piRNAs from complementary sequences. Previously, it was proposed that the ping-pong cycle requires two types of piRNA precursors: cluster transcripts and transcripts from active transposons provided in trans. The current results indicate that the ping-pong cycle can be activated by inherited piRNAs derived from the very same locus, provided that it is bidirectionally transcribed. Importantly, with the exception of one locus, all major piRNA clusters in the D. melanogaster germline are transcribed from both genomic strands, providing an abundant source of complementary transcripts to be used by the ping-pong process (Le Thomas, 2014).

    The major players in ping-pong processing in Drosophila are two Piwi proteins, AUB and AGO3, while the third Piwi protein, PIWI itself, is not involved in this process. AUB and AGO3 colocalize in cytoplasmic nuage granules, where the ping-pong processing is believed to take place. Therefore, the effect of inherited piRNAs on ping-pong processing impacts a late step of piRNA biogenesis after piRNA precursor transcripts are exported to the cytoplasm (Le Thomas, 2014).

    Although enhancing the ping-pong processing is clearly an important mechanism by which transgenerationally inherited piRNAs boost piRNA biogenesis, it cannot explain all of the observations, suggesting the existence of another mechanism. This study found that maternal piRNAs are also required for the biogenesis of PIWI-associated piRNAs, although those are not generated by ping-pong processing. Using several genetic systems, it was shown that inheritance of piRNAs leads to an increase of the H3K9me3 mark on regions homologous to the piRNAs. Importantly, acquisition of the H3K9me3 mark by genomic regions that did not previously produce piRNAs triggered piRNA generation in two transgenic systems. In contrast, the absence of inherited piRNAs led to a decreased H3K9me3 signal on homologous regions and a concomitant decrease of the corresponding piRNAs. These results suggest that modification of the chromatin structure of homologous genomic regions is the other mechanism by which transgenerationally inherited piRNAs turn on piRNA biogenesis in the progeny. Counterintuitively, it was found that enrichment of the H3K9me3 mark, which is generally assumed to be repressive, strongly correlates with enhanced piRNA biogenesis. In agreement with these results, a previous study showed that biogenesis of piRNAs from double-stranded clusters requires Eggless/SETDB1, one of the methyltransferases responsible for installation of the H3K9me3 mark (Le Thomas, 2014).

    Analysis of several transgenic piRNA clusters revealed differences in the impact of inherited piRNAs on the level of the H3K9me3 mark. The inherited piRNAs seem indispensable to maintain high H3K9me3 signal on the transgenic T1 and BX2* loci. However, the absence of maternal piRNAs leads to a relatively mild decrease in H3K9me3 on the telomeric piRNA cluster in the P1152 strain. These results indicate that natural piRNA clusters are able to maintain a certain level of the H3K9me3 mark in a piRNA-independent fashion. This is not unexpected, as natural piRNA clusters are located close to heterochromatin, which is known to have a high level of H3K9me3 signal. In contrast, the T1 and BX2 transgenes are inserted in a euchromatic site that is normally lacking this mark. Overall, the data strongly support an essential role of the H3K9me3 mark in piRNA generation. They further reveal that enrichment of this mark on regions that generate piRNAs at least partially depends on the inheritance of homologous piRNAs from the previous generation. Finally, acquisition of the H3K9me3 mark by a naive locus as a result of exposure to homologous piRNAs strongly correlates with the initiation of de novo primary piRNA biogenesis from such a locus. The exact mechanism for piRNA-dependent deposition of the H3K9me3 mark on piRNA regions remains to be elucidated; however, recent studies suggest that it might occur through recognition of nascent transcripts by the nuclear PIWI/piRNA complex, which is known to be deposited by the mother into the developing egg and has been shown to install H3K9me3 on its genomic targets (Le Thomas, 2014).

    The proposal that inherited piRNAs trigger piRNA biogenesis by changing the chromatin structure of homologous sequences raises the question of how the piRNAs distinguish a genuine transposon, a target that needs to be silenced, from a piRNA cluster that needs to be activated. Surprisingly, the results indicate that targeting by piRNAs leads to simultaneous repression of the target and activation of piRNA biogenesis from the same sequence. It was found that targeting of a unique sequence by piRNAs triggers accumulation of the H3K9me3 mark, a decrease in target expression, and activation of piRNA biogenesis. Importantly, the target-derived piRNAs are not generated by the ping-pong mechanism (which would be a trivial explanation), as they are present in complex with PIWI, which does not participate in ping-pong processing. The similarity between transposon targets and piRNA-producing regions is supported by recent work that demonstrated that new transposon insertions in euchromatin start to generate piRNAs; i.e., they are becoming de novo piRNA clusters. Indeed, careful consideration suggests that the requirement to 'silence' a genuine transposon target versus 'activate' a piRNA cluster is a false dichotomy: If nascent transcripts generated from piRNA target loci are channeled into the piRNA processing machinery instead of the standard mRNA processing pathway, the transcript will be effectively silenced, since no full-length mRNA will accumulate. The idea that the target of piRNA repression becomes a source of new piRNAs makes the distinction between piRNA clusters (source of piRNAs) and targets obsolete. Furthermore, the results expose a case in which the same genomic region is 'silenced' and 'activated' at the same time, depending on the exact output the researcher is looking at (generation of full-length mRNA or piRNAs). Similar phenomena might be more widespread than previously suspected, as studies in yeast suggest a very similar model in which centromeric repeats are 'silenced' and generate siRNAs at the same time (Le Thomas, 2014).

    How can the high level of the allegedly repressive H3K9me3 mark enhance piRNA biogenesis? The results show that the H3K9me3 mark provides a platform for the binding of Rhino, a chromodomain protein that shows specific enrichment over piRNA clusters. As high levels of the H3K9me3 mark are also present in other genomic regions, it is possible that recognition of H3K9me3 is not sufficient for Rhino’s stable binding and that it interacts with other proteins to achieve its localization on chromatin of double-stranded clusters. Rhino forms a complex with Cutoff, a protein that is also required for piRNA biogenesis. The H3K9me3 mark, Rhino, and Cutoff colocalize at double-stranded piRNA clusters, and Cutoff is de-localized from nuclear foci in rhino mutants, suggesting that it is recruited to piRNA clusters through its interaction with Rhino. Taken together, these results suggest that Rhino and Cutoff, which were previously shown to be indispensable for piRNA generation from double-stranded piRNA clusters, are recruited to cluster chromatin through the H3K9me3 mark (Le Thomas, 2014).

    The exact molecular mechanism by which the Rhino/Cutoff complex activates piRNA biogenesis in the nucleus remains to be elucidated; however, two not necessarily mutually exclusive hypotheses can be proposed. First, Cutoff might bind and target nascent transcripts generated from piRNA clusters to the piRNA processing machinery instead of the normal pre-mRNA processing. In support of this idea, this study found that the association of Cutoff with chromatin is RNA-dependent. It has been shown previously that inserting intron-containing heterologous gene sequences into piRNA clusters results in abundant piRNAs from both the exonic and intronic sequences, indicating that normal splicing is perturbed. According to the second hypothesis, the Rhino/Cutoff complex might enhance transcription of piRNA clusters, hence providing more precursors for piRNA biogenesis. Indeed, the run-on experiment showed that Rhino is required for efficient transcription of dual-stranded piRNA clusters. Furthermore, in agreement with an effect on transcription, this study found that, in the cutoff mutant, both siRNAs and piRNAs as well as long RNAs are eliminated from double-stranded piRNA clusters, arguing against a role of Cutoff exclusive to piRNA processing (Le Thomas, 2014).

    The counterintuitive idea that the H3K9me3 mark might enhance rather than suppress transcription through binding of nonconventional epigenetic 'readers' has interesting parallels in yeast. In Schizosaccharomyces pombe, H3K9 methylation induces binding of Swi6/HP1, which then recruits the Jumonji protein Epe1 that promotes nucleosome turnover, resulting in increased transcription of heterochromatic repeats and generation of siRNAs. One possible mechanism by which Cutoff might enhance cluster transcription is by suppressing RNA polymerase II (Pol II) termination. Indeed, transgenic insertions that contain polyA cleavage/termination signals into piRNA clusters generate piRNAs downstream from the polyA signal, indicating that not only splicing but transcription termination is also suppressed in piRNA clusters. Ignoring transcription termination signals is likely an important feature of piRNA clusters, as otherwise, multiple signals within transposon sequences present in the clusters would terminate transcription and disrupt piRNA generation (Le Thomas, 2014).

    Overall, these data revealed that regions that produce piRNAs in Drosophila germ cells are defined by the epigenetic process of the transgenerational inheritance of cognate small RNAs. It was found that inherited piRNAs trigger piRNA generation in the progeny by two mechanisms that seem to work simultaneously and cooperate to shape the final piRNA population. In the nucleus, inherited piRNAs mark genomic regions that will give rise to new piRNAs and enhance early steps of piRNA biogenesis. In the cytoplasm, inherited piRNAs further trigger the post-transcriptional processing of cluster transcripts through the ping-pong amplification loop (Le Thomas, 2014).

    Panoramix enforces piRNA-dependent cotranscriptional silencing

    The Piwi-interacting RNA (piRNA) pathway is a small RNA-based innate immune system that defends germ cell genomes against transposons. In Drosophila ovaries, the nuclear Piwi protein is required for transcriptional silencing of transposons, though the precise mechanisms by which this occurs are unknown. This study shows that the CG9754 protein is a component of Piwi complexes that functions downstream of Piwi and its binding partner, Asterix, in transcriptional silencing. Enforced tethering of CG9754 to nascent messenger RNA transcripts causes cotranscriptional silencing of the source locus and the deposition of repressive chromatin marks. CG9754 has been named 'Panoramix,' and it is proposed that this protein could act as an adaptor, scaffolding interactions between the piRNA pathway and the general silencing machinery that it recruits to enforce transcriptional repression (Yu, 2015).

    The Piwi interacting RNA (piRNA) pathway controls transposons through a number of distinct, but likely interlinked, mechanisms. Whereas cytoplasmic Piwi proteins silence their targets posttranscriptionally through piRNA-directed cleavage and the ping-pong cycle, nuclear Piwi-piRNA complexes function at the transcriptional level. Piwi-directed repression of transcription is thought to be dependent on piRNA-guided recognition of nascent transposon transcripts. Transcriptional gene silencing (TGS) correlates with the presence of histone H3 lysine 9 trimethylation (H3K9me3) marks, yet the mechanism through which Piwi binding promotes the deposition of these marks remains enigmatic. With the exception of the zinc finger protein Asterix (also known as DmGTSF1), the components of Piwi effector complexes at target loci are largely unexplored (Yu, 2015).

    This study systematically mined candidate genes from RNA interference (RNAi) screens for potential TGS effector proteins and identified CG9754 in three independently published screens as being critical in both the germ cells and follicle cells for transposon silencing. Loss of CG9754 had essentially no effect on the abundance or content of piRNA populations or on the nuclear localization of Piwi protein, suggesting that it is probably an effector component. CG9754 encodes a ~60-kD nuclear protein with no identifiable domains. The expression of CG9754 is restricted to the female gonads, as is seen for other core piRNA pathway components such as Asterix (Yu, 2015).

    To examine global effects on transposon expression, RNA sequencing (RNA-seq) was used to measure steady-state RNA levels from ovaries with germline-specific knockdowns of either CG9754 or Piwi. Piwi knockdown caused a sharp rise in transposon transcripts, with minimal effects on protein-coding gene expression. Knockdown of CG9754 caused effects very similar to those of Piwi, with most transposon targets being shared. Changes in steady-state RNA levels could have resulted from alterations in either element transcription or the stability of transposon mRNAs. Global run-on sequencing (GRO-seq) was used to measure nascent RNA synthesis following gene knockdown. Loss of either CG9754 or Piwi produced very similar profiles, suggesting that CG9754 is specifically required for transcriptional silencing of transposons targeted by Piwi (Yu, 2015).

    Piwi-mediated TGS correlates with the presence of H3K9me3 marks at silenced transposons. Depletion of either CG9754 or Piwi resulted in nearly identical losses of H3K9me3 over transposons. Four independent frameshift mutations of CG9754 generated via the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein system were isolated. A consistent global up-regulation of transposable elements and the corresponding loss of H3K9me3 marks in CG9754 mutant ovaries were observed without changes in piRNA levels. Similarly to other core piRNA pathway mutants, female flies lacking CG9754 were sterile. Moreover, flies double-mutant for CG9754 and Asterix showed transposon derepression comparable to that of flies with either single mutation, suggesting that both genes act in the same pathway. Thus, CG9754 functions along with Piwi and Asterix in the repression of transposon transcription (Yu, 2015).

    Next it was asked whether the presence of CG9754 at a target locus might be sufficient to induce its silencing. Because piRNAs likely direct binding to nascent RNAs rather than to the DNA of their targets, CG9754 was delivered via protein-RNA interactions. A series of luciferase reporters was constructed with BoxB sites in their 3' untranslated regions (UTRs) and they were used to create transgenic reporter flies. BoxB sites are bound by the λN protein, which can also bring other components to the RNA as part of a fusion. Flies were also generated expressing λN proteins fused to CG9754, Asterix, nuclear Piwi, and, as a negative control, a cytoplasmic Piwi missing its nuclear localization signal (dN-Piwi). When coexpressed with the reporter, the dN-Piwi fusion failed to induce any change in luciferase expression. Of the remainder, only the CG9754 fusion considerably reduced luciferase activity. Silencing appeared to be dosage-dependent, as the degree of repression correlated with the number of BoxB binding sites inserted into the reporter mRNA. Consistent with a role for CG9754 in transcriptional silencing, the abundance of the reporter mRNAs was significantly reduced upon tethering (Yu, 2015).

    Although CG9754-triggered repression appeared to be independent of chromatin context, integration of the reporter into genomic DNA appeared to be critical for repression. Transient cotransfection of reporter constructs into the OSS cell line, which contains an active piRNA pathway, resulted in little to no detectable silencing. In contrast, tethering Drosophila Ago1 (λN-dAgo1) to the luciferase reporter mRNA in OSS cells caused substantial repression of the same reporter. These results indicate that CG9754 can function properly only in the context of chromatin, likely acting at the transcriptional level, by interacting with nascent transcripts (Yu, 2015).

    To test the hypothesis that λN-CG9754 acts on nascent transcripts, a reporter was generated for which the BoxB binding sites were located within the intron of the primary transcript. λN-CG9754 maintained the ability to repress this reporter but not a similar transcript carrying BoxB sites in the antisense orientation integrated into the same genomic locus. Because the spliced, mature reporter transcripts lack the BoxB sites, it was reason that λN-CG9754 must be able to exert its effects by binding to unspliced precursor mRNAs. Given that splicing occurs cotranscriptionally, this implies that CG9754 confers its effects by interaction with the nascent transcript (Yu, 2015).

    If CG9754 mediates Piwi-dependent transcriptional silencing, delivery of CG9754 alone might recapitulate hallmarks of piRNA-directed repression. Tethering of CG9754 had a highly specific effect, changing levels of only the reporter mRNA. Repression occurred at the transcriptional level, as GRO-seq indicated a loss of nascent RNA from the integrated reporter. Repression by CG9754 also correlated with specific deposition of H3K9me3 marks over the reporter locus. Tethering of CG9754 failed to trigger piRNA production from the reporter, as has been seen previously for some loci that become targets of the piRNA pathway. Of note, spreading of H3K9me3 marks to other regions of the reporter gene was observed, as described previously for regions flanking piRNA-targeted transposon insertions. Thus, delivering CG9754 to the nascent RNA causes repression of a locus in a manner that mimics targeting by the piRNA pathway (Yu, 2015).

    Whether CG9754 might be a component of Piwi complexes, as predicted by epistasis experiments, was tested. Functional GFP-Piwi fusion proteins copurified with hemagglutinin (HA)-tagged CG9754 from OSS cells, but not with a negative-control fusion (HA-mKate2). Conversely, Flag-tagged CG9754 was able to specifically precipitate endogenous Piwi proteins from OSS cell lysates, confirming the interaction between these two proteins. Given its properties, CG9754 was named 'Panoramix,' after the mentor who empowers the French comic book character Asterix to perform his feats of strength (Yu, 2015).

    The identification of Panoramix as a key mediator of piRNA-directed TGS presented an opportunity to use the tethering assay to dissect the mechanism of transcriptional silencing. RNAi was used to deplete selected piRNA pathway genes in flies in which λN-Panoramix was tethered to the luciferase-BoxB reporter. Knockdown of Panoramix itself weakened the repression significantly, as compared with a control knockdown (mCherry). Silencing of factors required for piRNA biogenesis (Zuc and Armi) or those that are expected to act upstream of Panoramix (Piwi and Asterix) did not significantly affect repression. Depletion of dLSD1/Su(var)3-3 and its cofactor, CoREST, which normally form a complex that removes H3K4me2 marks from promoters, had significant effects on the ability of Panoramix to repress the reporter. Because H3K4me2 marks actively transcribed genes, it is possible that dLSD1-mediated removal of these marks is a key step in Panoramix-mediated transcriptional silencing. This raises a potential parallel with piRNA-directed silencing in mice, wherein engagement by DNMT3L, which is necessary for piRNA-induced DNA methylation, requires removal of such marks. Similarly, knockdown of HP1a caused derepression, in agreement with its role as a constitutive heterochromatin component required for transposon silencing and with the observation that the presence of Panoramix is correlated with the deposition of H3K9me3 marks at target loci. The H3K9 methyltransferase Eggless/dSETDB1 and its cofactor Windei appeared to be required specifically for Panoramix-mediated silencing, as knockdown of G9a, another H3K9 methyltransferase, showed no effect on the reporter. In eggless mutants, essentially complete derepression of the reporter was observed, despite Panoramix tethering. In contrast, the piRNA biogenesis mutant zuc showed little to no effect on the repression of the reporter, as also observed in zuc RNAi experiments. These data raise the possibility that Eggless could be one of the enzymes responsible for the deposition of H3K9me3 marks over silenced transposons in a Piwi-targeted fashion (Yu, 2015).

    Panoramix functions downstream of Piwi and Asterix and is both necessary and sufficient to elicit transcriptional repression when bound to nascent transcripts. Panoramix represents an example in metazoans of a protein inducing cotranscriptional silencing when recruited to the nascent transcript from a locus. In fact, only cotranscriptional silencing can resolve the conundrum of a target being transcriptionally repressed while transcripts from that target locus are responsible for recruiting their own repressors. Orthologs of some of the general silencing factors that act with Panoramix to deposit and interpret repressive chromatin marks have also been implicated in mammalian transposon silencing, in which the pathway functions by causing heritable DNA methylation. Though one cannot identify a mammalian ortholog of Panoramix based on primary sequence alone, the overall conservation of the piRNA-mediated transcriptional machinery suggests that a protein with an equivalent function likely exists in mammals (Yu, 2015).

    The HP1 homolog Rhino anchors a nuclear complex that suppresses piRNA precursor splicing

    piRNAs guide an adaptive genome defense system that silences transposons during germline development. The Drosophila HP1 homolog Rhino is required for germline piRNA production. Rhino is shown to bind specifically to the heterochromatic clusters that produce piRNA precursors; binding directly correlates with piRNA production. Rhino colocalizes to germline nuclear foci with Rai1/DXO-related protein Cutoff (Cuff) and the DEAD box protein UAP56, which are also required for germline piRNA production. RNA sequencing indicates that most cluster transcripts are not spliced and that rhino, cuff, and uap56 mutations increase expression of spliced cluster transcripts over 100-fold. LacI::Rhino fusion protein binding suppresses splicing of a reporter transgene and is sufficient to trigger piRNA production from a trans combination of sense and antisense reporters. It is therefore proposed that Rhino anchors a nuclear complex that suppresses cluster transcript splicing and it is speculated that stalled splicing differentiates piRNA precursors from mRNAs (Zhang, 2014).

    The piRNA pathway has an evolutionarily conserved role in transposon control during germline development and is essential for transmission of the inherited genetic complement. In the Drosophila ovary, unique piRNAs are concentrated in 'clusters' composed of complex arrays of nested transposon fragments that are generally localized to pericentromeric or subtelomeric heterochromatin. These loci fall into two classes, based on strand bias. Clusters that produce piRNAs from both genomic strands (dual-strand clusters) are dominant in the germline, while clusters that are expressed on only one genomic strand (uni-strand clusters) produce most of the piRNAs in somatic follicle cells that surround the germline. Primary piRNAs from dual-strand clusters, bound to Piwi proteins, appear to drive a ping-pong cycle that amplifies the silencing RNA pool. Primary piRNAs that initiate the amplification cycle, by definition, are produced by a ping-pong-independent mechanism. Similarly, ping-pong amplification is not required for production of piRNAs from uni-strand clusters. These observations suggest a simple model in which primary piRNAs from uni-strand and dual-strand clusters are produced by a common mechanism, and dual-strand clusters are equivalent to convergently transcribed uni-strand clusters. However, uni-strand cluster piRNA production is independent of rhi, uap56, and cuff, which are essential for production of piRNAs that map uniquely to dual-strand clusters. In addition, this study showed that Rhi-dependent piRNA production from an ectopic locus requires a combination of transgenes expressing complementary transcripts. Primary piRNA production by dual-strand and uni-strand clusters thus appear to proceed by distinct mechanisms (Zhang, 2014).

    These findings also suggest that piRNA production by dual-strand clusters requires complementary precursors. The role of complementary RNAs in the germline piRNA biogenesis pathway, however, remains to be determined (Zhang, 2014).

    piRNA pathway mutations increase expression of a subset of transposons by over 200-fold, but do not alter germline gene expression. This remarkable specificity is almost certainly essential to gamete production, but how piRNA precursors are differentiated from mRNAs is not understood. The vast majority of protein coding premRNAs are efficiently spliced, exported from the nucleus, and translated in the cytoplasm. By contrast, splicing is suppressed at a transgene inserted into the Drosohila X-TAS piRNA cluster, and transcriptome wide studies indicate that rapidly evolving HP1 homolog Rhi, the Rai1-related protein Cuff, and the DEAD box protein UAP56 suppress slicing at resident consensus donor and acceptor sites in germline clusters. This is most clearly illustrated at the sox102F locus, which produces efficiently spliced pre-mRNAs in the soma but is the source of piRNAs from unspliced primary transcripts in the germline. Significantly, accumulation of both unspliced transcripts and piRNAs requires rhi, cuff, and uap56, and tethering a LacI::Rhi fusion to a intron-containing reporter transgene suppresses splicing and is sufficient to trigger de novo piRNA production from a trans combination of sense and antisense transgenes. It is therefore proposed that Rhi functions with Cuff and UAP56 to suppress cluster transcript splicing and that the stalled splicing intermediates are the precursors for primary piRNAs (Zhang, 2014).

    Cuff is a homolog of Rai1/DXO, which binds and degrades mRNAs carrying incomplete cap structures. This would appear to support a role for Cuff in destabilizing spliced cluster transcripts, but the residues required for the 5' to 3' exonuclease activity of Rai1/DXO are not conserved in Cuff. Murine DXO has been cocrystallized with an uncapped RNA, and with a cap analog. The crystal structures reveal the protein residues that interact with the RNA backbone, and indicate that the cap is bound in a pocket in the interior of the protein. The sequences of Drosophila Cuff was aligned with the murine DXO. Twenty one percent of the positions are identical, and conserved amino acids are present throughout the entire alignment. Overall protein fold of Cuff is therefore highly likely to resemble DXO. Therefore the Modeler and I-TASSER algorithms were used to build a homology model of Cuff based on the Murine DXO structures. Essentially, all of the RNA-binding interactions are preserved in the homology model of Cuff. It is therefore proposed that Cuff is not a catalytically active nuclease, but binds to uncapped RNA ends as they emerge from Pol2 or to newly capped cluster transcripts (Zhang, 2014).

    Cap binding by the nuclear Cap Binding Complex (CBC) is required for splicing and polyadenylation. It is therefore proposed that Cuff binds to Rhi and cotranscriptionally associates with cluster transcripts, preventing capping or recognition of capped RNAs by the CBC, which blocks splicing. Previous work has shown that UAP56 immunoprecipitation significantly enriches for cluster transcripts, not for mRNAs, and that RNAs mapping to the major 42AB cluster are the most highly enriched species in the immunoprecipitated pool. The point mutation in uap56 that specifically blocks piRNA biogenesis disrupts a salt bridge predicted to stabilize the ATP and RNA bound form of the protein. These observations suggest that stable cluster transcript binding by UAP56 is required for piRNA biogenesis. Mutations in the yeast cap-binding complex lead to arrest at an early step in the splicing pathway, with the U2 snRNP bound to primary transcripts. UAP56 was identified as a binding partner of the U2 snRNP protein U2AF65 (Zhang, 2014).

    Cuff binding to capped cluster transcripts may therefore prevent cap recognition by the CBC, arresting splicing with UAP56 stably bound. This aberrant stable complex could differentiate piRNA precursors from pre-mRNAs. While this model is highly speculative, it makes several clear predictions and should therefore serve as a useful starting point to additional studies. Adaptation to transposon invasion by the piRNA pathway appears to be initiated by insertion of the invading element into a cluster. This speculate model, with the observation that Rhi can spread from anchor sites, suggest an adaption model in which Rhi spreads into active transposons that insert into clusters, leading to Cuff binding to capped transcripts from transposon promoters. This would block processing and promote production of new piRNAs, thus coordinately silence the inserted element and produce the transsilence species that control dispersed active elements (Zhang, 2014).

    Studies in the pathogenic yeast Crypotoccous provide evidence for a direct link between stalled splicing and transposon silencing by the siRNA pathway. It has been shown that splicing factors associate with the Crypotoccous siRNA biogenesis machinery and siRNAs are produced from unspliced transposon transcripts. In addition, intron removal reduces siRNA production, and splice site mutations that reduce splicing efficiency increase siRNA production. Furthermore, recent genome-wide screens have implicated splicing factors in transposon silencing, and the splicing and small RNA-silencing pathways appears to be coevolving. These findings, with the the current studies, suggest that stalled splicing generates a conserved molecular signature for potentially deleterious RNAs, which directs these transcripts to small silencing RNA biogenesis pathways. Retrotransposons and retroviruses encode essential spliced transcripts, but splicing must be suppressed to produce full-length genomic RNAs. This novel feature of the retroviral life cycle may have driven evolution of silencing systems that use stalled splicing as a hallmark of pathogenic RNAs (Zhang, 2014).

    A transgenerational process defines piRNA biogenesis in Drosophila virilis

    Piwi-interacting (pi)RNAs repress diverse transposable elements in germ cells of Metazoa and are essential for fertility in both invertebrates and vertebrates. The precursors of piRNAs are transcribed from distinct genomic regions, the so-called piRNA clusters; however, how piRNA clusters are differentiated from the rest of the genome is not known. To address this question, piRNA biogenesis was studied in two D. virilis strains that show differential ability to generate piRNAs from several genomic regions. That active piRNA biogenesis was found to correlate with high levels of histone 3 lysine 9 trimethylation (H3K9me3) over genomic regions that give rise to piRNAs. Furthermore, piRNA biogenesis in the progeny requires the transgenerational inheritance of an epigenetic signal, presumably in the form of homologous piRNAs that are generated in the maternal germline and deposited into the oocyte. The inherited piRNAs enhance piRNA biogenesis through the installment of H3K9me3 on piRNA clusters (Le Thomas, 2014: PubMed).

    piRNAs are associated with diverse transgenerational effects on gene and transposon expression in a hybrid dysgenic syndrome of D. virilis

    Hybrid dysgenic syndromes are a strong form of genomic incompatibility that can arise when transposable element (TE) family abundance differs between two parents. When TEs inherited from the father are absent in the mother's genome, TEs can become activated in the progeny, causing germline damage and sterility. Studies in Drosophila indicate that dysgenesis can occur when TEs inherited paternally are not matched with a pool of corresponding TE silencing PIWI-interacting RNAs (piRNAs) provisioned by the female germline. Using the D. virilis syndrome of hybrid dysgenesis as a model, this study characterize the effects that divergence in TE profile between parents has on offspring. Overall, this study shows that divergence in the TE landscape is associated with persisting differences in germline TE expression when comparing genetically identical females of reciprocal crosses and these differences are transmitted to the next generation. Moreover, chronic and persisting TE expression coincides with increased levels of genic piRNAs associated with reduced gene expression. Gene expression is idiosyncratically influenced by differences in the genic piRNA profile of the parents that arise though polymorphic TE insertions. Overall, these results support a model in which early germline events in dysgenesis establish a chronic, stable state of both TE and gene expression in the germline that is maintained through adulthood and transmitted to the next generation (Erwin, 2015).

    Somatic primary piRNA biogenesis driven by cis-acting RNA elements and trans-acting Yb

    Primary piRNAs in Drosophila ovarian somatic cells arise from piRNA cluster transcripts and the 3' UTRs of a subset of mRNAs, including Traffic jam (Tj) mRNA. However, it is unclear how these RNAs are determined as primary piRNA sources. This study identified a cis-acting 100-nt fragment in the Tj 3' UTR that is sufficient for producing artificial piRNAs from unintegrated DNA. These artificial piRNAs were effective in endogenous gene transcriptional silencing. The Tudor domain RNA helicase Yb, a core component of primary piRNA biogenesis center Yb bodies, directly binds the Tj-cis element. Disruption of this interaction markedly reduces piRNA production. Thus, Yb is the trans-acting partner of the Tj-cis element. Yb-CLIP revealed that Yb binding correlates with somatic piRNA production but Tj-cis element downstream sequences produced few artificial piRNAs. It is thus proposed that Yb determines primary piRNA sources through two modes of action: primary binding to cis elements to specify substrates and secondary binding to downstream regions to increase diversity in piRNA populations (Ishizu, 2015).

    PIWI-interacting RNAs (piRNAs) interact with PIWI proteins to form piRNA-induced silencing complexes (piRISCs), which repress target genes, mostly transposons, either transcriptionally or at the post-transcriptional level by cleaving transcripts in the cytoplasm. Interestingly, not all cells in the gonads use both mechanisms. Follicle cells in Drosophila ovaries use transcriptional silencing but lack piRISC-mediated post-transcriptional silencing, while germ cells possess both transcriptional and post-transcriptional piRISC machineries. In Bombyx ovaries, only posttranscriptional silencing occurs. This variation largely depends on which PIWI proteins are expressed in a given cell type; transcriptional silencing requires nuclear PIWI proteins while post-transcriptional silencing requires cytoplasmic PIWI proteins (Ishizu, 2015).

    Primary piRNAs are produced from single-stranded long noncoding RNAs transcribed from piRNA clusters in a Dicer-independent manner. The Drosophila genome contains 142 piRNA clusters, whose expression is regulated differently in different cell types. flamenco (flam), a representative of unidirectional piRNA clusters, is expressed only in follicle cells, whereas the bidirectional cluster 42AB is expressed specifically in nurse cells. The types of transposon fragments inserted in individual piRNA clusters also vary; therefore, piRNA populations differ among cell types. piRNAs in nurse cells are rather complex because primary piRNAs are amplified through the amplification loop, yielding secondary piRNAs. Recent studies showed that secondary piRNAs further produce phased trailer piRNAs. Follicle cells do not use this amplification system and thus only contain primary piRNAs (Ishizu, 2015).

    The biogenesis of somatic primary piRNAs has been studied using ovaries and an ovarian somatic cell (OSC). A current model suggests that upon transcription flam-piRNA precursors are localized to perinuclear Flam bodies and processed at adjacent Yb bodies. Yb bodies contain many piRNA factors besides Yb. Zucchini (Zuc), an endonuclease required for processing piRNA intermediates into mature piRNAs, is localized on the surface of mitochondria. Yb bodies tend to be observed in inter-mitochondrial regions. This arrangement of organelles appears crucial for accelerating piRNA processing because it centralizes all the necessary factors in the cytoplasm. Upon maturation, piRNAs associate with Piwi, a Drosophila PIWI protein, to form piRISCs, which are then translocated to the nucleus to implement nuclear transposon silencing through chromatin modifications on target transposon loci with support from co-factors such as GTSF1/ Asterix and Maelstrom (Ishizu, 2015).

    flam is the major source of primary piRNAs in OSCs and follicle cells in the ovaries. flam is largely occupied by transposon remnants, whose orientation predominantly opposes that of the parental transposons; thus, most primary piRNAs arising from the piRNA cluster act as antisense oligos to repress parental transposons. Some protein-coding genes such as Traffic jam (Tj) also act as primary piRNA sources, and genic piRNA sources express proteins in OSCs and follicle cells. The TJ protein, encoded by Tj, is a large Maf transcriptional factor necessary for controlling gonad morphogenesis (Li, 2003). Loss of Tj function abolishes Piwi expression in follicle cells. However, Piwi expression in nurse cells is not influenced by TJ loss. Thus, the dependence of Piwi expression on TJ differs between follicle cells and germ cells (Ishizu, 2015).

    Only a limited number of transcripts serve as somatic primary piRNA precursors. However, the mechanism underlying the recognition and selection of these transcripts as piRNA precursors is poorly understood. To better understand the mechanism, the Tj 3' UTR was used as representative of somatic primary piRNA sources to identify a cis element and its trans-acting partner necessary for producing primary piRNAs in OSCs (Ishizu, 2015).

    Yb bodies and Flam bodies in OSCs are considered to be the centers for primary piRNA maturation/piRISC formation and piRNA intermediate storage, respectively, and exist in close proximity. The formation of both bodies depends on the Yb protein, particularly its RNA-binding activity (Murota, 2014). In the absence of this, piRNA processing fails, resulting in piRNA loss, although piRNA intermediates and processing factors are present in the cytosol. Thus, Yb binding to piRNA sources centralizes all necessary ingredients for piRNA biogenesis, which is crucial for primary piRNA production. This study discovered that the direct association of Yb with a specific ~100-nt element (i.e., cis element) within the piRNA precursors provokes somatic primary piRNA biogenesis from downstream regions. Insertion of the Yb-binding element within RNA molecules that do not otherwise serve as piRNA precursors converts the RNA transcripts into piRNA sources. Artificial primary piRNAs were mapped only downstream, but not upstream, of regions of the Yb-binding element. Previous studies demonstrated that natural genic piRNAs mostly arise from 3' UTRs rather than mRNA CDS or 5' UTRs. The present study also showed that few Tj-piRNAs mapped to the Tj CDS, and that few Yb-CLIP tags were also found in the Tj CDS. Thus, Yb determines not only substrate specificity but also processing directionality in the somatic primary piRNA biogenesis pathway. This may occur through the Yb- controlled recruitment of other piRNA factors, such as another putative RNA helicase Armi and endonuclease Zuc, only to downstream sequences (Ishizu, 2015).

    Yb-CLIP tags greatly overlap with primary piRNA-producing loci in the genome. This strongly supports the idea that Yb is the central player in determining substrates in the piRNA pathway. An unexpected but intriguing observation in this study is that Tj-R1 and Tj-R2 in the Tj 3' UTR show strong Yb-binding marks, as does the Tj-cis element, but provoked very little artificial piRNA production in contrast to the Tj-cis element. Yb-CLIP experiments showed that Yb binding to Tj-R1 and Tj-R2 within the Drosophila genome largely depends on Yb binding to its upstream Tj-cis element. Therefore, a model is proposed in which Yb determines primary piRNA sources by two sequential modes of action: primary binding to cis elements that represents selection of piRNA precursors among cellular RNAs, then secondary binding to downstream regions, representing the defining domains to be processed by precursors. This complexity in determining piRNA precursors could ensure the high diversity in piRNA populations, which is a unique feature of piRNAs (Ishizu, 2015).

    The RNA-binding activity of Yb is required for primary piRNA production in OSC. Yb mutants carrying a point mutation within the DEAD box showed little RNA binding activity. When these Yb mutants were expressed individually in OSC lacking endogenous Yb, piRNA precursors were not accumulated in Flam bodies, and few piRNAs were produced. As a consequence, transposons were de-silenced. Therefore, there is little doubt that the RNA-binding activity of Yb through the DEAD-box is indispensable for primary piRNA production. HITS-CLIP experiments clarified direct interaction of Yb with piRNA sources, including Tj mRNA. Insertion of a particular Yb-bound RNA element within Tj mRNA, i.e., the Tj-cis element, upstream of any given RNA molecule enables the arbitrary sequences to produce artificial piRNAs. Deletion of the Tj-cis element from the Drosophila genome significantly abolished piRNA production from its downstream region spanning at least ~200 nt. These observations strongly support the proposed model, in which Yb is the trans-acting factor that recognizes and binds cis elements within piRNA precursors to provoke primary piRNA biogenesis in ovarian somatic cells. However, it does not exclude the possibility that Yb collaboratively achieves this task with unknown factors. Moreover, it is not certain if Yb is the uppermost factor in the cytoplasmic phase of the biogenesis pathway upon nuclear transport of piRNA precursors (Ishizu, 2015).

    Yb integrates piRNA intermediates and processing factors into perinuclear bodies to enhance piRISC assembly

    PIWI-interacting RNAs (piRNAs) direct Piwi to repress transposons and maintain genome integrity in Drosophila ovarian somatic cells. piRNA maturation and association with Piwi occur at perinuclear Yb bodies, the centers of piRNA biogenesis. This study shows that piRNA intermediates arising from the piRNA cluster flamenco (flam) localize to perinuclear foci adjacent to Yb bodies, termed Flam bodies. RNAi-based screening of piRNA factors revealed that Flam body formation depends on Yb, the core component of Yb bodies, while Piwi and another Yb body component, Armitage, are dispensable for formation. Abolishing the RNA-binding activity of Yb disrupts both Flam bodies and Yb bodies. Yb directly binds flam, but not transcripts from neighboring protein-coding genes. Thus, Yb integrates piRNA intermediates and piRNA processing factors selectively into Flam bodies and Yb bodies, respectively. It is suggested that Yb is a key upstream factor in the cytoplasmic phase of the piRNA pathway in ovarian somatic cells (Murota, 2014).

    This study visualized flam-piRNA intermediates in OSCs and follicle cells using RNA-FISH and EM-ISH and revealed that they concentrate at perinuclear Flam bodies. Flam bodies locate in very close proximity to Yb bodies, the sites of piRNA maturation and piRISC formation. It is postulated that flam signals might also be detectable within Yb bodies. However, this was not the case. The simplest explanation for this observation is that piRNA processing at Yb bodies occurs so quickly, and the processed piRNAs localize to the nucleus as piRISCs so immediately, that the flam signal at Yb bodies was below the level of detection at Yb bodies (Murota, 2014).

    In Zuc-depleted cells, flam transcripts were detected predominantly as flam-piRNA intermediates, being several hundred to 4,000 nt in length, while the full transcriptional unit of flam is estimated to be over 180 kb. Both Yb body and Flam body formation require Yb, or more precisely, its RNA-binding activity through its NTD. Yb binds flam-piRNA intermediates directly. Based on these findings, a new model is proposed for primary piRNA biogenesis in ovarian soma, in which the association of Yb with piRNA intermediates, which most likely occurs in the cytoplasm because Yb is a cytoplasmic protein, is the initiation point of the cytoplasmic phase of piRNA biogenesis. This follows the nuclear phase of piRNA biogenesis: flam transcription and nuclear export of flam transcripts through the nuclear pores. flam transcription is initiated by RNA polymerase II and requires the transcriptional factor Cubitus interruptus. However, it is not known by which export factors and in what lengths the flam transcripts are exported from the nucleus. Further investigation will be required for a detailed understanding of the nuclear phase of piRNA biogenesis (Murota, 2014).

    The locations of the genomic flam loci in the nucleus and Flam bodies do not seem to be arranged to be close to each other, meaning that the flam transcripts move a long distance to arrive at Flam bodies. Do the flam transcripts move within the nucleus to get closer to Flam bodies before export to the cytoplasm? Alternatively, does nuclear export occur first and then flam transcripts are localized to Flam bodies? Yb localization in the cytoplasm seems to be so dynamic that a point mutation in Yb that disrupts the RNA-binding capacity of Yb drastically changes the subcellular localization of Yb, causing it to be scattered evenly in the cytosol. Thus, the latter scenario appears more likely, in which Yb plays a crucial role; upon nuclear export, Yb captures flam transcripts through direct binding and localizes them, as flam-piRNA intermediates, to Flam bodies. Flam body formation depends on the RNA-binding activity of Yb, a cytoplasmic protein; this notion further supports the idea that Flam bodies are cytoplasmic structures (Murota, 2014).

    Unlike flam transcripts, DIP1 mRNAs were virtually undetectable in Yb-CLIP tags, although the DIP1 protein-coding gene and the flam piRNA cluster are neighbors on chromosome X and DIP1 is expressed in OSCs. The sequences of Yb-CLIP tags were examined closely, but no obvious consensus sequences were found. Yb may recognize binding substrates owing to higher-order structures. Immunoelectron microscopy using an anti-Yb antibody showed that Yb bodies are often attached to mitochondria, to which Zuc endoribonuclease, the piRNA intermediate processor, anchors on the surface to face into cytoplasmic Yb bodies. This peculiar spatial arrangement of Zuc and Yb bodies, along with Flam bodies, integrates all the ingredients necessary for primary piRNA production locally, enhancing the rates of piRISC assembly. Another virtue of this perinuclear arrangement is that it enables assembled piRISCs to be immediately imported into the nucleus, where the RNP complex (i.e., the PIWI-piRNA complex) exerts its nuclear-specific function of silencing transposon transcription. How does Yb decide where within the perinuclear region to integrate all the materials necessary for primary piRNA biogenesis? Reconstitution of the whole machinery in, for instance, nongonadal somatic Schneider2 cells, in which no primary piRNAs are otherwise expressed, might address this fundamental question (Murota, 2014).

    Splicing-independent loading of TREX on nascent RNA is required for efficient expression of dual-strand piRNA clusters in Drosophila

    The conserved THO/TREX (transcription/export) complex is critical for pre-mRNA processing and mRNA nuclear export. In metazoa, TREX is loaded on nascent RNA transcribed by RNA polymerase II in a splicing-dependent fashion; however, how TREX functions is poorly understood. This study shows that Thoc5 and other TREX components are essential for the biogenesis of piRNA, a distinct class of small noncoding RNAs that control expression of transposable elements (TEs) in the Drosophila germline. Mutations in TREX lead to defects in piRNA biogenesis, resulting in derepression of multiple TE families, gametogenesis defects, and sterility. TREX components are enriched on piRNA precursors transcribed from dual-strand piRNA clusters and colocalize in distinct nuclear foci that overlap with sites of piRNA transcription. The localization of TREX in nuclear foci and its loading on piRNA precursor transcripts depend on Cutoff, a protein associated with chromatin of piRNA clusters. Finally, it was shown that TREX is required for accumulation of nascent piRNA precursors. These data reveal a novel splicing-independent mechanism for TREX loading on nascent RNA and its importance in piRNA biogenesis (Hur, 2016).

    Thoc5 mRNA loading piRNA biogenesis transcriptional regulation THO is a multiprotein complex, which itself is part of the TREX (transcription/export) complex. The THO complex was first identified in Saccharomyces cerevisiae and is conserved throughout metazoan evolution. In metazoans, THO contains six proteins; three proteins are homologous to yeast proteins (Hpr1/Thoc1, Thoc2, and Thoc3/Tex1), and three are unique (Thoc5/FMIP, Thoc6, and Thoc7). In addition to the THO subunits, the complete TREX contains two proteins conserved between yeasts and mammals: Yra1, also known as REF/Aly, and Sub2, also known as UAP56. In yeast, the THO/TREX complex participates in transcription, pre-mRNA processing, and nuclear mRNA export of the majority of genes. Yeast TREX associates with transcribing RNA polymerase II (Pol II) and is necessary for transcription elongation. TREX is loaded onto nascent transcripts during transcription and is essential for mRNA export from the nucleus, as the Yra1 subunit of TREX recruits the Mex67p protein, which interacts with nucleoporins and mediates passage of complexes of mRNAs and proteins (mRNPs) through the nuclear pore (Hur, 2016).

    Despite the conservation of the THO/TREX complex in yeast and metazoa, there are important differences in its function in different organisms. As it is in yeast, metazoan TREX is loaded onto nascent transcripts; however, it does not associate with RNA polymerase. Instead, association of TREX with pre-mRNA depends on splicing of pre-mRNA and requires the interaction of several TREX subunits with the nuclear cap-binding proteins CBC20 and CBC80. Most metazoan genes contain introns, leading to effective splicing-dependent TREX loading onto mRNAs. Furthermore, TREX is recruited to several intronless genes in Drosophila and mammals. For some of these genes, TREX recruitment is mediated by sequence-specific RNA-binding proteins that recognize distinct motifs on the mRNAs; in other cases, the mechanism of splicing-independent loading is not understood (Hur, 2016).

    The conserved composition of TREX suggests that it plays the same function in the nuclear export of mRNPs in metazoa as it does in yeast. Indeed, in Drosophila, depletion of Sub2/UAP56 causes accumulation of polyadenylated RNAs within the nucleus. In contrast, however, depletion of other TREX components has a surprisingly mild effect on mRNA export and gene expression in Drosophila and mammals. Depletion of the THO complex in Drosophila S2 cells results in changes in expression of only a small fraction of genes. Similarly, in HeLa cells deficient in Thoc5, no significant accumulation of poly(A) RNAs is observed in the nucleus. Transcriptome analyses of mouse embryonic fibroblasts in which thoc5 expression was inhibited showed down-regulation of only 143 genes, and these were efficiently spliced but retained in the nucleus. In Drosophila, thoc5 mutants are viable but have spermatogenesis defects (Hur, 2016 and references therein).

    This study shows that Thoc5 and other THO subunits are required for female fertility, oocyte patterning, and transposable element (TE) repression in the Drosophila germline. Thoc5 is required for biogenesis of piRNAs, a distinct class of small noncoding RNAs (ncRNAs) that are expressed in germ cells and guide transposon silencing. Mature 23- to 28-nucleotide (nt) piRNAs are processed from long, noncoding transcripts (piRNA precursors) generated from distinct genomic regions dubbed piRNA clusters. Most piRNA clusters in the Drosophila genome are transcribed from both genomic strands and are therefore called dual-strand clusters; the rarer unistrand clusters are transcribed from one strand. A group of proteins composed of the HP1 homolog Rhino (Rhi), the RNA helicase UAP56, and two proteins of unknown function, Cutoff (Cuff) and Deadlock (Del), were shown to be essential for piRNA biogenesis from dual-strand but not unistrand clusters, indicating that piRNA biogenesis from these two types of clusters is quite different. Rhi, Del, and Cuff form the RDC complex that associates with chromatin of dual-strand but not unistrand clusters. The chromodomain of Rhi directly binds the H3K9me3 mark, which is enriched in the chromatin of dual-strand clusters (Hur, 2016).

    Thoc5 localizes in distinct nuclear foci that overlap with sites of piRNA transcription. In addition, it was observed that Thoc5 is highly enriched on noncoding piRNA transcripts compared with protein-coding pre-mRNAs. Binding of Thoc5 to unspliced piRNA precursors and localization to nuclear foci require Cuff protein that is present on the chromatin of piRNA clusters. The data reveal a novel, splicing-independent mechanism of TREX loading on nascent RNA. Thoc5 was also found to be necessary for accumulation of nascent piRNA precursors, indicating that TREX has a role in transcriptional control (Hur, 2016).

    This study found that Thoc5 and other components of the THO complex are required for Drosophila ovarian germ cell development and fertility. Further experiments revealed that THO is necessary for repression of multiple TE families. In agreement with othese results, several components of the THO complex were identified in a genome-wide RNAi screen for factors involved in TE repression in germ cells (Hur, 2016 and references therein).

    TE derepression in Thoc5-deficient ovaries is caused by a decrease in the abundance of piRNAs, ncRNAs that guide transcriptional and post-transcriptional repression of TEs in germ cells. Multiple lines of evidence suggest that THO is directly involved in piRNA biogenesis. First, several THO subunits (Thoc5, Thoc2, and Thoc7) localize at distinct nuclear foci that coincide with Rhi, a factor that is enriched on piRNA clusters and required for piRNA biogenesis. Second, two THO components (Thoc5 and Thoc7) were found to be enriched on long piRNA precursor transcripts and are necessary for their expression (Hur, 2016).

    In yeast and mammals, THO, the RNA helicase UAP56 (called Sub2 in yeast), and Aly/Ref together form the TREX complex, which is loaded on nascent RNA. UAP56 is required for piRNA biogenesis and binds to piRNA precursors. This study found that Thoc5 forms a complex with UAP56 in an RNA-independent fashion. Furthermore, Thoc5 and UAP56 colocalize in nuclear foci, and their localization, but not protein stability, is interdependent. These data suggest that a preformed TREX complex is loaded on piRNA precursors. Although TREX is considered to be a general factor, which is loaded on nascent RNA transcribed by RNA Pol II, the current RIP-seq data showed that association of TREX with different nascent transcripts is quantitatively different. Indeed, while <5% of mRNAs display greater than twofold enrichment in Thoc5 RIP, transcripts from all dual-strand piRNA clusters are enriched. Enrichment of TREX on piRNA precursors raises the question about the mechanism for its specific loading to these transcripts. This question is particularly intriguing, since association of TREX with nascent RNAs in metazoa was previously shown to be dependent on splicin, but piRNA precursors are not spliced (Zhang, 2014). Furthermore, splicing of piRNA precursors induced by depletion of Rhi was shown to have a negative effect on piRNA biogenesis. The clue to this puzzle was provided by the fact that both Thoc5 and UAP56 are required for piRNA biogenesis from only a subset of all genomic regions that generate piRNA; namely, dual-strand piRNA clusters. The same exclusive effect on dual-strand clusters was previously shown for Rhi and Cuff, two proteins that form a complex on chromatin of dual-strand clusters due to direct binding of the H3K9me3 histone mark by the chromodomain of Rhi. This study found that TREX and Cuff physically and genetically interact. First, Cuff is required for localization of both Thoc5 and UAP56 to nuclear foci, the sites of nascent piRNA precursor transcription. Second, Cuff copurifies with Thoc5 from cellular extract, and this interaction is at least partially RNA-independent, indicating that Cuff can bind the TREX complex either directly or through other proteins. Importantly, a close paralog of Cuff, CG9125, does not form a complex with TREX, supporting the specificity of the interaction. Finally, tethering of Cuff to an artificial intronless reporter enhances binding of TREX to the reporter transcript. This experiment provides direct evidence that the presence of Cuff in the vicinity of a nascent transcript stimulates the association of TREX with the RNA. Together, these results suggest that Cuff and TREX form a complex on dual-strand piRNA loci, and Cuff enhances loading of TREX on nascent RNA (Hur, 2016).

    Together with previously reported results, the current data can be integrated into a model that suggests that interaction with chromatin-bound Cuff helps to recruit TREX to dual-strand piRNA cluster loci in the nucleus to ensure its binding with nonspliced nascent piRNA transcripts. Previous studies showed that Cuff is enriched on chromatin of dual-strand piRNA clusters, likely through formation of the complex with Rhi that directly binds the histone H3K9me3. This study proposes that, similar to the result that were obtained using an artificial reporter, Cuff enhances loading of TREX on nascent piRNA precursors. Unfortunately, it is impossible to test the association of TREX with piRNA precursors in cuff mutants, as cuff deficiency leads to almost complete elimination of these RNAs. The current model proposes a novel splicing-independent mechanism of TREX loading in which a chromatin-binding protein (Rhi) and an adapter protein (Cuff) result in locus-specific, but sequence-independent, loading of an RNA-binding protein complex on nascent transcripts. It will be interesting to explore whether other proteins that associate with specific chromatin marks mediate loading of TREX on other intronless transcripts. It is hypothesized that chromatin structure and associated chromatin proteins might play crucial roles in guiding loading of other non-sequence-specific RNA-binding proteins on nascent RNA. Several RNA-binding proteins are cotranscriptionally loaded on nascent RNA and may even shuttle with processed mRNA to the cytoplasm. Therefore, the origin of an RNA in a particular chromatin environment might have long-lasting and far-reaching effects on its stability, subcellular localization, and translation efficiency. Quantitative profiling of the association between RNA-binding proteins and RNA originating from different chromatin environments will provide the ultimate test of this hypothesis (Hur, 2016).

    TREX was proposed to play roles in post-transcriptional pre-mRNA processing and nuclear export of mRNA. However, the current results showed that only a small number of genes change their expression in TREX mutants. Similar results were previously reported using knockdown of TREX components in fly and mammalian cells, suggesting that the function of TREX in mRNA biogenesis in metazoa might be redundant with functions of other factors. Using two independent approaches, analysis of chromatin-associated nascent transcripts and in situ RNA hybridization, this study showed that TREX is required for the accumulation of nascent piRNA precursors at sites of transcription. These experiments cannot formally rule out the possibility that TREX stabilizes nascent piRNA transcripts and prevents their cotranscriptional degradation. However, the decrease in Pol II occupancy on piRNA clusters in the thoc5e/1 mutant suggests that TREX might be required for efficient transcription of piRNA precursors. TREX has previously been shown to prevent formation of RNA–DNA hybrids (R loops) between the nascent transcript and the DNA template in mammalian cells, establishing a possible mechanism by which TREX could facilitate transcription. The impact of TREX on transcription does not eliminate the possibility that it also functions on later steps of piRNA biogenesis. Particularly, it will be important to determine whether TREX plays a role in the export of piRNA precursors from the nucleus to the cytoplasm, where final processing into mature piRNAs takes place (Hur, 2016).

    Temperature-responsive miRNAs in Drosophila orchestrate adaptation to different ambient temperatures

    The majority of Drosophila genes are expressed in a temperature-dependent manner, but the way in which small RNAs may contribute to this effect is completely unknown, since ideas of how small RNA transcriptomes change as a function of temperature are lacking. Applying high throughput sequencing techniques complemented by quantitative real-time PCR experiments, this study demonstrates that altered ambient temperature induces drastic, but reversible changes in sequence composition and total abundance of both, miRNA- and piRNA populations. Further, mRNA sequencing reveals that the expression of miRNAs and their predicted target transcripts correlates inversely, suggesting that temperature-responsive miRNAs drive adaptation to different ambient temperatures on the transcriptome level. Finally, shifts in temperature were shown to affect both, primary and secondary piRNA pools, and the observed aberrations are consistent with altered expression levels of the involved Piwi-pathway factors. It was further reasoned that enhanced ping-pong processing at 29 ° C is driven by dissolved RNA secondary structures at higher temperatures, uncovering target sites that are not accessible at low temperatures. Together, these results show that small RNAs are an important part of epigenetic regulatory mechanisms that ensure homeostasis and adaptation under fluctuating environmental conditions (Fast, 2017).

    A heterochromatin-dependent transcription machinery drives piRNA expression

    Nuclear small RNA pathways safeguard genome integrity by establishing transcription-repressing heterochromatin at transposable elements. This inevitably also targets the transposon-rich source loci of the small RNAs themselves. How small RNA source loci are efficiently transcribed while transposon promoters are potently silenced is not understood. This study show that, in Drosophila, transcription of PIWI-interacting RNA (piRNA) clusters-small RNA source loci in animal gonads-is enforced through RNA polymerase II pre-initiation complex formation within repressive heterochromatin. This is accomplished through Moonshiner, a paralogue of a basal transcription factor IIA (TFIIA) subunit, which is recruited to piRNA clusters via the heterochromatin protein-1 variant Rhino. Moonshiner triggers transcription initiation within piRNA clusters by recruiting the TATA-box binding protein (TBP)-related factor TRF2, an animal TFIID core variant. Thus, transcription of heterochromatic small RNA source loci relies on direct recruitment of the core transcriptional machinery to DNA via histone marks rather than sequence motifs, a concept that is a recurring theme in evolution (Andersen, 2017).

    This study has identified a heterochromatin-dependent transcription machinery in Drosophila that allows piRNA precursor production despite potent silencing of transposon-encoded promoters and enhancers. Moonshiner-dependent transcription, which cannot rely on recognition of DNA motifs because of their inaccessibility in heterochromatin, achieves locus specificity through Rhino, an HP1 variant that binds H3K9me3 marks at piRNA clusters. Thereby the cell allows transcription of transposon-rich loci into piRNA precursors while transcription of the same loci into functional transposon mRNAs is suppressed via heterochromatin-mediated exclusion of sequence-specific transcription factors (Andersen, 2017).

    Small RNA source loci embedded in heterochromatin and transcribed on both genomic strands are also a hallmark of genome defence pathways in plants and fungi. In fission yeast, a 'passive' mode of small RNA expression has been proposed, where Pol II transcribes small RNA precursors from pericentromeric regions during G1/S phases when heterochromatin is less condensed. In contrast, an active recruitment mode with conceptual similarities to the Moonshiner pathway occurs in plants. Here SHH1, a reader of H3K9me marks, recruits the plant-specific RNA polymerase IV to heterochromatin to transcribe small RNA precursors. Although SHH1 and Rhino both bind H3K9me residues, the two proteins are unrelated, suggesting that specification of small RNA source locus transcription via heterochromatin readers has evolved independently in animals and plants. Also in plants, small RNA precursor transcription initiates at 'YR' initiator sites dispersed on both genomic strands. Whether Moonshiner-mediated transcription, like that of plant Pol IV, depends on collaboration with nucleosome remodellers to access heterochromatic target loci is unclear. The reported interaction of TRF2 with the NURF chromatin remodelling complex supports this possibility. The recurring evolution of small RNA source locus transcription specified by chromatin marks rather than DNA sequence suggests that this constitutes a common alternative mode of transcriptional activation. The DNA inaccessibility of heterochromatin is thereby transformed into a specificity mark for non-canonical transcription activation. It is noted that the major Drosophila somatic piRNA cluster, flamenco, is transcribed from a single defined enhancer-driven promoter and avoids piRNA-mediated silencing because of the antisense orientation of the vast majority of the contained transposons. The production of plant siRNAs from Pol IV transcripts initiates a positive feedback loop: siRNA-mediated targeting leads to DNA methylation, which in turn increases H3K9 methylation, thereby bringing in SHH1 and Pol IV. In a similar fashion, production of Moonshiner-dependent piRNA precursors leads to generation of Piwi-bound piRNAs, which in turn guide H3K9 methylation and thereby Rhino recruitment. This explains how Piwi-mediated transcriptional silencing 'transforms' active transposon insertions into heterochromatic piRNA source loci with bidirectional transcription (Andersen, 2017).

    Rhino and the associated factors Deadlock and Cutoff are required for transcription of dual-strand piRNA clusters. Owing to its ability to inhibit co-transcriptional processes such as splicing and transcription termination, Cutoff has been suggested to be the main effector of this complex. Such an inhibition of termination is supported by data on cluster38C1, where transcription from defined promoters results in 10-15 kb transcripts in a Rhino-, Deadlock-, and Cutoff-dependent manner. Cutoff also interacts with the transcription/export (TREX) complex, which orchestrates several co-transcriptional processes and which is required for transcription of Rhino-dependent piRNA source loci. Together with the identification of Moonshiner/TRF2 as piRNA cluster transcription initiation factors, this suggests that Rhino acts as a molecular hub for several effector proteins that stimulate different (co)-transcriptional processes. Though Rhino is not conserved outside drosophilids, data from mouse studies support a conserved role of TRF2 in transcription of germline heterochromatin. In summary, this study has uncovered the molecular mechanism by which heterochromatic piRNA loci are transcribed in Drosophila and propose that the identified coupling of chromatin readers to basal transcription factors is a recurring theme in eukaryotic heterochromatin biology (Andersen, 2017).

    Diversity of P-element piRNA production among M' and Q strains and its association with P-M hybrid dysgenesis in Drosophila melanogaster

    Transposition of P elements in the genome causes P-M hybrid dysgenesis in Drosophila melanogaster. For the P strain, the P-M phenotypes are associated with the ability to express a class of small RNAs, called piwi-interacting small RNAs (piRNAs), that suppress the P elements in female gonads. However, little is known about the extent to which piRNAs are involved in the P-M hybrid dysgenesis in M' and Q strains, which show different abilities to regulate the P elements from P strains. To elucidate the molecular basis of the suppression of paternally inherited P elements, this study analyzed the mRNA and piRNA levels of P elements in the F1 progeny between males of a P strain and nine-line females of M' or Q strains (M' or Q progenies). M' progenies showed the hybrid dysgenesis phenotype, while Q progenies did not. Consistently, the levels of P-element mRNA in both the ovaries and F1 embryos were higher in M' progenies than in Q progenies, indicating that the M' progenies have a weaker ability to suppress P-element expression. The level of P-element mRNA was inversely correlated to the level of piRNAs in F1 embryos. Importantly, the M' progenies were characterized by a lower abundance of P-element piRNAs in both young ovaries and F1 embryonic bodies. The Q progenies showed various levels of piRNAs in both young ovaries and F1 embryonic bodies despite all of the Q progenies suppressing P-element transposition in their gonad. These results are consistent with an idea that the level of P-element piRNAs is a determinant for dividing strain types between M' and Q and that the suppression mechanisms of transposable elements, including piRNAs, are varied between natural populations (Wakisaka, 2017).

    piRNA-mediated regulation of transposon alternative splicing in the soma and germ line

    Transposable elements can drive genome evolution, but their enhanced activity is detrimental to the host and therefore must be tightly regulated. The Piwi-interacting small RNA (piRNA) pathway is vital for the regulation of transposable elements, by inducing transcriptional silencing or post-transcriptional decay of mRNAs. This study shows that piRNAs and piRNA biogenesis components regulate precursor mRNA splicing of P-transposable element transcripts in vivo, leading to the production of the non-transposase-encoding mature mRNA isoform in Drosophila germ cells. Unexpectedly, it was shown that the piRNA pathway components do not act to reduce transcript levels of the P-element transposon during P-M hybrid dysgenesis, a syndrome that affects germline development in Drosophila. Instead, splicing regulation is mechanistically achieved together with piRNA-mediated changes to repressive chromatin states, and relies on the function of the Piwi-piRNA complex proteins Asterix (also known as Gtsf1) and Panoramix (Silencio), as well as Heterochromatin protein 1a [HP1a; encoded by Su(var)205]. Furthermore, this machinery, together with the piRNA Flamenco cluster, not only controls the accumulation of Gypsy retrotransposon transcripts but also regulates the splicing of Gypsy mRNAs in cultured ovarian somatic cells, a process required for the production of infectious particles that can lead to heritable transposition events. These findings identify splicing regulation as a new role and essential function for the Piwi pathway in protecting the genome against transposon mobility, and provide a model system for studying the role of chromatin structure in modulating alternative splicing during development (Teixeira, 2017).

    Hybrid dysgenesis is a syndrome that affects progeny in a non-reciprocal fashion, being normally restricted to the offspring of crosses in which males carry transposable elements but which females lack. In Drosophila, the dysgenic traits triggered by the P-element DNA transposon are restricted to the germ line and include chromosomal rearrangements, high rates of mutation, and sterility. The impairment is most prominent when hybrids are grown at higher temperatures, with adult dysgenic females being completely sterile at 29°C. Despite the severe phenotypes, little is known about the development of germ cells during P-M dysgenesis. To address this, germline development was characterized in the progeny obtained from reciprocal crosses between w1118 (P-element-devoid strain) and Harwich (P-element-containing strain) flies at 29°C. In non-dysgenic progeny, germline development progressed normally throughout embryonic and larval stages, leading to fertile adults. Although the development of dysgenic germline cells was not disturbed during embryogenesis, germ cells decreased in number during early larval stages, leading to animals with no germ cells by late larval stages. These results indicate that the detrimental effects elicited by P-element activity are triggered early on during primordial germ cell (PGC) development in dysgenic progeny, leading to premature germ cell death (Teixeira, 2017).

    Maternally deposited small RNAs cognate to the P-element are thought to provide the 'P-cytotype' by conferring the transgenerationally inherited ability to protect developing germ cells against P-elements. Small RNA-based transposon regulation is typically mediated by either transcriptional silencing or post-transcriptional clearance of mRNAs, both of which result in a decrease in the accumulation of transposon mRNA. To understand how maternally provided small RNAs control P-elements in germ cells, this study focused on embryonic PGCs sorted from 4- to 20-h-old embryos generated from reciprocal crosses between w1118 and Harwich strains. Surprisingly, the accumulation of P-element RNA as measured by quantitative reverse transcription PCR (RT-qPCR) showed no change in dysgenic PGCs when compared to non-dysgenic PGCs. This indicates that P-cytotype small RNAs exert their function by means other than regulating P-element mRNA levels (Teixeira, 2017).

    P-element activity relies on production of a functional P-element transposase protein, the expression of which requires precursor mRNA (pre-mRNA) splicing of three introns. To analyse P-element RNA splicing in germ cells during hybrid dysgenesis, primers were designed that specifically anneal to spliced mRNA transcripts. The accumulation of spliced forms for the first two introns (IVS1 and IVS2) did not show changes in dysgenic PGCs when compared to non-dysgenic PGCs. By contrast, the accumulation of spliced transcripts for the third intron (IVS3) was substantially increased in dysgenic germ cells. Given that the overall accumulation of P-element mRNA showed no changes, the results indicate that the maternally provided P-cytotype can negatively regulate P-element IVS3 splicing and therefore inhibits the production of functional P-transposase in germ cells (Teixeira, 2017).

    Analysis of publically available small RNA sequencing data from 0-2-h-old embryos laid by Harwich females indicated that two classes of small RNAs cognate to the P-element are maternally transmitted: small interfering RNAs (siRNAs, 20-22-nucleotides long) and piRNAs (23-29 nucleotides long). To test the role of distinct small RNA populations on P-element expression, mutants were analyzed uniquely affecting each small RNA biogenesis pathway in the Harwich background. Mutations that disrupt siRNA biogenesis components Dicer-2 (Dcr-2) and Argonaute 2 (AGO2), or mutations ablating components of the piRNA biogenesis pathway, such as the Argonautes piwi, aubergine (aub), and Argonaute 3 (AGO3), as well as the RNA helicase vasa (vas) and spindle E (spn-E), did not affect P-element mRNA accumulation in adult ovaries as measured by RT-qPCR. However, mutations that disrupted piRNA biogenesis, and not the siRNA pathway, led to a strong and specific increase in the accumulation of IVS3-spliced mRNAs. RNA sequencing (RNA-seq) analysis on poly(A)-selected RNAs from aub and piwi mutant adult ovaries confirmed the specific effect on IVS3 splicing. To examine transposon expression in tissue, RNA fluorescent in situ hybridization (FISH) was performed using probes specific for the P-element and for the Burdock retrotransposon, a classic target of the germline piRNA pathway. In mutants affecting piRNA biogenesis, increased abundance of Burdock RNA was readily observed in germline tissues, with most of the signal accumulating close to the oocyte. By contrast, no difference was detected in the P-element RNA FISH signal in piRNA biogenesis mutants compared to control. Nuclear RNA foci observed in nurse cells were of similar intensity and number regardless of the genotype, and cytoplasmic signal showed no detectable difference. Therefore, the results indicate that in germ cells, piRNAs specifically modulate IVS3 splicing. This regulation is reminiscent of the well-documented mechanism that restricts P-element activity to germline tissues, which involves the expression of a host-encoded RNA binding repressor protein that negatively regulates IVS3 splicing in somatic tissues (Teixeira, 2017).

    In somatic tissues, P-element alternative splicing regulation is mediated by the assembly of a splicing repressor complex on an exonic splicing silencer element directly upstream of IVS3. To test whether the P-element IVS3 and flanking exon sequences were sufficient to trigger the piRNA-mediated splicing regulation in germ cells, a transgenic reporter system for IVS3 splicing was used in which a heterologous promoter (Hsp83) drives the expression of an IVS3-lacZ-neo fusion mRNA specifically in the germ line. Using RT-qPCR, the F1 progeny from reciprocal crosses between w1118 and Harwich flies were analyzed in the presence of the hsp83-IVS3-lacZ-neo reporter. The fraction of spliced mRNAs produced from the transgenic reporter was substantially increased in dysgenic compared to non-dysgenic adult ovaries, in agreement with previously reported results. Most importantly, genetic experiments confirmed that the repression of IVS3 splicing in germ cells relies on piRNA biogenesis, as the splicing repression observed with this reporter in non-dysgenic progeny was specifically abolished in adult ovaries of aub and vas mutants (Teixeira, 2017).

    Mechanistically, piRNA-mediated splicing regulation may be achieved through direct action of piRNA complexes on target pre-mRNAs carrying the IVS3 sequence or indirectly by piRNA-mediated changes in chromatin states. Piwi-interacting proteins such as Asterix (Arx) and Panoramix (Panx) are dispensable for piRNA biogenesis but are essential for establishing Piwi-mediated chromatin changes, possibly by acting as a scaffold to recruit histone-modifying enzymes and chromatin-binding proteins to target loci. To test the role of these chromatin regulators on P-element splicing, germline-specific RNA interference (RNAi) knockdown experiments were performed in the Harwich background. Similar to what was observed for the piRNA biogenesis components, germline knockdown of Arx and Panx showed no change in the accumulation of P-element RNA, but a strong and specific effect on IVS3 splicing in adult ovaries. The same pattern on IVS3 splicing was observed in the germline knockdown of HP1a and Maelstrom (Mael), both of which act downstream of Piwi-mediated targeting to modulate chromatin structure. The same genetic requirement for Panx for IVS3 splicing control was also confirmed when using the transgenic IVS3 splicing reporter, further indicating that Piwi-mediated chromatin changes at the target locus are involved in IVS3 splicing regulation. At target loci, Piwi complexes are known to mediate the deposition of the classic heterochromatin mark histone H3 lysine 9 trimethylation (H3K9me3). To assess the effect of piRNA-targeting on P-element chromatin marks directly, H3K9me3 chromatin immunoprecipitation was performed followed by sequencing (ChIP-seq) or quantitative PCR on adult ovaries of progeny from reciprocal crosses between w1118 and Harwich strains (to avoid developmental defects, ChIP was performed on F1 progeny raised at 18°C. This analysis revealed a specific loss of global H3K9me3 levels over P-element insertions in dysgenic progeny when compared to non-dysgenic progeny (Teixeira, 2017).

    To analyse the chromatin structure at individual P-element insertions, DNA sequencing (DNA-seq) data was used to identify all euchromatic insertions in the Harwich strain, and RNA-seq analysis was used to define transcriptionally active insertions. At transcriptionally active P-element euchromatic insertions, the spreading of H3K9me3 into the flanking genomic regions was readily observed in non-dysgenic progeny, but was completely absent in dysgenic offspring. Similarly, a reduction in H3K9me3 modification levels was also observed over the IVS3 transgenic reporter in dysgenic progeny when compared to non-dysgenic progeny. Interestingly, euchromatic insertions with no evidence of transcriptional activity were devoid of an H3K9me3 signal in both non-dysgenic and dysgenic crosses, providing further evidence for a model initially suggested in yeast and more recently proposed for Drosophila and mammals, in which H3K9me3 deposition by piRNA complexes would require transcription of the target loci. Mechanistically different from the well-described somatic repression, the results uncovered the existence of an unexpected piRNA-mediated, chromatin-based mechanism regulating IVS3 alternative splicing in germ cells (Teixeira, 2017).

    To expand the analysis, the literature was searched for other cases of transposon splicing regulation. Drosophila Gypsy elements are retrotransposons that have retrovirus-like, infective capacity owing to their envelope (Env) protein. These elements are expressed in somatic ovarian cells, in which they are regulated by the flamenco locus, a well-known piRNA cluster that is a soma-specific source of antisense piRNAs cognate to Gypsy. Interestingly, it has been shown that mutations in flamenco not only elicited the accumulation of Gypsy RNA, but also modulated pre-mRNA splicing, favouring the production of the env mRNA and therefore germline infection. To test whether the piRNA pathway, in addition to its role in regulating the accumulation of Gypsy RNA, is also responsible for modulating the splicing of Gypsy elements in somatic tissues, publically available RNA-seq data from poly(A)-selected RNAs extracted from in vivo cultures of ovarian somatic cells (OSCs) was analyzed. The analysis indicates that piwi knockdown was sufficient to modulate Gypsy splicing, favouring the accumulation of env-encoding mRNA. In agreement with a chromatin-mediated regulation of alternative splicing, RNAi depletion of Arx, Panx, HP1a and Mael, as well as knockdown of the histone linker H1, was sufficient to favour Gypsy splicing, recapitulating the effect caused by Piwi depletion. Notably, this was also the case for the H3K9 methyltransferase Setdb1, but not for the H3K9 methyltransferases Su(var)3-9 and G9a, indicating specific genetic requirements. Taken together, the results indicate that the piRNA pathway, through its role in mediating changes in chromatin states, regulates the splicing of transposon pre-mRNAs in both somatic and germline tissues (Teixeira, 2017).

    Using P-M hybrid dysgenesis as a model, this study hasa uncovered splicing regulation elicited by chromatin changes as a previously unknown mechanism by which the piRNA pathway protects the genome from the detrimental effects of transposon activity. Splicing control at piRNA-target loci is likely to be mechanistically different from what has been observed for germline piRNA clusters given the low enrichment of the HP1 homologue Rhino (also known as HP1D) protein, which is required for piRNA cluster RNA processing, over the endogenous P-element insertions in the Harwich genome or over the transgenic IVS3 splicing reporter in non-dysgenic and dysgenic progeny (as measured by ChIP-qPCR). Because small RNA-based systems leading to chromatin mark changes at target loci are pervasive in eukaryotes, it is expected that this new type of targeted regulation is of importance in settings far beyond the scope of the piRNA pathway and Drosophila. Indeed, small RNA-guided DNA methylation over the LINE retrotransposon Karma was recently shown to modulate alternative splicing in oil palm, disrupting nearby gene expression and ultimately affecting crop yield. In this context, small RNA-based control of chromatin structure may be crucially important in genomes with a high content of intronic transposon insertions, such as the human genome, by providing a mechanism to suppress exonization of repeat elements. Although the means by which piRNA-mediated changes in chromatin states could regulate alternative splicing remain to be determined, it is tempting to speculate that piRNA pathway components do so by co-transcriptionally modulating interactions between RNA polymerase II and the spliceosome (Teixeira, 2017).

    Spontaneous gain of susceptibility suggests a novel mechanism of resistance to hybrid dysgenesis in Drosophila virilis

    Syndromes of hybrid dysgenesis (HD) have been critical for understanding of the transgenerational maintenance of genome stability by piRNA. HD in D. virilis represents a special case of HD since it includes simultaneous mobilization of a set of TEs that belong to different classes. The standard explanation for HD is that eggs of the responder strains lack an abundant pool of piRNAs corresponding to the asymmetric TE families transmitted solely by sperm. However, there are several strains of D. virilis that lack asymmetric TEs, but exhibit a "neutral" cytotype that confers resistance to HD. To characterize the mechanism of resistance to HD, a comparative analysis of the landscape of ovarian small RNAs was performed in strains that vary in their resistance to HD mediated sterility. It was demonstrated that resistance to HD cannot be solely explained by a maternal piRNA pool that matches the assemblage of TEs that likely cause HD. In support of this, a cytotype shift from neutral (N) to susceptible (M) was observed in a strain devoid of all major TEs implicated in HD. This shift occurred in the absence of significant change in TE copy number and expression of piRNAs homologous to asymmetric TEs. Instead, this shift is associated with a change in the chromatin profile of repeat sequences unlikely to be causative of paternal induction. Overall, these data suggest that resistance to TE-mediated sterility during HD may be achieved by mechanisms that are distinct from the canonical syndromes of HD (Funikov, 2018).

    Recurrent gene duplication diversifies genome defense repertoire in Drosophila

    Transposable elements (TEs) comprise large fractions of many eukaryotic genomes and imperil host genome integrity. The host genome combats these challenges by encoding proteins that silence TE activity. Both the introduction of new TEs via horizontal transfer and TE sequence evolution requires constant innovation of host-encoded TE silencing machinery to keep pace with TEs. One form of host innovation is the adaptation of existing, single-copy host genes. Indeed, host suppressors of TE replication often harbor signatures of positive selection. Such signatures are especially evident in genes encoding the piwi-interacting-RNA pathway of gene silencing, for example, the female germline-restricted TE silencer, HP1D/Rhino. Host genomes can also innovate via gene duplication and divergence. However, the importance of gene family expansions, contractions, and gene turnover to host genome defense has been largely unexplored. This study functionally characterize Oxpecker, a young, tandem duplicate gene of HP1D/rhino. Oxpecker was shown to support female fertility in Drosophila melanogaster and silences several TE families that are incompletely silenced by HP1D/Rhino in the female germline. It was further shown that, like Oxpecker, at least ten additional, structurally diverse, HP1D/rhino-derived daughter and “granddaughter” genes emerged during a short 15-million year period of Drosophila evolution. These young paralogs are transcribed primarily in germline tissues, where the genetic conflict between host genomes and TEs plays out. These findings suggest that gene family expansion is an underappreciated yet potent evolutionary mechanism of genome defense diversification (Levine, 2016).


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    date revised: 25 June 2018

    Zygotically transcribed genes

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