The Interactive Fly

Transposons

  • Transposable elements in Drosophila
  • Mechanisms of LTR-Retroelement Transposition: Lessons from Drosophila melanogaster
  • In between: gypsy in Drosophila melanogaster reveals new insights into endogenous retrovirus evolution.
  • The centrosomal protein CP190 is a component of the gypsy chromatin insulator
  • Distribution, evolution, and diversity of retrotransposons at the flamenco locus reflect the regulatory properties of piRNA cluster
  • The somatic piRNA pathway controls germline transposition over generations
  • RNA editing regulates transposon-mediated heterochromatic gene silencing
  • The Drosophila fragile X mental retardation protein participates in the piRNA pathway
  • Unique transposon landscapes are pervasive across Drosophila melanogaster genomes
  • Antisense transcription of retrotransposons in Drosophila: The origin of endogenous small interfering RNA precursors
  • piRNAs are associated with diverse transgenerational effects on gene and transposon expression in a hybrid dysgenic syndrome of D. virilis
  • Drosophila IRBP bZIP heterodimer binds P-element DNA and affects hybrid dysgenesis
  • Production of small non-coding RNAs from the flamenco locus is regulated by the gypsy retrotransposon of Drosophila melanogaster
  • Natural variation of piRNA expression affects immunity to transposable elements
  • Spontaneous gain of susceptibility suggests a novel mechanism of resistance to hybrid dysgenesis in Drosophila virilis
  • piRNA-mediated regulation of transposon alternative splicing in the soma and germ line
  • Conserved noncoding elements influence the transposable element landscape in Drosophila
  • Double insertion of transposable elements provides a substrate for the evolution of satellite DNA
  • Extensive exchange of transposable elements in the Drosophila pseudoobscura group
  • Antisense transcription of retrotransposons in Drosophila: The origin of endogenous small interfering RNA precursors
  • The evolution of small-RNA-mediated silencing of an invading transposable element
  • Stress affects the epigenetic marks added by natural transposable element insertions in Drosophila melanogaster
  • Subcellular localization and Egl-mediated transport of telomeric retrotransposon HeT-A ribonucleoprotein particles in the Drosophila germline and early embryogenesis
  • Drosophila small ovary gene is required for transposon silencing and heterochromatin organisation and ensures germline stem cell maintenance and differentiation
  • A robust transposon-endogenizing response from germline stem cells
  • Pathogenic tau-induced piRNA depletion promotes neuronal death through transposable element dysregulation in neurodegenerative tauopathies
  • Population specific dynamics and selection patterns of transposable element insertions in European natural populations
  • SQuIRE reveals locus-specific regulation of interspersed repeat expression
  • Dynamic Interactions between the genome and an endogenous retrovirus: Tirant in Drosophila simulans wild-type strains
  • Stress response, behavior, and development are shaped by transposable element-induced mutations in Drosophila
  • Maelstrom represses canonical Polymerase II transcription within bi-directional piRNA clusters in Drosophila melanogaster
  • Transposon silencing in the Drosophila female germline is essential for genome stability in progeny embryos
  • The integrity of piRNA clusters is abolished by insulators in the Drosophila germline
  • Dynamics of transposable element invasions with piRNA clusters
  • Cellular labeling of endogenous retrovirus replication (CLEVR) reveals de novo insertions of the gypsy retrotransposable element in cell culture and in both neurons and glial cells of aging fruit flies

    The centrosomal protein CP190 is a component of the gypsy chromatin insulator

    Chromatin insulators, or boundary elements, affect promoter-enhancer interactions and buffer transgenes from position effects. The gypsy insulator of Drosophila, a component of the gypsy long terminal repeat (LTR) retrotransposon, is bound by a protein complex with two characterized components, the zinc finger protein Suppressor of Hairy-wing [Su(Hw)] and Mod(mdg4)2.2, which is one of the multiple spliced variants encoded by the modifier of mdg4 [mod(mdg4)] gene. A genetic screen for dominant enhancers of the mod(mdg4) phenotype identified the Centrosomal Protein 190 (CP190) as an essential constituent of the gypsy insulator. The function of the centrosome is not affected in CP190 mutants whereas gypsy insulator activity is impaired. CP190 associates physically with both Su(Hw) and Mod(mdg4)2.2 and colocalizes with both proteins on polytene chromosomes. CP190 does not interact directly with insulator sequences present in the gypsy retrotransposon but binds to a previously characterized endogenous insulator, and it is necessary for the formation of insulator bodies. The results suggest that endogenous gypsy insulators contain binding sites for CP190, which is essential for insulator function, and may or may not contain binding sites for Su(Hw) and Mod(mdg4)2.2 (Pai, 2004).

    A genetic screen for dominant enhancers of mod(mdg4) has resulted in the identification of CP190 as a third component of the gypsy insulator. CP190 is present at gypsy retrotransposon insulator sites and overlaps extensively with Su(Hw) and Mod(mdg4)2.2 at presumed endogenous insulators. CP190 displays a specific distribution pattern on polytene chromosomes, showing significant overlap with Su(Hw) and Mod(mdg4)2.2 at the junctions between transcriptionally inert bands and transcriptionally active interbands. Similar localization patterns have been reported for other insulators. For example, the faswb insulator at the notch locus and the BEAF-32 protein of the scs' insulator are also present at the boundaries between bands and interbands. Results suggest that CP190 can bind DNA on its own or can be tethered to the chromosome through interactions with Su(Hw). Mutations in the CP190 gene impair the function of the insulator present in the gypsy retrotransposon without affecting the presence of Su(Hw) and Mod(mdg4)2.2, suggesting an essential task for CP190 in the activity of this insulator. In addition, the lethality of CP190 mutants suggests a critical role for the CP190 protein in the function of gypsy endogenous insulators. This essential role may be a consequence of the requirement of CP190 for the formation of insulator bodies in the nuclei of diploid cells (Pai, 2004).

    The insulator present in the gypsy retrotransposon contains only Su(Hw) binding sites, and CP190 is present in this insulator through direct interactions with Su(Hw). The gypsy insulator contains 12 Su(Hw) binding sites, and at least four are needed for insulator activity. However, clusters of three or more Su(Hw) binding sites are rare in the genome. Therefore, a critical question is whether the sites of Su(Hw) and Mod(mdg4)2.2 localization present throughout the genome truly function as insulators. The presence of CP190 at these sites and its ability to bind DNA might explain this apparent paradox. For example, the endogenous insulator present in the yellow-achaete region has only two binding sites for Su(Hw). Nevertheless, the y454 fragment containing this insulator is able to bind CP190, suggesting that this protein might act in concert with Su(Hw) to confer insulator activity. It is therefore possible that endogenous gypsy insulators are composed of binding sites for Su(Hw) and/or for CP190 and, together with Mod(mdg4)2.2, form a complex. Endogenous gypsy insulators may have few or no Su(Hw) binding sites, and they may rely on CP190 to bind DNA and tether other insulator components such as Mod(mdg4)2.2 via protein-protein interactions (Pai, 2004).

    Previous studies have suggested that gypsy insulators separated at a distance in the genome may come together and form large insulator bodies in the nucleus during interphase. These aggregates represent higher order structures of chromatin and are implicated in the regulation of gene expression by compartmentalizing the genome into transcriptionally independent domains. The formation of these aggregates appears to require Mod(mdg4) function because the large aggregates are missing in mod(mdg4) mutants. The formation of gypsy insulator bodies is severely impaired also in CP190 mutants, suggesting that CP190 plays an essential role in the formation of these bodies and in the establishment of the chromatin domain organization mediated by gypsy endogenous insulators. It is possible that the BTB/POZ protein-protein interaction domains of both CP190 and Mod(mdg4)2.2 are required for and contribute to the stability of the interactions among insulator sites. In vitro-expressed CP190 lacking the BTB/POZ domain is soluble, whereas the wt protein is not, further suggesting that CP190 might exist as a complex with itself or other proteins in vivo, and the formation of this complex is likely mediated by the BTB/POZ domain. However, because CP190 is present at the gypsy insulator in the absence of Mod(mdg4)2.2 protein, the interaction between these two proteins may not be crucial for CP190 recruitment to the insulator (Pai, 2004).

    Previous studies have identified CP190 as a centrosome-specific protein during mitosis that also associates with chromatin during interphase. Although many of these studies have focused on the possible role of CP190 during cell division, the current results suggest that centrosomal function and cell division are not affected in CP190 mutants. This conclusion is supported by independent studies of CP190 function during the cell cycle. The main function of CP190 might then be to regulate chromosome-related processes during interphase. Several lines of evidence suggest that this role is related to the function of the gypsy insulator: mutations in CP190 alter gypsy-induced phenotypes; CP190 colocalizes with Su(Hw) and Mod(mdg4)2.2 on polytene chromosomes and in diploid cell nuclei, and CP190 associates physically with gypsy insulator components in vitro and in vivo. However, the centrosomal localization of CP190 might also be important for its role in the gypsy insulator despite being unnecessary for cell cycle progression. The centrosome could either be a temporary storage site for CP190 during mitosis, or a site for a mitosis-specific modification that could be important for CP190 reassociation with chromosomes later in the cell cycle. The presence of CP190 in the centrosome could also be related to the regulation of the level of this protein in the cell. In fact, it has been shown that some chromatin-binding proteins are targeted to the centrosome for degradation. Alternatively, the presence of CP190 at the centrosome might be related to a possible role in the ubiquitin modification pathway. Recent findings have linked BTB/POZ domain proteins to ubiquitin E3 ligase function, some of which are known to be present at the centrosome. CP190 may be involved in similar types of interactions as an adaptor for ubiquitin E3 ligases and might target associated insulator proteins to the centrosome during mitosis for ubiquitination and/or degradation, which in turn may be required for properly reestablishing chromosome domain boundaries after mitosis (Pai, 2004).

    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).

    The somatic piRNA pathway controls germline transposition over generations

    Transposable elements (TEs) are parasitic DNA sequences that threaten genome integrity by replicative transposition in host gonads. The Piwi-interacting RNAs (piRNAs) pathway is assumed to maintain Drosophila genome homeostasis by downregulating transcriptional and post-transcriptional TE expression in the ovary. However, the bursts of transposition that are expected to follow transposome derepression after piRNA pathway impairment have not yet been reported. This study shows, at a genome-wide level, that piRNA loss in the ovarian somatic cells boosts several families of the endogenous retroviral subclass of TEs, at various steps of their replication cycle, from somatic transcription to germinal genome invasion. For some of these TEs, the derepression caused by the loss of piRNAs is backed up by another small RNA pathway (siRNAs) operating in somatic tissues at the post transcriptional level. Derepressed transposition during 70 successive generations of piRNA loss exponentially increases the genomic copy number by up to 10-fold (Barckmann, 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. 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).

    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).

    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).

    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).

    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).

    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).

    Francis, M. J., Roche, S., Cho, M. J., Beall, E., Min, B., Panganiban, R. P. and Rio, D. C. (2016). Drosophila IRBP bZIP heterodimer binds P-element DNA and affects hybrid dysgenesis. Proc Natl Acad Sci U S A 113(46): 13003-13008. PubMed ID: 27799520

    Drosophila IRBP bZIP heterodimer binds P-element DNA and affects hybrid dysgenesis

    In Drosophila, P-element transposition causes mutagenesis and genome instability during hybrid dysgenesis. The P-element 31-bp terminal inverted repeats (TIRs) contain sequences essential for transposase cleavage and have been implicated in DNA repair via protein-DNA interactions with cellular proteins. The identity and function of these cellular proteins were unknown. Biochemical characterization of proteins that bind the TIRs identified a heterodimeric basic leucine zipper (bZIP) complex between an uncharacterized protein that is termed 'Inverted Repeat Binding Protein (IRBP) 18' and its partner Xrp1. The reconstituted IRBP18/Xrp1 heterodimer binds sequence-specifically to its dsDNA-binding site within the P-element TIRs. Genetic analyses implicate both proteins as critical for repair of DNA breaks following transposase cleavage in vivo. These results identify a cellular protein complex that binds an active mobile element and plays a more general role in maintaining genome stability (Francis, 2016).

    A role for the IRBP complex in the P-element transposition reaction has been postulated since initial identification of its sequence-specific DNA-binding properties (Staveley, 1995; Beall, 1994; Beall, 1996; Rio, 1988). The organizational overlap between IRBP DNA binding and transposase cleavage sites makes the IRBP complex a prime cellular candidate to influence some aspect of the P-element transposition cycle. Several putative IRBP proteins copurified with observed IRBP DNA-binding activity or unambiguously promoted DNA repair posttransposase P-element cleavage (Ku 70 and mus309/DmBLM). None, however, could reconstitute site-specific DNA binding to the P-element 31-bp TIRs (Francis, 2016).

    This report identifies a bZIP heterodimer between a C/EBP family member IRBP18 and a drosophilid-specific protein Xrp1 as the sequence-specific DNA-binding subunits of a larger multiprotein IRBP complex that binds to the P-element TIRs. These proteins work in concert to facilitate efficient DNA repair following P-element transposase-mediated DNA cleavage. Finally, these proteins are required more generally in the cellular DNA damage response and DSB repair in the absence of P elements (Francis, 2016).

    Repair of P-element~induced DNA breaks occurs predominantly through two distinct DSB repair pathways: Non-homologous end joining (NHEJ) or a variant of classical homologous recombinational repair, synthesis-dependent strand annealing (SDSA), The choice between these two pathways is dictated by cell-cycle location, the availability of pathway substrates, and tissue type. Because the IRBP homozygous mutant males are sterile, it is not possible at present to use any of the established DNA repair reporter strains to determine in which DNA repair pathway IRBP18 and Xrp1 participates. Future would should determine if the IRBP18/Xrp1 heterodimer can bind directly to different DNA repair intermediates and thus provide a direct link between the bZIP heterodimer and DNA repair (Francis, 2016).

    A role for bZIP proteins and specifically mammalian C/EBP proteins in DNA repair is well established. In human and mouse keratinocytes, UV-B UV DNA damage induces p53-dependent transcriptional activation of both the C/EBPα and β genes. C/EBPα expressed in prostate cancer cells where it interacts with the DNA repair proteins Ku p70/p80 heterodimer and the poly (ADP ribose) polymerase 1 (PARP-1). Notably, Drosophila p53 up-regulates three bZIP proteins (CG6272/IRBP18, CG17836/Xrp1, and CG15479/Mabiki) upon DNA damage. CG15479/Mabiki is a novel regulator of caspase-independent cell death of excess cells in the expanded head region of 6x-bcd embryos and is thought to work in concert with other caspase-independent cell death mechanisms to ensure proper development. Genetic deletion of the CG17836/Xrp1 gene resulted in a DNA repair phenotype when challenged with ionizing radiation (Akdemir, 2007). Additionally, the Dmp53 DNA damage-induced apoptotic response was unaffected in the Xrp1 mutants, suggesting that Xrp1 functions to preserve genome stability through a pathway independent of apoptosis (Akdemir, 2007). Although this study has demonstrated that IRBP18 and Xrp1 share a similar function in DNA repair, more experiments are needed to understand how these proteins work downstream of p53 transactivation (Francis, 2016).

    D. melanogaster uses multiple endogenous mechanisms to limit P-element transposition. Expression of catalytically active transposase is restricted to the germline by tissue-specific pre-mRNA splicing regulation. The germline piwi-interacting RNA pathway has been demonstrated to repress transposition in trans and plays a critical role in host adaptation to newly invaded P elements. It is proposde that the IRBP18/Xrp1 heterodimer recognizes new P elements and that its native function is to facilitate repair of breaks to maintain genomic stability during a genotoxic event such as ionizing radiation or the massive P-element mobilization that occurs following a hybrid dysgenic cross (Francis, 2016).

    bZIP proteins are well suited to recognize newly invaded foreign DNA due to their inherit ability to form multiple heterodimers. IRBP18 and Xrp1, for example, form heterodimers with other several other bZIP proteins; the net result is expansion of the repertoire of DNA sequences that can be bound. This library of bZIP dimers can be deployed to recognize foreign DNA as part of a survival mechanism against the genome instability created by foreign DNA invasion. In humans, mice and Drosophila p53 transactivate steady-state levels of several bZIP proteins in response to DNA damage. It is unclear how changes in steady-state levels of these DNA repair proteins determine dimer formation or function. What is clear is that bZIP proteins are important players in DNA repair and maintenance of genome stability. In this respect, the IRBP18/Xrp1 heterodimer is a newly identified component of the interconnected pathways to combat the genotoxic effects of mass invasion/mobilization of transposons (Francis, 2016).

    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, 2016).

    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).

    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).

    Conserved noncoding elements influence the transposable element landscape in Drosophila

    Highly conserved noncoding elements (CNEs) constitute a significant proportion of the genomes of multicellular eukaryotes. The function of most CNEs remains elusive, but growing evidence indicates they are under some form of purifying selection. Noncoding regions in many species also harbor large numbers of transposable element (TE) insertions, which are typically lineage specific and depleted in exons because of their deleterious effects on gene function or expression. However, it is currently unknown whether the landscape of TE insertions in noncoding regions is random or influenced by purifying selection on CNEs. This study combine comparative and population genomic data in Drosophila melanogaster to show that abundance of TE insertions in intronic and intergenic CNEs is reduced relative to random expectation, supporting the idea that selective constraints on CNEs eliminate a proportion of TE insertions in noncoding regions. However, no evidence was found for differences in the allele frequency spectra for polymorphic TE insertions in CNEs versus those in unconstrained spacer regions, suggesting that the distribution of fitness effects acting on observable TE insertions is similar across different functional compartments in noncoding DNA. The results provide evidence that selective constraints on CNEs contribute to shaping the landscape of TE insertion in eukaryotic genomes, and provide further evidence that CNEs are indeed functionally constrained and not simply mutational cold spots (Manee, 2018).

    Double insertion of transposable elements provides a substrate for the evolution of satellite DNA

    Eukaryotic genomes are replete with repeated sequences in the form of transposable elements (TEs) dispersed across the genome or as satellite arrays, large stretches of tandemly repeated sequences. Many satellites clearly originated as TEs, but it is unclear how mobile genetic parasites can transform into megabase-sized tandem arrays. Comprehensive population genomic sampling is needed to determine the frequency and generative mechanisms of tandem TEs, at all stages from their initial formation to their subsequent expansion and maintenance as satellites. The best available population resources, short-read DNA sequences, are often considered to be of limited utility for analyzing repetitive DNA due to the challenge of mapping individual repeats to unique genomic locations. A new pipeline called ConTExt has been developed that demonstrates that paired-end Illumina data can be successfully leveraged to identify a wide range of structural variation within repetitive sequence, including tandem elements. By analyzing 85 genomes from five populations of Drosophila melanogaster, it was discovered that TEs commonly form tandem dimers. The results further suggest that insertion site preference is the major mechanism by which dimers arise and that, consequently, dimers form rapidly during periods of active transposition. This abundance of TE dimers has the potential to provide source material for future expansion into satellite arrays, and one such copy number expansion of the DNA transposon hobo was found to approximately 16 tandem copies in a single line. The very process that defines TEs-transposition-thus regularly generates sequences from which new satellites can arise (McGurk, 2018).

    Extensive exchange of transposable elements in the Drosophila pseudoobscura group

    As species diverge, so does their transposable element (TE) content. Within a genome, TE families may eventually become dormant due to host-silencing mechanisms, natural selection and the accumulation of inactive copies. The transmission of active copies from a TE families, both vertically and horizontally between species, can allow TEs to escape inactivation if it occurs often enough, as it may allow TEs to temporarily escape silencing in a new host. Thus, the contribution of horizontal exchange to TE persistence has been of increasing interest. This study annotated TEs in five species with sequenced genomes from the D. pseudoobscura species group, and curated a set of TE families found in these species. Compared to host genes, many TE families showed lower neutral divergence between species, consistent with recent transmission of TEs between species. Despite these transfers, there are differences in the TE content between species in the group. It is concluded that the TE content is highly dynamic in the D. pseudoobscura species group, frequently transferring between species, keeping TEs active. This result highlights how frequently transposable elements are transmitted between sympatric species and, despite these transfers, how rapidly species TE content can diverge (Hill, 2018).

    The evolution of small-RNA-mediated silencing of an invading transposable element

    Transposable elements (TEs) are genomic parasites that impose fitness costs on their hosts by producing deleterious mutations and disrupting gametogenesis. Host genomes avoid these costs by regulating TE activity, particularly in germline cells where new insertions are heritable and TEs are exceptionally active. However, the capacity of different TE-associated fitness costs to select for repression in the host, and the role of selection in the evolution of TE regulation more generally, remain controversial. This study used forward, individual-based simulations to examine the evolution of small-RNA-mediated TE regulation, a conserved mechanism for TE repression that is employed by both prokaryotes and eukaryotes. To design and parameterize a biologically realistic model, this study drew on an extensive survey of empirical studies of the transposition and regulation of P-element DNA transposons in Drosophila melanogaster. Even under conservative assumptions, where small-RNA-mediated regulation reduces transposition only, repression evolves rapidly and adaptively after the genome is invaded by a new TE in simulated populations. It was further shown that the spread of repressor alleles through simulated populations is greatly enhanced by two additional TE-imposed fitness costs: dysgenic sterility and ectopic recombination. Finally, it was demonstrated that the adaptive mutation rate to repression is a critical parameter that influences both the evolutionary trajectory of host repression and the associated proliferation of TEs after invasion in simulated populations. These findings suggest that adaptive evolution of TE regulation may be stronger and more prevalent than previously appreciated, and provide a framework for interpreting empirical data (Kelleher, 2018).

    Stress affects the epigenetic marks added by natural transposable element insertions in Drosophila melanogaster

    Transposable elements are emerging as an important source of cis-acting regulatory sequences and epigenetic marks that could influence gene expression. However, few studies have dissected the role of specific transposable element insertions on epigenetic gene regulation. Bari-Jheh is a natural transposon that mediates resistance to oxidative stress by adding cis-regulatory sequences that affect expression of nearby genes. This work, integrated publicly available ChIP-seq and piRNA data with chromatin immunoprecipitation experiments to get a more comprehensive picture of Bari-Jheh molecular effects. Bari-Jheh was shown to be enriched for H3K9me3 in nonstress conditions, and for H3K9me3, H3K4me3 and H3K27me3 in oxidative stress conditions, which is consistent with expression changes in adjacent genes. It was further shown that under oxidative stress conditions, H3K4me3 and H3K9me3 spread to the promoter region of Jheh1 gene. Finally, another insertion of the Bari1 family was associated with increased H3K27me3 in oxidative stress conditions suggesting that Bari1 histone marks are copy-specific. It is concluded that besides adding cis-regulatory sequences, Bari-Jheh influences gene expression by affecting the local chromatin state (Guio, 2018).

    Subcellular localization and Egl-mediated transport of telomeric retrotransposon HeT-A ribonucleoprotein particles in the Drosophila germline and early embryogenesis

    The study of the telomeric complex in oogenesis and early development is important for understanding the mechanisms which maintain genome integrity. Telomeric transcripts are the key components of the telomeric complex and are essential for regulation of telomere function. The biogenesis of transcripts generated by the major Drosophila telomere repeat HeT-A in oogenesis and early development was studied with disrupted telomeric repeat silencing. In wild type ovaries, HeT-A expression is downregulated by the Piwi-interacting RNAs (piRNAs). By repressing piRNA pathway, this study showed that overexpressed HeT-A transcripts interact with their product, RNA-binding protein Gag-HeT-A, forming ribonucleoprotein particles (RNPs) during oogenesis and early embryonic development. Moreover, during early stages of oogenesis, in the nuclei of dividing cystoblasts, HeT-A RNP form spherical structures, which supposedly represent the retrotransposition complexes participating in telomere elongation. During the later stages of oogenesis, abundant HeT-A RNP are detected in the cytoplasm and nuclei of the nurse cells, as well as in the cytoplasm of the oocyte. Further on, it was demonstrate that HeT-A products co-localize with the transporter protein Egalitarian (Egl) both in wild type ovaries and upon piRNA loss. This finding suggests a role of Egl in the transportation of the HeT-A RNP to the oocyte using a dynein motor. Following germline piRNA depletion, abundant maternal HeT-A RNP interacts with Egl resulting in ectopic accumulation of Egl close to the centrosomes during the syncytial stage of embryogenesis. Given the essential role of Egl in the proper localization of numerous patterning mRNAs, it is suggested that its abnormal localization likely leads to impaired embryonic axis specification typical for piRNA pathway mutants (Kordyukova, 2018).

    Drosophila small ovary gene is required for transposon silencing and heterochromatin organisation and ensures germline stem cell maintenance and differentiation

    Self-renewal and differentiation of stem cells is one of the fundamental biological phenomena relying on proper chromatin organisation. This study describes a novel chromatin regulator encoded by the Drosophila small ovary (sov) gene. sov was shown to be required in both the germline stem cells (GSCs) and the surrounding somatic niche cells to ensure GSC survival and differentiation. Sov maintains niche integrity and function by repressing transposon mobility, not only in the germline, but also in the soma. Protein interactome analysis of Sov revealed an interaction between Sov and HP1a. In the germ cell nuclei, Sov co-localises with HP1a, suggesting that Sov affects transposon repression as a component of the heterochromatin. In a position effect variegation assay, a dominant genetic interaction was found between sov and HP1a, indicating their functional cooperation in promoting the spread of heterochromatin. An in vivo tethering assay and FRAP analysis revealed that Sov enhances heterochromatin formation by supporting the recruitment of HP1a to the chromatin. A model is proposed in which sov maintains GSC niche integrity by regulating transposon silencing and heterochromatin formation (Jankovics, 2018).

    A robust transposon-endogenizing response from germline stem cells

    The heavy occupancy of transposons in the genome implies that existing organisms have survived from multiple, independent rounds of transposon invasions. However, how and which host cell types survive the initial wave of transposon invasion has remained unclear. This study shows that the germline stem cells can initiate a robust adaptive response that rapidly endogenizes invading P element transposons by activating the DNA damage checkpoint and piRNA production. Temperature modulates the P element activity in germline stem cells, establishing a powerful tool to trigger transposon hyper-activation. Facing vigorous invasion, Drosophila first shut down oogenesis and induce selective apoptosis. Interestingly, a robust adaptive response occurs in ovarian stem cells through activation of the DNA damage checkpoint. Within 4 days, the hosts amplify P element-silencing piRNAs, repair DNA damage, subdue the transposon, and reinitiate oogenesis. It is proposed that this robust adaptive response can bestow upon organisms the ability to survive recurrent transposon invasions throughout evolution (Moon, 2018).

    Considered as 'selfish DNA sequences,' transposons have heavily accumulated in the genome of nearly all organisms during evolution. Although capable of fueling genomic divergence, the transposon invasion process is disruptive to host cells and often severely impacts host fertility or even survival. Therefore, taming invading transposons is an essential and endless task for the host organism. In this study, by using P element invasion as a model, temperature shifting was established as a powerful tool to adjust the intensity of transposon invasion. By investigating the response from the Drosophila adult ovaries, in which P element activity and germ cell development can be measured in detail, a robust transposon-endogenizing mechanism from the germline stem cells was uncovered. Centered on the key DNA damage checkpoint component, Chk2, this robust adaptive response renders hosts the ability to permanently silence invading transposons within just 4 days (Moon, 2018).

    GFP::Vasa mobilization assay shows that the P element actively hops in germline stem cells. Does the P element also mobilize in other ovarian cells? Since nurse cells are polyploid and the developing oocytes are transcriptionally inactive, the current assay could not faithfully monitor P element mobilization in them. However, previous study shows that nurse cells express the protein P-element somatic inhibitor (PSI), which can block intron removal of P element transcripts and lead to the production of inactive transposases. Therefore, it is unlikely that P elements mobilize within developing egg chambers. As a type of DNA transposon, which employs the cut-and-paste mechanism for transposition, P elements cannot directly increase their copy number through mobilization. Instead, the propagation is likely achieved via homologous repair from the sister DNA strand during S-phase of the cell cycle. Hence, to amplify themselves during Drosophila oogenesis, perhaps P elements evolved to preferentially mobilize in the dividing germline stem cells but not in the developing oocytes, which are under cell cycle arrest (Moon, 2018).

    By investigating adult oogenesis of Drosophila, this study uncovered the Chk2-mediated adaptive response from germline stem cells upon P element transposon invasion (Moon, 2018).

    Interestingly, it appears that arrested germ cells are not equally capable of taming transposons, and Chk2 activation promotes adaptation by eliminating the cells with lower competency. Several lines of evidence support the occurrence of selective cell elimination. First, a significant increase in cell death was detected once P elements became hyperactive after the temperature shift. Second, although GFP-negative egg chambers directly connected to germaria at 25°C were occasionally observed from the GFP::Vasa mobilization assay, no GFP-negative cells were detected in later stage egg chambers at any time points. This suggests that the germ cells that maintained high P element activity, and were presumably less competent to adapt, were eliminated at early stages of oogenesis. Third, the number of new P element insertion events declined to 44% in recovered ovaries after adaptation. This dramatic decline indicates that only the stem cells that had lower transposition rates survived the selection. Therefore, it is tempting to speculate that not all germ cells are created equal and that in addition to germarial arrest, the Chk2-mediated DNA break checkpoint also has a role in selecting the survivors from P element invasion and promoting adaptation (Moon, 2018).

    In the surviving ovarian stem cells, Chk2-mediated oogenesis arrest provides a critical time window to propel piRNA generation from the paternally inherited clusters, initiating the amplification cycles for piRNA biogenesis. With at least two piRNA clusters containing P element sequences in the paternally inherited genome, invaded progeny are capable of generating P element-silencing piRNAs de novo. Although it is still unclear when the clusters become active during pre-adult development, it has been shown that the primordial germ cells in larval ovaries can already initiate de novo piRNA production. Consistently, low levels of piRNAs were detected corresponding to P element before adaptation. However, it appears that the amount of piRNAs produced at this stage is too scarce to silence invading P elements. Their activation results in sterility and triggers the Chk2-dependent acute adaptive response from germline stem cells. Subsequently, the Chk2-mediated arrest blocks differentiation, which would allow the newly produced P element-silencing piRNAs to quickly reach a concentration sufficient for Ping-Pong amplification. Finally, these newly produced piRNAs silence transposons at the post transcriptional level and also initiate transcriptional silencing (Moon, 2018).

    Besides promoting piRNA production, the arrest period also allows germ cells to repair DNA lesions before reinitiating oogenesis, thereby preventing the proliferation of cells with DNA damage and defective differentiation. Having the ability to repair damage and endogenize invading transposons in germline stem cells ensures permanent restoration of robust oogenesis and protection of all daughter cells from transposon activation (Moon, 2018).

    Pathogenic tau-induced piRNA depletion promotes neuronal death through transposable element dysregulation in neurodegenerative tauopathies

    Transposable elements, known colloquially as 'jumping genes', constitute approximately 45% of the human genome. Cells utilize epigenetic defenses to limit transposable element jumping, including formation of silencing heterochromatin and generation of piwi-interacting RNAs (piRNAs), small RNAs that facilitate clearance of transposable element transcripts. This study utilize Drosophila melanogaster and postmortem human brain samples to identify transposable element dysregulation as a key mediator of neuronal death in tauopathies, a group of neurodegenerative disorders that are pathologically characterized by deposits of tau protein in the brain. Mechanistically, it was found that heterochromatin decondensation and reduction of piwi and piRNAs drive transposable element dysregulation in tauopathy. A significant increase is reported in transcripts of the endogenous retrovirus class of transposable elements in human Alzheimer's disease and progressive supranuclear palsy, suggesting that transposable element dysregulation is conserved in human tauopathy. Taken together, these data identify heterochromatin decondensation, piwi and piRNA depletion and consequent transposable element dysregulation as a pharmacologically targetable, mechanistic driver of neurodegeneration in tauopathy (Sun, 2018).

    This study uncovered a therapeutically targetable mechanism whereby pathogenic tau drives neuronal death. Specifically, these studies identified dysregulation of transposable elements as a consequence of pathogenic tau and a driver of aberrant cell cycle activation in neurons and subsequent neuronal death. Genetic, dietary and pharmacological approaches were taken to reduce transposable element dysregulation and suppress tau-induced neurotoxicity in Drosophila. An unbiased transcriptomic approach was taken to extend these findings to postmortem human brains, and differentially expressed transposable elements were identified in Alzheimer's disease and progressive supranuclear palsy (Sun, 2018).

    Because the complexity and repetitive nature of transposable elements present challenges to RNA-seq analysis, which is associated with a greater frequency of false positives and negatives compared to analysis of canonical messenger RNAs, this study performed secondary validation of differentially expressed transposable element transcripts in tau transgenic Drosophila by NanoString. While NanoString data showed a similar expression trend for most of the transposable elements that were identified as differentially expressed in tau transgenic Drosophila by RNA-seq, some of the elements were not confirmed as differentially expressed by NanoString. These data reveal the limitations of each assay when analyzing transposable element transcripts and stress the importance of rigorous secondary validation. Since many members of the copia family are increased at the transcript level in both RNA-seq and NanoString analyses, it is speculated that gypsy-TRAP reporter activation is a result of copia insertion into the <i>ovo locus, rather than gypsy. Attempts to sequence de novo copia insertions within the <i>ovo locus in homogenates prepared from tau transgenic Drosophila heads resulted in a high frequency of mismatches, which is likely a result of the stochastic nature of transposable element insertion (Sun, 2018).

    According to current understanding, cells have two layers of defense against potentially deleterious transposable element activation: transposable element transcription is limited by heterochromatin-mediated silencing, and transposable element transcripts are cleared from the cell by piRNA-mediated degradation. Both mechanisms of transposable element suppression were compromised in tauopathy. It is speculated that tau-induced heterochromatin decondensation facilitates active transcription of transposable elements and that tau-induced piwi and piRNA reduction allows those transcripts to persist. While these results are consistent with the effects of heterochromatin decondensation and piwi reduction on transposable element expression that have been reported in the Drosophila germline, these studies reveal a previously undocumented role for heterochromatin- and piRNA-mediated transposable element silencing in the brain. On the basis of studies in the germline reporting a direct interaction between piwi and HP140 and a requirement for Rhino, a member of the HP1 subfamily, for piRNA production, it is possible that a direct interaction between piwi and HP1 is also required to silence transposable elements in the brain (Sun, 2018).

    Among upregulated transposable elements in human tauopathy, the human endogenous retrovirus (HERV) family, including HERV-K, was significantly over-represented. Elevated HERV-K transcripts are associated with amyotrophic lateral sclerosis (ALS)8 and many human cancers, including melanoma, breast cancer, germ cell tumors, renal cancer and ovarian cancer. A causal association between HERV-K and neuronal dysfunction has previously been established, as ectopic expression of HERV-K or the retroviral envelope protein that it encodes decreases synaptic activity and induces progressive motor dysfunction in mice. Antiretroviral reverse transcriptase inhibitors inhibit HERV-K activation in cultured cells and are now in clinical trials for the treatment of ALS. On the basis of the data presented in this study, reverse transcriptase inhibitors have significant potential as a therapeutic strategy for the treatment of neurodegenerative tauopathies, including Alzheimer's disease (Sun, 2018).

    The ability of flamenco loss-of-function mutations to enhance tau-induced neurotoxicity and the ability of piwi overexpression, dietary restriction and inhibition of reverse transcriptase to reduce transposable element dysregulation and suppress tau-induced neurotoxicity suggest that tau-induced transposable element dysregulation is deleterious to neuronal survival. In addition to the detrimental effects of transposable element jumping, double-stranded RNAs produced by transposable element transcripts, including HERVs, can trigger a type I interferon response through the innate immune system. In light of the HERV increase in human tauopathy and the involvement of the innate immune response as a disease-promoting mechanism in Alzheimer's disease, it is tempting to speculate that expression of endogenous retroviruses in human tauopathy contributes to neuroinflammation, in addition to promoting genomic instability. In future studies, it will be important to investigate a potential effect of transposable element activation on the innate immune response in the context of tauopathy (Sun, 2018).

    Population specific dynamics and selection patterns of transposable element insertions in European natural populations

    Transposable elements (TEs) are ubiquitous sequences in genomes of virtually all species. While TEs have been investigated for several decades, only recently has the opportunity appeared to study their genome-wide population dynamics. Most of the studies so far have been restricted either to the analysis of the insertions annotated in the reference genome or to the analysis of a limited number of populations. Taking advantage of the European Drosophila population genomics consortium (DrosEU) sequencing dataset, this study has identified and measured the dynamics of TEs in a large sample of European Drosophila melanogaster natural populations. The mobilome landscape is population specific and highly diverse depending on the TE family. In contrast with previous studies based on SNP variants, no geographical structure was observed for TE abundance or TE divergence in European populations. De novo individual insertions were also identified using two available programs and, as expected, most of the insertions were present at low frequencies. Nevertheless, a subset of TEs present at high frequencies was identified and located in genomic regions with a high recombination rate. These TEs are candidates for being the target of positive selection, although neutral processes should be discarded before reaching any conclusion on the type of selection acting on them. Finally, parallel patterns of association between the frequency of TE insertions and several geographical and temporal variables were found between European and North American populations, suggesting that TEs can be potentially implicated in the adaptation of populations across continents (Lerat, 2018).

    SQuIRE reveals locus-specific regulation of interspersed repeat expression

    Transposable elements (TEs) are interspersed repeat sequences that make up much of the human genome. Their expression has been implicated in development and disease. However, TE-derived RNA-seq reads are difficult to quantify. Past approaches have excluded these reads or aggregated RNA expression to subfamilies shared by similar TE copies, sacrificing quantitative accuracy or the genomic context necessary to understand the basis of TE transcription. As a result, the effects of TEs on gene expression and associated phenotypes are not well understood. This study presents Software for Quantifying Interspersed Repeat Expression (SQuIRE), the first RNA-seq analysis pipeline that provides a quantitative and locus-specific picture of TE expression. SQuIRE is an accurate and user-friendly tool that can be used for a variety of species. SQuIRE was applied to RNA-seq from normal mouse tissues and a Drosophila model of amyotrophic lateral sclerosis. In both model organisms, previously reported TE subfamily expression levels were recapitulated and locus-specific TE expression was revealed. Differences were identified in TE transcription patterns relating to transcript type, gene expression and RNA splicing that would be lost with other approaches using subfamily-level analyses. Altogether, these findings illustrate the importance of studying TE transcription with locus-level resolution (Yang, 2019).

    Dynamic Interactions between the genome and an endogenous retrovirus: Tirant in Drosophila simulans wild-type strains

    All genomes contain repeated sequences that are known as transposable elements (TEs). Among these are endogenous retroviruses (ERVs), which are sequences similar to retroviruses and are transmitted across generations from parent to progeny. These sequences are controlled in genomes through epigenetic mechanisms. At the center of the epigenetic control of TEs are small interfering RNAs of the piRNA class, which trigger heterochromatinization of TE sequences. The tirant ERV of Drosophila simulans displays intra-specific variability in copy numbers, insertion sites, and transcription levels, providing a well-suited model to study the dynamic relationship between a TE family and the host genome through epigenetic mechanisms. tirant transcript amounts and piRNA amounts are positively correlated in ovaries in normal conditions, unlike what was previously described following divergent crosses. In addition, tirant insertion polymorphism in the genomes of three D. simulans wild-type strains is described, revealing a limited number of insertions that may be associated with gene transcript level changes through heterochromatin spreading having phenotypic impacts. Taken together, these results participate in the understanding of the equilibrium between the host genome and its TEs (Fablet, 2019).

    Stress response, behavior, and development are shaped by transposable element-induced mutations in Drosophila

    Most of the current knowledge on the genetic basis of adaptive evolution is based on the analysis of single nucleotide polymorphisms (SNPs). Despite increasing evidence for their causal role, the contribution of structural variants to adaptive evolution remains largely unexplored. This work analyzed the population frequencies of 1,615 Transposable Element (TE) insertions annotated in the reference genome of Drosophila melanogaster, in 91 samples from 60 worldwide natural populations. A set of 300 polymorphic TEs were identified that are present at high population frequencies, and located in genomic regions with high recombination rate, where the efficiency of natural selection is high. The age and the length of these 300 TEs are consistent with relatively young and long insertions reaching high frequencies due to the action of positive selection. Besides, a set of 21 fixed TEs also likely to be adaptive was identified. Indeed evidence has been found of selection for 84 of these reference TE insertions. The analysis of the genes located nearby these 84 candidate adaptive insertions suggested that the functional response to selection is related with the GO categories of response to stimulus, behavior, and development. It was further shown that a subset of the candidate adaptive TEs affects expression of nearby genes, and five of them have already been linked to an ecologically relevant phenotypic effect. These results provide a more complete understanding of the genetic variation and the fitness-related traits relevant for adaptive evolution. Similar studies should help uncover the importance of TE-induced adaptive mutations in other species as well (Rech, 2019).

    Maelstrom represses canonical Polymerase II transcription within bi-directional piRNA clusters in Drosophila melanogaster

    In Drosophila, 23-30 nt long PIWI-interacting RNAs (piRNAs) direct the protein Piwi to silence germline transposon transcription. Most germline piRNAs derive from dual-strand piRNA clusters, heterochromatic transposon graveyards that are transcribed from both genomic strands. These piRNA sources are marked by the heterochromatin protein 1 homolog Rhino (Rhi), which facilitates their promoter-independent transcription, suppresses splicing, and inhibits transcriptional termination. This study reports that the protein Maelstrom (Mael) represses canonical, promoter-dependent transcription in dual-strand clusters, allowing Rhi to initiate piRNA precursor transcription. Mael also represses promoter-dependent transcription at sites outside clusters. At some loci, Mael repression requires the piRNA pathway, while at others, piRNAs play no role. It is proposed that by repressing canonical transcription of individual transposon mRNAs, Mael helps Rhi drive non-canonical transcription of piRNA precursors without generating mRNAs encoding transposon proteins (Chang, 2018).

    Fly piRNA clusters must solve a gene expression paradox. They record the ancient and contemporary exposure of the animal to transposon invasion, and this information must be copied into RNA in order to generate protective, anti-transposon piRNAs. However, recent transposon insertions retain the ability to produce mRNA encoding proteins required for their transposition. In flies, dual-strand piRNA clusters solve this paradox by using Rhi to initiate non-canonical transcription of unspliced RNA from both genomic strands, generating piRNA precursors, while repressing promoter-initiated, canonical transcription. These data suggest that Mael is required for this second process, allowing dual-strand piRNA clusters to safely generate piRNA precursor transcripts without risking production of transposon mRNAs. Within dual-strand clusters, Mael is likely guided to its targets by the piRNA pathway. However, the current analyses also predict that for some loci, Mael functions to repress canonical transcription in heterochromatin separately from the piRNA pathway, probably via one or more proteins that direct Mael to specific genomic sites (Chang, 2018).

    In maelM391/r20 ovaries, piRNAs mapping to dual-strand clusters decrease, despite a concomitant increase in canonical transcription from these same loci. The data suggest that in maelM391 mutants, Rhi-mediated non-canonical transcription, cluster transcript export, and ping-pong amplification become uncoupled. Perhaps, canonical transcription and Rhi-mediated non-canonical transcription compete for Pol II. Instead of fueling piRNA production, the canonical transcripts from dual-strand piRNA clusters produced in the absence of Mael are translated into protein. Mael therefore contributes to dual-strand cluster piRNA production by tipping the balance toward non-canonical transcription (Chang, 2018).

    The data indicate that in addition to relying on the piRNA pathway, Mael can also be guided to its targets by piRNA-independent mechanisms. Moreover, piRNA-dependent repression by Mael may be widespread outside of flies: although Drosophila melanogaster piwi is found only in the gonads, piwi is expressed broadly in the soma of most arthropods. In fact, 12 arthropods with somatic piRNAs also express mael in the soma, while 3 arthropods with no detectable piRNAs outside the gonads have low or undetectable somatic mael mRNA (Chang, 2018).

    In male mice, loss of MAEL also leads to loss of piRNAs, germline transposon derepression, and sterility. As in flies, loss of MAEL in mice does not trigger loss of heterochromatin: DNA methylation of L1 elements is unchanged (Chang, 2018).

    Because Mael is conserved from protists to humans, it is hypothesized that in different organisms Mael may be co-opted by different pathways to repress transcription of various targets. Prior studies suggest a model for how Mael confers repression. In protists, the MAEL domain was predicted to degrade RNA and may directly destroy nascent transcripts. In insects, the MAEL domain interacts with single-stranded RNA; it is speculated that fly Mael may have retained a role in destabilizing RNA. In this view, Mael may promote premature termination or degradation of nascent transcripts. In addition, because fly Mael has a partial HMG domain, it may also directly bind to DNA and repress transcription by preventing canonical core transcription factors from binding to promoters. Another possibility is that fly Mael may play a role in establishing or maintaining chromatin modifications not monitored in this study. Consistent with all of these possible mechanisms, non-canonical transcription mediated by Rhi is expected to be unaffected by Mael because the Rhi allows transcriptional initiation in dual-strand clusters without need for promoters and prevents degradation of unspliced piRNA precursor transcripts (Chang, 2018).

    Transposon silencing in the Drosophila female germline is essential for genome stability in progeny embryos

    The Piwi-interacting RNA pathway functions in transposon control in the germline of metazoans. The conserved RNA helicase Vasa is an essential Piwi-interacting RNA pathway component, but has additional important developmental functions. This study addresses the importance of Vasa-dependent transposon control in the Drosophila female germline and early embryos. Transient loss of vasa expression during early oogenesis leads to transposon up-regulation in supporting nurse cells of the fly egg-chamber. Elevated transposon levels have dramatic consequences, as de-repressed transposons accumulate in the oocyte where they cause DNA damage. Suppression of Chk2-mediated DNA damage signaling in vasa mutant females restores oogenesis and egg production. Damaged DNA and up-regulated transposons are transmitted from the mother to the embryos, which sustain severe nuclear defects and arrest development. These findings reveal that the Vasa-dependent protection against selfish genetic elements in the nuage of nurse cell is essential to prevent DNA damage-induced arrest of embryonic development (Durdevic, 2018).

    This study shows that a transient loss of vas expression during early oogenesis leads to up-regulation of transposon levels and compromised viability of progeny embryos. The observed embryonic lethality is because of DNA DSBs and nuclear damage that arise as a consequence of the elevated levels of transposon mRNAs and proteins, which are transmitted from the mother to the progeny. This study thus demonstrates that transposon silencing in the nurse cells is essential to prevent maternal transmission of transposons and DNA damage, protecting the progeny from harmful transposon-mediated mutagenic effects (Durdevic, 2018).

    The finding that suppression of Chk2-mediated DNA damage signaling in loss-of-function vas mutant flies restores oogenesis, and egg production demonstrates that Chk2 is epistatic to vas. However, hatching is severely impaired, because of the DNA damage sustained by the embryos. The defects displayed by vas, mnk double mutant embryos resembled those of PIWI (piwi, aub, and ago3) single and mnk; PIWI double mutant embryos. Earlier observation that inactivation of DNA damage signaling does not rescue the development of PIWI mutant embryos led to the assumption that PIWI proteins might have an essential role in early somatic development, independent of cell cycle checkpoint signaling. By tracing transposon protein and RNA levels and localization from the mother to the early embryos, it is suggested that, independent of Chk2 signaling, de-repressed transposons are responsible for nuclear damage and embryonic lethality. This study indicates that transposon insertions occur in the maternal genome where they cause DNA DSBs that together with transposon RNAs and proteins are passed on to the progeny embryos. Transposon activity and consequent DNA damage in the early syncytial embryo cause aberrant chromosome segregation, resulting in unequal distribution of the genetic material, nuclear damage and ultimately embryonic lethality. This study shows that early Drosophila embryos are defenseless against transposons and will succumb to their mobilization if the first line of protection against selfish genetic elements in the nuage of nurse cell fails (Durdevic, 2018).

    A recent study showed that in p53 mutants, transposon RNAs are up-regulated and accumulate at the posterior pole of the oocyte, without deleterious effects on oogenesis or embryogenesis. It is possible that the absence of pole plasm in vas mutants results in the release of the transposon products and their ectopic accumulation in the oocyte. Localization of transposons to the germ plasm may restrict their activity to the future germline and protect the embryo soma from transposon activity. Transposon-mediated mutagenesis in the germline would produce genetic variability, a phenomenon thought to play a role in the environmental adaptation and evolution of species. It would therefore be of interest to determine the role of pole plasm in transposon control in the future (Durdevic, 2018).

    Transposon up-regulation in the Drosophila female germline triggers a DNA damage-signaling cascade. In aub mutants, before their oogenesis arrest occurs, Chk2-mediated signaling leads to phosphorylation of Vasa, leading to impaired grk mRNA translation and embryonic axis specification. Considering the genetic interaction of vas and mnk (Chk2) and the fact that Vasa is phosphorylated in Chk2-dependent manner, it is tempting to speculate that phosphorylation of Vasa might stimulate piRNA biogenesis, reinforcing transposon silencing and thus minimizing transposon-induced DNA damage. The arrest of embryonic development as a first, and arrest of oogenesis as an ultimate response to DNA damage, thus, prevents the spreading of detrimental transposon-induced mutations to the next generation (Durdevic, 2018).

    The integrity of piRNA clusters is abolished by insulators in the Drosophila germline

    Piwi-interacting RNAs (piRNAs) control transposable element (TE) activity in the germline. piRNAs are produced from single-stranded precursors transcribed from distinct genomic loci, enriched by TE fragments and termed piRNA clusters. The specific chromatin organization and transcriptional regulation of Drosophila germline-specific piRNA clusters ensure transcription and processing of piRNA precursors. TEs harbour various regulatory elements that could affect piRNA cluster integrity. One of such elements is the suppressor-of-hairy-wing (Su(Hw))-mediated insulator, which is harboured in the retrotransposon gypsy. To understand how insulators contribute to piRNA cluster activity, the effects were studied of transgenes containing gypsy insulators on local organization of endogenous piRNA clusters. Transgene insertions interfere with piRNA precursor transcription, small RNA production and the formation of piRNA cluster-specific chromatin, a hallmark of which is Rhino, the germline homolog of the heterochromatin protein 1 (HP1). The mutations of Su(Hw) restored the integrity of piRNA clusters in transgenic strains. Surprisingly, Su(Hw) depletion enhanced the production of piRNAs by the domesticated telomeric retrotransposon TART, indicating that Su(Hw)-dependent elements protect TART transcripts from piRNA processing machinery in telomeres. A genome-wide analysis revealed that Su(Hw)-binding sites are depleted in endogenous germline piRNA clusters, suggesting that their functional integrity is under strict evolutionary constraints (Radion, 2019).

    Dynamics of transposable element invasions with piRNA clusters

    In mammals and invertebrates the proliferation of an invading transposable element (TE) is thought to be stopped by an insertion into a piRNA cluster. This study explored the dynamics of TE invasions under this trap model using computer simulations. piRNA clusters were found to confer a substantial benefit, effectively preventing extinction of host populations from a proliferation of deleterious TEs. TE invasions consists of three distinct phases: first the TE amplifies within the population, next TE proliferation is stopped by segregating cluster insertions and finally the TE is inactivated by fixation of a cluster insertion. Suppression by segregating cluster insertions is unstable and bursts of TE activity may yet occur. The transposition rate and the population size mostly influence the length of the phases but not the amount of TEs accumulating during an invasion. Solely the size of piRNA clusters was identified as a major factor influencing TE abundance. A single non-recombining cluster is more efficient in stopping invasions than clusters distributed over several chromosomes. Recombination among cluster sites makes it necessary that each diploid carries, on the average, four cluster insertions to stop an invasion. Surprisingly, negative selection in a model with piRNA clusters can lead to a novel equilibrium state, where TE copy numbers remain stable despite only some individuals in a population carrying a cluster insertion. In Drosophila melanogaster the trap model accounts for the abundance of TEs produced in the germline but fails to predict the abundance of TEs produced in the soma (Kofler, 2019).

    Cellular labeling of endogenous retrovirus replication (CLEVR) reveals de novo insertions of the gypsy retrotransposable element in cell culture and in both neurons and glial cells of aging fruit flies

    Evidence is rapidly mounting that transposable element (TE) expression and replication may impact biology more widely than previously thought. Investigation of the biological impact of mobile elements in somatic cells will be greatly facilitated by the use of donor elements that are engineered to report de novo events in vivo. In multicellular organisms, reporter constructs demonstrating engineered long interspersed nuclear element (LINE-1; L1) mobilization have been in use for quite some time, and strategies similar to L1 retrotransposition reporter assays have been developed to report replication of Ty1 elements in yeast and mouse intracisternal A particle (IAP) long terminal repeat (LTR) retrotransposons in cultivated cells. This study describes a novel approach termed cellular labeling of endogenous retrovirus replication (CLEVR), which reports replication of the gypsy element within specific cells in vivo in Drosophila. The gypsy-CLEVR reporter reveals gypsy replication both in cell culture and in individual neurons and glial cells of the aging adult fly.The gypsy-CLEVR replication rate is increased when the short interfering RNA (siRNA) silencing system is genetically disrupted. This CLEVR strategy makes use of universally conserved features of retroviruses and should be widely applicable to other LTR retrotransposons, endogenous retroviruses (ERVs), and exogenous retroviruses (Chang, 2019).


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