InteractiveFly: GeneBrief

aubergine: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - aubergine

Synonyms -

Cytological map position - 32C5

Function - translational regulation

Keywords - pole cell formation, translational regulation, translational initiation, post-transcriptional gene silencing, Repeat-associated small interfering RNAs (rasiRNAs), retrotransposon silencing

Symbol - aub

FlyBase ID: FBgn0000146

Genetic map position - 2-39

Classification - PAZ domain protein

Cellular location - cytoplasmic

NCBI links:  Entrez Gene
aub orthologs: Biolitmine
Recent literature
Barckmann, B., Pierson, S., Dufourt, J., Papin, C., Armenise, C., Port, F., Grentzinger, T., Chambeyron, S., Baronian, G., Desvignes, J. P., Curk, T. and Simonelig, M. (2015). Aubergine iCLIP reveals piRNA-dependent decay of mRNAs involved in germ cell development in the early embryo. Cell Rep 12: 1205-1216. PubMed ID: 26257181
The Piwi-interacting RNA (piRNA) pathway plays an essential role in the repression of transposons in the germline. Other functions of piRNAs such as post-transcriptional regulation of mRNAs are now emerging. This study perform iCLIP with the PIWI protein Aubergine (Aub) and identified hundreds of maternal mRNAs interacting with Aub in the early Drosophila embryo. Gene expression profiling reveals that a proportion of these mRNAs undergo Aub-dependent destabilization during the maternal-to-zygotic transition. Strikingly, Aub-dependent unstable mRNAs encode germ cell determinants. iCLIP with an Aub mutant that is unable to bind piRNAs confirms piRNA-dependent binding of Aub to mRNAs. Base pairing between piRNAs and mRNAs can induce mRNA cleavage and decay that are essential for embryonic development. These results suggest general regulation of maternal mRNAs by Aub and piRNAs, which plays a key developmental role in the embryo through decay and localization of mRNAs encoding germ cell determinants.

Vourekas, A., Alexiou, P., Vrettos, N., Maragkakis, M. and Mourelatos, Z. (2016). Sequence-dependent but not sequence-specific piRNA adhesion traps mRNAs to the germ plasm. Nature 531: 390-394. PubMed ID: 26950602
The conserved Piwi family of proteins and piwi-interacting RNAs (piRNAs) have a central role in genomic stability, which is inextricably linked to germ-cell formation, by forming Piwi ribonucleoproteins (piRNPs) that silence transposable elements. In Drosophila melanogaster and other animals, primordial germ-cell specification in the developing embryo is driven by maternal messenger RNAs and proteins that assemble into specialized messenger ribonucleoproteins (mRNPs) localized in the germ (pole) plasm at the posterior of the oogenesis. Maternal piRNPs, especially those loaded on the Piwi protein Aubergine (Aub), are transmitted to the germ plasm to initiate transposon silencing in the offspring germ line. The transport of mRNAs to the oocyte by midoogenesis is an active, microtubule-dependent process; mRNAs necessary for primordial germ-cell formation are enriched in the germ plasm at late oogenesis via a diffusion and entrapment mechanism, the molecular identity of which remains unknown. Aub is a central component of germ granule RNPs, which house mRNAs in the germ plasm, and interactions between Aub and Tudor are essential for the formation of germ granules. This study shows that Aub-loaded piRNAs use partial base-pairing characteristics of Argonaute RNPs to bind mRNAs randomly in Drosophila, acting as an adhesive trap that captures mRNAs in the germ plasm, in a Tudor-dependent manner. Notably, germ plasm mRNAs in drosophilids are generally longer and more abundant than other mRNAs, suggesting that they provide more target sites for piRNAs to promote their preferential tethering in germ granules. Thus, complexes containing Tudor, Aub piRNPs and mRNAs couple piRNA inheritance with germline specification. These findings reveal an unexpected function for piRNP complexes in mRNA trapping that may be generally relevant to the function of animal germ granules.

Lewis, S. H., Salmela, H. and Obbard, D. J. (2016). Duplication and diversification of Dipteran Argonaute genes, and the evolutionary divergence of Piwi and Aubergine. Genome Biol Evo [Epub ahead of print]. PubMed ID: 26868596

Genetic studies of Drosophila melanogaster have provided a paradigm for RNAi in arthropods, in which the miRNA and antiviral pathways are each mediated by a single Argonaute (Ago1 and Ago2) and germline suppression of transposable elements is mediated by a trio of Piwi-subfamily Argonaute proteins (Ago3, Aub and Piwi). Without a suitable evolutionary context, deviations from this can be interpreted as derived or idiosyncratic. This study analysed the evolution of Argonaute genes across the genomes and transcriptomes of 86 Dipteran species, showing that variation in copy number can occur rapidly, and that there is constant flux in some RNAi mechanisms. The lability of the RNAi pathways is illustrated by the divergence of Aub and Piwi (182-156 million years ago), independent origins of multiple Piwi-family genes in Aedes mosquitoes (less than 25mya), and the recent duplications of Ago2 and Ago3 in the tsetse fly Glossina morsitans. In each case the tissue-specificity of these genes has altered, suggesting functional divergence or innovation, and consistent with the action of dynamic selection pressures across the Argonaute gene family. There are large differences in evolutionary rates and gene turnover between pathways, and paralogues of Ago2, Ago3 and Piwi/Aub show contrasting rates of evolution after duplication. This suggests that Argonautes undergo frequent evolutionary expansions that facilitate functional divergence (Lewis, 2016).

Hayashi, R., Schnabl, J., Handler, D., Mohn, F., Ameres, S. L. and Brennecke, J. (2016). Genetic and mechanistic diversity of piRNA 3'-end formation. Nature [Epub ahead of print]. PubMed ID: 27851737
Small regulatory RNAs guide Argonaute (Ago) proteins in a sequence-specific manner to their targets and therefore have important roles in eukaryotic gene silencing. Of the three small RNA classes, microRNAs and short interfering RNAs are processed from double-stranded precursors into defined 21- to 23-mers by Dicer, an endoribonuclease with intrinsic ruler function. PIWI-interacting RNAs (piRNAs)-the 22-30-nt-long guides for PIWI-clade Ago proteins that silence transposons in animal gonads-are generated independently of Dicer from single-stranded precursors. piRNA 5' ends are defined either by Zucchini, the Drosophila homologue of mitoPLD - a mitochondria-anchored endonuclease, or by piRNA - guided target cleavage. Formation of piRNA 3' ends is poorly understood. This study report that two genetically and mechanistically distinct pathways generate piRNA 3' ends in Drosophila. The initiating nucleases are either Zucchini or the PIWI-clade proteins Aubergine (Aub) or Ago3. While Zucchini-mediated cleavages directly define mature piRNA 3' ends, Aub/Ago3-mediated cleavages liberate pre-piRNAs that require extensive resection by the 3'-to-5' exoribonuclease Nibbler (Drosophila homologue of Mut-7). The relative activity of these two pathways dictates the extent to which piRNAs are directed to cytoplasmic or nuclear PIWI-clade proteins and thereby sets the balance between post-transcriptional and transcriptional silencing. Notably, loss of both Zucchini and Nibbler reveals a minimal, Argonaute-driven small RNA biogenesis pathway in which piRNA 5' and 3' ends are directly produced by closely spaced Aub/Ago3-mediated cleavage events. These data reveal a coherent model for piRNA biogenesis, and should aid the mechanistic dissection of the processes that govern piRNA 3'-end formation.
Ma, X., Zhu, X., Han, Y., Story, B., Do, T., Song, X., Wang, S., Zhang, Y., Blanchette, M., Gogol, M., Hall, K., Peak, A., Anoja, P. and Xie, T. (2017). Aubergine controls germline stem cell self-renewal and progeny differentiation via distinct mechanisms. Dev Cell 41(2): 157-169.e155. PubMed ID: 28441530
Piwi family protein Aubergine (Aub) maintains genome integrity in late germ cells of the Drosophila ovary through Piwi-associated RNA-mediated repression of transposon activities. Although it is highly expressed in germline stem cells (GSCs) and early progeny, it remains unclear whether it plays any roles in early GSC lineage development. This study reports that Aub promotes GSC self-renewal and GSC progeny differentiation. RNA-iCLIP results show that Aub binds the mRNAs encoding self-renewal and differentiation factors in cultured GSCs. Aub controls GSC self-renewal by preventing DNA-damage-induced Chk2 activation and by translationally controlling the expression of self-renewal factors. It promotes GSC progeny differentiation by translationally controlling the expression of differentiation factors, including Bam. Therefore, this study reveals a function of Aub in GSCs and their progeny, which promotes translation of self-renewal and differentiation factors by directly binding to its target mRNAs and interacting with translational initiation factors.
Rojas-Rios, P., Chartier, A., Pierson, S. and Simonelig, M. (2017). Aubergine and piRNAs promote germline stem cell self-renewal by repressing the proto-oncogene Cbl. EMBO J 36(21): 3194-3211. PubMed ID: 29030484
PIWI proteins play essential roles in germ cells and stem cell lineages. In Drosophila, Piwi is required in somatic niche cells and germline stem cells (GSCs) to support GSC self-renewal and differentiation. Whether and how other PIWI proteins are involved in GSC biology remains unknown. This study shows that Aubergine (Aub), another PIWI protein, is intrinsically required in GSCs for their self-renewal and differentiation. Aub needs to be loaded with piRNAs to control GSC self-renewal and acts through direct mRNA regulation. The Cbl proto-oncogene, a regulator of mammalian hematopoietic stem cells, was identified as a novel GSC differentiation factor. Aub stimulates GSC self-renewal by repressing Cbl mRNA translation and does so in part through recruitment of the CCR4-NOT complex. This study reveals the role of piRNAs and PIWI proteins in controlling stem cell homeostasis via translational repression and highlights piRNAs as major post-transcriptional regulators in key developmental decisions.
Dufourt, J., Bontonou, G., Chartier, A., Jahan, C., Meunier, A. C., Pierson, S., Harrison, P. F., Papin, C., Beilharz, T. H. and Simonelig, M. (2017). piRNAs and Aubergine cooperate with Wispy poly(A) polymerase to stabilize mRNAs in the germ plasm. Nat Commun 8(1): 1305. PubMed ID: 29101389
Piwi-interacting RNAs (piRNAs) and PIWI proteins play a crucial role in germ cells by repressing transposable elements and regulating gene expression. In Drosophila, maternal piRNAs are loaded into the embryo mostly bound to the PIWI protein Aubergine (Aub). Aub targets maternal mRNAs through incomplete base-pairing with piRNAs and can induce their destabilization in the somatic part of the embryo. Paradoxically, these Aub-dependent unstable mRNAs encode germ cell determinants that are selectively stabilized in the germ plasm. This study shows that piRNAs and Aub actively protect germ cell mRNAs in the germ plasm. Aub directly interacts with the germline-specific poly(A) polymerase Wispy, thus leading to mRNA polyadenylation and stabilization in the germ plasm. These results reveal a role for piRNAs in mRNA stabilization and identify Aub as an interactor of Wispy for mRNA polyadenylation. They further highlight the role of Aub and piRNAs in embryonic patterning through two opposite functions.
Blom-Dahl, D. and Azpiazu, N. (2017). The Pax protein Eyegone (Eyg) interacts with the pi-RNA component Aubergine (Aub) and controls egg chamber development in Drosophila. Dev Biol [Epub ahead of print]. PubMed ID: 29278721
The eyegone (eyg) gene encodes Eyg, a transcription factor of the Pax family with multiple roles during Drosophila development. Eyg has been shown to be nuclear in the cells where it functions. This report describes a new functional cytoplasmic distribution of Eyg during egg chamber development in the female ovarioles. The protein is present from the germarium until stage 10 of cyst development. The majority of egg chambers that develop in the absence of Eyg arrest their development before stage 10, show augmented levels of the telomeric retro-transposon TART-A and low levels of heterochromatin marks in the oocyte nucleus. During the maternal to zygotic transition (MTZ) Eyg seems to play a role in destabilizing germ cell less (gcl) and oo 16 RNA binding protein (orb) mRNAs. Eyg interacts with Aubergine (Aub), a component of the pi-RNA pathway during egg chamber development. This interaction could be essential for Eyg to be retained in the cytoplasm and fulfill its functions there.
Huang, Y. C., Moreno, H., Row, S., Jia, D. and Deng, W. M. (2018). Germline silencing of UASt depends on the piRNA pathway. J Genet Genomics. PubMed ID: 29789221
One of the most extensively used techniques in Drosophila is the Gal4/UAS binary system, which allows tissue-specific misexpression or knockdown of specific genes of interest. The original UAS vector, UASt, can only be activated for transgene expression in somatic tissues but not in the germline cells. This study found that the piwi-interacting RNA (piRNA) pathway is involved in suppressing UASt expression in ovarian germline cells. Individually knocking down or mutating components of the piRNA biogenesis pathway (e.g., Piwi, AGO3, Aub, Spn-E, Su(var)2-10, Hsp83, and Vasa) resulted in the expression of the UASt-reporter (GFP or RFP) in the germline. An RNA-seq analysis of small RNAs revealed that the hsp70 promoter of UASt is targeted by piRNAs, and in the aub mutant ovary, the amount of piRNAs targeting the hsp70 promoter is reduced by around 40 fold. In contrast, the SV40 3'UTR of the UASt, which happens to be targeted by the nonsense-mediated RNA decay (NMD) pathway, is not responsible for germline UASt suppression, as UASt-reporters with NMD-insensitive 3'UTR fail to show germline expression. Taken together, these studies reveal a crucial role of the piRNA pathway, potentially via the suppression of the hsp70 promoter, in germline UASt silencing in Drosophila ovaries.
Theron, E., Maupetit-Mehouas, S., Pouchin, P., Baudet, L., Brasset, E. and Vaury, C. (2018). The interplay between the Argonaute proteins Piwi and Aub within Drosophila germarium is critical for oogenesis, piRNA biogenesis and TE silencing. Nucleic Acids Res. PubMed ID: 30113668
Transposable elements (TEs) have invaded most genomes and constitute up to 50% of the human genome. Machinery based on small non-coding piRNAs has evolved to inhibit their expression at the transcriptional and post-transcriptional levels. Surprisingly, this machinery is weakened during specific windows of time in mice, flies or plants, allowing the expression of TEs in germline cells. The function of this de-repression remains unknown. In Drosophila, it has been shown that this developmental window is characterized by a reduction of Piwi expression in dividing germ cells. This study shows that the unique knock-down of Aub in these cells leads to female sterility. It correlates with defects in piRNA amplification, an increased Piwi expression and an increased silencing of transcriptionally silenced TEs. These defects are similar to those observed when Aub is depleted in the whole germline which underlies the crucial role of this developmental window for both oogenesis and TE silencing. This study further showed that, with age, some fertility is recovered which is concomitant to a decrease of Piwi and TE silencing. These data pinpoint the Pilp (Piwi-less pocket - a region of the oocyte lacking Piei) as a tremendously important step for female fertility and genome stability. They further show that such a restricted developmental niche of germ cells may sense environmental changes, such as aging, to protect the germline all along the life.
Wakisaka, K. T., et al. (2018). Novel roles of Drosophila FUS and Aub responsible for piRNA biogenesis in neuronal disorders. Brain Res. PubMed ID: 30578769
This study examined the possible roles of Aubergine (Aub), a Piwi-family protein (PIWI) responsible for piRNAs biogenesis, in the neuronal disorders, using the Cabeza (Caz) knockdown Drosophila. Caz is a Drosophila homologue of FUS, which is one of the genes causing amyotrophic lateral sclerosis (ALS). Aub overexpression enhanced the mobility defects accompanied by anatomical defects in motoneurons at neuromuscular junctions induced by the neuron-specific knockdown of Caz. In order to elucidate the underlying mechanisms, this study examined pre-piRNA and mature-size piRNA levels under these conditions. qRT-PCR and RNA-seq analyses revealed that the Caz knockdown increased pre-piRNA levels, but reduced mature-size piRNA levels in the central nervous system (CNS), suggesting a role in the pre-piRNAs production. Aub overexpression did not increase mature-size piRNA levels. These results suggest that the accumulated pre-piRNAs are abnormal abortive pre-piRNAs that cannot be further processed by slicers, including Aub. A relationship between Caz and pre-piRNAs in the CNS was demonstrated by RNA immunoprecipitation. Aub overexpression induced the abnormal cytoplasmic localization of Caz. Based on these results, a model is proposed in which Caz knockdown-induced abnormal pre-piRNAs associate with Caz, then translocate and accumulate in the cytoplasm, a process that may be mediated by Aub.
Bergalet, J., Patel, D., Legendre, F., Lapointe, C., Benoit Bouvrette, L. P., Chin, A., Blanchette, M., Kwon, E. and Lecuyer, E. (2020). Inter-dependent centrosomal co-localization of the Cen and Ik2 cis-Natural Antisense mRNAs in Drosophila. Cell Rep 30(10): 3339-3352. PubMed ID: 32160541
Overlapping genes are prevalent in most genomes, but the extent to which this organization influences regulatory events operating at the post-transcriptional level remains unclear. Studying the Cen and Ik2 genes of Drosophila melanogaster, which are convergently transcribed as cis-natural antisense transcripts (cis-NATs) with overlapping 3' UTRs, it was found that their encoded mRNAs strikingly co-localize to centrosomes. These transcripts physically interact in a 3' UTR-dependent manner, and the targeting of Ik2 requires its 3' UTR sequence and the presence of Cen mRNA, which serves as the main driver of centrosomal co-localization. The Cen transcript undergoes localized translation in proximity to centrosomes, and its localization is perturbed by polysome-disrupting drugs. By interrogating global fractionation-sequencing datasets generated from Drosophila and human cellular models, this study found that RNAs expressed as cis-NATs tend to co-localize to specific subcellular fractions. This work suggests that post-transcriptional interactions between RNAs with complementary sequences can dictate their localization fate in the cytoplasm.
Wang, L., Barbash, D. A. and Kelleher, E. S. (2020). Adaptive evolution among cytoplasmic piRNA proteins leads to decreased genomic auto-immunity. PLoS Genet 16(6): e1008861. PubMed ID: 32525870
The piRNA pathway acts as a genomic immune system, employing small RNA-mediated silencing to defend host DNA from the harmful effects of transposable elements (TEs). This study used interspecific complementation to test for functional differences between Drosophila melanogaster and D. simulans alleles of three adaptively evolving piRNA pathway proteins: Armitage, Aubergine and Spindle-E. In contrast to piRNA-mediated transcriptional regulators examined in previous studies, these three proteins have cytoplasmic functions in piRNA maturation and post-transcriptional silencing. Across all three proteins interspecific divergence was observed in the regulation of only a handful of TE families, which were more robustly silenced by the heterospecific piRNA pathway protein. This unexpected result suggests that unlike transcriptional regulators, positive selection has not acted on cytoplasmic piRNA effector proteins to enhance their function in TE repression. Rather, TEs may evolve to "escape" silencing by host proteins. It was further discovered that D. simulans alleles of aub and armi exhibit enhanced off-target effects on host transcripts in a D. melanogaster background, as well as modest reductions in the efficiency of piRNA biogenesis, suggesting that promiscuous binding of D. simulans Aub and Armi proteins to host transcripts reduces their participation in piRNA production. Avoidance of genomic auto-immunity may therefore be a critical target of selection. These observations suggest that piRNA effector proteins are subject to an evolutionary trade-off between defending the host genome from the harmful effect of TEs while also minimizing collateral damage to host genes.
Iki, T., Kawaguchi, S. and Kai, T. (2023). miRNA/siRNA-directed pathway to produce noncoding piRNAs from endogenous protein-coding regions ensures Drosophila spermatogenesis. Sci Adv 9(29): eadh0397. PubMed ID: 37467338
PIWI-interacting RNA (piRNA) pathways control transposable elements (TEs) and endogenous genes, playing important roles in animal gamete formation. However, the underlying piRNA biogenesis mechanisms remain elusive. This study shows that endogenous protein coding sequences (CDSs), which are normally used for translation, serve as origins of noncoding piRNA biogenesis in Drosophila melanogaster testes. The product, namely, CDS-piRNAs, formed silencing complexes with Aubergine (Aub) in germ cells. Proximity proteome and functional analyses show that CDS-piRNAs and cluster/TE-piRNAs are distinct species occupying Aub, the former loading selectively relies on chaperone Cyclophilin 40. Moreover, Argonaute 2 (Ago2) and Dicer-2 activities were found critical for CDS-piRNA production. We provide evidence that Ago2-bound short interfering RNAs (siRNAs) and microRNAs (miRNAs) specify precursors to be processed into piRNAs. This study further demonstrates that Aub is crucial in spermatid differentiation, regulating chromatins through mRNA cleavage. Collectively, these data illustrate a unique strategy used by male germ line, expanding piRNA repertoire for silencing of endogenous genes during spermatogenesis.
Ho, S., Rice, N. P., Yu, T., Weng, Z. and Theurkauf, W. E. (2023). Aub, Vasa and Armi localization to phase separated nuage is dispensable for piRNA biogenesis and transposon silencing in Drosophila. bioRxiv. PubMed ID: 37546958
From nematodes to placental mammals, key components of the germline transposon silencing piRNAs pathway localize to phase separated perinuclear granules. In Drosophila, the PIWI protein Aub, DEAD box protein Vasa and helicase Armi localize to nuage granules and are required for ping-pong piRNA amplification and phased piRNA processing. Drosophila piRNA mutants lead to genome instability and mnk double mutants, we show that Chk2 activation disrupts nuage localization of Aub and Vasa, and that the HP1 homolog Rhino, which drives piRNA precursor transcription, is required for Aub, Vasa, and Armi localization to nuage. However, these studies also show that ping-pong amplification and phased piRNA biogenesis are independent of nuage localization of Vasa, Aub and Armi. Dispersed cytoplasmic proteins thus appear to mediate these essential piRNA pathway functions.
Kiuchi, T., Shoji, K., Izumi, N., Tomari, Y., Katsuma, S. (2023). Non-gonadal somatic piRNA pathways ensure sexual differentiation, larval growth, and wing development in silkworms. PLoS Genet, 19(9):e1010912 PubMed ID: 37733654
PIWI-interacting RNAs (piRNAs) guide PIWI proteins to target transposons in germline cells, thereby suppressing transposon activity to preserve genome integrity in metazoans' gonadal tissues. Piwi, one of three Drosophila PIWI proteins, is expressed in the nucleus and suppresses transposon activity by forming heterochromatin in an RNA cleavage-independent manner. Recently, Piwi was reported to control cell metabolism in Drosophila fat body, providing an example of piRNAs acting in non-gonadal somatic tissues. However, mutant flies of the other two PIWI proteins, Aubergine (Aub) and Argonaute3 (Ago3), show no apparent phenotype except for infertility, blurring the importance of the piRNA pathway in non-gonadal somatic tissues. The silkworm, Bombyx mori, possesses two PIWI proteins, Siwi (Aub homolog) and BmAgo3 (Ago3 homolog), whereas B. mori does not have a Piwi homolog. Siwi and BmAgo3 are mainly expressed in gonadal tissues and play a role in repressing transposon activity by cleaving transposon RNA in the cytoplasm. This study generated Siwi and BmAgo3 loss-of-function mutants of B. mori and found that they both showed delayed larval growth and failed to become adult moths. They also exhibited defects in wing development and sexual differentiation. Transcriptome analysis revealed that loss of somatic piRNA biogenesis pathways results in abnormal expression of not only transposons but also host genes, presumably causing severe growth defects. These results highlight the roles of non-gonadal somatic piRNAs in B. mori development.

In Drosophila oocytes, activation of Oskar translation from a transcript localized to the posterior pole is an essential step in the organization of the pole plasm, specialized cytoplasm that contains germline and abdominal body patterning determinants. Oskar is a component of polar granules, large particles associated with the pole plasm and the germline precursor pole cells of the embryo. aubergine mutants fail to translate oskar mRNA efficiently and are thus defective in posterior body patterning and pole cell formation. Aubergine protein is related to eukaryotic translation initiation factor 2C. In addition, Aubergine is recruited to the posterior pole in a vasa-dependent manner and is itself a polar granule component. Consistent with its presence in these structures, Aubergine is required for pole cell formation independently of its initial role in oskar translation. Unlike two other known polar granule components, Vasa and Oskar, Aubergine remains cytoplasmic after pole cell formation, suggesting that the roles of these proteins diverge during embryogenesis (Harris, 2001).

aubergine has also been implicated in regulation of post-transcriptional gene silencing in Drosophila. aubergine and spindle-E mutations cause a relief of Stellate and Su(Ste) silencing (Schmidt, 1999). Stellate derepression in the presence of the intact Su(Ste) locus has been observed as a result of aubergine and spindle-E (spn-E) mutations, also known as sting and homeless, respectively. The Aubergine protein has homologs involved in post-transcriptional gene silencing (PTGS) and double stranded RNA interference in plants, fungi, and animals. The spn-E gene encodes a putative RNA helicase that is also proposed as a participant in dsRNA-mediated silencing (Schmidt, 1999; Aravin, 2001).

Relief of Stellate< silencing occurs as a result of the spn-E1 mutation. This was confirmed by studying the expression of the Ste-lacZ reporter construct in the spn-E1/+ and spn-E1/spn-E1 males. The expression of ß-galactosidase in testes is greatly enhanced in spn-E1/spn-E1 males as compared to the heterozygous ones. The effects of the aubsting-1 and spn-E1 mutations on the level of sense and antisense Suppressor of Stellate [Su(Ste)] transcripts were assessed. Both mutations, when homozygous, have no effect on the level of antisense Su(Ste) transcripts, but increase the level of sense Su(Ste) RNA. Thus, a common mechanism, assisted by the Aubergine and Spindle-E proteins is operated in Su(Ste) dsRNA-mediated silencing of Stellate and sense Su(Ste) expression. The effect of the spn-E1 mutation is restricted to the germline, since no increase in the level of sense Su(Ste) transcripts in the heads of homozygous flies was observed (Schmidt, 1999; Aravin, 2001).

Meiotic drive is defined as any alteration of meiosis or subsequent production of gametes that results in the biased transmission of a particular genotype. In other words, meiotic drive results in an excess of one type of gamete, X for example, over the production of the alternative gamete (Y in this case) instead of the usual ratio of 1:1 that is normally produced. Thus, meiotic drive is defined as any alteration in the meiotic process that distorts the normal 1:1 ratio of reciprocal gametes. Aubergine, in disguise as the mutation sting, is involved in crystal formation and meiotic drive in the male germ line of Drosophila. The sting mutation, caused by a P element inserted into polytene region 32D, was isolated by a screen for male sterile insertions in Drosophila melanogaster. This sterility correlates with the presence of crystals in spermatocytes and spermatids that are structurally indistinguishable from those produced in males carrying a deficiency of the Y-linked crystal (cry, also known as Suppressor of Stellate) locus. In addition, their morphology is needle-like in Stellate (Ste) plus flies and star-shaped in Ste flies, once again as observed in cry males. The sting mutation leads to meiotic drive of the sex chromosome. The sting mutation results in an excess of X over Y sperm in the one hand and 0 over XY sperm on the other. The strength of the meiotic drive phenomenon is correlated with the copy number of the repetitive Ste locus. The same correlation is also true for the penetrance of the male sterile mutation. A presumptive sti null allele results in male sterility and lethal maternal effect. The gene is expressed only in the germline of both sexes. The interaction of sting with the Ste locus can also be demonstrated at the molecular level. While an unprocessed 8-kb Ste primary transcript is expressed in wild-type males, in X/Y homozygous sti males, as in X/Y cry- males, a 0.7-kb mRNA is produced (Schmidt, 1999).

Previous analysis of aub implicated it in two processes: the translational activation of a narrowly restricted subset of ovarian mRNAs, including osk and grk, and the subsequent localization of nos mRNA (Wilson, 1996). To investigate the molecular function of aub, the locus was positionally cloned. aub maps by meiotic recombination to genetic position 39 on the second chromosome. aub was subsequently mapped between P elements reported to be inserted in polytene regions 31E and 33A. RFLP mapping as well as clones and sequence information from the Berkeley Drosophila Genome Project were used to narrow the location of the aub gene to a small region at the boundary of polytene divisions 32C and 32D (Harris, 2001).

This region contains sting (Schmidt, 1999), a member of an ancient gene family that includes the gene for the eukaryotic translation initiation factor eIF2C (Zou, 1998). With support from three lines of evidence, aub was identified and it was concluded that aub and sting are the same gene. (1) In each of three aub mutant chromosomes, mutations predicted to truncate the Aub/Sting protein were identified. (2) Antibodies raised against a bacterially expressed C-terminal portion of Aub/Sting recognize a band of approximately 105 kDa in extracts from wild-type ovaries but not ovaries from aub mutants. A band corresponding to a similar molecular weight is produced by in vitro transcription and translation of an aub/sting cDNA. (3) Two genomic DNA fragments which include the aub/sting gene rescue aub mutants: aub- mothers carrying a single copy of either transgene are fertile and produce viable offspring. Following tradition, the original name, aubergine, is used for this locus (Harris, 2001).

The Drosophila genome contains four other members of the eIF2C-like gene family. One of these is piwi, a close chromosomal neighbor of aub that acts in germline stem cell maintenance. Two additional members, Argonaute 2 and AGO1, are reported in the genome annotation. The latter is the closest known relative of eIF2C in flies and is presumably the Drosophila eIF2C homolog. The fifth family member, corresponding to the genomic sequence AE003107 and EST clot 2083, was identifed by tBLASTn searches of the BDGP databases using parts of Aub protein as the query sequence (Harris, 2001).

An early analysis of aub implicated it in two processes: the translational activation of a narrowly restricted subset of ovarian mRNAs, including osk and grk, and the subsequent localization of nos mRNA (Wilson, 1996). The latter function could be indirect, with Aub activating translation of a factor directly involved in nos mRNA localization, or Aub might itself play a more direct role in the process. The demonstration that Aub is related to the translation initiation factor eIF2C lends further support for a direct role of Aub in translational activation. Evidence that Aub is a component of polar granules and is required for pole cell formation adds an additional function and also suggests that its later role in localization of nos mRNA may be relatively direct (Harris, 2001).

Although the genetic pathways in which these eIF2C/piwi family members act have been identified, the direct activity through which any plays a role in development or RNAi has not been determined to date. The only gene product in the family for which a specific biochemical activity has been demonstrated is the translation initiation factor eIF2C (formerly Co-eIF-2A) (Zou, 1998). eIF2C purified from rabbit reticulocytes has two related activities that affect the ternary complex, which is composed of initiator methionine tRNA, GTP and eIF-2. The ternary complex binds the 40S ribosomal subunit to allow scanning for AUG codons in mRNA. Purified eIF2C stimulates formation of the ternary complex from components present at physiological levels, and it stabilizes the complex against dissociation in the presence of natural mRNAs (Roy, 1981; Bagchi, 1985; Chakravarty, 1985; Roy, 1988). The known activity of eIF2C offers a trivial explanation of the roles of other family members in multiple processes: each could simply enhance the translation of a protein required for that particular process. This seems unlikely, in large part because Piwi has been shown to be nuclear in both the germline and somatic cells in which its activity is required. Furthermore, Aub may play a direct role in nos mRNA localization. Nevertheless, the conserved domains that define this protein family should be expected to perform similar functions in the different proteins, and so some similarity to eIF2C function among other family members would not be surprising (Harris, 2001).

Aub protein may have biochemical activities similar to those of eIF2C. While eIF2C is predicted to play a global role in translation, mutants of aub only detectably affect the translation of a limited number of transcripts, revealing specificity in Aub function. Two broad classes of models are proposed for the role of Aub in message-specific translational activation. (1) Aub may simply elevate the level of eIF2C-like activity in the ovary, leading to enhanced translation of mRNAs that depend most on this initiation factor. Differential dependence would presumably reflect some feature of mRNA structure, such as folding or AUG sequence context, or association of the mRNA with other proteins or regulatory factors. A variation of this model invokes association of Aub with dependent mRNAs, either directly through an RNA-binding activity or indirectly via binding to other factors, effectively elevating the local concentration of the eIF2C-like activity. (2) Aub may perform an activity distinct from that of eIF2C but still be involved in some aspect of translation. As with the first model, the activity could be concentrated on dependent mRNAs through direct or indirect binding. A GFP-tagged form of Aub is not concentrated at the site of grk mRNA localization, and it has been argued that posterior concentration of Aub is not required for osk mRNA translation. Thus, if Aub does associate with dependent mRNAs as part of its role in translation, the interaction is likely to be transient (Harris, 2001).

Efforts to define the feature of transcripts that makes them dependent on Aub are constrained by the limited number of such mRNAs that have been identified: at present only osk and grk have been found. For osk mRNA, aub dependence is conferred in part by the 3' UTR (Wilson, 1996), a region known to bind multiple proteins and mediate both mRNA localization and translational repression. Therefore, Aub-dependence may be conferred by some aspect of translational repression or mRNA localization. The features of grk mRNA that confer aub-dependence are unknown (Harris, 2001).

osk mRNA is repressed by Bru and BicC, probably in concert with other factors. Bru also binds to grk, and this interaction has been suggested to mediate repression. If repression by Bru is indeed a shared feature of the osk and grk mRNAs, then Aub might inhibit or override this process. However, elimination of Bru-mediated repression by mutation of the BREs, the Bru-binding sites in the osk mRNA 3' UTR, does not abrogate the requirement for aub in translation of posteriorly localized osk mRNA (Wilson, 1996). Thus, Aub does not simply alleviate Bru-dependent repression at the posterior pole of the oocyte (although it may contribute to this process) (Harris, 2001).

One feature that is unquestionably shared by the osk and grk mRNAs is localization to specific domains within the oocyte. Perhaps tenacious binding of localization factors places unusual constraints on the translational apparatus that can only be overcome through the action of Aub. Supporting this possibility is the curious requirement for Aub in translation of posteriorly localized oskBRE- mRNA, but not in the precocious translation that occurs prior to localization, when oskBRE- mRNA is present throughout much of the oocyte (Wilson, 1996). Thus, posterior localization of osk mRNA correlates with the Aub-dependent phase of its translation. However, anterior localization of osk mRNA, achieved by exchanging the osk 3' UTR with the bcd mRNA localization signal, obviates the need for Aub. Therefore, mRNA localization in and of itself need not impose the requirement for Aub, although different localization signals may have unique properties that affect the relative translatability of their transcripts (Harris, 2001).

GFP-Aub colocalizes with Osk in polar granules in the pole plasm and pole cells. Moreover, since Osk and Vas are also largely colocalized in polar granules, it is inferred that the Aub-containing particles also include Vas. GFP-Aub is also highly enriched in the perinuclear zones of the nurse cells, a distribution shared by Vas and Tud, another polar granule component whose distribution was not monitored in these experiments. Notably, vas is required for the presence of normal levels of GFP-Aub in both the polar granules and the perinuclear zones. Pole plasm components have been suggested to be transported in particles from the nurse cells to the posterior ooplasm, and the results are consistent with such a scheme. The defect in GFP-Aub accumulation at the posterior in vas mutants may be a direct result of the initial failure to recruit Aub to the periphery of the nurse cell nuclei, a process that may well involve the assembly of complexes containing both Vas and Aub. In contrast, Stau is not required for the perinuclear localization of either Vas or GFP-Aub, and thus may only act later in the recruitment of particles to the posterior pole of the oocyte (Harris, 2001).

In the embryo, polar granules are present in the cytoplasm of pole cells, and nuclear bodies that are similar in appearance form in the nuclei of these cells. Evidence supporting a structural relationship between the two classes of particle has come from the demonstration that Vas is present in both. This work provides additional supporting evidence of such a relationship: further indication of structural similarity comes from the demonstration that Osk and Vas are colocalized in both nuclear and cytoplasmic aggregates, some of which have the peculiar donut-like shape that is characteristic of a subset of polar granules and nuclear bodies. However, the data also reveal that the cytoplasmic and nuclear particles are not identical in composition, since the nuclear bodies are distinguished from the polar granules by the absence of GFP-Aub. Nevertheless, Aub, like Osk and Vas, is involved in pole cell formation, and all are predominantly or exclusively cytoplasmic during that process. It is possible that later aspects of germ cell development require certain polar granule components, such as Osk or Vas, to act in the nucleus. Future experiments in which the selective partitioning of the various polar granule and nuclear body components is perturbed may provide insight into how pole cell fates are specified and maintained or lost during development (Harris, 2001).

Binding of guide piRNA triggers methylation of the unstructured N-terminal region of Aub leading to assembly of the piRNA amplification complex

PIWI proteins use guide piRNAs to repress selfish genomic elements, protecting the genomic integrity of gametes and ensuring the fertility of animal species. Efficient transposon repression depends on amplification of piRNA guides in the ping-pong cycle, which in Drosophila entails tight cooperation between two PIWI proteins, Aub and Ago3. This study shows that post-translational modification, symmetric dimethylarginine (sDMA), of Aub is essential for piRNA biogenesis, transposon silencing and fertility. Methylation is triggered by loading of a piRNA guide into Aub, which exposes its unstructured N-terminal region to the PRMT5 methylosome complex. Thus, sDMA modification is a signal that Aub is loaded with piRNA guide. Amplification of piRNA in the ping-pong cycle requires assembly of a tertiary complex scaffolded by Krimper, which simultaneously binds the N-terminal regions of Aub and Ago3. To promote generation of new piRNA, Krimper uses its two Tudor domains to bind Aub and Ago3 in opposite modification and piRNA-loading states. These results reveal that post-translational modifications in unstructured regions of PIWI proteins and their binding by Tudor domains that are capable of discriminating between modification states is essential for piRNA biogenesis and silencing (Huang, 2021).

The PIWI-interacting RNA (piRNA) pathway acts as a conserved defensive system that represses the proliferation of transposable elements (TEs) in the germline of sexually reproducing animals. Loss of PIWI proteins causes derepression of transposons associated with gametogenesis failure and sterility in flies and mice. PIWI proteins recognize transposon targets with help of the associated small (23-30 nt) non-coding RNA guides, piRNAs (Huang, 2021).

PIWI proteins belong to the conserved Argonaute protein family present in all domains of life. Argonautes bind nucleic acid guides and share common domain architecture, all containing the conserved N, PAZ, MID, and PIWI domains. The MID and PAZ domains bind the 5' and 3' ends of the guide RNA, respectively. The PIWI domain contains an RNase-H-like fold with a conserved amino acid tetrad that endows Argonautes with endonuclease activity for precise cleavage of the target. The degradation of complementary target mRNA by PIWI proteins can trigger the generation of new RNA guides in a process termed the ping-pong cycle. Ping-pong requires cooperativity between two PIWI molecules as the product resulting from target cleavage by one protein is passed to the other and is converted to a new piRNA guide. In Drosophila, two distinct cytoplasmic PIWI proteins, Aub and Ago3, cooperate in the ping-pong cycle with each protein generating an RNA guide that is loaded into its partner. Amplification of piRNA guides through the ping-pong cycle is believed to be essential for efficient transposon repression as it allows the pathway to mount an adaptive response to actively transcribed transposons (Huang, 2021).

In addition to four conserved domains, eukaryotic members of the Argonaute family, including PIWI proteins, contain an N-terminal extension region of various lengths with low sequence conservation. Structural studies of Agos suggest that the N-terminal regions adopt a disordered conformation. Despite low overall conservation, the N-terminal region of the majority of PIWI proteins harbors arginine-rich (A/G)R motifs. In both insects and mammals, these motifs were shown to be substrates for post-translational modification by the PRMT5 methyltransferase, which produces symmetrically dimethylated arginine (sDMA) residues. Loss of Prmt5 (encoded by the Csul and Vls genes) in Drosophila leads to reduced piRNA level and accumulation of transposon transcripts in germ cells, suggesting that sDMA modification of PIWIs plays an important role in the piRNA pathway (Huang, 2021).

Multiple Tudor domain-containing proteins (TDRDs) can bind to sDMA modifications. Aromatic residues in binding pocket of Tudor domains form cation-π interactions with sDMA. Studies in Drosophila and mouse revealed that several TDRDs interact with PIWIs and are involved in piRNA-guided transposon repression, although their specific molecular functions remain poorly understood. Previously it was found that the Tudor-domain containing protein Krimper is required for ping-pong piRNA amplification and is capable of both self-interactions and binding of the two ping-pong partners, Aub and Ago3. Krimper co-localizes with Aub and Ago3 in nuage, a membraneless perinuclear cytoplasmic compartment where piRNA-guided target degradation and ping-pong are proposed to take place. Ago3 requires Krimper for recruitment into this compartment, though Aub does not. These results led to the proposal that Krimper assembles a complex that brings Ago3 to Aub and coordinates ping-pong in nuage. However, the architecture of the ping-pong piRNA processing (4 P) complex and the extent to which Krimper regulates ping-pong remained unresolved (Huang, 2021).

Both the ping-pong cycle and sDMA modification of PIWI proteins are conserved features of the piRNA pathway, found in many organisms, suggesting that these processes are essential for pathway functions. sDMA modification of PIWIs provides a binding platform for interactions with Tudor-domain proteins, however, its biological function and regulation are not known. Despite the essential role of ping-pong in transposon repression, there is little understanding of the molecular mechanisms that control this process. This study revealed the biological function of Aub and Ago3 sDMA modifications and show that it plays an essential role in orchestrating assembly of the 4 P complex in the ping-pong cycle. The modification signals whether PIWI proteins are loaded with guide piRNA, and this information is used to assemble a ping-pong complex that is receptive for directional transfer of RNA to an unloaded PIWI protein (Huang, 2021).

Although PIWI proteins and piRNAs share many similarities with other Agos and their RNA guides, the piRNA pathway has evolved unique features that are essential for its function as an adaptive genome defense system. One such unique property is the amplification of piRNAs that target active transposons in the ping-pong cycle. Ping-pong employs the intrinsic RNA binding and processing capabilities of Ago proteins, however, it creates new functionality through the cooperation between two PIWI proteins. The results indicate that the ping-pong cycle and sDMA-modification are tightly linked and that the modification status of PIWI proteins regulates the assembly of the ping-pong processing complex (Huang, 2021).

Several lines of evidence suggest that sDMA modification of Aub is induced by the binding of a piRNA guide. First, Aub mutants that are deficient in piRNA binding due to mutation in either the RNA 5' or the 3' end binding pocket have a decreased level of sDMA modification. Second, disruption of piRNA biogenesis diminishes methylation of wild-type Aub. Finally, the loading of chemically synthesized RNA into Aub promotes its association with the methylosome complex and sDMA modification. In contrast, sDMA modification of Aub is not required for its loading with piRNA and for its slicer activity. Together, these results suggest that sDMA modification of Aub acts as a signal of its piRNA-bound state (Huang, 2021).

The results suggest that piRNA loading induces sDMA methylation through a conformational change that makes the N-terminal sequence accessible to the methylation enzyme. While unloaded Aub is poorly methylated, the N-terminal sequence alone is a good substrate for methylation. Insertion of a sequence between the N-terminal region and the rest of the protein also promotes methylation (despite the protein not being able to bind piRNA), suggesting that other parts of the protein inhibit modification. Finally, partial proteolysis indicates that Aub undergoes a conformation change upon piRNA loading. Combined, these experiments suggest that the N-terminal sequence is poorly accessible to the modifying enzyme until Aub binds a guide RNA, inducing a conformation change that exposes its N-terminus (Huang, 2021).

Structural differences between empty and loaded states were reported for several prokaryotic and eukaryotic Agos, corroborating the idea that binding to guide RNA induces conformational change. The PAZ domain of Agos exhibit a high level of flexibility upon loading of guide RNA/DNA. During the recognition of target RNA, the PAZ domain undergoes a conformational transition that releases the 3' end of the guide and facilitates downstream guide-target base pairing. The results indicate that binding of the 3' end by the PAZ domain is critical for sDMA modification of Aub's N-terminal region. Unfortunately, the N-terminal extension region was often truncated to facilitate Ago expression and crystallization and thus reported structures do not provide information about the N-terminal extension region. If the N-terminal region is preserved, it exists in an unstructured conformation that remains unresolved by crystallography. However, piRNA loading of the nuclear PIWI protein in Drosophila was shown to induce a conformational change that exposes the nuclear localization sequence (NLS) located in its N-terminus and to enable its binding to importin. Thus, two PIWI clade proteins, Aub and Piwi, harbor an N-terminal sequence that becomes accessible upon piRNA loading and its exposure promotes interactions with other factors and regulates protein function. Similar to Aub, the N-terminal extension region of Ago3 also harbors a (G/A)R motif that can be modified. Considering that piRNA binding triggers exposure of the N-terminus in Aub and Piwi, a similar process might occur in Ago3. Indeed, previous studies and the current results revealed that, unlike the bulk of the cellular Ago3 pool, Krimper-bound Ago3 is both unloaded and unmethylated, indicating that piRNA binding and modification are correlated for Ago3 as well as for Aub (Huang, 2021).

The results demonstrate the importance of the N-terminal region in the function of PIWI proteins. Unlike other domains (PAZ, MID, PIWI) of Argonautes with well-characterized functions in RNA guide binding and target cleavage, the N-terminal region has received little attention due to its disordered conformation and its low conservation between different Agos. The results suggest that the low conservation and absence of a fixed structure are in fact important features of the N-terminal region that are critical for PIWI proteins function. The flexible structure of this region might provide sensitivity to changes in overall protein conformation, such as the changes triggered by guide RNA binding. In Aub and Ago3, the modification and binding of sDMA sites to other proteins, as well as NLS-mediated interaction of Piwi require only a short linear motif, and thus the N-terminal region does not require a strongly conserved sequence or rigid folding. In agreement with this, the presence of a (G/A)R motif in Aub and Ago3 proteins is conserved in other Drosophila species, however, the specific position and sequence context of the motif is diverse. The poor similarity between N-terminal sequences of different Agos might endow them with distinct functions. It might be worth exploring whether signaling of the guide-loading state through exposure of the N-terminal region is also conserved in Ago-clade proteins and whether it regulates their function (Huang, 2021).

The central feature of ping-pong is that the cleavage of target RNA by one PIWI protein results in the transfer of the cleaved product to another PIWI protein. Although the original model of ping-pong did not provide information on the molecular complex and interactions within the complex, ping-pong intuitively implies physical proximity between the two PIWI proteins followed by complex molecular rearrangements. This study found that, although sDMA modification does not affect slicer activity of Aub, information about the piRNA-loading state of PIWI proteins signaled by their sDMA modifications is used to assemble a complex that enables the transfer of the processed RNA from Aub to Ago3 (Huang, 2021).

While previous findings strongly suggest that Krimper plays a role in the assembly of the ping-pong piRNA processing (4 P) complex in which Aub and Ago3 are brought into close physical proximity (Webster, 2015), the architecture of this complex and the extent to which Krimper regulates ping-pong remained unknown. The current results indicate that a single Krimper molecule interacts simultaneously with Aub and Ago3, suggesting that ping-pong takes place within a tertiary complex containing one molecule of each protein. Krimper actively selects the two ping-pong partners using the distinct specificities of its two Tudor domains: eTud1 uniquely binds Ago3, while eTud2 recognizes modified Aub. This study found that in vitro the eTud2 domain is capable of binding both sDMA-modified Aub and Ago3 peptides, however, in vivo Krimper complexes were reported to contain exclusively unmodified Ago3, suggesting that in the proper cellular context eTud2 only binds sDMA-Aub. Thus, the domain architecture of Krimper ensures that tertiary complexes contain Aub-Ago3 partners rather than random pairs. This finding is in line with the observation that ping-pong occurs predominantly between Aub and Ago3, although, in principle, ping-pong can take place between two identical proteins, and a small level of homotypic Aub/Aub ping-pong was previously detected. Thus, these results suggest that the propensity for heterotypic ping-pong is, at least in part, due to Krimper (Huang, 2021).

Ping-pong not only requires the physical proximity of two PIWI proteins but also that they have opposite piRNA-loading states: one protein induces piRNA-guided RNA cleavage (and therefore has to be loaded with a piRNA guide), while the other accepts the product of this reaction (and therefore has to be free of piRNA). The results suggest that the opposite binding preference of the two Tudor domains towards sDMA ensures that the tertiary complex contains PIWI proteins in opposite RNA-loading states. While the overall fold structure of the two Tudor domains is similar, they have critical differences responsible for their distinct binding preferences. The binding pocket of eTud2 is similar to that of other Tudor domains and contains four aromatic residues that interact with sDMA. As sDMA modification of Aub signals its piRNA-binding status, the binding of eTud2 to modified Aub ensures that the complex contains Aub/piRNA. The structural studies and in vitro binding assays revealed that Ago3 binds to eTud1 in its unmethylated state and sDMA modification of any of the Arg residues within its (A/G)R motif prevents this interaction. The unusual binding preference of eTud1 is reflected in its non-canonical binding pocket, which lacks three of the four conserved aromatic residues. The binding of methylated Aub and unmethylated Ago3 ensures that Aub has a guide piRNA and Ago3 is free, thus enabling loading of Ago3 with RNA generated by Aub/piRNA-induced cleavage (Huang, 2021).

The architecture of the tertiary complex assembled by Krimper permits Aub-dependent generation and loading of RNA into Ago3. However, the ping-pong cycle also includes the opposite step, Ago3-dependent generation of Aub piRNA (henceforth these steps were termed 'ping' and 'pong'). The results suggest that the ping and pong steps require the assembly of two distinct complexes discriminated by the modification status of Aub and Ago3 (Huang, 2021).

As a single Krimper simultaneously binds Aub and Ago3, Krimper dimerization might be dispensable for ping-pong, raising the question of what the function of Krimper self-interaction is. Previous findings suggest that Krimper forms a scaffold for assembly of nuage, a membraneless organelle (MLO) that surrounds nuclei of nurse cells and resembles other MLO possibly formed through liquid-liquid phase separation. Several lines of evidence point at Krimper as an essential component of nuage that acts as a scaffold for its assembly and the recruitment of client components. First, unlike other nuage components, FRAP measurements show very little Krimper exchange between nuage and the dispersed cytoplasmic compartment. Second, wild-type, but not mutant Krimper that lacks the self-interaction domain, forms cytoplasmic granules upon expression in heterologous cells that do not contain other nuage proteins. In contrast, other nuage components including Aub and Ago3 are dispersed in the cytoplasm when expressed in a similar setting, suggesting that they do not form condensates on their own and rely on other components for recruitment to nuage. Krimper recruits both Aub and Ago3 into MLO that it forms in heterologous cells. Combined, these data indicating that Krimper works as a scaffold, and Ago3 and Aub as its clients for nuage assembly. Thus, the interactions between Krimper and the N-terminal regions of Aub and Ago3 is not only essential for the assembly of the tertiary molecular complex but is also responsible for the recruitment of these proteins into membraneless cellular compartment (see Model for sDMA regulation and its function in ping-pong cycle). The high local concentration of proteins and RNA involved in the piRNA pathway in nuage might enhance the efficiency of ping-pong as well as the recognition of RNA targets by Aub and Ago3 (Huang, 2021).

The PIWI protein Aubergine recruits eIF3 to activate translation in the germ plasm

Piwi-interacting RNAs (piRNAs) and PIWI proteins are essential in germ cells to repress transposons and regulate mRNAs. In Drosophila, piRNAs bound to the PIWI protein Aubergine (Aub) are transferred maternally to the embryo and regulate maternal mRNA stability through two opposite roles. They target mRNAs by incomplete base pairing, leading to their destabilization in the soma and stabilization in the germ plasm. This study reports a function of Aub in translation. Aub is required for translational activation of nanos mRNA, a key determinant of the germ plasm. Aub physically interacts with the poly(A)-binding protein (PABP) and the translation initiation factor eIF3. Polysome gradient profiling reveals the role of Aub at the initiation step of translation. In the germ plasm, PABP and eIF3d assemble in foci that surround Aub-containing germ granules, and Aub acts with eIF3d to promote nanos translation. These results identify translational activation as a new mode of mRNA regulation by Aub, highlighting the versatility of PIWI proteins in mRNA regulation (Ramat, 2020).

Translational control is a widespread mechanism to regulate gene expression in many biological contexts. This regulation has an essential role during early embryogenesis, before transcription of the zygotic genome has actually started. In Drosophila, embryonic patterning depends on the translational control of a small number of maternal mRNAs, among them, nanos (nos) mRNA encodes a key posterior determinant required for abdominal segmentation and development of the germline. nos mRNA is present in the whole embryo, but a small proportion accumulates at the posterior pole in the germ plasm, a specialized cytoplasm in which the germline develops. Localization and translational control of nos mRNA are linked, such that the pool of nos mRNA present in the bulk of the embryo is translationally repressed, whereas the pool of nos mRNA localized in the germ plasm is translationally activated to produce a Nos protein gradient from the posterior pole. Both repression of nos mRNA translation in the bulk of the embryo and activation in the germ plasm are required for embryonic development (Ramat, 2020).

The coupling between mRNA localization and translational control depends in part on the implication of the same factors in both processes. The Smaug (Smg) RNA binding protein specifically recognizes nos mRNA through binding to two Smaug recognition elements (SRE) in its 3'UTR. Smg is both a translational repressor of nos, and a localization factor through its role in mRNA deadenylation and decay in the bulk of the embryo, by recruitment of the CCR4-NOT deadenylation complex. Smg directly interacts with the Oskar (Osk) protein that is specifically synthesized at the posterior pole of oocytes and embryos and drives germ plasm assembly. Smg interaction with Osk prevents Smg binding to nos mRNA, thus contributing to relieving both Smg-dependent translational repression and mRNA decay in the germ plasm. Osk is therefore a key player in the switch of nos and other germ cell mRNA regulation between soma and germ plasm of the embryo (Ramat, 2020).

A role of Aubergine (Aub) in the localization of germ cell mRNAs to the germ plasm has been demonstrated. Aub is one of the three PIWI proteins in Drosophila. PIWI proteins belong to a specific clade of Argonaute proteins that bind 23-30 nucleotides (nt)-long small RNAs referred to as Piwi-interacting RNAs (piRNAs). piRNAs and PIWI proteins have an established role in the repression of transposable elements in the germline of animals. piRNAs target transposable element mRNAs through complementarity and guide interaction with PIWI proteins that, in turn, cleave targeted mRNAs through their endonucleolytic activity. In addition to this role, piRNAs have a conserved function in the regulation of cellular mRNAs in various biological contexts. In the Drosophila embryo, Aub loaded with piRNAs produced in the female germline is present both at low levels in the bulk of the embryo and at higher levels in the germ plasm. Aub binds maternal germ cell mRNAs through incomplete base pairing with piRNAs. Aub binding to these mRNAs induces their decay in the bulk of the embryo, either by direct cleavage or recruitment together with Smg of the CCR4-NOT deadenylation complex. In contrast, in the germ plasm Aub recruits Wispy, the germline-specific cytoplasmic poly(A) polymerase, leading to poly(A) tail elongation and stabilization of Aub-bound mRNAs. Thus, Aub and piRNAs play a central role in the localization of germ cell mRNAs through two opposite functions in mRNA stability: mRNA destabilization in the bulk of the embryo and stabilization in the germ plasm. The role of piRNAs and PIWI proteins in cellular mRNA regulation in other contexts, including mouse spermiogenesis and sex determination in Bombyx, also depends on their function in the regulation of mRNA stability (Ramat, 2020).

This study describes translational activation as a new mechanism of mRNA regulation by piRNAs and PIWI proteins. Using ectopic expression of Osk in the whole embryo to mimic the germ plasm, this study shows that Aub and piRNAs are required for nos mRNA translation. Mass spectrometry analysis of Aub interactors in early embryos identifies several components of the translation machinery, including translation initiation factors. This study finds that Aub physically interacts with the poly(A)-binding protein (PABP) and several subunits of the translation initiation complex eIF3. Furthermore, PABP and eIF3d accumulate in foci that assemble around and partially overlap with Aub-containing germ granules in the germ plasm. Polysome gradient analysis indicates that Aub activates translation at the initiation step. Finally, functional experiments involving the concomitant decrease of Aub and eIF3d show that both proteins act together in nos mRNA translation in the germ plasm. These results identify translational activation as a new level of mRNA regulation by PIWI proteins. Moreover, they expand the role of the general eIF3 translation initiation complex in translation regulatory mechanisms required for developmental processes (Ramat, 2020).

Several studies have reported the role of PIWI proteins in cellular mRNA regulation at the level of stability. piRNA-dependent binding of mRNAs by PIWI proteins leads to their decay in different biological systems. In addition, in Drosophila embryos, mRNA binding by the PIWI protein Aub also leads to their stabilization in a spatially regulated manner. This study reports a novel function of Aub in direct translational control of mRNAs. Using nos mRNA as a paradigm, it was shown that Aub is required for nos mRNA translation. Nos protein levels are also strongly reduced in armi mutant, in which piRNA biogenesis is massively affected, suggesting that Aub loading with piRNAs is necessary for its function in translational activation. Consistent with this, deletion of two piRNA target sites in nos mRNA decreases its translation. Importantly, Nos levels are not affected in a panx mutant background. Panx is a piRNA factor required for transcriptional repression of transposable elements, but has no function in piRNA biogenesis. In addition, as is the case for aub and armi mutants, panx mutant embryos do not develop. Finally, Nos levels are similar in unfertilized eggs and embryos overexpressing Osk, demonstrating that Nos protein synthesis is independent of embryonic development. Together, these results strongly argue for a direct role of Aub and piRNAs in nos mRNA translational control, independently of their role in transposable element regulation or developmental defects in piRNA pathway mutants (Ramat, 2020).

Mass spectrometry analysis of Aub interactors points to a strong link with the translation machinery. In addition, polysome gradient analyses reveal Aub association with actively translated mRNAs in polysomal fractions. A link has been reported previously between the PIWI proteins Miwi and Mili and the translation machinery in mouse testes, where Miwi and Mili were found to associate with the cap-binding complex. However, the role of Miwi and Mili in translational control has not been characterized. This study has deciphered the molecular mechanisms of Aub function in translational activation of germ cell mRNAs in the Drosophila embryo. This study demonstrates a physical interaction between Aub and the translation initiation factors PABP, eIF4E and subunits of the eIF3 complex. These interactions are in agreement with polysome gradient analyses in WT and aub mutant backgrounds that indicate a role of Aub in translation initiation (Ramat, 2020).

Recent data have identified specific roles of eIF3 in the regulation of translation. eIF3 is the most elaborate of translation initiation factors containing twelve subunits and an associated factor, eIF3j. This complex promotes all steps of translational initiation and does so in part through direct association with other translation initiation factors, contributing to their functional conformations on the small ribosomal subunit surface. In addition to this role in basal translation, the eIF3a, b, d and g subunits were shown to directly bind 5'UTR of specific mRNAs, leading to cap-dependent translation activation or repression. The eIF3d subunit that attaches to the edge of the complex appears to play an especially important role in various modes of eIF3-dependent translational control: (1) eIF3d is involved in the translational repression of Drosophila sex-lethal mRNA through binding to its 5'UTR. (2) eIF3d was reported to directly bind the cap structure of specific mRNAs in mammalian cells, thus bypassing the requirement of eIF4E binding to the cap for translation initiation. (3) In the same line, eIF3d was involved in cap-dependent translational activation of specific mRNAs for neuronal remodeling in Drosophila larvae, in a context where eIF4E is blocked by 4E-binding protein (4E-BP). Other studies have reported the role of eIF3 in promoting cap-independent translation, thus highlighting eIF3 functional versatility in the control of translation. eIF3 was shown to directly bind methylated adenosine m6A, in mRNA 5'UTRs to induce cap-independent translation under stress conditions. Furthermore, PABP bound to the poly(A) tail was also shown to cooperate with eIF3 for its binding to mRNA 5'UTR triggering cap-independent translation (Ramat, 2020).

This study describes a new mode of eIF3-dependent translational activation through its recruitment by the PIWI protein Aub. Based on previous information on the nos translation repressor complex and data presented in this study on translational activation, the following model is proposed. nos mRNA translation is repressed in the somatic part of the embryo by two mechanisms. First, the 4E-BP protein Cup in complex with Smg binds to eIF4E and prevents eIF4G recruitment and cap-dependent translation. The detailed mechanism of Cup recruitment to the repressor complex has not been clarified, but Cup was shown to directly associate with the Not1 subunit of the CCR4-NOT complex and this interaction might stabilize Cup association with eIF4E. CCR4-NOT itself is recruited to nos mRNA by Smg and Aub. Second, two translational repressors, the RNA helicase Me31B (Drosophila DDX6) and its partner Tral coat the length of nos mRNA and prevent translation through a cap-independent mechanism. Again the mode of Me31B/Tral specific recruitment to nos mRNA has not been determined, but the CCR4-NOT complex might also be involved since DDX6 directly binds the Not1 subunit of CCR4-NOT. Aub coprecipitation with components of the nos translational repressor complex is consistent with its association with the CCR4-NOT complex in the soma and suggests that Aub might be involved in translational repression, in addition to mRNA decay. In the germ plasm, Osk interaction with Smg prevents Smg binding to nos mRNA9 and this contributes to CCR4-NOT displacement from the mRNP complex. Consistent with this, CCR4 is depleted in the germ plasm. The lack of CCR4-NOT on nos mRNA might preclude the recruitment of Me31B/Tral and relieve the cap-independent mechanism of translational repression. This study finds that Aub physically interacts with PABP and several subunits of eIF3. It is proposed that these associations would lead to translational activation independently of eIF4E through binding of eIF3 to nos 5'UTR, followed by direct recruitment of the 40 S ribosome by eIF3 and PABP, as previously reported for translation of XIAP mRNA. Alternatively, eIF3 might act through direct binding of eIF3d to the cap structure; however, this hypothesis is not favored. Indeed, if eIF3d interaction with the cap was involved, overexpression of the point mutant eIF3dhelix11 that is unable to bind the cap, would be expected to induce negative dominant defects, due to the lack of translation mediated by this interaction. However, overexpression of eIF3dhelix11 with the nos-Gal4 driver did not induce any defects in embryonic development or Nos protein synthesis (Ramat, 2020).

Germ granules coordinate germ cell mRNA regulation with piRNA inheritance through the role of PIWI proteins in both processes. Recent studies in C. elegans have shown that piRNA/PRG1-dependent mRNA accumulation in germ granules prevent their silencing, strengthening the function of piRNAs in germ granules for mRNA storage and surveillance. In Drosophila, Aub mediates the link between piRNAs and mRNA regulation in germ granules since Aub localization to germ granules depends on its loading with piRNAs and Aub/piRNAs play a general role in the localization and stabilization of germ cell mRNAs in germ granules. How do germ granules accommodate translational control has remained more elusive. In Drosophila embryos, germ granules contain mRNAs that are translated sequentially. This study demonstrates a direct role of Aub in translational activation. Strikingly, PABP and eIF3d tend to colocalize with Aub at the periphery of germ granules. This is reminiscent of a study analyzing translational control in relation to RNA granules in Drosophila oocytes, in which translational repressors such as Me31B were found to concentrate in the granule core with repressed mRNAs, whereas the translational activator Orb was localized at the edge of the granules where mRNAs docked for translation. Similarly, germ granules in embryos might be partitioned into functional subdomains involved in various steps of mRNA regulation, including storage (in an internal region of granules) and translational activation (at the granule periphery). This work reveals the central role of Aub in activation of translation. Future studies will undoubtedly address the complexity of mRNA regulation by PIWI proteins in relation with germ granules (Ramat, 2020).

While this manuscript was under review, a role of Miwi and piRNAs in translational activation during mouse spermiogenesis has been demonstrated (Dai, 2019). Miwi was shown to be in complex with PABP and several subunits of eIF3 for its function in translational activation, which is required for spermatid development. This reveals a striking evolutionary conservation of PIWI protein function in translational control for key developmental processes (Ramat, 2020).

Protein components of ribonucleoprotein granules from Drosophila germ cells oligomerize and show distinct spatial organization during germline development

The assembly of large RNA-protein granules occurs in germ cells of many animals and these germ granules have provided a paradigm to study structure-functional aspects of similar structures in different cells. Germ granules in Drosophila oocyte's posterior pole (polar granules) are composed of RNA, in the form of homotypic clusters, and proteins required for germline development. In the granules, Piwi protein Aubergine binds to a scaffold protein Tudor, which contains 11 Tudor domains. Using super-resolution microscopy, this study showed that surprisingly, Aubergine and Tudor form distinct clusters within the same polar granules in early Drosophila embryos. These clusters partially overlap and, after germ cells form, they transition into spherical granules with the structural organization unexpected from these interacting proteins: Aubergine shell around the Tudor core. Consistent with the formation of distinct clusters, this study showed that Aubergine forms homo-oligomers and using all purified Tudor domains, it was demonstrated that multiple domains, distributed along the entire Tudor structure, interact with Aubergine. These data suggest that in polar granules, Aubergine and Tudor are assembled into distinct phases, partially mixed at their 'interaction hubs', and that association of distinct protein clusters may be an evolutionarily conserved mechanism for the assembly of germ granules (Vo, 2019).

This work focuses on the high-resolution imaging and in vitro analysis of the assembly of interacting proteins, Piwi family protein Aub and scaffold protein Tud, in the RNA-protein granules (polar granules) in the germline. Aub and Tud are the principal components of the granules and since they interact, it was expected that they would be homogenously distributed within the granules. Surprisingly, it was found that, while in the same granules and partially overlapping, these proteins form distinct mutually exclusive clusters in the germ plasm before germ cell formation. In addition, after germ cells form, Aub and Tud show even more striking segregation pattern within the large sphere-like granules that form in the cytoplasm of germ cells. In these granules, Aub is at the surface of the sphere, wrapping around a large Tud core (Vo, 2019).

Classic EM images of the germ granules in Drosophila germ cells from Mahowald's lab demonstrated large spherical cytoplasmic organelles, which in EM sections, appeared as donut- or ring-like structures with electron-dense rim around an electron-lucid core. These granules had the diameter of 0.75 μm - 1 μm in EM sections and are in good agreement with the Aub-Tud large granules described in this study (1.13 μm on average), suggesting that one of the components of the electron-dense rim is Aub. Furthermore, consistent with these data, similar donut-like Aub granules were described in germ cells using confocal microscopy. Although these large spherical granules are quite prominent, they are not as abundant as much more numerous germ plasm-like small polar granules in the cytoplasm of germ cells, therefore, the test of their functional significance awaits further investigation (Vo, 2019).

Interestingly, other proteins can assemble at the surface of large granules, that are different from polar granules, with ring-like distribution visualized in optical sections which resemble electron-dense rim of the corresponding granules imaged with EM. In particular, in Drosophila melanogaster, while Tud and Aub are exclusively cytoplasmic, germ granule proteins Oskar (Osk) and Vasa (Vas) can form granules in the nuclei of germ cells, which are referred to as nuclear bodies. These nuclear bodies are similar in EM sections to cytoplasmic large polar granules described in this study and also show an electron-dense rim with Osk and Vas ring-like distribution with the characteristic donut-like morphology in optical sections. Furthermore, in C. elegans germ granules (P granules), using high-resolution lattice light sheet microscopy, MEG-3 protein was shown to be assembled at the surface of the PGL-3 protein core and these proteins only partially overlap within the P granules. MEG-3/PGL-3 distribution in P granules resembles Aub/Tud assembly in the large cytoplasmic granules in Drosophila germ cells and suggests conservation in the molecular mechanisms of protein assembly in the large germ granules (Vo, 2019).

Interestingly, other 'non-germ' RNP granules, including mammalian P-bodies, stress granules and nucleoli show heterogeneity in their protein distribution within the granules. In particular, there is evidence that Xenopus nucleoli consist of different immiscible liquid-like phases that form core-shell arrangement, with Nucleophosmin NPM1 protein phase (shell) enveloping the FIB1 protein clusters (core). What may be responsible for the shell/core arrangement of the germ and nucleolus RNP granules? Analysis of the nucleolus protein phases provided evidence that their different hydrophobicity and surface tensions result in their distinct incorporation into the nucleolus structure which can be mimicked using different types of oil mixed with water. However, the FIB1 clusters can also 'age' and transition to solid-like state over time. Another mechanism to form distinct compartments within the RNP granules is based on the reentrant phase transition of RNPs which can be controlled by RNA. In this case, titration of an RNA-binding protein with increasing RNA concentration can initially result in the formation of a positively charged RNP droplet (due to a positively charged RNA-binding protein amino acid residues) which subsequently, at higher RNA concentration, leads to the change of the RNP charge from positive to negative. This charge inversion of the RNP granule causes the eventual dissolution of the granule. However, during this process, internal compartments form inside the RNP granules and at certain RNA concentrations these granules can exist for more than two hours, which can be sufficiently long for the time scale of many cellular and developmental processes. The physical principles described above, based on different liquid phases' surface tensions in aqueous environment and the reentrant phase transition, were used to describe behavior of spherical granules and may be contributing to distinct cluster and core-shell architecture of Aub-Tud RNP polar granules reported in this study. In fact, Tud scaffold core may stabilize Aub shell to prevent that from dissolution. However, some important aspects of Tud and Aub distribution as distinct clusters in polar granules before germ cell formation, when these proteins are not embedded into spherical granules but rather overlap in 'interaction hubs' forming amorphous diverse granules, await further biochemical and biophysical analysis (Vo, 2019).

The data show that Aub forms homo-oligomers under native conditions. It remains to be determined whether this oligomerization property of Aub is required for the assembly of polar granules. Interestingly, similarly to Aub homo-oligomerization, nucleolar shell NPM1 protein forms pentameric homo-oligomers and this oligomerization is required for NPM1 to form the protein liquid droplets in vitro and to efficiently localize to the nucleolus (Vo, 2019).

This study also demonstrates that Aub homo-oligomers are insensitive to Aub methylation status and form when Aub contains sDMAs required for interaction with Tud domains of Tud protein. This may indicate that in polar granules, Tud binds to methylated Aub oligomers rather than to monomeric Aub. Furthermore, six Tud domains (1, 3, 4, 6, 9 and 11) of Tud can bind to Aub. Consistent with previous methylated Aub peptide-Tud domains 7-11 binding studies, no interaction of domain 10 to full-length Aub was detected presumably due to this domain's incomplete sDMA-binding pocket, and Aub interaction was shwon with domains 9 and 11. Although Aub binding to the other five Tud domains of Tud protein was not detected, it was previously shown that methylated Aub peptides can bind to Tud domains 7 and 8. Therefore, based on this and previous data, it is proposed that one molecule of Tud protein contains at least eight Tud domains that may potentially bind to multiple Aub homo-oligomers. However, future research will be required to determine the precise stoichiometry of Tud/Aub complexes in polar granules (Vo, 2019).

The formation of distinct Tud and Aub clusters in the same polar granules is intriguing and was unexpected since these proteins are bona fide interacting granule proteins. Tud and Aub may undergo phase transitions during the assembly of polar granules, forming, together with their interacting ligands, two distinct phases. The data are consistent with the model that while these Tud and Aub phases are immiscible with the surrounding cytoplasm, they may be partially mixing and wetting each other, thereby partially overlapping in the 'interaction hubs'. Non-spherical shape of Aub- and Tud-labeled polar granules in the germ plasm before germ cells formation may indicate that Aub and Tud phases, at least to some degree, have transitioned to gel-like or solid-like state. In support of this model, it was observed that polar granules in the germ plasm of early embryos are more structured than liquid droplets and seem to contain both liquid and hydrogel-like regions (Vo, 2019).

After germ cells form in the embryo's posterior, the formation of large granules with Aub shell and Tud core may be driven by the concentration-dependent assembly since germ cells actively accumulate polar granule components using dynein-dependent transport. During this process Aub homo-oligomers (which themselves form even at low concentration) may be interacting with each other to stabilize the shell of the granule and free Aub oligomers may be recruited to assemble around Tud scaffold (Vo, 2019).

Previous imaging analysis of the polar granules demonstrated that RNA granule components form homotypic clusters in the granules. This study has shown that interacting protein components of polar granules form distinct clusters within the same granules which can be subsequently distributed into the large shell-core architecture during development. Future studies will establish how the RNA and protein clusters together determine the granule morphology and how this clustering contributes to the function of polar granules in germ cell development (Vo, 2019).

Distinct functions for the Drosophila piRNA pathway in genome maintenance and telomere protection

Transposons and other selfish DNA elements can be found in all phyla, and mobilization of these elements can compromise genome integrity. The piRNA (PIWI-interacting RNA) pathway silences transposons in the germline, but it is unclear if this pathway has additional functions during development. This study shows that mutations in the Drosophila piRNA pathway genes, armi, aub, ago3, and rhi, lead to extensive fragmentation of the zygotic genome during the cleavage stage of embryonic divisions. Additionally, aub and armi show defects in telomere resolution during meiosis and the cleavage divisions; and mutations in ligase-IV, which disrupt non-homologous end joining, suppress these fusions. By contrast, lig-IV mutations enhance chromosome fragmentation. Chromatin immunoprecipitation studies show that aub and armi mutations disrupt telomere binding of HOAP, which is a component of the telomere protection complex, and reduce expression of a subpopulation of 19- to 22-nt telomere-specific piRNAs. Mutations in rhi and ago3, by contrast, do not block HOAP binding or production of these piRNAs. These findings uncover genetically separable functions for the Drosophila piRNA pathway. The aub, armi, rhi, and ago3 genes silence transposons and maintain chromosome integrity during cleavage-stage embryonic divisions. However, the aub and armi genes have an additional function in assembly of the telomere protection complex (Khurana, 2010).

Drosophila piRNAs have been implicated in transposon silencing and maintenance of genome integrity during female germline development. However, piRNA pathway mutations lead to complex developmental phenotypes, and piRNAs have been implicated in control of gene expression. Furthermore, the majority of piRNAs in other systems, including mouse testes, are not derived from repeated elements. The full extent of piRNA functions thus remains to be explored (Khurana, 2010).

Mutations in the majority of Drosophila piRNA pathway genes disrupt asymmetric localization of RNAs along the axes of the oocyte, and lead to maternal effect embryonic lethality. The axis specification defects linked to several of piRNA pathway mutations are dramatically suppressed by a null mutation in mnk, which encodes a Checkpoint kinase 2 (Chk2) homolog required for DNA damage signaling, indicating that the loss of asymmetric RNA localization is downstream of DNA damage. Oocyte patterning defects generally lead to embryonic lethality, but the mnk allele that suppresses the axis specification defects associated with piRNA mutations does not suppress embryonic lethality. piRNAs thus have an essential function during embryogenesis that is independent of Chk2 activation and DNA damage signaling. To gain insight into potential new functions for the piRNA pathway, the embryonic lethality associated with four piRNA pathway mutations was characterized. These studies reveal a novel function for a subset of piRNA genes in assembly of the telomere protection complex, and suggest that this process is directed by a subpopulation of 19-22 nt piRNAs (Khurana, 2010). The armi and aub genes encode a putative RNA helicase and a piRNA binding PIWI Argonaute protein, and recent studies suggest that they have distinct functions in piRNA biogenesis. Mutations in aub dramatically reduce piRNA species that overlap by 10 nt, which is characteristic of ping-pong amplification, while armi mutations reduce total piRNA production but enhance the ping-pong signature. Mutations in aub and armi lead to maternal-effect embryonic lethality, however, suggesting that these genes share an essential function. To gain insight into the lethality associated with these mutations, DNA break accumulation during oogenesis was analyzed. Germline-specific DNA breaks normally form during early oogenesis, as meiosis is initiated. In several piRNA mutants, however, DNA breaks persist, which could compromise the female pronucleus and thus lead to genetic instability in the early zygote. DNA breaks trigger phosphorylation of histone H2Av, producing γ-H2Av foci near the break sites. In wild-type ovaries, γ-H2Av foci begin to accumulate in region 2 of the germarium, as meiotic breaks are formed. These foci are significantly reduced in stage 2 egg chambers, which have completed meiotic repair and budded from the germarium. Later in oogenesis, γ-H2Av foci accumulate in the nurse cell nuclei, which undergo endoreduplication. However, these foci remain undetectable in the oocyte. In ovaries mutant for aub or armi, γ-H2Av foci appear in germarium region 2, but persist in nurse cells and the oocyte through stage 4. By stage 5, however, γ-H2Av foci are undetectable in 50% of armi and aub mutant oocytes, and are significantly reduced in the remaining oocytes. Both armi and aub mutations thus increase DNA damage during early oogenesis, but most of the damage in the oocyte appears to be repaired as oogenesis proceeds (Khurana, 2010).

As wild type oocytes mature and initiate meiotic spindle assembly, the major chromosomes form a single mass at the spindle equator and the non-exchange 4th chromosomes move toward the poles. In OregonR, distinct 4th chromosomes were observed in 79% of stage 13 oocytes. In stage 13 aub and armi mutants, by contrast, distinct 4th chromosomes were observed in only 11% and 18% of stage 13 oocytes, respectively. However, a single primary mass of chromatin was always observed. These observations are consistent with γ-H2Av data suggesting that DNA breaks formed during early oogenesis are often repaired as the oocyte matures. In addition, both aub and armi mutations appear to inhibit separation of the small 4th chromosomes, although it is also possible that this small chromosome is fragmented and thus difficult to detect cytologically (Khurana, 2010).

Drosophila oocytes are activated as they pass through the oviduct, which triggers completion of the meiotic divisions. The first meiotic division is completed in the oviduct, but meiosis II can be observed in freshly laid eggs and is characterized by four well-separated meiotic products on tandem spindles. In aub and armi mutant embryos, the meiotic chromatin was either stretched across the paired meiotic spindles, or fragmented and spread over both spindles. No wild type meiotic figures were observed. Breaks thus appear to persist in some stage 14 oocytes, although this does not disrupt the karyosome organization during earlier stages. However, other oocytes appear to have intact chromosomes that fail to resolve during the meiotic divisions (Khurana, 2010).

Chromatin fragmentation could result from replication of broken chromosomes inherited from the female, or from post-fertilization fragmentation of the zygotic genome. To directly assay zygotic genome integrity, mutant females were mated to wild type males and dual-label FISH was used to monitor physically separate regions of the Y chromosome. In male embryos derived from wild type females, the two Y chromosome probes always co-segregated through anaphase and telophase. Mutant embryos showing chromatin fragmentation, by contrast, contained chromatin clusters that did not label for either Y chromosome probe, or that labeled for only one of the two probes. In mutant embryos that proceeded through cleavage stage mitotic cycles, the majority of segregating chromatids retained both Y chromosome markers, indicating that chromosome continuity had been maintained. Chromatids with only one of two markers were observed, however, indicating that breaks had separated regions on a Y chromosome arm from the centromere. The axial patterning defects associated with piRNA mutations are suppressed by mutations in mnk, but mnk did not suppress either the chromatin fragmentation or segregation defects linked to aub and armi. Mutations in aub and armi thus destabilize the genome of the zygote and disrupt chromosome resolution during the cleavage divisions through processes that are independent of DNA damage signaling (Khurana, 2010).

Mutations in the armi and aub genes disrupt piRNA production and transposon silencing, but have also been reported to inhibit homology dependent target cleavage by siRNAs. In addition, null mutations in argonaute2 (ago2), which block siRNA based silencing, have been reported to disrupt mitosis during the syncytial blastoderm stage. These observations raise the possibility that chromatin fragmentation and fusion in aub and armi mutants result from defects in the siRNA pathway. Therefore, cleavage was analyzed in embryos from females homozygous for null mutations in ago2 and dcr2, which block siRNA production and silencing. Consistent with previous studies, it was found that embryos from ago2 and dcr2 mutant females are viable. However, neither chromosome fragmentation nor a statistically significant increase in anaphase bridge formation was found relative to wild type controls. The loquacious (loqs) gene encodes a Dicer-1 binding protein required for miRNA production, and it was found that embryos from loqs mutant females also proceed through normal cleavage stage divisions. Chromosome segregation and maintenance of zygotic genome integrity during early embryogenesis thus appear to be independent of the siRNA and miRNA pathways, but require at least two components of the piRNA pathway (Khurana, 2010).

In S. pombe, mutations in ago1, dcr1 and rdp1 disrupt kinetochore assembly and thus lead to lagging mitotic chromosomes due to defects in centromere movement to the spindle poles. To determine if Drosophila piRNA mutations disrupt kinetochore assembly, dual label FISH was performed for centromeric dodeca-satellite sequences and the telomere-specific transposon HeT-A. In aub and armi mutants, centromeric sequences segregated to the spindle poles in essentially every anaphase figure, but telomere specific sequences were consistently present at the chromatin bridges. These observations indicate that armi and aub are not required for kinetochore assembly, but are needed for telomere resolution (Khurana, 2010).

Telomeres are protected from recognition as DNA double strand breaks by the telomere-protection complex (TPC), and defects in telomere protection thus lead to covalent ligation of chromosome ends by the non-homologous end-joining (NHEJ) pathway. DNA Ligase IV is required for NHEJ, and ligase IV mutations suppress fusions that result from covalent joining of unprotected chromosome ends. To determine if chromosome fusions in aub and armi are due to NHEJ, ligIV;aub and ligIV;armi double mutant females were generated and chromosome segregation was analyzed in the resulting embryos. In aub single mutant embryos, 50% of anaphase figures show bridges, but anaphase bridges are present in only 15% of ligIV;aub double mutants. By contrast, the fraction of embryos showing chromosome fragmentation increases in ligIV;aub double mutants. Chromosome fragmentation also increased in ligIV;armi mutant embryos, and as a result morphologically normal anaphase figures could not be observed. These findings strongly suggest that lagging chromosomes result from covalent ligation of chromosome ends by the NHEJ pathway, while chromatin fragmentation results from DNA breaks that are repaired by NHEJ. Mutations in armi and aub lead to significant over-expression of transposable elements, including DNA elements that are mobilized by a 'cut and paste' mechanism that directly produces double strand breaks. In addition, NHEJ pathway has been implicated in repair of gapped retroviral integration intermediates. Chromosome fragmentation may therefore result from transposon over-expression and mobilization, which induces breaks that overwhelm the NHEJ pathway. Telomere fusions, by contrast, appear to result from defects in telomere protection, which lead to chromosome end recognition by the NHEJ pathway (Khurana, 2010).

The Drosophila TPC includes HOAP and Modigliani (Moi), which may function only at chromosome ends, and HP1a and the MRN complex, which have additional roles in heterochromatic silencing and DNA repair. To directly assay for TPC recruitment, chromatin immunoprecipitation (ChIP) was used to measure HP1a and HOAP binding to the telomere specific transposon HeT-A. In wild type ovaries, HOAP and HP1a bind to multiple regions of HeT-A. In armi and aub mutants, by contrast, HOAP and HP1a binding to the Het-A 5'-UTR and ORF are significantly reduced. The 5' end of Het-A is oriented toward the chromosome end, and is therefore likely to lie at the telomere. Ovarian tissue consists of germ cells with a surrounding layer of somatic cells, which complicates interpretation of these biochemical studies. However, ChIP on 0-3 hour old embryos from aub and mnk,aub mutant females revealed significant reduction in HOAP binding at the HeT-A 5'-UTR. The aub and armi genes thus appear to be required for TPC recruitment, consistent with ligation of chromosome ends in mutant embryos (Khurana, 2010).

To determine if other piRNA pathway mutations disrupt telomere protection, the cleavage stage embryonic divisions was analyzed in ago3 and rhi mutants. The ago3 locus encodes a PIWI clade protein that primarily binds sense strand piRNAs, and rhi encodes a rapidly evolving HP1 homologue required for production of precursor RNAs from a subset of piRNA clusters. Essentially all of the rhi and ago3 mutant embryos showed chromatin fragmentation, as observed in the majority of aub and armi mutants. Therefore TPC assembly was analyzed in ovarian chromatin using ChIP for HOAP and HP1a. Surprisingly, neither ago3 nor rhi mutations disrupt HOAP or HP1a binding to Het-A, and rhi mutants show greater than wild type levels of HOAP binding to Het-A. By contrast, these rhi alleles reduce total piRNA production by 10 fold. The ago3 mutations appear to be null, and the rhi mutations are strong hypomorphc alleles. Assembly of the TPC in the ago3 and rhi mutants is therefore unlikely to be mediated by residual protein. Instead, these findings strongly suggest that aub and armi have a function in telomere protection that is not shared by ago3 or rhi (Khurana, 2010).

In Drosophila, chromosome breaks can be converted to stable telomeres, called terminal deletions, which accumulate additional copies of the telomeric elements HeT-A and TART. When terminal deletions are passaged in animals heterozygous for aub or the piRNA pathway gene spnE, the number of terminal TART repeats increase. The defects in TPC assembly in aub and armi could therefore be triggered by increased HeT-A and TART copy number, which could titrate TPC components. Therefore telomeric transposon copy number was assayed in aub and armi mutants, which show defects in TPC assembly, and in rhi and ago3 mutants, which do not. Telomeric transposon copy number and mitotic chromosome segregation was also analyzed in a wild-type variant, Gaiano, that has been reported to carry additional HeT-A repeats. Consistent with previous reports, it was found that Gaiano has 10 to 15 fold more HeT-A copies than OregonR controls. Despite the increase in telomere length, this stock is viable and fertile, and no telomere fusions or lagging chromosomes were observed during the cleavage stage embryonic divisions. In addition, it was found that aub mutants that show defects in TPC assembly do not accumulate additional copies of HeT-A or TART, while rhi and ago3 mutants that are wild type for TPC binding show an increase in telomere-specific transposon copy number. Assembly of the TPC is therefore independent of telomere specific transposon copy number (Khurana, 2010).

piRNAs are proposed to direct PIWI clade proteins to targets through sequence specific interactions. The current observations raised the possibility that armi and aub promote production of piRNAs that direct the telomere protection complex to transposons that make up chromosome ends. Published small RNA deep sequencing data was analyzed for species derived from a fourth chromosome cluster, defined by a high density of uniquely mapping piRNAs, containing multiple repeats of the telomeric transposons. This bioinformatic analysis showed that 70-80% of telomere specific piRNAs match this cluster. Length histograms for small RNAs from wt, rhi, ago3, aub and armi mutant ovaries map to this cluster. Significantly, aub and armi mutations lead to a preferential loss of shorter piRNAs mapping to the minus genomic strand. Loss of these shorter RNAs highlights the peak at 21 nt, which is retained in all of the mutants and likely represent endogenous siRNAs. The telomeric elements (HeT-A and TART) are almost exclusively on the minus genomic strand in this cluster, and the RNAs that are lost in aub and armi thus correspond to the sense strand of the target elements. Ovaries mutant for ago3 and rhi, by contrast, retain these shorter sense strand RNAs (Khurana, 2010).

The relative abundance of typical 23-29nt long piRNAs and the shorter 19-22nt species were quantified, excluding the 21nt endo-siRNA peak. All four mutations significantly reduce 23 to 29 nt piRNAs, although rhi mutants retain approximately 50% of wild type minus strand species. Loss of these piRNAs is consistent with over-expression of transposons matching this cluster in all four mutants. By contrast, the shorter minus strand RNAs are reduced by 3 to 10 fold in armi and aub, but are expressed at 80% to 95% of wild type levels in ago3 and rhi. In addition, short piRNA species from the telomeric cluster co-immunoprecipitate with Piwi protein, which localizes to the nucleus and is a likely effector of chromatin functions for the piRNA pathway. Binding of this subpopulation of piRNAs by Piwi is retained in ago3 mutants, which assemble the TPC, but significantly reduced in armi mutants, which block assembly of the TPC (Khurana, 2010).

Taken together, these observations suggest that the piRNA pathway has two genetically distinct functions during oogenesis and early embryogenesis. The pathway prevents DNA damage during oogenesis and maintains the integrity of the zygotic genome during the embryonic cleavage divisions, which likely reflects the established role for piRNAs in transposon silencing. This function requires aub, armi, rhi and ago3, which are also required for wild type piRNA production. In addition, these studies reveal a novel function for the piRNA genes aub and armi in telomere protection, whch may be mediated by a novel class of short RNAs that bind to Piwi. Consistent with this hypothesis, it has been reported that germline clones of piwi null alleles do not significantly disrupt oogenesis, but lead to maternal effect embryonic lethality and severe chromosome segregation defects during the cleavage division. A subpopulation of Piwi-bound piRNAs may therefore direct assembly of the TPC (Khurana, 2010).

Genetic and molecular characterization of sting, a gene involved in crystal formation and meiotic drive in the male germ line of Drosophila melanogaster

sting mutation leads to a combination of a male sterile and maternal lethal effect in Drosophila melanogaster. In particular, the male sterility depends on a spectrum of meiotic abnormalities that closely resemble those produced by the deficiency of the Y-linked crystal locus. Cytological and genetic analyses show that the sti mutation induces the same three phenomena as in X/Y, cry- males: (1) the formation of crystalline aggregates in primary spermatocytes; (2) meiotic nondisjunction of sex chromosomes and autosomes, and (3) meiotic drive of sex chromosomes and autosomes. As in the case of X/Y, cry- males, the meiotic defects induced by the sti gene result from the activation of both the heterochromatic and euchromatic Ste sequence clusters, leading to the production of a protein that, depending on its amount, self-aggregates in needle- or star-shaped crystals (Schmidt, 1999).

The similarity of the meiotic effects to the situation in X/Y, cry- males goes even further. In both cases, sex chromosome nondisjunction is correlated to Ste copy number, but this is not found with respect to drive. Thus, the Ste-sti and Ste-cry interactions are a system in which sex chromosome disjunction and drive are uncoupled, in contrast to the deficiencies of the X-Y pairing sites of the X heterochromatin that cause strongly correlated nondisjunction and meiotic drive (Schmidt, 1999).

The Northern analysis of the Ste expression in sti mutant flies led to the discovery that the Ste sequences are basically transcribed in several tissues of males and females that produce high-molecular-weight transcripts. The observations that the high-molecular-weight Ste transcripts are present in males carrying the W12 X chromosome and lacking the euchromatic Ste cluster strongly suggest that these transcripts correspond to the heterochromatic Ste cluster. Unfortunately, at the present time, a viable X chromosome with a deletion of the heterochromatic Ste cluster is not available, it cannot be determined whether transcripts from the euchromatic cluster are also part of the high-molecular-weight fraction. Interestingly, a concomitant decrease of this fraction occurs with the production of the 750- to 850-nt Ste transcripts, which is more evident in the testes of cry- males independent of the Ste allelic status or in the testes of cry+ homozygous sti males when they carry a Ste allele. This indicates that at least for the heterochromatic Ste sequences, the regulation of their expression in X/Y, cry- testes could be post-transcriptional. It has already been shown that crystal can regulate the expression of Ste sequences at the transcriptional and splicing levels (Schmidt, 1999).

With the present results, it is not unreasonable to suggest that these two different levels of regulation reflect the fact that the heterochromatic and euchromatic clusters are under different regulatory mechanisms, namely that the euchromatic Ste sequences are under transcriptional control, while the heterochromatic sequences are mainly regulated at the post-transcriptional level (Schmidt, 1999).

The data clearly show that a gene has been identified that somehow interferes with the Ste-cry interaction system. Alternative hypotheses can be proposed to explain the interference of sti with the Ste-cry system. It is possible that both cry and sti are part of the same machinery that exerts control of Ste expression and, perhaps, additional genes in meiotic cells. However, it is possible that sti misfunction affects chromosome organization in meiotic cells, leading to the inactivation of the crystal locus, thus triggering Ste expression. In this case, there are at least two ways in which chromosomal alterations formally inactivate crystal. The sti mutation may induce Y chromosome loss or produce an alteration in the chromatin conformation of the crystal region. Careful analysis of homozygous sti male testes has revealed the presence of the Y loops in all mature spermatocytes, thus ruling out a sti-induced chromosome loss. Regarding the possible alteration in chromatin conformation, it is very difficult to perform a significant assay because the chromosomes are already altered as a result of the action of Ste protein. Therefore, at this time, there are no strong indications favoring one of the two alternative hypotheses (Schmidt, 1999).

The interaction between the autosomal sti and the X-chromosomal Ste gene opens the question of how these two partners have evolved. It is known, by Southern hybridizations, that the repetitive X-chromosomal Stellate gene exists only in D. melanogaster and its closest relatives, D. mauritiana and D. simulans (Livak, 1984). However, sequences homologous to at least parts of the Sti protein could be detected in a variety of organisms, ranging from the unicellular to higher plants and animals. Therefore, sti sequences should be detectable in more Drosophila species than those in which Ste sequences are found. This is indeed the case. The sting gene hybridizes even under stringent conditions with DNA from all species of the melanogaster subgroup, i.e., D. erecta, D. mauritiana, D. simulans, D. teissieri, and D. yacuba. Thus, the Ste-sti interaction is apparently a rather recent acquisition in some species of the melanogaster subgroup (Schmidt, 1999).

It is tempting to speculate that the primary sti function is related to the observed maternal lethality, and that this essential function is the one responsible for the conservation of Sti protein. Its function during spermatogenesis would then only be a secondary one. The modular structure of the upstream controlling regions favors this hypothesis; in these regions, the sequences necessary for expression in the ovary can clearly be separated from those that regulate expression in the male gonad. What, then, could be the primary function of the Sti protein? One possibility is that the wild-type function represses general or specific transcript processing. This is strongly supported by the Northern analysis of Ste expression, where processed Ste mRNA is found in mutant males. By this view, an induced out-of-phase gene expression should be responsible for the observed lethal maternal effect (Schmidt, 1999).


Effects on oskar translation

Although translational regulation of maternal mRNA is important for proper development of the Drosophila embryo, few genes involved in this process have been identified. The role of aubergine in oskar translation has been examined. aubergine has been implicated in dorsoventral patterning, since eggs from aubergine mutant mothers are ventralized and seldom fertilized (Schüpbach, 1991). Two new alleles of aubergine have been isolated in a novel genetic screen. aubergine has been shown to also be required for posterior body patterning, since the small fraction of eggs from aubergine minus mothers that are fertilized develop into embryos that lack abdominal segmentation. Although aubergine mutations do not appear to affect the stability of either oskar mRNA or protein, the level of Oskar protein is significantly reduced in aubergine mutants. Thus, aubergine is required to enhance oskar translation. While aubergine-dependence is conferred upon oskar mRNA by sequences in the oskar 3' UTR, aubergine may influence oskar translation through an interaction with sequences upstream of the oskar 3' UTR (Wilson, 1996).

The characterization of maternal-effect mutants that produce embryos with abdominal defects led to the discovery of the 'posterior group' genes, which act in posterior body patterning. Additional maternally acting genes are likely to participate in this process, but they may not have been identified as posterior group genes because they function at multiple stages of development, with the resulting phenotypes obscuring any posterior patterning defects. An approach to identify such genes was suggested by the finding that the bicaudal phenotype resulting from over- or mis-expression of osk could be partially suppressed if the mothers were also heterozygous for a mutation in one of the posterior group genes, such as vas or tud. Similarly, the BicaudalD phenotype is suppressed in vas minus and tud minus heterozygotes. Apparently, low levels of these proteins are limiting in the formation of bicaudal embryos. A screen for mutations which, when heterozygous, partially suppresses the formation of bicaudal embryos might therefore uncover additional genes involved in posterior body patterning. Such a screen was performed using the OB1 transgene to produce a bicaudal phenotype (Wilson, 1996).

The OB1 transgene consists of a slightly modified osk coding region fused to the bcd 3' UTR. Since the bcd 3' UTR contains the bcd mRNA localization signal, the encoded Osk protein is misexpressed at the anterior pole of the oocyte and embryo. Among the mutants recovered were two new alleles of aubergine. aub has been identified as one of a class of female-sterile mutants in which the eggshell is ventralized to variable extents (Schüpbach, 1991). Eggs laid by these mutants are typically not fertilized and consequently do not secrete cuticles, making it difficult to detect any additional patterning defects. The partial suppression of the bicaudal phenotype in aub minus heterozygotes led to an examination of the eggs laid by aub minus mothers (for simplicity, embryos are referred to by the genotype of their mothers). Approximately 2% of aub mutant eggs are fertilized. All of the resulting embryos lack abdominal segments, the classic posterior group mutant phenotype. (At least half of these cuticles display additional abnormalities, which presumably reflect substantial defects in both dorsoventral and anteroposterior body patterning). Thus aub is required for posterior patterning of the embryo. The fertilized eggs of aub mutant mothers lack pole cells; the same is true for most other posterior group mutants (Wilson, 1996).

Two events in which aub acts in posterior body patterning, and one in which aub acts in dorsoventral body patterning, have been documented. Considered in most detail is the enhancement of osk translation by aub. Curiously, the effect of aub mutations on osk translation does not appear to be related to the partial suppression of the OB1 bicaudal phenotype (Wilson, 1996).

A simple explanation for the action of aub in anteroposterior and dorsoventral patterning is that aub plays a role in a process that these two systems utilize in common. mRNA localization is one such process. Therefore the distributions of several localized mRNAs were compared in wild-type and aub mutant ovaries. The distributions of bcd and grk mRNA are wild-type in aub mutants. The early phases of osk mRNA localization, including concentration in the oocyte of early egg chambers and the initial localization to the posterior pole during stages 7-8, are also not affected by aub mutations. In contrast, aub mutants are defective in maintenance of osk mRNA localization. Normally, osk mRNA remains tightly localized to the posterior cortex of later-staged oocytes and early embryos. In aub mutants, osk mRNA prematurely diffuses from the posterior of oocytes and can only rarely be detected at the posterior of embryos (Wilson, 1996).

Although the results presented above could suggest that aub acts differently in anteroposterior and dorsoventral patterning, it is also possible that the primary role is in a single type of process, and that the requirement for aub in osk mRNA maintenance is indirect. One possibility is that aub mutants are defective in the accumulation of specific proteins. One affected protein would be Osk, since Osk protein acts to maintain osk mRNA localization, and another would be a protein required for dorsoventral patterning. To test this idea, the levels of Osk protein were compared in wild type and aub mutants. Despite normal amounts of osk mRNA, the levels of Osk protein are substantially reduced. At least two protein products of the osk gene are detected on Western blots: an abundant protein of approximately 55,000 Mr and a rarer protein of about 70,000 Mr. The amounts of both Osk proteins are decreased in aub mutants, although the 55,000 Mr protein is affected to a greater degree. Immunohistochemistry was used to monitor Osk protein. Under normal staining conditions, very little Osk protein is detected in aub mutant ovaries, and its appearance is slightly delayed relative to wild-type. By extending the staining reaction some Osk protein was detected in almost all aub mutant ovaries, with the protein appearing dispersed over the posterior third of the oocyte, rather than tightly concentrated at the posterior cortex as in wild-type ovaries (Wilson, 1996).

The level of Grk protein is also greatly reduced in aub mutants, while the level of grk mRNA remains normal. This decrease in the amount of Grk protein may give rise to the dorsoventral patterning defects of aub mutants. Nevertheless, the Grk protein present in aub mutants is apparently sufficient for establishment of correct anteroposterior polarity, since aub mutants do not exhibit general defects in mRNA localization or in migration of the oocyte nucleus to the anterior, two processes which rely on grk. In addition, aub is not generally required for the accumulation of proteins during oogenesis, since aub mutations do not affect the levels of any of several other ovarian proteins tested, including Vas, Exu, Kel, and Sn (Wilson, 1996).

Because osk mRNA levels remain normal in aub mutants, but Osk protein is reduced, aub must influence either the synthesis or stability of Osk protein. Evidence that argues strongly against a role for aub in stabilizing Osk protein comes from experiments employing the OB1 transgene. Osk protein is normally detected at both poles of OB1 transgenic embryos; the anterior protein is from the transgene, while the posterior protein is from the endogenous osk gene. In aub;OB1 embryos, the posterior Osk protein is largely missing, however, the anterior Osk protein encoded by the OB1 mRNA remains present at a high level. A similar effect is found in aub;OB1 ovaries. To rule out the possibility that the slight modification of the Osk protein encoded by the OB1 transgene affects the accumulation of anterior Osk protein in aub mutants, these experiments were repeated using the mob transgene. Like OB1, this transgene misexpresses Osk protein at the anterior pole of oocytes and embryos. However, mob encodes a wild-type Osk protein. The distributions of osk protein in aub;mob ovaries and embryos is indistinguishable from that in aub;OB1 animals (Wilson, 1996).

Because the proteins encoded by osk and mob are identical (the genes differ only in their 3' UTRs), aub is unlikely to affect the stability of Osk protein. Thus, aub appears to enhance translation of osk mRNA (Wilson, 1996).

The differential dependence of osk and OB1 mRNAs on aub for translation also provides some insight into which features confer aub-dependence on the osk mRNA. Because the mRNAs differ only in their 3' UTRs, it can be concluded that the requirement for aub is conferred by the osk mRNA 3' UTR. Further results suggest that aub may interact, directly or indirectly, with sequences outside of the osk 3' UTR to influence translation. While translation of osk mRNA is reduced in aub mutants, translation of the OB1 message is increased. Although only low levels of Osk protein are detected in the oocytes of early-staged, OB1 egg chambers, high levels of Osk protein are seen when these transgenic ovaries are additionally mutant for aub. Furthermore, the few, small patches of anteriorly localized Osk protein that are first detected in OB1 egg chambers in stages 7 and 8 are expanded to outline the anterior margin of aub;OB1 egg chambers. This effect is both substantial and highly reproducible. It is unlikely that the increased translation of OB1 mRNA in aub mutants is mediated through the bcd 3' UTR, which is present in the OB1 message, since the levels of Bcd protein (determined by whole-mount immunostaining and Western analysis) are not affected by aub mutations. Instead, wild-type aub function appears to modestly inhibit the translation of the hybrid message, and this effect must be mediated by sequences in the osk mRNA 5' UTR or coding region (Wilson, 1996).

Bruno protein binds sequences in the osk 3' UTR termed BREs and represses osk translation. One potential role for aub, as a positive regulator of osk translation, could be to override this known form of repression. If so, an osk mRNA not repressed by Bruno would not require aub for the normal high levels of translation. To test this possibility, the P[oskBRE minus] transgene, which differs from wild-type osk only by point mutations in the BREs, was used. The P[oskBRE minus] and control P[osk plus ] transgenes were introduced into aub minus flies. Comparing the levels of Osk protein in aub; P[oskBRE minus ] and aub; P[osk plus] ovaries by Western analysis, no difference was found. Thus the absence of Bruno-mediated repression does not eliminate the requirement for aub in osk translation (Wilson, 1996).

These results clearly implicate aub in translation of osk. However, they do not fully explain the posterior body patterning defects caused by the absence of aub for two reasons. (1) The reduced levels of Osk protein found in aub mutants might not be expected to eliminate abdominal segmentation completely, since Osk protein normally appears to be present in substantial excess. Instead, the dispersal of Osk protein across much of the posterior third of the oocyte in aub mutants might be expected to cause ectopic posterior body patterning. Nevertheless, there is little or no abdominal segmention in embryos from aub minus mothers. (2) While the OB1 bicaudal phenotype is partially suppressed in aub minus heterozygotes, translation of the OB1 transgene does not require aub: anterior Osk protein accumulates in OB1 ovaries (and also in embryos), even when both alleles of aub are mutant (Wilson, 1996).

An explanation for the extreme posterior patterning defects of aub mutants, and the partial suppression of the OB1 bicaudal phenotype in aub minus heterozygotes, is revealed by further analysis of embryos from OB1 or aub;OB1 mothers. In OB1 embryos, anterior Osk protein ectopically activates nos and results in the production of bicaudal embryos. Although anterior Osk protein is also present in aub;OB1 embryos, there is no corresponding anterior recruitment of nos mRNA or accumulation of Nos protein, despite normal levels of nos mRNA (Wilson, 1996).

Not surprisingly, the resulting embryos lack abdominal segmentation. Therefore aub is also required for a second event in posterior body patterning, intermediate between osk mRNA translation and nos mRNA localization. Again, this event might involve translational regulation. Candidate mRNAs for this second regulatory event are encoded by the vas and tud genes, both of which are conventionally portrayed as intermediate between osk and nos in the posterior body patterning hierarchy and are also required for the activity of anteriorly localized Osk protein. By Western analysis, however, both proteins remain at wild-type levels in aub mutant ovaries. Consequently, the second requirement for aub in posterior body patterning remains incompletely defined (Wilson, 1996).

Posterior localization of Aubergine

Vasa (Vas), a key protein in establishing the specialized translational activity of the Drosophila pole plasm, accumulates at the posterior pole of the developing oocyte. Mutation in gustavus (gus), a gene that encodes a protein that interacts with Vas, blocks posterior localization of Vas, as does deletion of a segment of Vas containing the GUS binding site. Like Vas, Gus is present in cytoplasmic ribonucleoprotein particles. Heterozygotes for gus or a deletion including gus produce embryos with fewer pole cells and posterior patterning defects. Therefore, Gus is essential for the posterior localization of Vas. However, gus is not required for the posterior localization of oskar (osk). The effects of the gusZ409 mutation on pole plasm assembly can mostly be explained through the failure of Vas to be deployed to the posterior pole plasm in this mutant. An exception, however, is the striking reduction of Osk level at the posterior of stage 10 gusZ409 oocytes, since vas alleles have little if any effect on Osk accumulation at this developmental stage. This observation can be explained if gus also affects the spatial distribution of Aub, a translational activator of osk that is a component of polar granules and probably of nuage particles. Posterior accumulation of Aub is vas dependent and therefore is presumably dependent on gus, but Aub-mediated translational activation of osk does not require it to be posteriorly deployed. However, if Aub is a component of nuage particles like Vas, which is suggested by the observation that vas mutations largely abolish perinuclear accumulation of Aub-containing particles in nurse cells, then the gusZ409 mutation could delay or block the movement of Aub through the ring canals and into the oocyte cytoplasm and render it less able to activate translation of osk, thus explaining the observation of decreased Osk at the posterior (Styhler, 2002).

Requirement for Pole Cell formation

Identification of Aub as a polar granule component suggests that it, like other such components, may be required for pole cell formation. Although aub mutants lack pole cells, this could be attributed to their deficiency in synthesis of Osk protein, a prerequisite for pole cell formation. Therefore, an osk-bcd transgene was used to bypass the requirement for aub in osk mRNA translation and to assess the role of aub in pole cell formation. In this transgene, the osk 3'UTR is replaced with the bicoid (bcd) localization signal, and the encoded mRNA is localized to the anterior of the oocyte. Eggs produced by aub+ females carrying an osk-bcd transgene synthesize Osk protein at both poles. In embryos produced by aub mutants carrying the osk-bcd transgene, Osk protein is absent from the posterior pole but efficiently translated at the anterior, reflecting a difference in the requirement for aub for translation of the transgenic and endogenous mRNAs (Wilson, 1996). However, while most of the Osk protein at the anterior pole is taken up in ectopic pole cells in embryos from aub heterozygous mothers, Osk protein particles become dispersed over the cortex in the anterior part of embryos from homozygous aub mutant mothers and fail to direct pole cell formation at the anterior pole. Thus, aub is required for the development of ectopic pole cells at the anterior of the embryo, and it is inferred that, like genes for other polar granule components, aub is involved in pole cell formation at the posterior as well (Harris, 2001).

Aub is found in particles that also contain Osk and are inferred to contain Vas, both known polar granule components. The Aub protein is located, like polar granules, at the posterior pole of the oocyte and embryo and are incorporated into pole cells. Although the posterior concentration of Aub in oocytes might seem to contribute to its role in translation of osk mRNA, which occurs predominantly at that site, several observations are at odds with this inference. In examination of more than 20 stage 8/9 egg chambers, it was discovered that the posterior accumulation of GFP-Aub is slightly delayed from the appearance of Osk protein in the same region. In the oocytes, significant Osk protein is often detected when very little GFP-Aub concentration at the posterior has occurred. In contrast, an egg chamber that had localized GFP-Aub but only low levels of Osk protein was never observed. Thus, it appears that Aub is not concentrated at the posterior before the onset of osk translation. No concentration of Aub is observed in the anterodorsal region of the oocyte where gurken (grk) mRNA is localized even though aub has also been implicated in the activation of grk translation (Harris, 2001).

Finally, it is possible to greatly reduce the posterior concentration of GFP-Aub and still initiate translation of Osk at normal levels. This situation occurs in vas mutants. Although previous reports have shown that vas mutant ovaries accumulate reduced levels of Osk protein, this study finds that Osk initially appears at normal levels in vas mutants. The previous analyses relied on Western blots of total ovarian protein, and so were unable to reveal any temporal specificity to the reduction. Confocal microscopy was used to compare Osk protein levels in ovaries and embryos from vas mutant and heterozygous females. Up to stage 11 of oogenesis, before deposition of the vitelline membrane interferes with whole-mount antibody staining, vas mutants accumulate normal amounts of Osk. By contrast, early embryos from vas mutant mothers display dramatic reductions in Osk protein. Thus, Osk protein levels are affected in vas mutants only in late stages of oogenesis, either through reduced translation or stability. The fact that normal levels of Osk appear in stage 9-11 vas mutant oocytes despite greatly lowered levels of GFP-Aub posterior accumulation, together with other observations, strongly suggest that the posterior concentration is not required for aub-dependent activation of osk translation (Harris, 2001).

Thus, the uniform low level of Aub found throughout the ooplasm appears to be sufficient for its action in osk and grk translation at the posterior pole and anterodorsal corner of the oocyte, respectively. The higher levels of Aub resulting from posterior recruitment presumably reflect other roles for Aub. These include two roles Aub shares with Vas: the posterior localization of nos mRNA (Wilson, 1996) and pole cell formation (Harris, 2001).

Piwi, Aubergine and Ago3 bind and cleave piwi-interacting RNAs to regulate of transposon activity in Drosophila

Drosophila Piwi-family proteins have been implicated in transposon control. This study examined piwi-interacting RNAs (piRNAs) associated with each Drosophila Piwi protein; Piwi and Aubergine were found to bind RNAs that are predominantly antisense to transposons, whereas Ago3 complexes contain predominantly sense piRNAs. As in mammals, the majority of Drosophila piRNAs are derived from discrete genomic loci. These loci comprise mainly defective transposon sequences, and some have previously been identified as master regulators of transposon activity. These data suggest that heterochromatic piRNA loci interact with potentially active, euchromatic transposons to form an adaptive system for transposon control. Complementary relationships between sense and antisense piRNA populations suggest an amplification loop wherein each piRNA-directed cleavage event generates the 5' end of a new piRNA. Thus, sense piRNAs, formed following cleavage of transposon mRNAs may enhance production of antisense piRNAs, complementary to active elements, by directing cleavage of transcripts from master control loci (Brennecke, 2007).

Mobile genetic elements, or their remnants, populate the genomes of nearly every living organism. Potential negative effects of mobile elements on the fitness of their hosts necessitate the development of strategies for transposon control. This is critical in the germline, where transposon activity can create a substantial mutational burden that would accumulate with each passing generation. Hybrid dysgenesis exemplifies the deleterious effects of colonization of a host by an uncontrolled mobile element. The progeny of intercrosses between certain Drosophila strains reproducibly show high germline mutation rates with elevated frequencies of chromosomal abnormalities and partial or complete sterility (Bucheton, 1990; Castro, 2004; Kidwell, 1977). Studies of the molecular basis of this phenomenon (Pelisson, 1981; Rubin, 1982) linked the phenotype to transposon mobilization (Brennecke, 2007).

Hybrid dysgenesis occurs when a transposon, carried by a male that has established control over that element, is introduced into a naive female that does not carry the element. The transposon becomes active in the progeny of the naive female, causing a variety of abnormalities in reproductive tissues that ultimately result in sterility. Since the dysgenic phenotype is often not completely penetrant, a fraction of the progeny from affected females may survive to adulthood. Such animals can develop resistance to the mobilized element, although in many cases, several generations are required for resistance to become fully established (Pelisson, 1987). Immunity to transposons can only be passed through the female germline (Bregliano, 1980), indicating that there are both cytoplasmic and genetic components to inherited resistance (Brennecke, 2007).

Studies of hybrid dysgenesis have served a critical role in revealing mechanisms of transposon control. In general, two seemingly contradictory models have emerged. The first model correlates resistance with an increasing copy number of the mobile element. A second model suggests that discrete genomic loci encode transposon resistance. The first model is supported by studies of the I element. Crossing a male carrying full-length copies of the I element to a naive female leads to I mobilization and hybrid dysgenesis. The number of I copies builds during subsequent crosses of surviving female progeny until it reaches an average of 1015 per genome. At this point, I mobility is suppressed, as the initially nave strain gains control over this element. Thus, a gradual increase in I element copy number over multiple generations was implicated in the development of transposon resistance (Brennecke, 2007).

The second model, which attributes transposon resistance to specific genetic loci, is illustrated by studies of gypsy transposon control (Bucheton, 1995). Genetic mapping of gypsy resistance determinants led to a discrete locus in the pericentric b-heterochromatin of the X chromosome that was named flamenco (Pelisson, 1994). Females carrying a permissive flamenco allele (one that allows gypsy activity) showed a dysgenic phenotype when crossed to males carrying functional gypsy elements. Permissive flamenco alleles exist in natural Drosophila populations but can also be produced by insertional mutagenesis of animals carrying a restrictive flamenco allele. Despite extensive deletion mapping over the flamenco locus, no transposon repressor from flamenco has been identified. For P elements, a repressor of transposition has been identified as a 66 kDa version of the P element transposase. Expression of the repressor was proposed to correlate with increasing P element copy number, leading to a self-imposed limitation on P element mobility. However, studies of resistance determinants indicated that control over P elements could also be established by insertion of P elements into specific genomic loci, arguing for an alternative, copy number-independent control pathway. Studies of inbred lines or of wild isolates with natural P element resistance indicated that P insertions near the telomere of X (cytological position 1A) were sufficient to confer resistance if maternally inherited. Additionally, several groups isolated insertions of incomplete P elements in this same cytological location that acted as dominant transposition suppressors. Importantly, these defective P elements lacked sequences encoding the repressor fragment of transposase (Brennecke, 2007 and references therein).

Both models of transposon resistance, those determined by specific genomic loci and those caused by copy number-dependent responses might be linked to small RNA-based regulatory pathways. Copy number-dependent silencing of mobile elements is reminiscent of copy number-dependent transgene silencing in plants (cosuppression) and Drosophila. In both cases, silencing occurs through an RNAi-like response where high-copy transgenes provoke the generation of small RNAs, presumably through a double-stranded RNA intermediate. Moreover, mutations in RNAi pathway genes impact transposon mobility in flies and C.elegans. Finally, small RNAs (rasiRNAs) corresponding to transposons and repeats have been isolated from flies and zebrafish (Brennecke, 2007 and references therein).

At the core of the RNAi machinery are the Argonaute proteins, which directly bind to small RNAs and use these as guides for the identification and cleavage of their targets. In animals, Argonautes can be divided into two clades (Carmell, 2002). One contains the Argonautes, which act with microRNAs and siRNAs to mediate gene silencing. The second contains the Piwi proteins. Genetic studies have implicated Piwi proteins in germline integrity. For example, piwi mutations cause sterility and loss of germline stem cells. aubergine is a spindle-class gene that is required in the germline for the production of functional oocytes. The third Drosophila Piwi gene, Ago3, has yet to be studied. Mutation of Piwi-family genes also affects mobile elements. For example, piwi mutations mobilize gypsy, and aubergine mutations impact TART and P elements. Finally, both Piwi and Aubergine bind rasiRNAs targeting a number of mobile and repetitive elements. These complexes are enriched for antisense small RNAs, as might be expected if they were actively involved in silencing transposons by recognition of their RNA products (Brennecke, 2007 and references therein).

Recently, a new class of small RNAs, the piRNAs, was identified through association with Piwi proteins in mammalian testes. These 26-30 nt RNAs are produced from discrete loci, generally spanning 50-100 kb. Interestingly, mammalian piRNAs are relatively depleted of transposon sequences. Despite apparent differences in the content of Piwi-associated RNA populations in mammals and Drosophila, Piwi-family proteins share essential roles in gametogenesis, with all three murine family members, Miwi2 , being required for male fertility. In order to probe mechanisms of transposon control in Drosophila and to understand the relationship between Piwi protein function in flies and mammals, a detailed analysis was undertaken of small RNAs associated with Piwi proteins in the Drosophila female germline. These studies indicate that Drosophila Piwi-family members function in a transposon surveillance pathway that not only preserves a genetic memory of transposon exposure but also has the potential to adapt its response upon contact with active transposons (Brennecke, 2007).

In C. elegans, effective RNAi depends upon an amplification mechanism. Small RNAs from the primary dsRNA trigger are largely dedicated to promoting the use of complementary targets as templates for RNA-dependent RNA polymerases (RdRPs) in the generation of secondary siRNAs. In Drosophila, no RdRPs have been identified. However, the ability of Piwi-mediated cleavage to prompt the production of new piRNAs could create an amplification cycle that serves the same purpose as the RdRP-driven secondary siRNA generation systems in worms (Brennecke, 2007).

The cycle, termed Ping-Pong amplification (Aravin, 2007; see Properties and biogenesis of piRNA) is initiated by generating primary piRNAs, sampled from the piRNA clusters that have been identified in this study. As these are composed mainly of defective transposon copies, they serve as a genetic memory of transposons to which the population has been exposed. piRNAs that are antisense to expressed, dispersed transposons would identify and cleave their targets, resulting in the genesis of a new, sense piRNA in an Ago3 complex. The Ago3 bound sense piRNA would then seek a target, probably a precursor transcript from a master control locus that contains antisense transposon sequences. Ago3-directed cleavage would then generate additional antisense piRNAs capable both of actively silencing their target element and reinforcing the cycle through the creation of additional sense piRNAs (Brennecke, 2007).

The existence of such an amplification cycle essentially permits sense and antisense piRNAs act in concert to increase production of silencing-competent RNAs in response to the activity of individual transposons. Since Argonautes act catalytically, a significant amplification of the response could be achieved by even a relatively low level of sense piRNAs in Ago3 complexes. This model predicts that piRNAs participating in this process, namely those with complementary partners, should be more abundant than piRNAs without detectable partners. In accord with this hypothesis, the most frequently cloned Aub and Ago3-associated piRNAs show an increased probability of having partners within the data set. Interestingly, Piwi-associated RNAs do not follow this pattern. Since the amplification cycle consumes target transposon transcripts as part of its mechanism, posttranscriptional gene silencing may be sufficient to explain transposon repression. However, the possibility that transcriptional silencing may also be triggered by Piwi-family RNPs cannot be ruled out (Brennecke, 2007).

The amplification cycle may not be absolutely essential for silencing of all elements, since loci such as flamenco may operate in somatic follicle cells where the absence of Aub and Ago3 forces it to act in a stoichiometric fashion. In this regard, flamenco is unusual in that the vast majority of transposon fragments within this locus exist in a common orientation, which can lead to the production of antisense primary piRNAs given a long, unidirectional, precursor transcript (Brennecke, 2007).

In contrast to flamenco, most piRNA loci appear to be both bidirectionally transcribed and contain transposon sequences in random orientation. Nevertheless, the marked asymmetry of Piwi/Aub and Ago3 complexes is conserved in piRNAs that uniquely map to clusters. Among piRNAs that match transposons, 77% and 79% of unambiguously cluster-derived Piwi- and Aub-associated RNAs are antisense, while 73% of those in Ago3 are sense. These observations strongly suggest that piRNA clusters themselves participate in choice. According to this model, the remarkable strand asymmetry in piRNA populations hinges on informative interactions between master control loci and active transposons, which by their nature produce sense RNAs. These observations identify Ago3 as the principal recipient of piRNAs derived from transposon mRNAs. Thus, as long as there is an input to the system from active transposon transcripts via Ago3 and a preferential relationship between Ago3 and Aub for generating secondary piRNAs in their reciprocal complexes, a strand bias can be maintained even if primary and secondary piRNAs can both be derived from master control loci (Brennecke, 2007).

The amplification cycle must be initiated by primary piRNAs. Presently, the biogenesis pathway that generates primary piRNAs from piRNA clusters remains obscure. The data suggest that the piRNA precursor is a long, single-stranded transcript that is cleaved, preferentially at U residues. Transcripts have been detected from piRNA loci by RT-PCR that encompass multiple transposon fragments and find numerous small RNAs that cross junctions between adjacent transposons. In the case of flamenco, P element insertions near the proximal end of the locus have a polar effect both on these long RNA transcripts and on flamenco piRNAs (Brennecke, 2007).

Equally mysterious is the generation of piRNA 3' ends. Mature piRNAs could arise by two cleavage events and subsequent loading into Piwi complexes. Alternatively, piRNAs could be created following 5' end formation and incorporation of a long RNA into Piwi by resection of their 3' ends. The latter model is attractive, since it could provide an explanation for observed size differences between RNAs bound to individual Piwi proteins, since piRNA size would simply reflect the footprint of each Piwi protein. Although de novo biogenesis mechanisms must exist, maternally inherited piRNA complexes could also serve to initiate the amplification cycle. All three Piwi proteins are loaded into the developing oocyte, and Piwi and Aub are concentrated in the pole plasm, which will give rise to the germline of the next generation. Inherited piRNAs could enhance resistance to transposons that are an ongoing challenge to the organism, augmenting zygotic production of primary piRNAs. Indeed, maternally loaded rasiRNAs were detected in early embryos, and their presence was correlated with suppression of hybrid dysgenesis in D. virilis (Brennecke, 2007).

These data point to a comprehensive strategy for transposon repression in Drosophila that incorporates both a long-term genetic memory and an acute response to the presence of potentially active elements in the genome. The model that emerges from these studies shows many parallels to adaptive immune systems. The piRNA loci themselves encode a diversity of small RNA fragments that have the potential to recognize invading parasitic genetic elements. Throughout the evolution of Drosophila species, a record of transposon exposure may have been preserved by selection for transposition events into these master control loci, since this is one key mechanism through which control over a specific element can be achieved. Evidence has already emerged that X-TAS can act as a transposition hotspot for P elements, raising the possibility the piRNA clusters in general may attract transposons. Once an element enters a piRNA locus, it can act, in trans, to silencing remaining elements in the genome, either directly through primary piRNAs or by engaging in the amplification model described above. A comparison of D. melanogaster piRNAs to transposons present in related Drosophilids shows a lack of complementarity when comparisons are made at high stringency. However, when even a few mismatches are permitted, it is clear that piRNA loci might have some limited potential to protect against horizontal transmission of these heterologous elements. The existence of a feed-forward amplification loop can be compared to clonal expansion of immune cells with the appropriate specificity following antigen stimulation, leading to a robust and adaptable response (Brennecke, 2007).

Small interfering RNAs function through the PIWI subfamily of Argonautes to ensure silencing of retrotransposons

In Drosophila, repeat-associated small interfering RNAs (rasiRNAs) are produced in the germ line by a Dicer-independent pathway and function through the PIWI subfamily of Argonautes to ensure silencing of retrotransposons. Small RNAs were sequenced associated with the PIWI subfamily member AGO3. Although other members of PIWI, Aubergine (Aub) and Piwi, associated with rasiRNAs derived mainly from the antisense strand of retrotransposons, AGO3-associated rasiRNAs arose mainly from the sense strand. Aub- and Piwi-associated rasiRNAs showed a strong preference for uracil at their 5' ends, and AGO3-associated rasiRNAs showed a strong preference for adenine at nucleotide. Comparisons between AGO3- and Aub-associated rasiRNAs revealed pairs of rasiRNAs showing complementarities in their first 10 nucleotides. Aub and AGO3 exhibited Slicer activity in vitro. These data support a model in which formation of a 5' terminus within rasiRNA precursors is guided by rasiRNAs originating from transcripts of the other strand in concert with the Slicer activity of PIWI (Gunawardane, 2007).

Small noncoding RNAs trigger various forms of sequence specific gene silencing, including RNA interference (RNAi), translational repression, and heterochromatin formation in a variety of eukaryotic organisms, commonly referred to as RNA silencing. Members of the Argonaute family of proteins are essential components of RNA silencing. In Drosophila, five genes encode distinct members of the Argonaute family: AGO1, AGO2, Aubergine (Aub), Piwi, and AGO3. AGO1 and AGO2 constitute the Argonaute (AGO) subfamily and bind microRNA (miRNA) and small interfering RNA (siRNA), respectively. Aub, Piwi, and AGO3 belong to the PIWI subfamily of the Argonaute family and are enriched in germline cells (Williams, 2002), and Aub and Piwi have been shown to play important roles in germline cell formation. They are involved in silencing retrotransposons and other repetitive elements and exhibit target RNA cleavage (slicing) activity in vitro . Both Aub and Piwi associate with repeat-associated siRNAs (rasiRNAs) (Vagin, 2006; Saito, 2006). Aub- and Piwi-associated rasiRNAs are derived mainly from the antisense strand of retrotransposons, with little or no phasing, and have a strong preference for uracil (U) at the 5' end. Small RNA processing factors such as Dicer and Drosha are known to cleave preferentially at the 5' side of U; however, rasiRNAs are thought to be produced by a Dicer-independent pathway (Vagin, 2006). The mechanisms governing rasiRNA production remain to be elucidated (Gunawardane, 2007).

Very little is known about the function of AGO3, the third member of the Drosophila PIWI subfamily. Full-length cDNA of AGO3 revealed that the AGO3 gene is 83 kb in length. Peptide sequence alignments among Drosophila Argonaute proteins revealed that AGO3 is most similar to Piwi. The Asp-Asp-His motif in the PIWI domain, originally identified as the catalytic center for Slicer activity in human AGO2, is conserved in AGO3 (Gunawardane, 2007).

Embryonic RNA expression patterns of AGO3 are very similar to those of Piwi and Aub; they are expressed maternally, but their expression disappears by embryonic stages 10 to 12. To confirm these results, a monoclonal antibody (mAb) to AGO3 was produced, that revealed that AGO3 is strongly expressed in earlier embryonic stages but decreases as development proceeds. AGO3 accumulates in the cytoplasm of germline cells including germline stem cells (GSCs), germline cyst cells, nurse cells, and oocytes at earlier stages. In testes, AGO3 is expressed in GSC, primary gonial cells, and early spermatocytes. Unlike Piwi, AGO3 expression was undetected in the hub, a tiny cluster of postmitotic somatic cells localized at the apical tip of the testis that functions as a niche for GSC). Thus, with respect to expression in germline cells, AGO3 is more similar to Aub than to Piwi (Gunawardane, 2007).

All of the other members of the fly Argonautes are specifically associated with a subset of small RNAs: siRNAs, miRNAs, or rasiRNAs. Therefore whether AGO3 also associates with small RNAs produced in the fly ovary was tested. Immunoprecipitation with AGO3 mAb from ovary lysate revealed small RNAs 23 to 26 nucleotides (nt) long. The size distribution of AGO3-associated small RNAs is similar to that of Aub-associated small RNAs; in both cases, the peak is 24 nt and the longest is 27 nt. Small RNAs associated with AGO3 are likely to lack either a 2' or 3' hydroxyl group, because they do not migrate faster after beta-elimination as opposed to a synthetic siRNA that has 2' and 3' hydroxyl groups at the 3' end, the latter being the hallmarks of Dicer cleavage. These results suggest that AGO3-associated small RNAs in the ovary are produced by a pathway similar to those involved in production of rasiRNAs that associate with Aub and Piwi (Gunawardane, 2007).

A cDNA library was constructed of small RNAs associated with AGO3 in the ovary. Of 420 clones sequenced, 410 matched Drosophila genomic sequences in a database search, and most were rasiRNAs (86%; 353 of 410), as in the case of Aub and Piwi. Like rasiRNAs associated with Aub or Piwi, rasiRNAs associated with AGO3 included various kinds of transposable elements, both LTR (long terminal repeat) retrotransposons and LINE (long interspersed nuclear element)like elements. rasiRNAs associated with Aub or Piwi in ovaries are derived mainly from the antisense strand of retrotransposons, and the 5' end is predominantly U. These characteristics were not found for rasiRNAs associated with AGO3. However, AGO3-associated rasiRNAs were derived mainly from the sense strand of retrotransposons (82%), and they showed a strong preference for adenine (A) at nucleotide 10, but no preference for U at the 5' end. These results suggest that AGO3-associated rasiRNAs belong to a subset of rasiRNAs that are distinct from Aub- and Piwi-associated rasiRNAs (Gunawardane, 2007).

Some Argonaute proteins exhibit Slicer activity that directs cleavage of its cognate mRNA target across from nucleotides 10 and 11, measured from the 5' end of the small RNA guide strand. Thus, these findings suggest a model for rasiRNA biogenesis, in which the 5' end of Aub- and Piwi-associated rasiRNAs is determined and cleaved by AGO3-rasiRNA complexes, and the 5' end of AGO3-associated rasiRNAs is determined by Aub- and Piwi-rasiRNA complexes through a similar rasiRNA-guided cleavage event. For instance, AGO3 associated with a rasiRNA with A at nucleotide 10 can target a long RNA molecule by Watson-Crick base pairing and cleave the target RNA, resulting in sliced RNAs with U at the 5' end. Similarly, when Aub or Piwi associated with rasiRNAs with U at the 5' end slices its cognate RNA target, the resulting cleaved RNA will have A at nucleotide 10 (Gunawardane, 2007).

To test this model, AGO3 was examined for Slicer activity by performing in vitro target RNA cleavage assays with glutathione S-transferase (GST)AGO3 fusions. The target RNA, luc passenger siRNA (21 nt long, 5' end labeled with 32P), was efficiently cleaved by GST-AGO3, as was the case for GST-AGO1 and GST-Aub. The size of the cleaved products (9 nt) indicated that they direct cleavage of target RNA across from nucleotides 10 and 11 as measured from the 5' end of the small RNA guide strand. Both GST-Aub and GST-AGO3 with a longer guide RNA (26 nt) were also able to cleave a long transcript (180 nt). Long precursors of rasiRNAs both in sense and antisense orientations appear to exist in fly ovaries. These results corroborate the model in which the 5' end of rasiRNAs within the precursors is determined by rasiRNAs and cleaved by members of PIWI that associate with these rasiRNAs (Gunawardane, 2007).

The model predicts that some AGO3-associated rasiRNAs should be complementary to the first 10 nt of Aub- and Piwi-associated rasiRNAs. Sequence comparison between AGO3- and Aub-associated rasiRNAs indeed revealed pairs of rasiRNAs that show complementarities at their first 10 nt. Sixteen of 353 AGO3-associated rasiRNAs had such pairs with 11 of 676 Aub-associated rasiRNAs. However, such pairings were only found between AGO3- and Aub-associated rasiRNAs, and no pairs were observed between AGO3- and Piwi-associated rasiRNAs (353 versus 330). Like Aub-associated rasiRNAs, Piwi-associated rasiRNAs arise mainly from the antisense strand and their 5' ends show a strong preference for U; thus, it is difficult to argue that Piwi is not involved in this type of rasiRNA biogenesis. One possible reason is that Piwi is nuclear, whereas AGO3 and Aub are cytoplasmic. This type of rasiRNA biogenesis may operate in the cytoplasm. Alternatively, formation of 5' ends of Piwi-associated rasiRNAs may occur only at an earlier time during germline development (Gunawardane, 2007).

RasiRNAs are involved in genome surveillance by silencing repetitive elements and controlling their mobilization in the Drosophila germ line. It was recently shown that rasiRNAs are produced by a mechanism that requires neither Dicer-1 nor Dicer-2 in flies. These data suggest that rasiRNAs in a sense orientation guide formation of the 5' end of rasiRNAs in an antisense orientation, and vice versa; as well, this cycle of mutual dependency elaborates optimal rasiRNA production. In this model, proteins of the PIWI subfamily function as Slicer for formation of the 5' end during rasiRNA biogenesis. This model requires that sliced rasiRNA precursors then be cleaved again at the 3' end by an as yet unidentified endonuclease (or nibbled by exonuclease) to produce mature rasiRNAs before or after loading of the resulting cleavage products onto another member of the PIWI. Once 'primary' complexes of rasiRNAs with proteins of PIWI are produced, these complexes will in turn function as the 'initiator' of secondary rasiRNA biogenesis, and so nascent rasiRNAs should be continuously supplied in the ovary and testis. Such a process may occur through rasiRNA germline transmission. Of the PIWI members, at least Aub is accumulated to the posterior pole in oocytes and remains in polar granules in early embryos. It is then incorporated in pole cells, the progenitor of the Drosophila germ line (10) (Gunawardane, 2007).

Without Argonaute3, Aubergine-bound piRNAs collapse but Piwi-bound piRNAs persist

Piwi-interacting RNAs (piRNAs) silence transposons in the germ line of animals. They are thought to derive from long primary transcripts spanning transposon-rich genomic loci, 'piRNA clusters.' piRNAs are proposed to direct an auto-amplification loop in which an antisense piRNA, bound to Aubergine or Piwi protein, directs the cleavage of sense RNA, triggering production of a sense piRNA bound to the PIWI protein Argonaute3 (Ago3). In turn, the new piRNA is envisioned to direct cleavage of a cluster transcript, initiating production of a second antisense piRNA. This study describes strong loss-of-function mutations in ago3, allowing a direct genetic test of this model. It was found that Ago3 acts to amplify piRNA pools and to enforce on them an antisense bias, increasing the number of piRNAs that can act to silence transposons. A second piRNA pathway was found centered on Piwi and functioning without benefit of Ago3-catalyzed amplification. Transposons targeted by this second pathway often reside in the flamenco locus, which is expressed in somatic ovarian follicle cells, suggesting a role for piRNAs beyond the germ line (Li, 2009).

The ability to tame transposons while retaining them in the genome is a particular specialty of eukaryotes. Transposons, repetitive sequences, and other forms of 'selfish' DNA comprise as much as 42% of the human genome and nearly 30% of the genome of Drosophila melanogaster. In metazoa, transposons are silenced by the piRNA pathway, which is guided by 23-30 nt RNAs. The piRNA pathway is distinct from other RNA silencing pathways in that its small RNA guides are not produced by dicing. In contrast, both small interfering RNAs (siRNAs) and microRNAs (miRNAs) are cleaved by double-stranded RNA-specific endonucleases, Dicers, to yield double-stranded intermediates-siRNA or miRNA/miRNA* duplexes-that are loaded into members of the Argonaute family of proteins. piRNAs, too, act as guides for Argonaute proteins, but they appear not to exist as stable double-stranded intermediates at any point in their biogenesis. piRNAs bind PIWI proteins, a sub-family of Argonaute proteins that are expressed in germ-line cells. PIWI proteins were first identified by their roles in maintaining and patterning Drosophila germ cells. The defects in the organization of embryonic pattern in piRNA pathway mutations are likely an indirect consequence of their larger role in maintaining genomic stability. The three Drosophila PIWI proteins, Piwi, Aubergine (Aub), and Argonaute3 (Ago3), are expressed in the male and female germ line cells (Li, 2009).

The prevailing model for piRNA biogenesis-the 'ping-pong' model-reflects the discovery that the first 10 nt of piRNAs bound to Aub or Piwi, which are largely antisense and typically begin with uridine, are often complementary to the first 10 nt of piRNAs bound to Ago3, which are largely sense and typically bear an adenosine at position 10. Many Argonaute proteins can act as RNA-guided RNA endonucleases, and all such Argonautes cut their target RNAs 5' to the base that pairs with the tenth nucleotide of the small RNA guide; all three fly PIWI proteins retain their endonuclease activity. Thus, the observed 10 nt 5' complementarity between piRNAs suggests that the 5' ends of piRNAs bound to Aub or Piwi are defined by Ago3-catalyzed cleavage, and, reciprocally, that the 5' ends of piRNAs bound to Ago3 are defined by piRNAs bound to Aub or Piwi. The ping-pong model seeks to explain these observations, as well as the role of piRNA cluster transcripts in piRNA biogenesis, the function of piRNAs in silencing transposons, and the extraordinary antisense bias of piRNAs generally. At its core, the model proposes that piRNAs participate in an amplification loop in which transposon sense transcripts (e.g., transposon mRNAs) trigger the production of new, antisense piRNAs. Ago3, guided by sense piRNAs, lies at the heart of the amplification loop (Li, 2009).

To test for the ping-pong model, strong loss-of-function mutations were isolated in ago3. This study report the detailed analysis of ago3 and aub mutant flies. The data provides strong support for an amplification cycle in which Ago3 amplifies piRNA pools and enforces on them a strong antisense bias, increasing the number of piRNAs that can act to destroy transposon mRNAs. Moreover, a second, perhaps somatic, piRNA pathway was detected, centered on Piwi and functioning without benefit of Ago3-catalyzed amplification. Most of the transposons targeted by this second pathway reside in the flamenco piRNA cluster, which was first identified as a repressor of transposon expression in somatic follicle cells (Li, 2009).

Because ovaries contain both germ-line and somatic cells, the data conflate two distinct cell lineages. Combining the data with extensive genetic studies of gypsy and other transposon families represented in the flamenco locus, this study has attempted to disentangle germ-line and somatic piRNA function. It is proposed that the somatic piRNA pathway is the more straightforward, involving only Piwi and not Ago3 or Aub. The data suggest that Piwi cannot act alone to amplify piRNAs. It is envisioned that Piwi-bound piRNAs in the soma are produced by a ribonuclease that randomly generates single-stranded guides that are subsequently loaded into Piwi and trimmed to length. Although Piwi-bound piRNAs generally begin with U and Piwi shows in vitro a preference for binding small RNA that begins with U, current evidence cannot distinguish between a putative piRNA-generating ribonuclease cleaving mainly at U and Piwi selecting U1 piRNAs from a set of RNAs with all possible 5' nucleotides (Li, 2009).

A model is presented for piRNA biogenesis. The Aub- and Ago3-dependent piRNA amplification cycle is envisioned to operate only in the germ line, whereas a Piwi-dependent, Aub- and Ago3-independent pathway is shown for somatic cells. In the germ line, Piwi can also partner with Ago3 to amplify piRNAs. Without an amplification cycle to ensure an antisense bias, some other mechanism must operate to explain why Piwi-bound piRNAs are overwhelmingly antisense. A plausible but somewhat unsatisfying explanation comes from flamenco itself, whose constituent transposons are nearly all oriented in a single direction, so that the ∼160 kb flamenco transcript is almost entirely antisense to the transposons. How such a non-random array of transposons could arise is unknown. Other non-randomly oriented piRNA clusters may explain the smaller number of transposons in group III that are not present in flamenco (Li, 2009).

The transposons in most piRNA clusters do not show such a pronounced non-random orientation. These likely act in the germ line to produce primary piRNAs that load into Aub. The observed antisense bias of Aub-bound piRNAs arises subsequently, when Aub generates Ago3-bound secondary piRNAs and Ago3 acts, in turn, to produce Aub-bound secondary piRNAs. It is proposed that in the absence of Ago3, the sense/antisense ratio of Aub-bound piRNAs reverts to the inherent sense/antisense bias of the transposable element sequences present in the transcripts of piRNA clusters (Li, 2009).

For this cycle to skew the Aub-bound piRNA population toward antisense, the substrate for cleavage by primary piRNA-bound Aub must be largely sense RNA. The best candidate for such sense RNA is mRNA derived from actively transcribed transposon copies. If such sense mRNA were largely found in the cytoplasm, it would be spatially segregated from the cluster transcripts, which is envisioned to be retained in the nucleus. Supporting this idea, sense transcripts from the group I transposon, I element, normally accumulate only in the nuclei of germ-line nurse cells (Chambeyron, 2008), likely because they are destroyed in the cytoplasm by Aub-bound primary piRNAs and Aub-bound piRNAs produced by Ago3-dependent amplification. In the absence of Aub, these sense transposon transcripts accumulate in the cytoplasm instead, consistent with the strong desilencing of I element in aub and ago3 mutants (Li, 2009).

The piRNA ping-pong hypothesis predicts a role for Ago3 in the production of Aub-bound antisense piRNAs, but the current finding that loss of Ago3 also reduced the abundance of Piwi-bound antisense piRNAs was unexpected. The majority of Aub and Ago3 is found in nuage and in the cytoplasm, but Piwi is predominantly nuclear. How then can Ago3 direct the production of Piwi-bound piRNAs? Perhaps Piwi transits the nuage en route from its site of synthesis, the cytoplasm, to where it accumulates, the nucleus. In this view, cytoplasmic Piwi is predicted to lack a small RNA guide. Piwi would then acquire its small RNA guide in the nuage, through a process that requires Ago3. Loading a piRNA into Piwi might then license it for entry into the nucleus, where it could act post-transcriptionally or transcriptionally to silence transposon expression. In this view, mutations in genes required for nuage assembly or stability, such as vasa, as well as genes required for Piwi loading would reduce the amount of nuclear Piwi. A similar mechanism may operate in mammals, where the PIWI protein MILI is found in cytoplasmic granules, whereas MIWI2 is nuclear. In the absence of MILI, MIWI2 delocalizes from the nucleus to the cytoplasm, although MIWI2 is not required for the localization of MILI (Li, 2009).

Such a model cannot explain the loading of Piwi in the somatic follicle cells, which contain little or no Ago3 or Aub and which do not contain nuage. A simple but untested hypothesis for these cells is that in the absence of nuage, empty Piwi readily enters the nucleus, where it obtains its small RNA guide. It might reasonably be expected that in germ cells the absence of nuage would impair the loading of Piwi by eliminating the Ago3-dependent, germ-line specific Piwi-loading process, but also facilitate entry of some empty Piwi into the nucleus, where it could obtain small RNA guides. Consistent with this idea, some Piwi is detected in the nucleus in ago3 ovaries. The simplicity of this hypothesis, of course, belies the complexity of testing it (Li, 2009).

Why are there two distinct piRNA production pathways? Retrotransposons 'reproduce' by producing sense RNA encoding transposases and other proteins that allow them to jump to new locations in the germ cell genome. The conservation of the piRNA ping-pong cycle in animals suggests that it is an ancient and conserved germ-line defense against retrotransposition. In flies, the gypsy family of retroelements appears to have moved its reproductive cycle to the somatic follicle cells adjacent to the germ line, which it infects using retrovirus-like particles. gypsy thus appears to avoid germ-line piRNA surveillance by transcribing and packaging its RNA in the soma. Perhaps expression of Piwi in Drosophila follicle cells reflects an adaptive evolutionary counter move to the gypsy reproductive strategy. The simplicity of the direct loading of Piwi with antisense piRNAs derived from flamenco may have made this counter defense more evolutionarily accessible than a strategy requiring expression of all the proteins needed for the Ago3:Aub ping-pong mechanism. In the future, more extensive analysis of the cellular and genetic requirements for ping-pong-independent and ping-pong-dependent piRNA mechanisms in Drosophila melanogaster and in more ancient animal species may provide a test for these ideas (Li, 2009).

Pimet, the Drosophila homolog of HEN1, mediates 2'-O-methylation of Piwi- interacting RNAs at their 3' ends

Piwi-interacting RNAs (piRNAs) consist of a germline-specific group of small RNAs derived from distinct intergenic loci in the genome. piRNAs function in silencing selfish transposable elements through binding with the PIWI subfamily proteins of Argonautes. This study shows that piRNAs in Drosophila are 2'-O-methylated at their 3' ends. Loss of Pimet/Hen1 (piRNA methyltransferase), the Drosophila homolog of Arabidopsis HEN1 methyltransferase for microRNAs (miRNAs), results in loss of 2'-O-methylation of fly piRNAs. Recombinant Pimet shows single-stranded small RNA methylation activity in vitro and interacts with the PIWI proteins within Pimet mutant ovary. These results show that Pimet mediates piRNA 2'-O-methylation in Drosophila (Saito, 2007; full text of article).

In Pimet mutant ovary, piRNAs associated with Aub and Piwi were not methylated at the 3' ends, most likely due to loss of Pimet expression. Whether GST-Pimet is able to methylate these piRNAs associated with the PIWI proteins from Pimet mutant ovary was investigated. Aub-piRNA complexes were immunopurified with a specific antibody against Aub and subjected to in vitro methylation assays. As a control, miRNAs associated with AGO1 were also obtained through immunoprecipitation using anti-AGO1 from ovary lysate. It was found that piRNAs were methylated even in a complex form with Aub. piRNA methylated in the assay showed resistance to oxidation and β-elimination treatment. Interestingly, miRNAs associated with AGO1 were not methylated, although these miRNAs are single-stranded in a complex form with AGO1. Confirmation that the miRNA levels were several-fold higher than those of piRNAs was provided by phosphorylation of these small RNAs. It seems that small RNA methylation by Pimet is largely influenced by the accessibility of the 3' ends of the substrates to Pimet itself. Structural analysis of Argonaute proteins suggests that the 5' end of the small guide RNA is anchored in a highly conserved pocket in the PIWI domain, whereas the 3' end of the small RNA is embedded in the PAZ domain. Taken together, these results suggest that the 3' ends of Aub-associated piRNAs are not tightly bound to the PAZ domain, but are exposed to the surface of the protein. In contrast, the 3' ends of AGO1-associated miRNAs are likely to be embedded in the PAZ domain and therefore are not exposed to the surface of the protein. Alternatively, but not mutually exclusively, it is conceivable that Pimet may interact only with PIWI proteins and not with AGO proteins, thereby methylating only small RNAs associated with PIWI proteins. To test this, whether Pimet associates with PIWI proteins was investigated. A GST pull-down assay was performed; GST-Pimet was first incubated with Pimet mutant ovary lysate, and after extensive washing the eluates were probed with PIWI protein antibodies. Aub, Piwi, and AGO3 were clearly detected in the bound fraction with GST-Pimet but not with GST itself. By contrast, AGO1 was not observed. These results indicated that Pimet is capable of physically interacting with PIWI proteins containing piRNAs that can serve as substrates for Pimet methylation. Addition of RNaseA did not affect the interaction of Pimet with Aub, suggesting that Pimet is able to associate directly with the PIWI proteins. In Drosophila, piRNA methylation may occur after matured piRNAs are loaded onto PIWI proteins. If so, it clearly differs from the case of miRNA methylation in plants, which likely occurs prior to miRNA loading onto the AGO proteins when miRNAs are still in a duplex form with the complementary miRNA* molecules (Saito, 2007).

Mutations in Arabidopsis hen1 cause reduced fertility. Thus, is the piRNA methylation by Pimet crucial in Drosophila? piRNAs function in genome surveillance in germlines in concert with PIWI proteins. Mutations in aub, piwi, and others like spindle-E (homeless) cause piRNAs not to be accumulated in gonads, and lead to germ cell malformation and sterility. This clearly indicates that piRNAs are necessary for perpetuation of organisms. However, the Pimet mutant fly seems to be viable and fertile. Steady-state levels of piRNAs in the methylation-defective mutant are also similar to those in wild type. Expression levels of retrotransposons do not seem to be changed by loss of Pimet expression. Thus, the function of 3' end methylation is currently unknown. Further investigation such as by immunohistochemistry may be required to obtain a more detailed morphology of the mutant. Extensive analyses of the mechanisms underlying piRNA methylation may also provide important clues to more fully elucidating piRNA biogenesis. Aub and AGO3, which determine and form the 5' end of piRNAs in piRNA biogenesis, were shown to be in the protein fraction associated with Pimet. Identifying more Pimet-associated proteins may reveal the factors required for formation of the 3' end of piRNAs (Saito, 2007).

Telomeric trans-silencing: an epigenetic repression combining RNA silencing and heterochromatin formation

The study of P-element repression in Drosophila led to the discovery of the telomeric Trans-Silencing Effect (TSE), a repression mechanism by which a transposon or a transgene inserted in subtelomeric heterochromatin (Telomeric Associated Sequence or TAS) has the capacity to repress in trans in the female germline, a homologous transposon, or transgene located in euchromatin. TSE shows variegation among egg chambers in ovaries when silencing is incomplete. This study reports that TSE displays an epigenetic transmission through meiosis, which involves an extrachromosomal maternally transmitted factor. This silencing is highly sensitive to mutations affecting both heterochromatin formation (Su(var)205 encoding Heterochromatin Protein 1 and Su(var)3-7) and the repeat-associated small interfering RNA (or rasiRNA) silencing pathway (aubergine, homeless, armitage, and piwi). In contrast, TSE is not sensitive to mutations affecting r2d2, which is involved in the small interfering RNA (or siRNA) silencing pathway, nor is it sensitive to a mutation in loquacious, which is involved in the micro RNA (or miRNA) silencing pathway. These results, taken together with the recent discovery of TAS homologous small RNAs associated to PIWI proteins, support the proposition that TSE involves a repeat-associated small interfering RNA pathway linked to heterochromatin formation, which was co-opted by the P element to establish repression of its own transposition after its recent invasion of the D. melanogaster genome. Therefore, the study of TSE provides insight into the genetic properties of a germline-specific small RNA silencing pathway (Josse, 2007; full text of article).

Repression of transposable elements (TEs) involves complex mechanisms that can be linked to either small RNA silencing pathways or chromatin structure modifications depending on the species and/or the TE family. Drosophila species are particularly relevant to the study of these repression mechanisms since some families of TEs are recent invaders, allowing genetic analysis to be carried out on strains with or without these TEs. In some cases, crossing these two types of strains induces hybrid dysgenesis, a syndrome of genetic abnormalities resulting from TE mobility. In D. virilis, repression of hybrid dysgenesis has been correlated to RNA silencing since small RNAs of the retroelement Penelope, responsible for dysgenesis, were detected in nondysgenic embryos but not in dysgenic embryos. In D. melanogaster, repression of retrotransposons can be established by noncoding fragments of the corresponding element (I factor, ZAM, and Idefix) and can be in some cases (gypsy, mdg1, copia, Het-A, TART, and ZAM, Idefix) sensitive to mutations in genes from the Argonaute family involved in small RNA silencing pathways. In the same species, strong repression of the DNA P TE, by a cellular state that has been called 'P cytotype', can be established by one or two telomeric P elements inserted in heterochromatic 'Telomeric Associated Sequences' (TAS) at the 1A cytological site corresponding to the left end of the X chromosome. This includes repression of dysgenic sterility resulting from P transposition. This P cytotype is sensitive to mutations affecting both Heterochromatin Protein 1 (HP1) (Ronsseray, 1996) and the Argonaute family member AUBERGINE (Reiss, 2004). P repression corresponds to a new picture of TE repression shown, using an assay directly linked to transposition, to be affected by heterochromatin and small RNA silencing mutants (Josse, 2007).

In the course of the study of P cytotype, a new silencing phenomenon has been discovered. Indeed, a P-lacZ transgene or a single defective P element inserted in TAS can repress expression of euchromatic P-lacZ insertions in the female germline in trans, if a certain length of homology exists between telomeric and euchromatic insertions. This homology-dependent silencing phenomenon has been termed Trans-Silencing Effect (TSE) (Roche, 1998). Telomeric transgenes, but not centromeric transgenes, can be silencers and all euchromatic P-lacZ insertions tested can be targets. TSE is restricted to the female germline and has a maternal effect since repression occurs only when the telomeric transgene is maternally inherited (Ronsseray, 2001). Further, when TSE is not complete, variegating germline lacZ repression is observed from one egg chamber to another, suggesting a chromatin-based mechanism of repression. Recently, an extensive analysis of small RNAs complexed with PIWI family proteins (AUBERGINE, PIWI, and AGO3) was performed in the Drosophila female germline. The latter study showed that most of the RNA sequences associated to these proteins derive from TEs. TSE corresponds likely to such a situation (Josse, 2007).

This study analyzed the genetic properties of TSE and shows that it has an epigenetic transmission through meiosis, which involves an extrachromosomal maternally transmitted stimulating component. Further, in order to investigate the mechanism behind TSE, a candidate gene analysis was performed to identify genes whose mutations impair TSE. It was found that TSE is strongly affected both by mutations in genes involved in heterochromatin formation and in the recently discovered small RNA silencing pathway called 'repeat-associated small interfering RNAs' (rasiRNA) pathway. In contrast, this study shows that TSE is not sensitive to genes specific to the classical RNA interference pathway linked to small interfering RNAs (siRNA) or to the micro RNA (miRNA) pathway. This suggests thus that TSE involves a rasiRNA pathway linked to heterochromatin formation and that such a mechanism, working in the germline, may underlie epigenetic transmission of repression through meiosis (Josse, 2007).

Arginine methylation of Piwi proteins catalysed by dPRMT5 is required for Ago3 and Aub stability

Piwi family proteins are essential for germline development and bind piwi-interacting RNAs (piRNAs). The grandchildless gene aub of Drosophila encodes the piRNA-binding protein Aubergine (Aub), which is essential for formation of primordial germ cells (PGCs). This study reports that Piwi family proteins of mouse, Xenopus laevis and Drosophila contain symmetrical dimethylarginines (sDMAs). Piwi proteins are expressed in Xenopus oocytes and numerous Xenopus piRNAs were identified. This paper reports that the Drosophila homologue of protein methyltransferase 5 (dPRMT5, capsuleen/dart5), which is also the product of a grandchildless gene, is required for arginine methylation of Drosophila Piwi, Ago3 and Aub proteins in vivo. Loss of dPRMT5 activity led to a reduction in the levels of piRNAs, Ago3 and Aub proteins, and accumulation of retrotransposons in the Drosophila ovary. These studies explain the relationship between aub and dPRMT5 (csul/dart5) genes by demonstrating that dPRMT5 is the enzyme that methylates Aub. These findings underscore the significance of sDMA modification of Piwi proteins in the germline and suggest an interacting pathway of genes that are required for piRNA function and PGC specification (Kirino, 2009).

Piwi family proteins are expressed in the germline and bind ~26 to ~30 nucleotide (nt) piRNAs. Drosophila express three Piwi proteins: Aub, Piwi and Ago3. Mice also express three Piwi proteins termed Miwi, Mili/PiwiL2 and Miwi2/PiwiL4. Tens of thousands of distinct piRNAs have been described and most of them are species-specific. In Drosophila , Piwi proteins and piRNAs (also known as rasiRNAs - repeat associated small interfering RNAs) silence transposons in the germline. A similar function has been found for a subset of mouse and zebrafish piRNAs. An amplification loop of piRNAs has been described but how primary piRNAs are generated is unknown. sDMA modifications occur in sequence motifs composed of arginines flanked by glycines (GRG) or alanines (GRA or ARG) that are often found as repeats. PRMT5 and its cofactors MEP50/WD45 and pICln form the methylosome that methylates Sm proteins. A highly specific monoclonal antibody (17.8) was produced that recognizes Mili by Western blot, immunoprecipitation and immunofluorescence microscopy. By serendipity it was discovered that the widely used Y12 monoclonal antibody recognizes mouse Mili and Miwi proteins and their bound piRNAs. The Sm proteins of spliceosomal small nuclear ribonucleoproteins (snRNPs) constitute the main antigen for Y12. piRNAs were not identified in immunoprecipitates of snRNPs, heterogeneous ribonucleoproteins (hnRNPs) or of the Survival of Motor Neurons (SMN) complex using various antibodies. By Northern blot analysis it was found that piR-1, but not miR-16, an abundant miRNA, was found in Y12 immunoprecipitates, suggesting that Y12 recognizes Piwi but not Ago proteins (Kirino, 2009).

The epitope that Y12 recognizes on Sm proteins consists of symmetrically dimethylated arginines, in the glycine-arginine rich regions of the proteins. It was reasoned that Y12 likely reacted with sDMA-containing epitopes in Mili and Miwi, and arginine residues were sought that could be symmetrically methylated. Intriguingly, it was found that most animal Piwi proteins contain sDMA motifs that are typically clustered close to the amino terminus, while no animal Ago proteins contained such motifs. However, it was found that four of ten Arabidopsis Ago proteins contained sDMA motifs (Kirino, 2009).

To test whether Miwi and Mili contain sDMAs, SYM11 and ASYM24 antibodies, which specifically recognize proteins containing sDMA-glycine or aDMA-glycine repeats, respectively, were used. SYM11, as well as Y12, reacted strongly with endogenous Miwi and Mili, while ASYM24 showed only faint reactivity towards endogenous Miwi. In contrast, recombinant Flag-Mili or Flag-Miwi purified from baculovirus-infected Sf9 cells, were not recognized by Y12 or SYM11 (or ASYM24). This is entirely consistent with the finding that recombinant human Sm proteins expressed in Sf9 cells also do not contain sDMAs because Sf9 cells do not express type II PRMTs and thus cannot produce sDMA modifications. These findings indicate that Mili and Miwi proteins contain sDMAs. The putative sDMA motifs of Miwi are concentrated very close to the amino terminus with the exception of one GRG triplet. Flag-tagged full-length Miwi and two truncated forms of Miwi (aa 68-862 or 1-212) were transfected in 293T cells, by Flag immunoprecipitation and subject to western blot with SYM11 antibody, SYM11 antibody recognizes the amino terminus of Miwi protein (Kirino, 2009).

Next it was asked whether the sDMA modification was conserved in Piwi family proteins from other species. A stumbling block in studying the molecular functions of Piwi proteins and piRNAs has been the lack of suitable cell culture systems. It was reasoned that Xenopus laevis oocytes might express Piwi proteins and piRNAs and thus prove very useful not only to confirm that sDMAs of Piwi proteins are conserved but also as a model to study the function of Piwi proteins and piRNAs. By searching the Gurdon EST database at Xenbase three Xenopus Piwi proteins were identified which were named Xili, Xiwi and Xiwi2. All three Xenopus Piwi proteins contain putative sDMA motifs. Immunoprecipitations with Y12 from X. laevis oocytes (defolliculated, mixed Dumont stages I-VI), testis and liver revealed the presence of two proteins at ~95 kDa and ~110 kDa specifically in the Y12 immunoprecipitates from oocytes and testis that were identified by mass spectrometry as Xiwi and Xili respectively. As shown in the western blots, Y12 recognizes both Xiwi and Xili, while anti-Mili (17.8) reacts only with Xili. In addition, both Xiwi and Xili are recognized by SYM11, indicating that Xiwi and Xili contain sDMAs (Kirino, 2009).

X. laevis piRNAs were isolated and analyzed from Y12 immunoprecipitates. ~26-29 nt piRNAs are present in the Y12 immunoprecipitates and their 3'-termini are not eliminated by periodate oxidation and are thus likely 2'-O-methylated, as seen in piRNAs from other species (Kirino, 2009).

Deep sequencing was performed of X. laevis piRNAs from Y12 immunoprecipitates of oocytes and testis. The nucleotide composition of X. laevis piRNAs shows enrichment of Uridine in the first nucleotide position and of Adenine in the tenth nucleotide position. There is also enrichment for piRNAs whose first 10 nucleotides are complementary to the first 10 nucleotide of other piRNAs. These features indicate that a fraction of X. laevis piRNAs target transposon transcripts and that they also participate in a piRNA amplification loop, as has been described for Drosophila and zebrafish piRNAs and prepachytene mouse piRNAs. By Northern blot XL-piR-3, a representative piRNA, is expressed specifically in oocytes and by in situ hybridization XL-piR-3 is localized predominantly in the cytoplasm of X. laevis oocytes and it is expressed in higher levels in immature oocytes (Kirino, 2009).

Genetic disruption of either Drosophila PRMT5 (dPRMT5; also know as capsuleen - csul- , and dart5) or its cofactor valois, (the Drosophila homolog of MEP50/WD45), results in complete loss of sDMA modifications of Sm proteins in ovaries. However, unlike the situation in mammals, the levels or function of Sm proteins is not affected by loss of sDMAs (Kirino, 2009).

Null or hypomorphic alleles of dPRMT5 (csul, dart5) phenocopy aub null alleles and it was reasoned that dPRMT5 might be the methyltransferase that produces sDMAs in Aub, Piwi and Ago3, in vivo. To test this, ovaries were used from csulRM50/Df(2R)Jp7 females, which give rise to embryos that are genetic nulls for dPRMT5 and w- was used as a wild-type control. Western blots of ovary lysates from wt and maternal null csul showed that there was near complete loss of SYM11 reactivity, indicating dramatic reduction of sDMA modified proteins in csul ovaries. There was no change in ASYM24 reactivity between wt and csul, indicating that aDMA modified proteins were not affected. These findings confirm that dPRMT5 (csul, dart5) activity is required specifically for sDMA modification. Piwi and Aub proteins were immunoprecipitated from wt and csul mutant ovaries and the immunoprecipitates were probed with SYM11 and ASYM24. SYM11 reacted very strongly with Aub and also with Piwi immunopurifed from wt but not csul ovaries; ASYM24 reacted only weakly with Aub from wt ovaries. Immunoprecipitates of Ago3 were also with SYM11 and ASYM24 and it was observed that only Ago3 from wt ovaries reacted with SYM11. These results indicate that, like the mouse and X. laevis Piwi family proteins, Drosophila Piwi, Aub and Ago3 contain sDMAs and that dPRMT5 is the methylase that produces sDMAs of these proteins (Kirino, 2009).

In Aub the four arginines that are putative substrates for symmetrical dimethylation are found in tandem very close to the amino terminus. Site-directed mutagenesis was used to change these arginines into lysines that are not subjected to methylation by PRMTs. Flag-tagged wild-type (WT) or mutant (M) Aub were stably transfected into Drosophila S2 cells (which express dPRMT5), the proteins were purified by Flag immunoprecipitation and subjected to western blot with Flag, SYM11 and ASYM24 antibodies. SYM11 antibody reacted only with wild-type Aub. Next the binding of wild-type and mutant Aub to a synthetic piRNA was assayed. Immunopurified, wild-type or mutant Flag-Aub were incubated with a 5'-end radiolabeled synthetic piRNA containing 4-thio-Uridine at the first position, followed by crosslinking with Ultraviolet light and NuPAGE analysis. There was similar binding between wild-type and mutant Aub proteins. These findings indicate that one or more of the four arginines in the amino terminus of Aub are symmetrically dimethylated and arginine methylation does not impact piRNA binding (Kirino, 2009).

Next, RNAs bound to Piwi and Aub were isolated and analyzed from wt or csul ovaries. piRNAs remain bound to Piwi and Aub proteins in the csul ovaries. There is mild reduction of Piwi-piRNAs and marked reduction of Aub-piRNAs in csul ovaries corresponding to concordant reduction of protein levels of Piwi and Aub. The Piwi associated piRNAs were gel purified and subjected to periodate oxidation followed by β-elimination and it was revealed that Piwi-associated piRNAs purified from csul ovaries retain 2'-O-methylation of their 3' termini. These findings indicate that the lack of sDMA modifications of Piwi and Aub in csul ovaries does not impair the methylation of piRNAs or their binding to Piwi and Aub (Kirino, 2009).

Next the protein levels of Piwi family proteins were compared between wt, heterozygous and homozygous csul ovaries. Western blot analysis showed that there was marked reduction of Aub and Ago3 protein levels and lesser reduction of Piwi levels in csul ovaries, whereas the levels of the miRNA binding protein Ago1 were not affected. Since mRNA levels of Aub, Piwi and Ago3 are the same between wt and csul ovaries, dPRMT5 activity might be required to stabilize the Aub, Ago3 and Piwi proteins most likely by symmetrically methylating their arginines. The level of a representative piRNA (roo-rasiRNA), was decreased in csul ovaries in accord with reduction of Piwi family proteins, while the level of a representative miRNA, miR-8, was not affected. The homozygous csul ovaries showed a 30-fold increase in the levels of the HeT-A retrotransposon transcript, whose expression is most sensitive to mutations that disrupt piRNA-directed silencing in the female germline. Collectively these results indicate that loss of dPRMT5 activity impairs the amounts of Piwi proteins and piRNAs, resulting in disruption of their function of transposon silencing (Kirino, 2009).

Next the localization of Ago3, Aub and Piwi was analyzed by confocal microscopy in wt and homozygous csul early stage egg chambers. Previous studies have shown that Piwi is localized predominantly in the nuclei of follicle and germ cells while Ago3 and Aub are localized in the cytoplasm of germ cells. In oocytes, Aub is concentrated in the germ (pole) plasm. Representative images reveal that the level of Ago3 is markedly reduced in csul early stage egg chambers, while there is only a mild reduction of Aub and Piwi protein levels (Kirino, 2009).

Germ cell (PGC) formation in Drosophila requires that cytoplasmic determinants are localized to the posterior pole of the embryo. Genetic screens have identified grandchildless maternal genes that are required for PGC specification and invariably the protein or RNA products of these genes are concentrated in the pole plasm and are incorporated into the PGCs. Among these genes are Aub, dPRMT5 (csul, dart5), and its cofactor valois and tudor, whose protein product contains eleven tudor domains. The localization of Aub was tested in csul oocytes by confocal microscopy. Representative results show that the levels of Aub in the pole plasm of stage 10 egg chambers are markedly reduced. Western blotting reveals marked reduction of Aub protein levels in csul ovaries while confocal microscopy shows that Aub levels are subtly reduced in early stage egg chambers but markedly reduced in later stage egg chambers, suggesting that lack of sDMAs affects Aub levels at later stages in oogenesis (Kirino, 2009).

These studies show that sDMA modification of Piwi family proteins is a conserved post-translational modification, and the methyltransferase PRMT5 (csul/dart5) is identified as the enzyme that catalyzes sDMAs of Piwi, Ago3 and Aub in Drosophila ovaries, in vivo. Both Aub and csul/dart5 (dPRMT5) are grandchildless genes and the finding that Aub is a substrate for dPRMT5, indicates that an important function of dPRMT5 in pole plasm function and PGC specification involves methylation of Aub. Intriguingly, tudor domains bind to sDMAs and Tudor protein is also a grandchildless gene that is required for pole plasm assembly and function. These findings suggest that pole plasm function may involve an interacting network of genes whose protein products contain sDMAs (Aub), the methylase (dPRMT5) and its cofactor (valois/dMEP50) that produce sDMAs and tudor domain (Tudor) proteins that may bind to sDMA-containing proteins. It is noted that both PRMT5 and tudor-domain-containing genes are found in all species that express Piwi family proteins and knockout of tudor domain containing 1/mouse tudor repeat 1 in mice leads to spermatogonial cell death and male sterility. Furthermore, it is noted that other Drosophila proteins whose genes are required for piRNA accumulation or function, such as Spindle-E/homeless, contain tudor domains. It will be interesting to test whether tudor domain containing proteins interact with sDMA-modified Piwi family proteins and to elucidate their function (Kirino, 2009).

Maternal mRNA deadenylation and decay by the piRNA pathway in the early Drosophila embryo

Piwi-associated RNAs (piRNAs), a specific class of 24- to 30-nucleotide-long RNAs produced by the Piwi-type of Argonaute proteins, have a specific germline function in repressing transposable elements. This repression is thought to involve heterochromatin formation and transcriptional and post-transcriptional silencing. The piRNA pathway has other essential functions in germline stem cell maintenance and in maintaining germline DNA integrity. This study uncovered an unexpected function of the piRNA pathway in the decay of maternal messenger RNAs and in translational repression in the early embryo. A subset of maternal mRNAs is degraded in the embryo at the maternal-to-zygotic transition. In Drosophila, maternal mRNA degradation depends on the RNA-binding protein Smaug and the deadenylase CCR4, as well as the zygotic expression of a microRNA cluster. Using mRNA encoding the embryonic posterior morphogen Nanos (Nos) as a paradigm to study maternal mRNA decay, it was found that CCR4-mediated deadenylation of nos depends on components of the piRNA pathway including piRNAs complementary to a specific region in the nos 3' untranslated region. Reduced deadenylation when piRNA-induced regulation is impaired correlates with nos mRNA stabilization and translational derepression in the embryo, resulting in head development defects. Aubergine, one of the Argonaute proteins in the piRNA pathway, is present in a complex with Smaug, CCR4, nos mRNA and piRNAs that target the nos 3' untranslated region, in the bulk of the embryo. It is proposed that piRNAs and their associated proteins act together with Smaug to recruit the CCR4 deadenylation complex to specific mRNAs, thus promoting their decay. Because the piRNAs involved in this regulation are produced from transposable elements, this identifies a direct developmental function for transposable elements in the regulation of gene expression (Rouget, 2010).

In Drosophila embryos, Nos is expressed as a gradient that emanates from the posterior pole and organizes abdominal segmentation. The majority of nos mRNA is distributed throughout the bulk cytoplasm, translationally repressed and subsequently degraded during the first 2-3h of development. This repression is essential for head and thorax segmentation. A small amount of nos transcripts, localized at the posterior pole of the embryo, escapes degradation and is actively translated, giving rise to the Nos protein gradient. nos mRNA decay in the bulk cytoplasm depends on the CCR4-NOT deadenylation complex and its recruitment onto nos by Smaug (Smg). This contributes to translational repression in the bulk of the embryo and is required for embryonic antero-posterior patterning (Zaessinger, 2006; Rouget, 2010 and references therein).

Smg has been suggested to be not the only activator of nos mRNA decay during early embryogenesis. Zygotically expressed miRNAs have been reported to activate maternal mRNA deadenylation in zebrafish embryos and decay in Drosophila embryos. This study investigated the potential involvement of other classes of small RNAs in mRNA deadenylation and decay before zygotic expression. Because piRNAs are expressed maternally in the germ line and are present in early embryos, the possible role of the piRNA pathway in maternal mRNA deadenylation was analyzed. Piwi, Aubergine (Aub) and Ago3 are specific Argonaute proteins, Armitage (Armi) and Spindle-E (Spn-E) are RNA helicases, and Squash (Squ) is a nuclease involved in piRNA biogenesis and function. Poly(A) test assays were performed to measure nos mRNA poly(A) tail length in embryos spanning 1-h intervals during the first 4 h of embryogenesis. In contrast to the progressive shortening of nos mRNA poly(A) tails observed in wild-type embryos correlating with mRNA decay during this period, nos poly(A) tail shortening was affected in embryos from females mutant for the piRNA pathway (herein referred to as mutant embryos). This defect in deadenylation correlated with higher amounts of nos mRNA in mutant embryos, as quantified by reverse transcription-quantitative PCR (RT-qPCR). In situ hybridization revealed stabilized nos mRNA in the bulk cytoplasm of mutant embryos where it is normally degraded in the wild type. Consistent with previous data showing that nos mRNA deadenylation is required for translational repression (Zaessinger, 2006), defective deadenylation in mutant embryos resulted in the presence of ectopic Nos protein throughout the embryo. The presence of Nos in the anterior region results in the repression of bicoid and hunchback mRNA translation and in affected head skeleton. It was found that the piwi1 mutant embryos that were able to produce a cuticle had head defects (Rouget, 2010).

The piRNA pathway has a role during early oogenesis in preventing DNA damage, possibly through the repression of transposable element transposition. DNA double-strand breaks arising in mutants of the piRNA pathway correlate with affected embryonic axis specification, and this developmental defect is suppressed by mutations in the Chk2 DNA-damage signal transduction pathway. This study found that defects in nos mRNA deadenylation and decay observed in aub or armi mutants were not suppressed by Chk2 (mnkP6) mutations, indicating that these defects did not result from activation of the Chk2 pathway earlier during oogenesis. Moreover, affected deadenylation of nos mRNA in piRNA pathway mutants did not depend on oskar (Rouget, 2010).

A potential direct role of the piRNA pathway in the regulation of nos mRNA deadenylation and decay in the embryo was addressed. Aub and Piwi accumulate in the pole plasm and in pole cells of the embryo. However, lower levels of Aub and Piwi are found throughout the entire embryo. Ago3 is also present throughout the embryo. Aub and Ago3 are found in the cytoplasm and accumulated in discrete foci, a distribution similar to that of CCR4 and Smg. CCR4 and Smg were reported to partially colocalize in small cytoplasmic foci (Zaessinger, 2006). Aub and Ago3 also partially colocalize with Smg and CCR4 in the bulk of syncytial embryos, in both cytoplasmic foci and a diffusely distributed cytoplasmic pool. Importantly, the distributions of CCR4 and Smg depended on the piRNA pathway; they are strongly affected in aub and spn-E mutant embryos. Although global amounts of CCR4 and Smg do not decrease in mutant embryos, CCR4 foci strongly increase in size, whereas Smg foci decreases in size or disappears. This suggests that subsets of CCR4 and Smg foci have different functions and that deadenylation may take place diffusely in the cytoplasm. These results demonstrate a functional link between CCR4-mediated deadenylation and the piRNA pathway (Rouget, 2010).

Co-immunoprecipitation experiments showed that Aub co-precipitate Smg, CCR4 and Ago3 in the absence of RNA, indicating the presence of these proteins in a common complex. Smg also co-precipitates CCR4, Aub and Ago3; however, Piwi is not found to co-precipitate Smg or CCR4. Importantly, Smg, CCR4 and Ago3 also co-precipitate with Aub in osk54 mutant embryos that are defective in pole plasm assembly, indicating the presence of this complex outside the pole plasm. Next it was shown that nos mRNA co-precipitate with Aub in both wild-type and osk54 embryos. The amount of nos mRNA is similar in Aub and Smg immunoprecipitates (Rouget, 2010).

These findings show that the Argonaute proteins Aub and Ago3 associate with Smg and the CCR4 deadenylase complex to directly regulate nos mRNA in the bulk cytoplasm of early embryos (Rouget, 2010).

The nos 3'untranslated region (UTR) contains Smg-binding sites located in its 5'-most region [referred to as the translational control element (TCE)]. piRNAs sequenced from early embryos and presumed capable of targeting nos 3'UTR were sought based on their sequence complementarity. Notably, a specific region located in the 3'-most part of the 3'UTR could be targeted by over 200 copies of piRNAs originating from two transposable elements, 412 and roo. piRNAs complementary to nos 3'UTR were visualized by northern blots. In addition, piRNAs predicted to target nos 3'UTR co-immunoprecipitated with Aub. nos genomic transgenes deleted for different parts of the 3'UTR were used to address the requirement of the corresponding regions for nos mRNA deadenylation. It was shown previously that the TCE (nucleotides 1-184) is required for nos mRNA poly(A) tail shortening, consistent with the role of Smg in this process. Deletion of region 184-403 (nos(delta1)) had no effect, whereas poly(A) tails from the transgene deleted for the region 403-618 (nos(delta2)) were elongated in 3-4-h embryos. This could indicate regulation by the miRNA predicted by miRBase to target this region. Deletion of 618-844 in the nos 3'UTR (nos(delta3)) had a strong effect on nos deadenylation. Consistent with this, nos mRNA levels produced by this transgene remained mostly stable. This resulted in defects in embryo patterning: a total of 35% of embryos from nos(delta3) females did not hatch and among them 86% showed head skeleton defects. Next specific sequences complementary to 412 (15 nucleotides) and roo (11 nucleotides) retrotransposon piRNAs were deleted. These short deletions, either independently or in combination, affected nos mRNA deadenylation (Rouget, 2010).

To support further the role of retrotransposon piRNAs in nos mRNA regulation, 412 and roo piRNAs were blocked by injecting specific 2'-O-methyl anti-piRNA in embryos, and cuticles were recorded as a functional assay of Nos ectopic synthesis at the anterior pole. Injection of anti-piRNA(412) or anti-piRNA(roo) resulted in specific head development defects (Rouget, 2010).

Together, these results provide strong evidence that an interaction between piRNAs and nos mRNA is required for nos mRNA deadenylation and translational repression in the first hours of embryogenesis (Rouget, 2010).

This study has identified a new function of the piRNA pathway in the regulation of maternal mRNAs. Recently, piRNAs derived from the 3'UTRs of cellular transcripts have been identified in gonadal somatic cells, although their biological role has not been clarified. It is proposed that piRNAs, in complex with Piwi-type Argonaute proteins Aub and Ago3, target nos maternal mRNAs and recruit or stabilize the CCR4-NOT deadenylation complex together with Smg. These interactions induce rapid mRNA deadenylation and decay. Thus, activation of mRNA deadenylation represents a new direct mechanism of action for the piRNA pathway with an essential developmental function during the first steps of embryogenesis (Rouget, 2010).

Smg is a general factor for mRNA decay during early embryogenesis. Because Aub and Ago3 are present in a complex with Smg in early embryos, a proportion of Smg mRNA targets could be regulated by the piRNA pathway. Consistent with this, other maternal mRNAs that are destabilized during early embryogenesis are targeted by abundant piRNAs and their deadenylation depends on the piRNA pathway (Rouget, 2010).

These piRNAs involved in gene regulation are generated from transposable element sequences. Although transposable elements have been described to be essential for genome dynamics and evolution, their immediate function within an organism has remained rather elusive. This study provides evidence for a co-evolution between transposable elements and the host genome and reveals the direct developmental function of transposable elements in embryonic patterning, through the regulation of gene expression (Rouget, 2010).

Aubergine is a component of a nanos mRNA localization complex

Localization of nanos (nos) mRNA to the posterior pole of the Drosophila oocyte is essential for abdominal segmentation and germline development during embryogenesis. Posterior localization is mediated by a complex cis-acting localization signal in the nos 3' untranslated region that comprises multiple partially redundant elements. Genetic analysis suggests that this signal is recognized by RNA-binding proteins and associated factors that package nos mRNA into a localization competent ribonucleoprotein complex. However, functional redundancy among localization elements has made the identification of individual localization factors difficult. Indeed, only a single direct-acting nos localization factor, Rumpelstiltskin (Rump), has been identified thus far (Jain, 2008). Through a sensitized genetic screen, the Argonaute family member Aubergine (Aub) has now been identified as a nos localization factor. Aub interacts with nos mRNA in vivo and co-purifies with Rump in an RNA-dependent manner. These results support a role for Aub, independent of its function in RNA silencing, as a component of a nos mRNA localization complex (Becalska, 2011).

Localization of nos mRNA to the posterior of the Drosophila embryo is critical for patterning of the A-P body axis. Although a cis-acting nos mRNA localization signal has been identified, the complement of trans-acting factors required for assembly of a nos RNP complex competent for posterior localization has remained elusive. From a sensitized genetic screen, Aub was identified as a novel nos mRNA localization factor, Aub interacts with nos mRNA in vivo. Importantly, nos localization was shown to be affected by aub acting downstream of osk mRNA localization, implying independent roles for aub in regulating these two transcripts. Although the role of aub in osk localization appears to be indirect, through a requirement in oocyte microtubule organization, these results suggest that aub plays a more direct role in regulating nos mRNA (Becalska, 2011).

A decrease in aub activity leads to defects in nos mRNA localization and, consequently, in patterning of the A-P axis when nos localization signal redundancy is reduced by removing two localization elements. A similar behavior is observed in rump mutants, which exhibit only weak segmentation defects unless redundantly acting elements are removed from the nos localization signal (Jain, 2008). Presumably, elimination of individual localization signal elements compromises localization by stripping away the contributions of nos localization factors with overlapping functions in nos RNP assembly. Conversely, elimination of multiple nos localization factors should lead to a more severe defect than elimination of an individual factor. Consistent with this prediction, decreasing aub gene dosage in rump mutants also leads to more severe loss of abdominal segments (Becalska, 2011).

In addition to allowing a requirement for aub to be uncovered, the sensitized nos+1+3 background has facilitated the separation of an indirect requirement for aub in osk localization from a more direct requirement in nos localization. Defects in osk regulation and abdominal segmentation are observed only when females are homozygous mutant for aub and not when they are heterozygous. By contrast, defects in nos+1+3 localization are observed when females are heterozygotes for aub mutations. These results are further supported by previous data showing that the ability of ectopically expressed Osk to recruit nos mRNA is compromised in aub/+ embryos (Becalska, 2011).

Aub has been implicated in the rasiRNA pathway that silences retrotransposons in the germline. However, mutation of squash, which encodes a rasiRNA pathway component that interacts with Aub, has no effect on the nos+1+3 transgene. Interestingly, another rasiRNA pathway component, piwi, has the opposite effect of aub on the nos+1+3 transgene, as heterozygosity for a piwi mutation results in increased segmentation in the sensitized background. Mutations that inactivate the rasiRNA pathway, including aub mutations, activate the DNA damage checkpoint, presumably due to unsuppressed transposon activity. Checkpoint activation disrupts microtubule organization and grk translation, resulting in a failure of axis specification that is thought to lead to subsequent defects in osk mRNA localization. However, the effect of aub mutation on nos+1+3 mRNA localization is independent of the DNA damage pathway, providing further evidence that Aub regulates nos independently of osk. Moreover, these results indicate that Aub function in nos localization is distinct from its function in RNA silencing (Becalska, 2011).

Biochemical experiments indicate nos mRNA forms a complex with Aub in vivo, although whether Aub interacts directly with nos mRNA, or is recruited to the complex by other proteins that bind directly to nos, is not yet clear. Soluble recombinant Aub necessary to distinguish between these possibilities has not been obtained. However, the RNA-dependent co-purification of Aub and Rump, combined with evidence for genetic interactions between aub and rump further supports the contribution of Aub to the formation and/or function of a nos localization RNP complex. Whereas Rump is not concentrated at the posterior of the oocyte, Aub-GFP is localized to the posterior during midoogenesis and continues to accumulate at the posterior pole throughout the later stages of oogenesis when nos becomes localized. Thus, the contributions of Rump and Aub to nos RNP complexes may be dynamic, with both proteins accompanying nos as it is dispersed throughout the oocyte during ooplasmic streaming, but only Aub remaining associated with the nos RNP upon its entrapment at the posterior. Isolation and characterization of the full complement of nos localization factors will be essential to dissect the assembly pathway for nos localization complexes. The isolation of aub in a sensitized genetic screen validates the use of such an approach, in addition to biochemical purification strategies that proved successful for isolation of Rump, for achieving this goal (Becalska, 2011).

Vreteno, a gonad-specific protein, is essential for germline development and primary piRNA biogenesis in Drosophila

In Drosophila, Piwi proteins associate with Piwi-interacting RNAs (piRNAs) and protect the germline genome by silencing mobile genetic elements. This defense system acts in germline and gonadal somatic tissue to preserve germline development. Genetic control for these silencing pathways varies greatly between tissues of the gonad. This study identified Vreteno (Vret), a novel gonad-specific protein essential for germline development. Vret is required for piRNA-based transposon regulation in both germline and somatic gonadal tissues. Vret, which contains Tudor domains, associates physically with Piwi and Aubergine (Aub), stabilizing these proteins via a gonad-specific mechanism that is absent in other fly tissues. In the absence of vret, Piwi-bound piRNAs are lost without changes in piRNA precursor transcript production, supporting a role for Vret in primary piRNA biogenesis. In the germline, piRNAs can engage in an Aub- and Argonaute 3 (AGO3)-dependent amplification in the absence of Vret, suggesting that Vret function can distinguish between primary piRNAs loaded into Piwi-Aub complexes and piRNAs engaged in the amplification cycle. It is proposed that Vret plays an essential role in transposon regulation at an early stage of primary piRNA processing (Zamparini, 2011).

Propagation of all sexually reproducing organisms depends upon the faithful development and function of reproductive organs. In Drosophila, oogenesis requires the coordinated differentiation of two distinct cell lineages, the germline and the gonadal somatic cells, to produce an egg. The germarium, where oogenesis initiates, contains both germline and somatic stem cells. Asymmetric cell division of germline stem cells (GSCs) within the germarium generates both a stem cell and a differentiated daughter cell, the cystoblast, which gives rise to a sixteen-cell interconnected cyst. One of the sixteen cells in the cyst differentiates into an egg and the remaining cells become nurse cells. Somatic cell populations are intimately associated with germ cells during oogenesis: niche cells provide GSC maintenance signals and are tightly connected to GSCs via adhesion and gap junctions; inner sheath cells (ISCs) intermingle with the differentiating cystoblast and early dividing cysts to promote formation of the sixteen-cell cyst; follicle stem cells and their progeny, the follicle cells, surround each germline cyst as it buds off from the germarium and provide the maturing egg chamber with the positional cues needed for establishment of anterior-posterior and dorsal-ventral polarity of the embryo (Zamparini, 2011).

In addition to germline development, genomic integrity must be preserved to generate viable progeny. In Drosophila, transposable elements occupy nearly one third of the genome and mobilization of even one of almost 150 transposon classes found can lead to defects in gametogenesis and sterility. Therefore, organisms have evolved small RNA-based defense systems to fight these elements (Malone, 2009). In Drosophila, both germline and somatic cells of the ovary rely on Piwi proteins and their 23-29 nt Piwi-interacting RNAs (piRNAs) to combat transposon activity. All three Drosophila Piwi proteins, Piwi, Aubergine (Aub) and Argonaute 3 (AGO3), are expressed in germline cells, whereas Piwi is also expressed in somatic gonadal cells. Interestingly, mutations in all known piRNA pathway components lead to oocyte and embryonic patterning defects and, ultimately, to sterility, believed to be an indirect consequence of transposon-induced genomic instability and activation of a DNA double-strand break checkpoint (Zamparini, 2011 and references therein).

In contrast to other small RNAs, such as microRNAs and siRNAs, which are produced from double-stranded RNA precursors, piRNAs are derived from single-stranded RNA precursors, independently of the endonuclease Dicer. piRNA precursors originate from either active transposon transcripts or discrete genomic loci known as 'piRNA clusters'. In Drosophila, piRNA clusters provide the primary source of antisense transposon transcripts, whereas active transposons predominantly provide sense transcripts. piRNAs associated with Piwi and Aub are mostly derived from piRNA clusters, mapping complementary to active transposons, whereas AGO3-bound piRNAs appear to be derived from the transposon itself. This relationship and a 10 nt overlap observed between sense and antisense piRNA pairs led to a model of piRNA amplification termed 'ping-pong', in which 5' ends of new piRNAs are generated through cleavage by the Piwi proteins themselves (Brennecke, 2007; Gunawardane, 2007). In the Drosophila ovary, piRNA 'ping-pong' is restricted to germline cells in which Piwi, Aub and AGO3 are present, although Piwi appears to be mostly dispensable for 'ping-pong' amplification (Malone, 2009). In gonadal somatic cells, in which only Piwi is expressed, an alternative pathway functions. Here, single-stranded piRNA clusters or gene transcripts are processed to produce 'primary' piRNAs that are directly loaded into Piwi, targeting active transposons or endogenous genes (Li, 2009; Malone, 2009; Saito, 2009). The overlapping genetic requirements for Piwi in the germline and ovarian somatic cells suggest that Piwi may also engage primary piRNAs in the germline. Like Piwi, the germline-specific Aub engages piRNAs complementary to transposons, but has not been directly linked to primary piRNAs. Therefore, the precise relationship between primary piRNAs and 'ping-pong' in the germline remains largely unknown (Zamparini, 2011).

The restriction of piRNA production and transposon control in gonadal tissues raises the question of how the piRNA biogenesis machinery has evolved specifically in the gonad. This study has identified Vreteno (Vret), a gonad-specific, Tudor domain-containing protein that functions specifically in the germline and somatic gonadal tissues during oogenesis. Vret broadly regulates transposon levels and has an essential role in primary piRNA biogenesis, leaving 'ping-pong' amplification intact (Zamparini, 2011).

This study identified a novel protein with critical roles in oocyte polarity, germline and soma differentiation, survival and transposon control. Vret, a Tudor-domain containing protein, associates with Piwi proteins in the cytoplasm of Drosophila ovarian cells and regulates their stability, as well as Piwi nuclear localization and localization of Aub to nuage. In the absence of Vret, piRNAs are dramatically reduced and transposons mobilized. By ordering the function of Vret within the network of the piRNA-transposon-based system, it is concluded that Vret functions in primary piRNA biogenesis at the stage of primary piRNA loading onto Piwi and Aub complexes (Zamparini, 2011).

Loss of Vret in the soma or germline has strikingly different morphological consequences. Molecular analysis, however, suggests the same underlying cause for these defects: a failure to produce biologically active piRNAs. Morphologically, the vret germline phenotype resembles that of mutants defective in germline piRNA biogenesis, such as aub, spnE and krimper. In these mutants, transposon mobilization activates a DNA damage checkpoint that leads to defects in transport and translation of maternal RNAs necessary for oocyte polarity and embryonic patterning. Interestingly, lack of vret in the soma resembles the piwi mutant phenotype, in which GSCs fail to differentiate as a consequence of somatic cell death, an event presumably associated with transposon misregulation. Thus, loss of vret in the germline and gonadal soma resembles loss of both Piwi and Aub. This, together with the findings that Vret associates with Piwi and Aub in ovarian extracts and affects the stability of both, strongly suggests that Vret regulates both proteins in a similar fashion (Zamparini, 2011).

Surprisingly, Vret is not required for piRNA 'ping-pong' amplification per se, suggesting that Vret might selectively interact with Aub and Piwi bound to primary piRNAs and not to those engaged in 'ping-pong'. In this scenario, it would be possible for maternally deposited Aub to initiate the 'ping-pong' cycle with AGO3, even in the absence of Vret (Brennecke, 2008). As some Aub protein remains in vret mutant ovaries, an active pool of Vret-independent Aub could maintain 'ping-pong' activity throughout the adult ovary. Therefore, it is proposed that a 'ping-pong'-independent pool of Aub within the cytoplasm depends upon primary piRNA loading, downstream of Vret function. It would be interesting to examine whether piRNAs associated with the Vret-dependent complex can, at any level, contribute to 'ping-pong', or whether Aub-bound primary piRNAs are functionally or enzymatically distinct from those involved in the piRNA amplification cycle (Zamparini, 2011).

In contrast to Aub, only a small subset of Piwi-bound piRNAs showed a 10 nt overlap with those bound to AGO3. Indeed, Piwi is genetically dispensible for 'ping-pong' and might be only marginally involved in 'ping-pong', if at all (Brennecke, 2007; Li, 2009). As Piwi slicer activity does not appear to be required for Piwi function (Saito, 2009), it seems most plausible that Piwi would act as a recipient, and not as an 'active' component of 'ping-pong' amplification. Regardless, the majority of Piwi-bound primary piRNAs act independently of 'ping-pong' and depend upon Vret for stability (Zamparini, 2011).

An ectopic expression experiment suggests that Piwi is not 'intrinsically unstable', but becomes unstable in the gonad in the absence of Vret. Furthermore, Vret is not required for Piwi or Aub transcription or translation. Vret, therefore, could either coordinate the process of biogenesis and loading of primary piRNAs into Piwi and Aub complexes or be involved in stabilizing the mature RISC (RNA-induced silencing complex). Armi, a putative helicase, and Zucchini (Zuc), a member of the phospholipase D (PLD) family of phosphodiesterases, act like Vret in the soma and germline; they specifically affect Piwi protein stability and primary piRNA levels leaving the 'ping-pong' cycle intact. Unlike Vret, the levels of unprocessed precursor RNA from flam are increased in zuc mutants implicating Zuc in piRNA cluster transcript processing. Therefore the hypothesis is favored that Vret, possibly together with Armi, is an essential component of Piwi and Aub RISC complexes. Vret is one of many Tudor domain proteins in Drosophila that affects piRNA biogenesis and contains conserved residues that are known to be required for binding of sDMAs found in Piwi proteins (Siomi, 2010). When mutated, each of these genes displays a rather distinct phenotype. Krimper and SpnE regulate transposon levels in the germline whereas fs(1)Yb is soma-specific. Vret is, at this point, the only Tudor domain protein known to be required in both tissues, suggesting a conserved and global role for this gene in piRNA regulation. It remains to be determined whether the mammalian Tudor homolog could fulfill a similar function (Zamparini, 2011).

PAPI, a novel TUDOR-domain protein, complexes with AGO3, ME31B and TRAL in the nuage to silence transposition

The nuage is a germline-specific perinuclear structure that remains functionally elusive. Recently, the nuage in Drosophila was shown to contain two of the three PIWI proteins - Aubergine and Argonaute 3 (AGO3) - that are essential for germline development. The PIWI proteins bind to PIWI-interacting RNAs (piRNAs) and function in epigenetic regulation and transposon control. This study reports a novel nuage component, PAPI (Partner of PIWIs), that contains a TUDOR domain and interacts with all three PIWI proteins via symmetrically dimethylated arginine residues in their N-terminal domain. In adult ovaries, PAPI is mainly cytoplasmic and enriched in the nuage, where it partially colocalizes with AGO3. The localization of PAPI to the nuage does not require the arginine methyltransferase dPRMT5 or AGO3. However, AGO3 is largely delocalized from the nuage and becomes destabilized in the absence of PAPI or dPRMT5, indicating that PAPI recruits PIWI proteins to the nuage to assemble piRNA pathway components. As expected, papi deficiency leads to transposon activation, phenocopying piRNA mutants. This further suggests that PAPI is involved in the piRNA pathway for transposon silencing. Moreover, AGO3 and PAPI associate with the P body component TRAL/ME31B complex in the nuage and transposon activation is observed in tral mutant ovaries. This suggests a physical and functional interaction in the nuage between the piRNA pathway components and the mRNA-degrading P-body components in transposon silencing. Overall, this study reveals a function of the nuage in safeguarding the germline genome against deleterious retrotransposition via the piRNA pathway (Liu, 2011).

Although the nuage has long been discovered in the germline of diverse organisms, little is known about its function. In this study identified and molecularly characterized a novel nuage component, PAPI. PAPI is a TUDOR-domain-containing protein that recruits PIWI proteins, especially AGO3, to the nuage and stabilizes them. The interaction between PAPI and AGO3 in the nuage is mediated by sDMAs in the N-terminal domain of AGO3 but is RNA independent. Previous studies have suggested the nuage as the cytoplasmic loci where post-transcriptional silencing of transposons occurs. In addition, loss of Drosophila TUDOR protein has been shown to affect the localization of AUB to the nuage and to alter the piRNA profile. The new findings of this study indicate that TUDOR-domain-containing proteins might serve as a platform for the recruitment of PIWI proteins to the nuage and for the assembly of piRNA pathway components. A subset of transposons are de-repressed in papi deficient ovaries, suggesting that PAPI is involved in transposon silencing in the nuage, just like other piRNA pathway components. This study thus reveals a function of the nuage in safeguarding the germline genome against deleterious retrotransposition via the piRNA pathway (Liu, 2011).

Furthermore, a physical association of PAPI and AGO3 with the TRAL/ME31B complex has been identifie along with their colocalization in the nuage, and the role of these P body proteins in silencing the expression of some transposons. The TRAL/ME31B complex has been shown to interact with CUP, which also associates with the nuclear pore complex component NUP154 (Grimaldi, 2007). The current findings reveal an exciting physical and functional link between the piRNA machinery and the P body components in the nuage and a mechanism for nuage localization to the nuclear periphery. The P body proteins are well known for their function in mRNA processing and degradation, yet the piRNA machinery regulates transposon silencing by reducing the level of their mRNAs. The physical interaction between these two machineries, with the functional relationship among known components of these two machineries in the nuage illustrated in a working model, raises the intriguing possibility that these two pathways work together in the nuage as a post-transcriptional mechanism to degrade transposon mRNAs, leading to transposon silencing. In addition, these data implicate the interaction of between the TRAL/ME31B complex and NUP154 via CUP as a mechanism of nuage localization to the nuclear periphery (Liu, 2011).

AGO3 Slicer activity regulates mitochondria-nuage localization of Armitage and piRNA amplification

In Drosophila melanogaster the reciprocal 'Ping-Pong' cycle of PIWI-interacting RNA (piRNA)-directed RNA cleavage catalyzed by the endonuclease (or 'Slicer') activities of the PIWI proteins Aubergine (Aub) and Argonaute3 (AGO3) has been proposed to expand the secondary piRNA population. However, the role of AGO3/Aub Slicer activity in piRNA amplification remains to be explored. This study shows that AGO3 Slicer activity is essential for piRNA amplification and that AGO3 inhibits the homotypic Aub:Aub Ping-Pong process in a Slicer-independent manner. It was also found that expression of an AGO3 Slicer mutant causes ectopic accumulation of Armitage, a key component in the primary piRNA pathway, in the Drosophila melanogaster germline granules known as nuage. AGO3 also coexists and interacts with Armitage in the mitochondrial fraction. Furthermore, AGO3 acts in conjunction with the mitochondria-associated protein Zucchini to control the dynamic subcellular localization of Armitage between mitochondria and nuage in a Slicer-dependent fashion. Collectively, these findings uncover a new mechanism that couples mitochondria with nuage to regulate secondary piRNA amplification (Huang, 2014).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Aubergine controls germline stem cell self-renewal and progeny differentiation via distinct mechanisms

Piwi family protein Aubergine (Aub) maintains genome integrity in late germ cells of the Drosophila ovary through Piwi-associated RNA-mediated repression of transposon activities. Although it is highly expressed in germline stem cells (GSCs) and early progeny, it remains unclear whether it plays any roles in early GSC lineage development. This study reports that Aub promotes GSC self-renewal and GSC progeny differentiation. RNA-iCLIP results show that Aub binds the mRNAs encoding self-renewal and differentiation factors in cultured GSCs. Aub controls GSC self-renewal by preventing DNA-damage-induced Chk2 activation and by translationally controlling the expression of self-renewal factors. It promotes GSC progeny differentiation by translationally controlling the expression of differentiation factors, including Bam. Therefore, this study reveals a function of Aub in GSCs and their progeny, which promotes translation of self-renewal and differentiation factors by directly binding to its target mRNAs and interacting with translational initiation factors (Ma, 2017).

Aub is an essential piRNA pathway component known to be important for silencing TEs to ensure normal late germ cell development in the Drosophila ovary. This study demonstrates that Aub is required intrinsically to maintain GSC self-renewal and promote their progeny differentiation in the Drosophila ovary. aub is required intrinsically in GSCs to maintain self-renewal by preventing DNA-damage-evoked Chk2 activation. Aub promotes cystoblast (CB) differentiation by maintaining Bam expression. In addition, Aub directly binds over 1,100 mRNAs in GSCs, some of which are known to be important for GSC maintenance and differentiation, suggesting that Aub can control GSC maintenance and progeny differentiation also by directly regulating gene expression. Aub directly binds bam, dnc, and Rm62 mRNAs, and regulates their expression at the translation level via 3' UTR, which can also help mechanistically explain how Aub controls GSC maintenance and progeny differentiation. Finally, Aub was shown to physically associated with the translation initiation eIF4 complex and pAbp, suggesting that it promotes gene expression via regulation of translation initiation. Therefore, this study has revealed new roles of Aub in promoting GSC self-renewal and GSC progeny differentiation through different mechanisms (Ma, 2017).

Although Aub represses TEs by controlling the 'ping-pong' piRNA amplification cycle in late germ cells of the Drosophila ovary, its importance in GSCs, CBs, and mitotic cysts has not been previously reported. THE genetic results show that Aub intrinsically maintains GSC self-renewal partly by preventing DNA-damage-evoked checkpoint activation. First, aub mutant ovaries have two GSCs at eclosion, but gradually lose their GSCs within 25 days. Second, marked aub mutant GSC clones are lost from the niche faster than the marked control GSCs. Third, aub mutant GSCs accumulate DNA damage recognized by γ-H2AvD, indicating that Aub is also important to produce piRNAs for repressing TEs, thereby preventing TE-induced DNA damage. Fourth, the GSC loss caused by aub mutations can also be partially and significantly rescued by Chk2 inactivation, indicating that DNA damage is one of the major causes for the loss of the aub mutant GSCs. This interpretation is supported by a recent study showing that Chk2 inactivation can drastically and significantly rescue the DNA-damage-induced GSC loss. This is also consistent with the previous finding that the dorsal-ventral polarity defect of aub mutant egg chambers can be drastically rescued by Chk2 inactivation. Finally, the loss of aub mutant GSCs could be caused primarily by differentiation, but might also be contributed by apoptosis. Taken together, these results demonstrate that Aub intrinsically controls GSC self-renewal partly by preventing DNA-damage-induced Chk2 activation, and have also confirmed that piRNAs are important in GSCs to repress TEs and thus prevent DNA damage. In addition, Aub regulates nanos (nos) mRNA localization in the oocyte and also controls the decay of maternal mRNAs in the early Drosophila embryo, including nos, in cooperation with piRNAs. This study has adopted the iCLIP experimental procedure to the in vitro cultured GSCs for the identification of potential mRNA targets of Aub in GSCs. These cultured GSCs can self-renew, and, more importantly, can also be induced to differentiate into 16-cell cysts upon Bam induction. iCLIP results show that Aub can bind more than 1,100 mRNAs in GSCs primarily via 5' UTR, 3' UTR, or both, 59 of which encode known GSC self-renewal factors, including BMP pathway components, E-cadherin, and chromatin remodeling factors (Ma, 2017).

This study has revealed another role of Aub in promoting GSC progeny differentiation by regulating the expression of differentiation factors via direct binding. First, Aub is required intrinsically to promote GSC progeny differentiation. The aub mutant ovaries accumulate excess undifferentiated CBs, whereas the marked mutant aub GSCs also produce excess undifferentiated CBs. Second, Aub sustains Bam protein expression in mitotic germ cells. Bam is a master regulator for driving GSC progeny differentiation. Genetically, aub interacts with bam to promote germ cell differentiation. Molecularly, Aub is required in mitotic cysts to sustain Bam protein expression, and its binding to the bam 3' UTR is important for regulating the levels of its protein but not mRNA, suggesting that Aub can regulate Bam expression at the translational level. In addition, bam transcription and mRNA levels are significantly lower in aub mutant ovaries than in the control ones, indicating that Aub can also regulate Bam at the transcriptional level. Thus, Aub can regulate Bam expression in early germ cells at the transcriptional and translational levels. Third, Aub is also required to maintain Rm62 expression in CBs and mitotic cysts via its 3' UTR. The deletion of the Aub-binding site from the Rm62 3' UTR significantly decreases its expression in CBs and mitotic cysts, indicating that Aub binding is critical for Rm62 protein expression in CBs and mitotic cysts. This study has confirmed the previously reported role of Rm62 in promoting GSC progeny differentiation. Taken together, this study demonstrates that Aub promotes GSC progeny differentiation at least in part by controlling the expression of differentiation factors at the translational level (Ma, 2017).

Previous studies have shown that Aub can regulate mRNA stability and localization in a piRNA-dependent manner. By examining the existence of the binding sites for known germline-specific piRNAs over the 3' UTRs of 1,189 mRNAs, no significant enrichment was seen of piRNA-binding sites in the Aub-binding regions of these Aub target mRNAs, indicating that Aub binding to its target mRNAs in GSCs is likely independent of piRNAs. More importantly, Aub promotes the expression of its target mRNAs at least partly at the translation level via direct 3' UTR binding in GSCs and their progeny, thereby promoting GSC maintenance, progeny differentiation, or both. Interestingly, co-immunoprecipitation experimental results show that Aub is physically associated with the translation initiation complex eIF4 and the poly(A)-binding protein pAbp in S2 cells. The eIF4 complex and pAbp are known to be able to interact with each other to bring the 5' UTR and 3' UTR in close proximity, facilitating the loading of eIF3 and the 40S ribosome subunit to initiate translation. However, the current findings do not contradict the previous finding that Aub can promote mRNA degradation in different developmental and cellular contexts. Many independent studies have shown that translation regulators can also control mRNA stability, and mRNA stability factors can also regulate translation. Based on these findings, it is proposed that Aub can regulate mRNA translation at the initiation step by facilitating the interaction between the eIF4 complex and pAbp in GSCs and their early progeny (Ma, 2017).


Northern analysis has revealed a polyadenylated RNA of ~3.0 kb length in accordance with the 2790 bp of the cDNA clone. In wild-type flies, the sti RNA accumulates exclusively in the gonads. Because of a more abundant expression in the ovaries, the sti transcript can be detected easily in total RNA isolated from whole females, but rarely in total RNA isolated from whole males. In homozygous mutant males and females, the expression is deregulated. As a consequence, the 3.0-kb RNA is also intensely transcribed outside the gonads. A larger 4.5-kb RNA also accumulates exclusively in the testes. The fact that the 3.0-kb RNA species continues to be transcribed is compatible with the theory that the integration site is most likely 5' to the transcriptional start site. The misexpression outside the gonads can already be seen in heterozygous flies. All attempts to clarify the structure of the new 4.5-kb testis-specific transcript were unsuccessful (Schmidt, 1999).

In the remobilization experiments mentioned above, new mutant alleles were also found. Most informative for the function of the sting gene was the mutation sti3a, a putative null allele. Because of a large deletion, not only the sti, but also the neighboring RpL9 expression was affected, resulting in severe impairment of vitality. The few escapers had minute bristles, were male and female sterile, and crystal needles could be found in spermatogenic stages. Heterozygous sti/sti3a flies were only male sterile and produced needles. Northern analysis with RNA isolated from a few homozygous sti3a escapers has indicated that sti was not expressed at all. This result was confirmed by the analysis of flies transgenic for a fragment that restored viability by introducing one copy of the RpL9 gene. These flies were still female sterile, thus demonstrating that the sterility was separable from the neighboring Minute gene. Males of the corresponding genotype were sterile needle producers. Hence, the null phenotype of the sti mutation is most likely sterility in both sexes, accompanied by the production of needles in spermatocytes and spermatids (Schmidt, 1999).

In Northern analysis, the 3.0-kb sti RNA could only be detected in 0- to 6-hr-old embryos, but not in older ones. Taking into account the large accumulation of transcripts during oogenesis, this could indicate that only maternally provided RNA is present in the embryos. It was therefore hypothesized that the observed female sterility instead represents a maternal lethality. To distinguish between these alternatives, homozygous sti3a females transgenic for the RpL9 gene were mated to wild-type flies. Those females laid eggs that started development. Embryogenesis, however, stopped at various stages, starting with late blastoderm (stage 5). Only very few embryos survived gastrulation (stage 8), but they died before germ band retraction (stage 12). This proves that the maternal contribution of sti mRNA is essential for early development; i.e., the observed female sterility is indeed a consequence of a maternal lethal effect (Schmidt, 1999).

Two different sting-lacZ fusion genes were introduced into the germline by P-element-mediated transformation to monitor the expression pattern in the gonads. The large construct should contain all regulatory elements because it starts in the neighboring gene. Furthermore, the 5' upstream regulatory sequences are identical to clone MR-3, a clone that can fully restore the wild type. The ß-galactosidase activity produced by the large construct should, therefore, reflect the normal activity of the sti gene. In the testes, enzymatic staining could be detected only at the very tip, i.e., in spermatogonia and early spermatocytes. This activity can also be demonstrated in the larval testis. This is almost complementary to the expression pattern in the original enhancer trap line, where the staining does not start at such early stages, but is observed only in late spermatocytes. If the enhancer trap expression pattern faithfully mirrors the expression of the endogenous sting gene, the apparent loss-of-function phenotype of the sting mutation can be explained easily. The gene is not transcribed, or at least not at the correct level, in the cells where it should be, i.e., early germ cells. Instead, the 3.0-kb mRNA accumulates primarily in late spermatocytes and at later stages, where the crystal needles can also be observed. The only synthesis in late spermatocytes might also be true for the 4.5-kb RNA, whose structure and function is, however, still unknown. The misexpression shown by the original enhancer trap line indicates that the P{lacW} insertion has separated or destroyed important regulatory elements. In accordance with this interpretation, the short construct with sequences from the 3' end of the P{lacW} to the transcriptional start site does not support any expression in testes (Schmidt, 1999).

In females transgenic for the large construct, expression is restricted to the ovary. There is a short, transient expression in the oocyte around stage 6 of oogenesis. At later stages, from stage 9 on, ß-galactosidase activity is found in the nurse cells. Later on, with the dumping of the nurse cell contents, this activity is detected in the oocyte and egg. Such a maternal contribution has to be postulated from the observed maternal lethal effect of a sti null allele. Although the ovary can not be reproducibly stained in the original male sterile enhancer trap line, the short construct does show ß-galactosidase activity in the ovary. Whether this difference is caused by activating or repressing elements that cannot function in one of the two transgenic lines is still an open question (Schmidt, 1999).

Isolation of new polar granule components in Drosophila reveals P body and ER associated proteins

Germ plasm, a specialized cytoplasm present at the posterior of the early Drosophila embryo, is necessary and sufficient for germ cell formation. Germ plasm is rich in mitochondria and contains electron dense structures called polar granules. To identify novel polar granule components, proteins were isolated that associate in early embryos with Vasa (Vas) and Tudor (Tud), two known polar granule associated molecules. Maternal expression at 31B (ME31B), eIF4A, Aubergine (AUB) and Transitional Endoplasmic Reticulum 94 (TER94) were identified as components of both Vas and Tud complexes and their localization to polar granules was confirmed by immuno-electron microscopy. ME31B, eIF4A and AUB are also present in processing (P) bodies, suggesting that polar granules, which are necessary for germ line formation, might be related to P bodies. The recovery of ER associated proteins TER94 and ME31B confirms that polar granules are closely linked to the translational machinery and to mRNP assembly (Thomson, 2008).

Little is understood of the molecular events that link the assembly of germ plasm to the formation of germ cells. There is a strong correlation between polar granule formation and germ cell formation, yet their functional relationship is still unclear. In an attempt to understand polar granule formation and function this study set out to isolate polar granule components with a biochemical approach; proteins common to both Tud and Vas complexes were isolated. These complexes were isolated by cross-linking proteins from early embryonic extracts followed by anti-Tud or anti-Vas immunoprecipitation; proteins found in both complexes were then immunolocalized using EM. Using this method it was confirmed that Aub is a polar granule component and three new polar granule components were identified: ME31B, TER94 and eIF4A. Through genetic interaction analysis in transheterozygous embryos it was shown that decreasing the levels of Vas or Tud along with either Aub, ME31B, Ter94 or eIF4A reduces germ cell number. This approach both identified novel polar granule components and implicated novel processes in germ cell formation (Thomson, 2008).

The presence of Aub, ME31B and eIF4A in polar granules supports the hypothesis that polar granules and P bodies are structurally, and perhaps functionally, related. Recovery of CUP, an ME31B-interacting protein, in the Tud complex further supports this. Similar parallels have recently been found for the mouse chromatoid body, an electron dense structure in the male germ line with similarities to Drosophila polar granules and nurse cell nuage. RNAs in P bodies are stored in a translationally quiescent state and can later be either degraded or translationally activated in response to physiological cues (Decker, 2006). Translational repression in P bodies occurs at the level of mRNA recruitment to the ribosome, and through miRNA silencing pathways. The polar granule components that were identified suggest their involvement in both types of post-transcriptional regulation. Aub has been implicated in processing of germ line specific piRNAs. Findings that Aub associates with polar granules implicates piRNAs in germ cell formation, as has a previous study. In contrast, Vas and eIF4A have been closely linked with translational regulation and are not known to participate in miRNA silencing pathways (Thomson, 2008).

The RNA-rich nature of early polar granules supports the idea that specific germ line-specific mRNAs are stored in polar granules in a translationally repressed state. Subsequently, these RNAs are translated and their function may be required for germ cell formation and further development. How could general translational repression mediated by polar granules be overcome? Conceivably Vas could be a key factor. Vas, a highly conserved polar granule component with homologues in other species involved in germ line formation, binds directly to eIF5B. Disrupting the eIF5B-Vas interaction abrogates germ cell formation, presumably due to the loss of the ability of Vas to initiate translation of yet unidentified mRNAs (Johnstone, 2004). Thus, Vas may act as a germ line specific mRNA translation derepression factor. Other tissue specific factors could adapt a P body to a specific function or cell line. Identification of mRNAs that localize in polar granules and are dependent on Vas for their translation will no doubt provide more insight into this mechanism (Thomson, 2008).

Ultrastructural analysis of proteins found in the Vas and Tud containing complexes revealed that polar granules were often in close proximity or in contact with ER. Supporting such a link, Ter94 and ME31B were present in both Tud and Vas complexes, and are enriched in polar granules. Further work is required to elucidate what proportion of polar granules associate with ER, and whether this association is stage dependent. The presence of Ter94, an ER exit site marker, with Vas, ME31B, Aub and eIF4A in the same structure suggests that ER exit sites directly associate with the translational machinery with both activating and repressing factors. Polar granules may form at ER exit sites, which could provide a mechanism for the localization and assembly of mRNPs required for the translational regulation of their constituent mRNAs. There is evidence that P bodies associate with ER exit sites. In the Drosophila ovary, Trailer Hitch (TRAL) associates with ER exit sites and associates with P body components such as ME31B and CUP. The C. elegans homologue of TRAL, Car-1, associates with DCAP-1, a P-body marker, and car-1 mutations affect ER assembly (Squirrell, 2006). A single TRAL peptide was recovered in one immunoprecipitation with Tud, perhaps lending additional support to an association between polar granules and ER exit sites (Thomson, 2008).

Repeated attempts to biochemically isolate polar granules were made over 30 years ago, before the advent of modern analytical techniques that allow the identification of very small amounts of protein. From this work a major polar granule component of approximately 95 kDa was identified. The nature of this protein was not determined although Ter94 has approximately the same molecular mass, as does PIWI, a 97-kDa likely polar granule component that has eluded the currently used screens. PIWI associates with Vas, a polar granule component, as well as with components of the miRNA machinery. PIWI RNA and protein are enriched in germ plasm and piwi mutants have defects in germ cell formation. The screen also did not identify Osk, which was shown by a yeast two-hybrid screen to bind directly to Vas. This may be because these proteins were not present in high enough abundance for detection. Alternatively, since the reactive ends of the cross-linkers that were used specifically cross-link cysteine residues, they would not stabilize a particular protein-protein interaction unless a pair of cysteine residues is within the range of the cross-linker. The work demonstrates that a molecular approach can be a powerful complement to genetics, and that purification schemes based on two independent reagents can reduce signal-to-noise problems that are inherent in co-immunoprecipitation experiments. Molecular approaches such as this one also have the capacity to identify proteins involved in a developmental process that are encoded by genes with multiple functions, or required for cellular viability, that will therefore elude phenotype-based genetic screens (Thomson, 2008).


The aub transcript is expressed at relatively high levels in the germarium, at lower levels during mid-oogenesis, and again at high levels in the nurse cells and oocyte from about stage 6 of oogenesis. Of greater interest is the distribution of Aub protein. Antibodies, which detect Aub on Western blots, fail to detect Aub protein in whole-mount ovaries. In both wild-type and aub mutant ovaries, a similar low level of uniform staining is observed. As an alternate approach to determine the distribution of Aub protein, the UAS/GAL4 system was used to express a GFP-tagged version of Aub in vivo. Flies carrying a uas-gfp-aub transgene were crossed to those that express a nos-gal4-vp16 transgene which drives expression of GAL4-VP16 in the female germline (Harris, 2001).

The temporal expression of the nos-gal4-vp16 driver parallels that of aub, and uas-gfp-aub driven by nos-gal4-vp16 is sufficient to fully rescue aub mutant defects, indicating both that the fusion protein is functional and that its distribution includes the pattern required of native Aub protein. GFP-Aub protein can be visualized throughout oogenesis. In the nurse cells, the protein is distributed evenly in the cytoplasm and in concentrated foci surrounding the nuclei. These presumptive perinuclear particles remain until around stage 10. In the oocyte, GFP-Aub is at first dispersed evenly in the cytoplasm. Later, beginning at stage 8/9, it becomes strikingly concentrated in the posterior pole plasm, where it largely overlaps the distribution of Osk protein. This posterior concentration appears to occur by recruitment of the GFP-Aub protein, since the aub mRNA is not itself localized within the oocyte (Harris, 2001).

The localization of Aub to the posterior pole of the oocyte raises the possibility that it is a component of polar granules. Polar granules are large, electron-dense structures found in the posterior pole plasm of the oocyte. In the embryo, these granules remain localized to the posterior pole where they are largely incorporated into the budding pole cells. Several other posteriorly localized proteins, including Tudor (Tud), Vas and Osk, have been shown by immunoelectron microscopy to be components of the polar granules. With fluorescence-based detection methods, polar granule components appear in particles, which have been presumed to be the polar granules. Colocalization of Osk and Vas (fused to GFP) in these particles supports this conclusion (Harris, 2001).

To determine if Aub is a polar granule component, confocal microscopy was used to characterize the subcellular distribution of GFP-Aub in embryos. Before pole cell formation, GFP-Aub is found throughout the cytoplasm but strongly concentrates at the posterior pole in distinct particles, most of which also contain Osk protein. The particles are predominantly incorporated into the pole buds, which form at the posterior pole of the embryo and initiate pole cell formation. At this time, the particles increase in size and decrease in number, suggesting that individual particles are fusing. Simultaneously, the level of dispersed, non-particulate GFP-Aub increases in the pole cell cytoplasm. Before cellularization, between nuclear division cycles 11 and 13, the particles often cluster in zones at opposite sides of the pole cell nuclei. Most of the particles continue to display colocalization of GFP-Aub and Osk. However, some Osk-containing particles now begin to appear in the pole cell nuclei, while GFP-Aub is not detectable in these particles and consistently remains excluded from the nuclei. By the time of cellularization, following nuclear division cycle 14, only a few large cytoplasmic particles persist in each pole cell, with much of the GFP-Aub distributed evenly throughout the cytoplasm. Osk continues to colocalize strongly with GFP-Aub in the particles, but most Osk staining is now detected in the nuclear particles and dispersed in the nucleoplasm. Both cytoplasmic and nuclear particles often appear in striking hollow spherical shapes (donut-like in optical sections), which are characteristic of some polar granule clusters as well as similar particles that are present in nuclei, called nuclear bodies. In comparable colocalization experiments, it was determined that Vas-GFP behaves very similarly to Osk: there is always a high level of colocalization in particles, both nuclear and cytoplasmic (Harris, 2001).

These data demonstrate that GFP-Aub is found in particles that (1) also contain Osk and are inferred to contain Vas, both known polar granule components; (2) are located, like polar granules, at the posterior pole of the oocyte and embryo and are incorporated into pole cells; (3) increase in size within the pole cells with similar timing to that previously described for polar granules, and (4) frequently appear, as do polar granules, in spherical structures that are donut-shaped in sections. From these results, it is concluded that Aub is a polar granule component. However, unlike Osk and Vas, which are largely nuclear after the cellular blastoderm stage, Aub is restricted to the cytoplasmic class of particles, indicating that the nuclear bodies and cytoplasmic polar granules do not have identical compositions (Harris, 2001).

Because GFP-Aub concentrates at the posterior pole of the oocyte and is found in polar granules, it was reasoned that Aub localization should depend on the activity of genes required for polar granule assembly. stau and vas are two such genes with somewhat different roles in this process. Stau acts primarily in the localization and translation of osk mRNA, prerequisites for polar granule formation. Although Stau is concentrated at the posterior pole of the oocyte, it is not incorporated into the polar granules early in embryogenesis. By contrast, Vas is a polar granule component and appears to be more directly involved in the formation and integrity of these structures (Harris, 2001).

Not surprisingly, GFP-Aub posterior localization is almost completely defective in stau mutants and greatly reduced in vas mutants, confirming that Aub is localized by the same general mechanism implicated in the localization of other polar granule components. In addition, a second and equally striking defect occurs in only the vas mutants: perinuclear localization of GFP-Aub in the nurse cells is almost entirely eliminated. Taken in conjunction with the known perinuclear concentration of Vas protein, this result raises the possibility that vas-dependent localization of Aub initiates in the nurse cells and involves a complex already containing both Vas and Aub proteins (Harris, 2001).

Much of Maelstrom localizes to highly abundant particles within germline cells. The frequency and distribution of Maelstrom particles are reminiscent of that previously described for nuage, to which Vasa localizes. Double labeling of Maelstrom and Vasa shows overlap in perinuclear germline granules from stem cells through stage 10 nurse cells. Double labeling of Maelstrom and a nuclear lamin shows that virtually all distinct Maelstrom particles are closely apposed to the cytoplasmic face of the nuclear envelope in nurse cells. Because nanos-GAL4-driven GFP-tagged Aubergine (AubGFP) localizes to nuage in late stage egg chambers, Aubergine localization was examined in combination with Vasa and Maelstrom immunostaining. Each discrete particle in the germarium and early egg chamber labels for Vasa, Maelstrom and AubGFP, a concordance that is also maintained in stages 7-10. (Owing to the discontinuous nature of the nanos driver, AubGFP is not highly expressed between approximately stages 3 and 6.) At the ultrastructural level, most nuage is lost from the oocyte by stage 1, prior to the formation of the karyosome. However, occasional particles of Vasa and Maelstrom can be detected in the ooplasm as late as stage 4. Although the most conspicuous localization of Maelstrom and Vasa is to nuage, each protein is also present within the nucleus and cytoplasm of all germline cells. Within the oocyte nucleus, both proteins localize to discrete regions in young egg chambers: in single confocal sections, Vasa often appears in discrete dot or dots, exclusive of, but adjacent to an 'aura' of concentrated Maelstrom. Maelstrom persists in the oocyte nucleus as diffuse staining through at least late stage 10B. After onset of pole plasm assembly (stage 8/9), Vasa accumulates in posterior region of the oocyte. Maelstrom, by contrast, never shows a posterior concentration in the ooplasm. Although Maelstrom is present in the mature egg and early embryo, its distribution is again uniform at these stages. Since neither the Maelstrom nor its RNA show preferential posterior accumulation in the ooplasm, Maelstrom is the first described nuage component that is not also concentrated in pole plasm (Findley, 2003).

Nuage, a germ line specific organelle, is remarkably conserved between species, suggesting that it has an important germline cell function. Very little is known about the specific role of this organelle, but in Drosophila three nuage components have been identified, the Vasa, Tudor and Aubergine proteins. Each of these components is also present in polar granules, structures that are assembled in the oocyte and specify the formation of embryonic germ cells. GFP-tagged versions of Vasa and Aubergine were used to characterize and track nuage particles and polar granules in live preparations of ovaries and embryos. Perinuclear nuage is a stable structure that maintains size, seldom detaches from the nuclear envelope and exchanges protein components with the cytoplasm. Cytoplasmic nuage particles move rapidly in nurse cell cytoplasm and passage into the oocyte where their movements parallel that of the bulk cytoplasm. These particles do not appear to be anchored at the posterior or incorporated into polar granules, which argues for a model where nuage particles do not serve as the precursors of polar granules. Instead, Oskar protein nucleates the formation of polar granules from cytoplasmic pools of the components shared with nuage. Surprisingly, Oskar also appears to stabilize at least one shared component, Aubergine, and this property probably contributes to the Oskar-dependent formation of polar granules. Bruno, a translational control protein, is associated with nuage, which is consistent with a model in which nuage facilitates post transcriptional regulation by promoting the formation or reorganization of RNA-protein complexes (Snee, 2004).

Perinuclear nuage contains, in addition to Vas and Aub, the Maelstrom (Mael), and Gustavus (Gus) proteins. Another component, Bruno (Bru), is a protein that acts in translational repression of osk and gurken (grk) mRNAs. By immunolocalization and expression of a GFP-tagged version of this protein, it was found that Bru is concentrated in perinuclear clusters, similar to the distribution of known nuage components. Double labelling experiments with GFPAub confirmed that Bru colocalizes with nuage. However, Bru is also present at high levels in the cytoplasm, raising the question of whether the colocalization reveals an association with nuage or simply reflects random overlap of an abundant protein with the more narrowly distributed nuage. Evidence that Bru is specifically associated with nuage comes from analysis of Bru distribution in vas mutants: as for other nuage components, the perinuclear clusters of Bru are strongly reduced. Given this identification of Bru as a nuage-associated protein, arrest (aret) mutants (the aret gene encodes Bru) were included in a genetic analysis of nuage. The other genes tested were vas, tud, aub and spindle E (spnE), each of which encodes a nuage component or has been shown to be required for nuage formation, or both (Snee, 2004).

Live imaging was used to better characterize the perinuclear nuage defects seen in static images and to extend the analysis to include cytoplasmic nuage particles. GFPAub was used as the nuage marker to test the role of vas, aret and tud, and VasGFP was used to test the roles of aub and spnE. The live imaging confirmed, for the most part, the basic observations from analysis of fixed samples. In vas mutants perinuclear nuage is almost completely absent, with only a few nuage clusters visible. Loss of spnE activity has a less extreme effect: the perinuclear nuage clusters are largely missing, but a perinuclear zone of VasGFP remains. Consistent with the results by using fixed samples, the persistent perinuclear zone of VasGFP is qualitatively different from wild type, appearing almost completely uniform and lacking any visible discontinuities. Similar results were obtained with the aub mutant, except that the VasGFP perinuclear clusters remain present up to stage 8 of oogenesis, after which they disappear. In aret and tud mutants no significant alteration of perinuclear nuage was detected (Snee, 2004).

In mutants whose perinuclear VasGFP is uniform (spnE- and later stage aub-), the protein undergoes rapid exchange with cytoplasmic pools, just as for VasGFP in perinuclear clusters of wild-type egg chambers. In photobleaching experiments the fluorescence-recovery half-time is 50 seconds in aub- and 48.5 seconds in spnE-, similar to the t1/2=59 seconds for wild type (Snee, 2004).

Cytoplasmic nuage particles are affected differently in the vas, aub and spnE mutants. The vas and spnE mutants have few or no cytoplasmic nuage particles. By contrast, aub mutants have no dramatic reduction in the abundance of cytoplasmic nuage particles, even at times well after the disappearance of perinuclear nuage clusters at stage 8, and the particles have a fairly typical size distribution. These particles do not simply represent the default appearance of VasGFP; they are absent in the spnE mutant. Thus, it seems unlikely that perinuclear nuage clusters are required for the formation of cytoplasmic nuage particles, a conclusion consistent with the observation that cytoplasmic particles are produced only infrequently by detachment of perinuclear nuage clusters (Snee, 2004).

The consequences of loss of vas activity were examined in the male germ line. Just as in nurse cells, Vas appears to be concentrated in nuage in spermatocytes. Given the crucial role for Vas in the nuage of other cell types, either male nuage must differ in this requirement or nuage is not essential in the male germ line for fertility. To distinguish between these possibilities vasAS spermatocytes were tested for the presence of nuage, using GFPAub as a marker. Although GFPAub was present in the cytoplasm, there were no visible perinuclear nuage clusters, indicating that nuage does not form in the vas mutant and is therefore not required for spermatocyte function. An alternate and less probable interpretation is that a rudimentary form of nuage, lacking Aub, is present and is sufficient to provide a minimal requirement for nuage in males (Snee, 2004).

In Drosophila, two types of function, not mutually exclusive, have been proposed for nuage. In one model nuage has been suggested to serve as a precursor to polar granules, a view initially based on ultrastructural similarities of the two organelles and supported by the identification of shared components. Another possible role for nuage is based on its position at the periphery of the nucleus, at or near nuclear pores. Specifically, nuage might act in some aspect of remodelling RNPs when RNAs are exported from the nucleus. Analysis of the movements and genesis of nuage particles provides two arguments against the first model: (1) the rate of release of perinuclear nuage clusters in the nurse cells is very low, much lower than expected if the clusters form polar granules; (2) no nuage particles arriving at the posterior pole of the oocyte and becoming incorporated into polar granules were detected. An additional observation that argues against a model where nuage is a precursor for polar granules, is the presence of cytoplasmic nuage particles in aub mutants, despite the fact that these mutants do not assemble polar granules. However, this evidence does not exclude the first model, because the nuage particles in the mutant might not be fully functional. A third argument is provided by the evidence that Osk cannot interact with nuage, leaving de novo assembly of polar granules as the only reasonable option. Overall, the results strongly suggest that nuage is not the precursor to polar granules, and it is believed that the shared features are simply indicative of similar biochemical activities, rather than a precursor-product relationship (Snee, 2004).

The data do not directly test the model that nuage might function as a transition zone in the movements of mRNAs from the nucleus to the cytoplasm, where RNP components might be exchanged or otherwise modified. However, new properties of nuage, and these relate to possible functions, have been identified. (1) It was found that Bruno, an RNA binding protein that acts as a translational repressor of osk and grk mRNAs, is associated with nuage. This extends the correlation of nuage components with factors that act in some aspect on mRNA localization or translational control. Of the previously identified nuage components, Vas and Gus are involved in the regulation of grk mRNA localization and translation, Aub is required for efficient translation of osk mRNA and has also been implicated in RNAi, and mael mutants display defects in the early stages of mRNA localization. Moreover, spnE, which is necessary for normal nuage formation, is required for the localization of multiple mRNAs and acts in RNAi. Thus, every known nuage component has a role in one or more types of post-transcriptional control of gene expression (Snee, 2004).

(2) The second property of nuage reported here, is the remarkably dynamic composition of perinuclear nuage clusters, despite their relatively fixed positions around the nucleus. This is in contrast to studies showing that general protein exchange is slow in mouse nuage. The rapid exchange of both Vas and Aub, the two proteins tested, suggests that the clusters are staging sites where these, and presumably additional proteins, become associated with other molecules and move off into the cytoplasm. Much like shuttling-proteins that escort RNAs in their travels from the nucleus to the cytoplasm, there might be a class of proteins that interact in nuage with newly exported RNAs and then facilitate post-transcriptional control events that occur in the cytoplasm. By this model nuage could be an organelle that concentrates and thus potentiates the activity factors normally present in all cells, but that must be especially active in germline cells because of their intensive reliance on post-transcriptional controls of gene expression (Snee, 2004).

It has been argued that nuage from the nurse cells is not used for polar granule assembly in the oocyte, yet these two subcellular structures clearly share components and may well have similar activities. One feature that clearly distinguishes polar granules from nuage is the presence of Osk protein. Under normal circumstances Osk is never in contact with nuage, because an elaborate set of post-transcriptional control mechanisms serves to prevent Osk accumulation in the nurse cells and to restrict the distribution of Osk protein within the oocyte to the posterior pole. The presence of Osk at this single location provides the cue for the assembly of polar granules, and misdirection of Osk to other sites in the oocyte leads to ectopic polar granule formation. Thus Osk is generally viewed as an anchor for the recruitment of the factors that form polar granules. Given the finding that polar granules are significantly more stable that perinuclear nuage clusters, it might be that Osk not only recruits other factors, but also strengthens their interactions. A further and unanticipated property of Osk was revealed in studies in which Osk was expressed precociously throughout the oocyte. Under these conditions GFPAub levels are substantially elevated in the oocyte. Two general explanations are possible. (1) Osk might stimulate the rate of transfer of GFPAub from the nurse cells to the oocyte. Such a model is not supported by any known property of Osk, and no increase in the rate at which GFPAub particles move into the oocyte was detected. Furthermore, GFPAub levels in the oocyte are enhanced even before the onset of known nurse cell to oocyte movements in the cytoplasm, and so Osk would have to dramatically alter the properties of the egg chamber under this model. (2) Osk could stabilize a normally labile pool of GFPAub in the oocyte. In the simplest form of this model, stabilization would occur as a consequence of the assembly into complexes, which could include factors other than Osk and GFPAub. This model appears to be most compatible with the data. In addition, such a model provides a possible explanation for the curious association of the Fat facets (Faf) protein, a deubiquitinating enzyme, with pole plasm. The role of Faf could be to stabilize one or more polar granule components, thereby enhancing the growth of polar granules (Snee, 2004).

Transposition-driven genomic heterogeneity in the Drosophila brain

Recent studies in mammals have documented the neural expression and mobility of retrotransposons and have suggested that neural genomes are diverse mosaics. This study found that transposition occurs among memory-relevant neurons in the Drosophila brain. Cell type-specific gene expression profiling revealed that transposon expression is more abundant in mushroom body (MB) αβ neurons than in neighboring MB neurons. The Piwi-interacting RNA (piRNA) proteins Aubergine and Argonaute 3, known to suppress transposons in the fly germline, are expressed in the brain and appear less abundant in αβ MB neurons. Loss of piRNA proteins correlates with elevated transposon expression in the brain. Paired-end deep sequencing identified more than 200 de novo transposon insertions in αβ neurons, including insertions into memory-relevant loci. These observations indicate that genomic heterogeneity is a conserved feature of the brain (Perrat, 2013).

Transposons constitute nearly 45% of the human genome and 15% to 20% of the fly genome. Mobilized transposons can act as insertional mutagens and create lesions where they once resided. Recombination between homologous transposons can also delete intervening loci. Specific regions of the mammalian brain, such as the hippocampus, might be particularly predisposed to transposition. LINE-1 (L1) retrotransposons mobilized during differentiation appear to insert in the open chromatin of neurally expressed genes. One such insertion in neural progenitor cells altered the expression of the receiving gene and the subsequent maturation of these cells into neurons. The mosaic nature of transposition could therefore provide additional neural diversity that might contribute to behavioral individuality and/or neurological disorders (Perrat, 2013).

The Drosophila melanogaster mushroom bodies (MBs) are brain structures critical for olfactory memory. The approximately 2000 intrinsic MB neurons are divisible into α'β', γ, and αβ according to their morphology and roles in memory processing. This study used cell type-specific gene expression profiling to gain insight into cellular properties of MB neurons. Intersectional genetics allowed the exclusive labeling of MB α'β', γ, and αβ neurons in the brain with green fluorescent protein (GFP). For comparison, a 'no MB' genotype, in which GFP labels other neurons in the brain but not MB neurons, was also examined. Sixty brains per genotype were dissected from the head capsule and dissociated by proteolysis and agitation; GFP-expressing single cell bodies were then collected by fluorescence-activated cell sorting (FACS). Total RNA was isolated from 10,000 cells per genotype, and polyadenylated RNA was amplified and hybridized to Affymetrix Drosophila 2.0 genome expression arrays. Each genotype was processed in four independent replicates (Perrat, 2013).

Routine statistical analysis for differentially expressed genes, including a multiple-testing correction across all 16 data sets, did not reveal significant differences at a false discovery rate (FDR) of <0.05. Therefore CARMAweb was used to identify 146 mRNAs whose average signal was >7 in αβ neurons and that were also higher than in α'β' neurons by a factor of >2. Of the top 60 transcripts from this list, 29 were significantly different from α'β' signals and represent transposons. Alignment of the corresponding values from the γ and no-MB profiles showed a similarly significant bias in transposon expression over these samples. Retrotransposons were identified that transpose via a replicative mechanism involving an RNA intermediate and DNA elements that use nonreplicative excision and repair. Retrotransposons can be subdivided into long-terminal repeat (LTR) elements and long interspersed nuclear elements (LINEs). Rleven LTR elements (Tabor, mdg1, roo, qbert, gypsy, invader3, gypsy2, microcopia, 412, accord, and blood), 11 LINE-like elements (G6, RT1b, HeT-A, Ivk, Cr1a, F element, Doc2, baggins, R2, Doc3, and Doc), and four DNA elements (Bari1, pogo, Tc3, and transib3) were identified (Perrat, 2013).

Fourteen transposons, representing the most abundant in each class, were further analyzed. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) of RNA from independently purified cell samples confirmed that transposon expression was significantly higher in αβ neurons than in other MB neurons. All transposons, other than R2, were also significantly higher in αβ neurons than in the rest of the brain. R2 is unique, because it exclusively inserts in the highly repeated 28S rRNA locus and heterochromatin (Perrat, 2013).

Transposition is ordinarily regulated by chromatin structure and posttranscriptional degradation of transposon mRNA guided by complementary RNAs. The small interfering RNA (siRNA) pathway has been implicated in somatic cell. In contrast, the Piwi-interacting RNA (piRNA) pathway has a more established role in the germline. The microarray analysis skewed attention toward piRNA because the expression level of the translocated Stellate locus, Stellate12D orphon (Ste12DOR) mRNA, was higher in αβ than in other MB neurons and the rest of the brain by a factor of >20. Stellate repeat transcripts are usually curtailed by piRNA, not siRNA. Stellate repeats encode a casein kinase II regulatory subunit, and piRNA mutant flies form Stellate protein crystals in testis. Immunostaining Stellate in the brain labeled puncta within αβ dendrites in the MB calyx, consistent with high Ste12DOR expression in wild-type αβ neurons (Perrat, 2013).

piRNAs are loaded into the Piwi clade argonaute proteins Piwi, Aubergine (Aub), and Argonaute 3 (Ago3). Piwi and Aub can amplify piRNA pools with Ago3. To investigate piRNA involvement in differential transposon expression, Piwi proteins and colocalized GFP were immunolocalized to assign signals to MB neuron type. Aub and Ago3 differentially labeled MB subdivisions in addition to structures throughout the brain, but no Piwi was detected. The ellipsoid body of the central complex stained strongly for Aub but not at all for Ago3, which suggests possible functional exclusivity of Piwi proteins in the brain (Perrat, 2013).

Differential Aub and Ago3 labeling was most evident within axon bundles in the peduncle and lobes, where MB neuron types are anatomically discrete. Aub protein colocalized with γ and α'β' neurons in the peduncle and lobes but was reduced in αβ neurons in both locations. Ago3 did not label MB lobes but colocalized with γ neurons in the peduncle. Ago3 labeled core αβ (αβc) neurons but did not label outer αβ neurons. Therefore, outer αβ neurons do not abundantly express Aub or Ago3, which implies that transposon suppression is relaxed. In contrast, γ neurons express Aub and Ago3, providing potential for piRNA amplification, and α'β' neurons express Aub. These patterns of Aub and Ago3 in the MB peduncle appear conserved in brains from D. erecta, D. sechellia, and the more distantly related D. pseudoobscura species (Perrat, 2013).

Loss of siRNA function elevates transposon expression in the head. These findings were replicated with ago2414 and dcr-2L811fsX mutant flies. In parallel, trans-heterozygous aub (aubHN2/aubQC42) and ago3 heads (ago3t2/ago3t3) and trans-heterozygous armitage heads (armi1/armi72.1) were used to test whether piRNA suppressed transposon expression. Levels of the 14 LTR, LINE-like, and TIR group transposons verified to be expressed in αβ neurons were assayed by qRT-PCR; of these 14 transposons, 13 were significantly elevated in siRNA-defective ago2 and dcr-2 mutants. The piRNA-defective aub, ago3, and armi mutants also exhibited significantly elevated levels of 9 of the 14 elements. Levels of the LTR elements gypsy, Tabor, and qbert; the LINE-like elements HeT-A, RT1b, and R2; and the TIR element pogo were higher in ago3 mutants. In addition, blood, Tabor, and R2 were elevated in aub mutants, and blood, gypsy, Tabor, invader3, qbert, HeT-A, and R2 were elevated in armi mutants. Therefore, the piRNA pathway contributes to transposon silencing in the brain, and low levels of Aub and Ago3 may permit expression in αβ neurons (Perrat, 2013).

To determine whether transposons are mobile, new insertions were mapped by deep sequencing of αβ DNA. αβ neurons were purified by FACS, as for transcriptome analysis, but isolated genomic DNA. Insertions were defined by paired-end reads in which one end mapped to the annotated genome and the other to the transposon sequence. To identify de novo transposition events in αβ neurons, the genomic position of transposons within the αβ sequence were compared to those located by sequencing DNA from genetically identical embryos. In addition, DNA was sequenced from the remainder of the brain tissue from the FACS separation of αβ neurons (Perrat, 2013).

These studies identified 3890 transposon insertions in embryo DNA that differed from the published Drosophila genome sequence. In comparison, αβ neuron DNA revealed 215 additional sites. The remaining brain tissue uncovered 200 new insertions, including 19 that were identical to those in αβ neurons. The sequencing depth for embryos was an order of magnitude greater than for neurons because embryo material could be collected more easily; hence, the αβ and other brain insertions are likely de novo. By randomly sampling reads to yield 1x genome coverage, 129 new transposon insertions were calculated per αβ neuron genome. Sequencing single neurons would reveal the exact cellular frequency and heterogeneity of transposition events (Perrat, 2013).

New αβ insertions occurred across all chromosomes, without obvious regional bias. In addition, insertions resulted from 49 different transposons representing LTR, LINE-like, TIR, and Foldback (FB) classes. They included 11 of the 29 transposons in the αβ transcriptome, and the number of insertions per class was consistent with their prevalence in the genome. Therefore, many transposons mobilize in αβ neurons (Perrat, 2013).

Of the 215 de novo αβ insertions, 108 mapped close to identified genes. Of these, 35 disrupted exons, 68 disrupted introns, and 5 fell in promoter regions (<1 kb from transcription start site). The remaining 107 insertions mapped to piRNA clusters or intergenic regions and were not assigned to a particular gene. A similar distribution was observed for the 200 new insertions in the rest of the brain. The reference fly genome has 258 transposon insertions in exonic regions, 11,110 insertions in intronic regions, 502 insertions in promoter regions, and 33,008 insertions in intergenic regions. Therefore, both groups of brain cells had a significantly larger fraction of insertions within exons, and fewer in intergenic regions, than the transposons that are annotated in the genome. To test whether such a distribution was unique to neurons, de novo insertions were analyzed in ovary DNA, again using embryo sequence as the comparison. New insertions in ovary DNA revealed a similar skew toward exons (Perrat, 2013).

In mammals, active L1 elements appear to disrupt neurally expressed genes. New αβ neuron insertions, but not those in other tissue, were significantly enriched in 12 Gene Ontology (GO) terms, all of which are related to neural functions. Moreover, promoter regions from 18 of 20 of the targeted genes drive expression in αβ neurons. Exonic insertions were found in gilgamesh, derailed, and mushroom body defect and intronic insertions in dunce and rutabaga, all of which have established roles in MB development and function. In addition, MB neurons are principally driven by cholinergic olfactory projection neurons and receive broad GABA-ergic inhibition and dopaminergic modulation through G protein-coupled receptors. Intronic insertions were in nicotinic Acetylcholine Receptor α 80B, G protein-coupled receptor kinase 1, and cyclic nucleotide gated channel-like and an exonic insertion in GABA-B-receptor subtype 1. Transposon-induced mosaicism could therefore alter integrative and plastic properties of individual MB αβ neurons (Perrat, 2013).

These data establish that transposon-mediated genomic heterogeneity is a feature of the fly brain and possibly other tissues. Together with prior work in rodents and humans, thede results suggest that genetic mosaicism may be a conserved characteristic of certain neurons. Work in mammals indicates that L1 expression occurs because the L1 promoter is released during neurogenesis. The data are consistent with such a model and also support the idea that transposons avoid posttranscriptional piRNA silencing in adult αβ neurons (Perrat, 2013).

A recent study described a role for piRNA in epigenetic control of memory-related gene expression in Aplysia neurons. It is therefore possible that MB neurons differentially use piRNA to control memory-relevant gene expression and that transposon mobilization is an associated cost. Because tansposon expression was found in αβ neurons of adult flies, it is conceivable that disruptive insertions accumulate throughout life, leading to neural decline and cognitive dysfunction. Alternatively, permitting transposition may confer unique properties across the 1000 neurons in the αβ ensemble and potentially produce behavioral variability between individual flies in the population (Perrat, 2013).


The original mutation was isolated in a screen for male sterile insertions among 2225 P{lacW} insertion lines that were generated by A. Beermann and C. Schultz in the laboratory of J. A. Campos-Ortega at the University of Cologne. The male sterile phenotype displays variable penetrance, leading sometimes to progeny from homozygous males. The P{lacW} insertion was localized by in situ hybridization to polytene region 32D on the left arm of chromosome 2, giving the mutation the provisional name ms(2)32D. Analysis of the spermatogenic stages in homozygous mutant males revealed the existence of needle-like structures in spermatocytes and spermatids. These needles strongly resemble those crystals found in X/0 males of Drosophila melanogaster, although the mutant males clearly have an X/Y genotype, as visualized by the presence of Y-chromosomal lampbrush loops (Schmidt, 1999).

An antibody against the Ste protein was tested to see whether these needles consist of the Ste protein despite the fact that they are produced in X/Y male germ cells. This is indeed the case. Furthermore, the crystal morphology is dependent on the Ste genotype. In mutant male flies carrying a Ste+-containing X chromosome, the crystals have a needle-like shape, whereas in a Ste background, they are similar to the star-shaped crystals found in Ste/cry- males. Thus, the crystals' phenotype is determined by the Ste locus in both the X/Y, cry+ homozygous sting mutant males and in X/Y, cry-; sting+ males. Because the processed Stellate transcript can also be detected in homozygous mutant males by Northern analysis, the gene was renamed sting (sti), as an acronym for Stellate-interacting gene and for the production of sting-like crystals (Schmidt, 1999).

To test the hypothesis that the phenotypes shown by the sti homozygous mutants are related to the Stellate system, the expression of the Stellate RNA was analyzed in these individuals by using different Ste alleles. Total RNA was extracted from the testes of X/Y males homozygous and heterozygous for the sti mutation carrying different Stellate alleles. The patterns of Stellate expression were compared with those of X/Y or X/0 testes by Northern hybridizations. The same smeared 750-nt Stellate RNA, typically present in X/Y, cry- testes, is also always present in testes of homozygous sti males independently of the Ste allele present. It is important to note that a high-molecular-weight RNA (of ~8 kb) is present in the testes of X/Y regular males and in X/Y, heterozygous sti males. This fragment, however, appears reduced or almost absent, concomitantly with the production of the 750-base fragment, in the testes of X/Y, cry+ homozygous sti males or X/Y, cry-; sti+ males, respectively. This suggests that, like the crystal locus (also known as Suppressor of Stellate), sting seems to control Stellate expression, mainly at post-transcriptional levels. The reduction of the high-molecular-weight RNA seen in the testes of X/0 males is observed in sting homozygous males only in combination with the mutant Stellate alleles, while it is not seen with the Ste+ alleles, unless the crystal deletion is also present. Intriguingly, the same high-molecular-weight Ste RNA fragment and another 1.2-kb fragment also are present in other tissues of males and females, such as the larval brains (where they are very abundant), salivary glands, and ovaries, irrespective of the sti genotypes or the absence of the Y chromosome. To determine the origin of these two types of constitutive transcripts, the Ste transcription pattern was analyzed in the testes of males carrying the W12 X chromosome, which lacks the euchromatic Ste cluster. In cry- male testes, the 8.0-kb transcripts decrease concomitantly with the production of the 750- to 850-base Ste transcripts. This suggests that these transcripts are processed in testes of cry- males, producing the 750-nt RNA. Therefore these data show that the Ste sequences are also normally transcribed in somatic tissues, and that the sti gene negatively controls the Ste expression only in male spermatocytes (Schmidt, 1999).

X/Y, cry- males show meiotic chromosome nondisjunction and meiotic drive. To test if the sti homozygous mutant males show the same spectrum of meiotic abnormalities correlated with the number of Ste repeats, three Ste+ alleles with low numbers and seven Ste alleles with different high numbers of Ste repeats, as estimated by slot blots, were chosen. Fertility and sex chromosome behavior were analyzed in sti homozygous or heterozygous males carrying Ste+ or Ste alleles. The derivation of the disjunctional parameter PXY (the probability of disjunction of the X and Y), the drive parameters RX (the recovery of X-bearing sperm) and RY (the recovery of Y-bearing sperm), and the statistical methods used have been described in detail. The data presented led to the following conclusions: (1) the sting mutation affects male fertility, and this is inversely correlated with the Ste repeat copy number; (2) the sting mutation induces meiotic nondisjunction, as seen in the high number of XY and 0 sperm (this is directly correlated with the Ste repeat copy number); (3) the sting mutation affects meiotic drive, since there is an excess of X over Y and 0 over XY sperm. Moreover, it is interesting to note that the spectrum of these defects is present also in the meiosis of heterozygous sti males carrying the Stellate X chromosomes. Taken together, the results of the statistical analysis described above show that sti affects fertility, disjunction, and drive, and that it is semidominant. As with cry1, however, PXY and fertility are much more sensitive to the Ste state (Ste vs. Ste+ or Ste copy number) than are RX or RY. A clear correlation between fertility and the total number of Stellate copies was also found (Schmidt, 1999 and references therein).

The effects of Ste copy number on autosomal behavior were also assessed. sti homozygous males carrying four different Ste alleles were crossed to females carrying the compound second chromosome C(2)EN. These crosses show the same basic pattern reported in previous work for Ste+ combined with synthetic, translocation-generated cry deficiencies or with the cry1Y chromosome. The main features of this pattern are: (1) the recovery of numerous diplo-2 and nullo-2 sperm; (2) a strong disruption of sex chromosome disjunction among these sperm, and (3) an excess recovery of nullo-2 sperm, probably reflective of an autosomal meiotic drive that is not correlated with Ste copy number. The observed drive of chromosome 2, when sex chromosome disjunction is regular, leads to the expectation of a similar preference for 0 over 22 sperm in those cases where the disjunction is destroyed. The lack of such an excess of 0;0 over 0;22 sperm suggests, therefore, that the disjunction of the sex chromosomes and autosomes is not completely independent in frequency, but that it is biased, to some extent, toward opposite poles (Schmidt, 1999).

The precise restriction of proteins to specific domains within a cell plays an important role in early development and differentiation. An efficient way to localize and concentrate proteins is by localization of mRNA in a translationally repressed state, followed by activation of translation when the mRNA reaches its destination. A central issue is how localized mRNAs are derepressed. Regulatory elements for both RNA localization and translational repression are situated in the 3' UTR of OSK mRNA, as they are in NOS. In the case of OSK, premature translation is prevented by Bruno, a 68-kD protein encoded by the arrest (aret) locus. Bruno recognizes a repeated conserved sequence (BRE, for Bruno response element) in the osk 3' UTR, and colocalizes with OSK mRNA to the posterior pole. In contrast to NOS, however, 3' UTR-mediated localization at the posterior pole is not sufficient for translation, as heterologous transcripts localized under the control of the full-length OSK 3' UTR are not translated. This indicates that the OSK 3' UTR, although it may participate, is not sufficient for translational activation, and that sequences elsewhere in the transcript are required for translation of OSK mRNA (Gunkel, 1998).

When OSK mRNA reaches the posterior pole of the Drosophila oocyte, its translation is derepressed by an active process that requires a specific element in the 5' region of the mRNA. This novel type of element is a translational derepressor element, whose functional interaction with the previously identified repressor region in the OSK 3' UTR is required for activation of Oskar mRNA translation at the posterior pole. The derepressor element only functions at the posterior pole, suggesting that a locally restricted interaction between trans-acting factors and the derepressor element may be the link between mRNA localization and translational activation. Specific interaction of two proteins with the OSK mRNA 5' region is shown; one of these also recognizes the 3' repressor element. p50 is a BRE binding protein that recognizes 3' repressor motifs similar to those recognized by Bruno. p50 functions as a second translational repressor independent of Bruno. The involvement of a second repressor protein in OSK translational control is not unexpected. Indeed, aubergine (aub), a gene required for efficient OSK mRNA translation, is required even when Bruno-mediated repression is alleviated by mutations in the BRE, leading to the suggestion that the aub gene product enhances translation by counteracting the action of a second repressor. It is interesting to note that the requirement for aub function in OSK translation is conferred not only by the OSK 3' UTR but also involves the 5' end of OSK mRNA. Consistent with this possible involvement of the OSK 5' end in translational repression, it is found that in transgenic flies containing an inefficient BRE, premature translation increases when the 5' end is truncated. Understanding the extent to which the 5' end of the OSKtranscript might contribute to overall translational repression will require mutations that selectively disrupt 5' repressor function without simultaneously affecting derepressor function (Gunkel, 1998).

RNAi is activated during Drosophila oocyte maturation in a manner dependent on aubergine and spindle-E

Gene silencing by double-stranded RNA is a widespread phenomenon called RNAi, involving homology-dependent degradation of mRNAs. RNAi is established in the Drosophila female germ line. mRNA transcripts are translationally quiescent at the arrested oocyte stage and are insensitive to RNAi. Upon oocyte maturation, transcripts that are translated become sensitive to degradation while untranslated transcripts remain resistant. Mutations in aubergine and spindleE, members of the PIWI/PAZ and DE-H helicase gene families, respectively, block RNAi activation during egg maturation and perturb translation control during oogenesis, supporting a connection between gene silencing and translation in the oocyte (Kennerdell, 2002).

To analyze the effects of dsRNA on mRNA stability in Drosophila oocytes, dsRNAs corresponding to the maternally expressed genes bicoid and hunchback were used. These genes were chosen because their mRNAs are synthesized, processed, and localized to the cytoplasm of oocytes during mid- to late oogenesis. To test the sensitivity of bicoid and hunchback to RNAi, fertilized eggs were initially injected with dsRNA. bicoid dsRNA reduces the expression of Bicoid protein and induces a bicoid loss-of-function phenotype in which embryos have partial transformation of anterior structures to posterior identities. The effect is robust enough that dsRNA-coated gold particles randomly introduced into fertilized eggs by a gene gun generate mutant phenotypes. hunchback dsRNA induces phenotypes in which embryos are missing thoracic and head segments. These phenotypes resemble mutant embryos generated when maternal and zygotic hunchback gene activity is reduced. To determine if dsRNA injection causes mRNA degradation, endogenous mRNA levels were measured using a semiquantitative RT-PCR assay. The level of bicoid mRNA was reduced about fourfold 40 min after injection of bicoid dsRNA. Likewise, injection of hunchback dsRNA resulted in a reduction of hunchback mRNA levels. Coinjection of a pan-specific ribonuclease inhibitor, vanadyl-ribonucleoside, with bicoid dsRNA results in no reduction of bicoid mRNA, indicating the effect requires a ribonuclease activity (Kennerdell, 2002).

Whether and when transcripts become sensitive to dsRNAs during oogenesis was determined. dsRNA was injected into staged oocytes and their consequent levels of bicoid and hunchback mRNAs were examined. Although oocytes earlier than stage 14 could not be injected, stage 14 oocytes could be examined for RNAi activity. Levels of bicoid and hunchback mRNAs were unchanged in stage 14 oocytes after injection of dsRNA, indicating that oocytes at this stage are unable to carry out RNAi (Kennerdell, 2002).

Oocytes of most animals arrest at species-specific stages of meiosis while differentiation of the oocytes occurs. Drosophila oocytes arrest transiently in prophase I while the oocytes are loaded with RNAs and proteins. Some of these molecules are differentially localized within the oocyte, imparting positional information to be used for embryonic axis formation. When Drosophila oocytes reach stage 14, they undergo meiotic arrest once more, this time at metaphase I. These arrested oocytes remain translationally quiescent in the ovary, potentially for weeks. Arrest is relieved as in most animal eggs by the process of maturation or activation that precedes fertilization. In the case of Drosophila, it appears that ovulation triggers activation of the oocyte to resume meiosis. When oocytes are activated, meiosis is completed and translation of maternal RNAs is dramatically elevated. Shortly thereafter, the oocyte is fertilized as it passes into the uterus (Kennerdell, 2002).

RNAi-like effects are not detected in arrested stage 14 oocytes injected with dsRNA. Was this a general feature of the female germ line? To explore this issue, dsRNA was injected into mature activated oocytes. Injection of dsRNA causes reduction in bicoid and hunchback mRNA levels comparable to those seen in embryos. To confirm that mRNA sensitivity to dsRNA is strictly coincident with oocyte maturation, arrested stage 14 oocytes were isolated from dissected ovaries and the oocytes were activated in vitro. This maturation procedure reactivates meiosis, mRNA translation, and vitelline membrane cross-linking. After maturation, oocytes were injected with bicoid dsRNA and assayed for bicoid mRNA levels. These oocytes showed a decrease in bicoid mRNA. Thus, immature Drosophila oocytes that are coordinately blocked for meiosis and translation are resistant to RNAi, and the block to these processes can be released by maturation or activation of oocytes (Kennerdell, 2002).

There are several possible ways in which RNAi might be blocked in arrested oocytes. One possibility is that an essential component of the RNAi machinery might be missing at this stage. Oocyte maturation would then involve synthesis of the component. To address if synthesis of a missing component is responsible, oocytes were activated in the presence of the protein synthesis inhibitor cycloheximide. Arrested stage 14 oocytes were preincubated with cycloheximide and then activated in vitro in the presence of cycloheximide. This treatment inhibits >95% of the protein synthesis that occurs during maturation. These oocytes were injected with bicoid dsRNA and, strikingly, they showed a decrease in bicoid mRNA levels that was comparable to that of normal mature oocytes. RNAi is established during oocyte maturation even when protein synthesis is blocked. Thus, RNAi establishment during oocyte maturation does not likely occur by synthesis of an essential protein component of the RNAi machinery (Kennerdell, 2002).

The stage 14 oocyte is coordinately blocked in both translation and RNAi. The two processes are released near simultaneously from this block, suggesting perhaps that a shared mechanism links their regulation. To test this possibility, the effectiveness of dsRNA was examined against a transcript that is present but not translated after oocyte maturation. The alphaTubulin67C gene encodes one of three alpha-tubulin proteins synthesized during oogenesis and embryogenesis. Transcript accumulates and is actively translated in early immature oocytes. However, after oocyte maturation, no translation of alphaTubulin67C mRNA occurs, even though transcripts at this stage are associated with ribosomes and are competent to drive translation in vitro. The stable pool of alphaTubulin67C mRNA is comparable to levels of bicoid and hunchback mRNA in mature oocytes. When two nonoverlapping dsRNAs against alphaTubulin67C transcript were independently injected into mature activated oocytes, no destruction of mRNA was detected. This suggests that the ability of dsRNAs to destroy transcripts during oogenesis is coupled to the translation activity of the transcript. Successful translation of transcripts is perhaps necessary to link a transcript to dsRNA-triggered degradation (Kennerdell, 2002).

Several Drosophila genes have been identified that affect translation of maternal mRNAs during oogenesis. One of these genes, aubergine (aub), encodes a protein with a PIWI and PAZ domain. To determine whether Aub has any role for RNAi in oocytes, the effect of aub mutations on RNAi activity was examined. bicoid and hunchback dsRNAs were injected into aub mutant oocytes that were activated in vitro. Degradation of bicoid and hunchback mRNAs was not observed in aub mutants, indicating that Aub is necessary for germ-line RNAi. Two independent aub alleles in heteroallelic combination produced the same result, indicating that the effect was not due to the influence of linked modifiers (Kennerdell, 2002).

The aub gene is a member of a family of genes implicated in RNAi and PTGS. Indeed, aub has been implicated in PTGS regulation of the Stellate repeats and Su(Ste) genes on X and Y chromosomes. Another member of the family, piwi, has been implicated in PTGS within somatic cells. A third family member, Ago2, is a subunit of the mRNA-cleaving complex that mediates RNAi in Drosophila embryonic cells. Thus, several members of this gene family in Drosophila have been implicated in RNAi and PTGS at various steps (Kennerdell, 2002).

It was of interest to determine if other translational regulatory genes are involved in RNAi. To test this possibility, two genes that possibly act through interactions with RNA were examined. vasa and spindle-E (spn-E) encode DexH-box RNA helicases. When activated spn-E mutant oocytes were injected with bicoid or hunchback dsRNAs, no reduction in cognate mRNA levels occurred. In contrast, activated vasa mutant oocytes injected with bicoid dsRNA were found to show transcript degradation comparable to wild type. It is concluded that activation of RNAi in oocytes is dependent on the activity of Spn-E but not Vasa (Kennerdell, 2002).

Arrested Drosophila oocytes are unable to generate RNAi silencing of endogenous maternal mRNAs, but selectively establish this capability upon egg maturation. How is RNAi activated by egg maturation? It is argued that RNAi is linked in some way to translation of maternal mRNAs, which is also specifically activated by egg maturation. Establishment of RNAi is probably not caused by translation of a missing RNAi component. Rather, the complete RNAi apparatus may be present and poised for action but is unable to target homologous substrate mRNAs until egg maturation. Translational masking of mRNAs, a mechanism that operates on maternal Drosophila gene expression, may conceivably be one way in which mRNA is blocked from RNAi attack. Alternatively, targeting of mRNA might require transcripts be assembled onto active polysomes. This may be the case, because siRNA-containing RISC complexes physically fractionate with polysomes, and siRNAs associate with polysomes in Trypanosoma brucei. There is no evidence to indicate that dsRNA-targeting requires ribosome translocation on transcripts, because it is found that cycloheximide inhibition of ribosome translocation does not block RNAi activity in activated mature oocytes (Kennerdell, 2002).

Coupling RNAi to translated mRNA might facilitate base-pairing interactions between siRNAs and an unfolded mRNA target, or it might simply be a means to mark RNAs to be scanned for destruction. The key evidence suggesting that transcript translation is linked to transcript degradation by RNAi comes from experiments in which dsRNA against the alphaTubulin67C message was tested. dsRNA is ineffective against the untranslated alphaTubulin67C transcript in mature activated oocytes, which are nevertheless competent to carry out RNAi against translated bicoid and hunchback transcripts. Thus, there is a correlation between the ability of a transcript to be translated and its ability to be destroyed by dsRNA (Kennerdell, 2002).

Aub and Spn-E are required for RNAi in Drosophila oocytes. They also regulate several features of germ-cell development, including translation of certain maternal mRNAs. Germ-line and stem-cell functions have been reported for orthologs of piwi and ago1 in a variety of species. The ego-1 gene of C. elegans is required for both germ-line development and germ-line PTGS. Finally, mutations in dcr-1, the C. elegans gene encoding Dicer, disrupt oogenesis in an unspecified manner. The developmental defects associated with mutations in these genes, including aub and spn-E, might reflect a loss of gene silencing important for oocyte development. Thus, Aub and Spn-E might play a specific role in gene silencing mechanisms, including RNAi, that nevertheless have a widespread impact on many features of development. Alternatively, Aub and Spn-E could be required for RNAi because they activate translation of germ-line transcripts including those for bicoid and hunchback. Although there is no evidence for translational control of bicoid mRNA in aub mutants, these mutants may perturb steps in the translation of transcripts that are essential for triggering RNAi. Future experiments should define the specific roles for Aub and Spn-E in dsRNA-mediated destruction and its relationship to translation control (Kennerdell, 2002).

Dissection of a natural RNA silencing process in the Drosophila melanogaster germ line; Mutations in aubergine impair silencing by eliminating short Su(Ste) RNA

To date, few natural cases of RNA-silencing-mediated regulation have been described. Repression has been analyzed of testis-expressed Stellate genes by the homologous Suppressors of Stellate [Su(Ste)] repeats that produce sense and antisense short RNAs. The Stellate promoter is dispensable for suppression, but local disturbance of complementarity between the Stellate transcript and the Su(Ste) repeats impairs silencing. Using in situ RNA hybridization, temporal control was found of the expression and spatial distribution of sense and antisense Stellate and Su(Ste) transcripts in germinal cells. Antisense Su(Ste) transcripts accumulate in the nuclei of early spermatocytes before the appearance of sense transcripts. The sense and antisense transcripts are colocalized in the nuclei of mature spermatocytes, placing the initial step of silencing in the nucleus and suggesting formation of double-stranded RNA. Mutations in the aubergine and spindle-E genes, members of the Argonaute and RNA helicase gene families, respectively, impair silencing by eliminating the short Su(Ste) RNA, but have no effect on microRNA production. Thus, different small RNA-containing complexes operate in the male germ line (Aravin, 2004).

A strong correlation is observed between Stellate silencing and the presence in testes of sense and antisense 25- to 27-nt RNAs homologous to Stellate and Su(Ste) sequences. The short RNAs are absent when Stellate genes are derepressed as a consequence of either a Su(Ste) locus deletion or mutations in the aub and spn-E genes. The cloning of short RNA from D. melanogaster testes also demonstrates the presence of short RNAs that are derived from Su(Ste) and are highly homologous to Stellate. A rigid size restriction of 21 to 23 nt has, however, been observed for siRNA in various in vitro studies of D. melanogaster RNAi. Examination of Dicer activity with different dsRNAs suggests a strong specificity of processing to 21- to 23-nt fragments in both Drosophila embryo extracts and cell culture. Furthermore, investigation of the functional anatomy of chemically synthesized siRNAs in embryo extracts defined the optimal length of siRNAs as 21 to 23 nt, while RNAs longer than 24 nt have practically no cognate-mRNA cleavage activity. It has been proposed that only RNAs that meet this size requirement can be loaded into the RISC. However, examples of the existence of two size classes of short RNAs (21 or 22 nt and 24 to 26 nt) involved in silencing have also been reported. Two different size variants of short RNAs were observed during artificial silencing in plants, with the short variant responsible for posttranscriptional gene silencing and the long one most likely participating in DNA methylation and spreading of the silencing signal. Furthermore, only RNAs from the long class have been detected that correspond to endogenous plant transposable elements. Two size classes of short RNAs are produced from dsRNA in plant extracts, and the activity of different Dicer proteins was shown to be responsible for producing each class. Cloning of endogenous short RNAs from D. melanogaster has also identified two size classes of short RNAs, with the short class (21 to 23 nt) including microRNAs and the long class (24 to 26 nt) comprising sequences derived from transcripts of transposable elements and other repetitive heterochromatic sequences (Aravin, 2004).

The larger size of the short Su(Ste) RNA may be explained by specific sequences affecting dsRNA processing by Dicer or by the presence in testes of specific factors that change the cleavage interval of dsRNA. However, exogenous Su(Ste) dsRNA is cleaved into 21- to 23-nt siRNA in testis extracts, most likely reflecting the activity of the same Dicer protein that acts in somatic tissues. The hypothesis is favored that the 25- to 27-nt Su(Ste) RNAs detected in vivo are produced by a mechanism at least partially different from conventional siRNA production. A clue to the origin of the short Su(Ste) RNAs comes from the finding that Su(Ste) dsRNA formation occurs in the nucleus, unlike that of artificial RNAi, in which dsRNA is believed to be processed in the cytoplasm. Both conventional-size siRNA and a longer short RNA have been observed during viroid replication in the plant nucleus. Two size classes of short RNAs may be produced in D. melanogaster by different Dicer proteins, as has been demonstrated in plants. Alternatively, specific nuclear factors may affect how a single Dicer protein processes dsRNA in the nucleus (Aravin, 2004).

Mutations in the aub and spn-E genes lead to elimination of short Su(Ste) RNA in testes. However, neither mutation affects processing of exogenously provided dsRNA to 21- to 23-nt siRNA in testis extracts. It has been observed that both aub and spn-E mutations block RNAi in oocytes produced by injected dsRNA. It has been proposed that both proteins affect RNAi because of their involvement in translational control, but the results suggest that Aub and Spn-E may be involved in the production and/or stabilization of siRNA. Similarly, the rde-1 and mut-7 genes of Caenorhabditis elegans are required for the production of siRNA in vivo but are dispensable for dsRNA processing in vitro. The corresponding proteins are required for long-term stabilization of siRNA rather than for dsRNA processing (Aravin, 2004).

The aub and spn-E mutations eliminate the short Su(Ste) RNA without affecting the abundance of two different microRNAs in testes. It is proposed that distinct protein complexes mediate production and/or stabilization of short Su(Ste) RNA and microRNAs in testes. Similarly, different members of the Argonaute family participate in artificial RNAi and in microRNA processing in C. elegans and plants, despite the central role of Dicer in both processes (Aravin, 2004).

Telomere elongation is under the control of the RNAi-based mechanism in the Drosophila germline; mutations in the spn-E and aub cause an increase in the frequency of telomeric element retrotransposition to a broken chromosome end

Telomeres in Drosophila are maintained by transposition of specialized telomeric retroelements HeT-A, TAHRE, and TART instead of the short DNA repeats generated by telomerase in other eukaryotes. This study implicates the RNA interference machinery in the control of Drosophila telomere length in ovaries. The abundance of telomeric retroelement transcripts is up-regulated owing to mutations in the spn-E and aub genes, encoding a putative RNA helicase and protein of the Argonaute family, respectively, which are related to the RNA interference (RNAi) machinery. These mutations cause an increase in the frequency of telomeric element retrotransposition to a broken chromosome end. spn-E mutations eliminate HeT-A and TART short RNAs in ovaries, suggesting an RNAi-based mechanism in the control of telomere maintenance in the Drosophila germline. Enhanced frequency of TART, but not HeT-A, attachments in individuals carrying one dose of mutant spn-E or aub alleles suggests that TART is a primary target of the RNAi machinery. At the same time, enhanced HeT-A attachments to broken chromosome ends were detected in oocytes from homozygous spn-E mutants. Double-stranded RNA (dsRNA)-mediated control of telomeric retroelement transposition may occur at premeiotic stages, resulting in the maintenance of appropriate telomere length in gamete precursors (Savitsky, 2006).

The problems of end-under-replication and stability of linear chromosomes are resolved by telomeres. The lengthening of terminal regions of linear eukaryotic chromosomes is often provided by RNA-templated addition of repeated DNA by reverse transcriptase enzyme, telomerase. In most eukaryotes, telomeric DNA is maintained by the action of telomerase, which is responsible for the synthesis of short 6-8-nucleotide (nt) arrays using an RNA component as a template. In contrast, telomeres of Drosophila are maintained as a result of retrotranspositions of specialized telomeric non-long-terminal repeat (LTR) HeT-A, TAHRE, and TART retrotranspositions (Biessmann, 1992b; Levis, 1993; for review, see Pardue, 2003; Abad, 2004b). Retrotransposons are also found in telomeric regions of such diverse organisms as Bombyx mori, Chlorella and Giardia lamblia. HeT-A, TAHRE, and TART are found at Drosophila telomeres in tandem arrays. HeT-A, the most abundant Drosophila telomeric element, contains a single ORF encoding a Gag-like RNA-binding protein, but lacks reverse transcriptase (RT). It is proposed that the RT necessary for its transposition might be provided in trans, perhaps by TART (Rashkova, 2002). TART ORF2 encodes a reverse transcriptase related to the catalytic subunit of telomerase. The recently discovered TAHRE element shows extensive similarity to HeT-A, but contains a second ORF, which encodes a reverse transcriptase (Abad, 2004b). A HeT-A promoter located in the 3' region of the element directs synthesis of a downstream neighbor (Danilevskaya, 1997). The TART element was shown to be transcribed bidirectionally using a putative internal sense promoter and antisense one that was localized within the 1-kb region of the TART 3' end (Danilevskaya, 1999). Maintenance of Drosophila telomere length is mediated by HeT-A and TART transpositions to chromosome ends as well as by terminal recombination/gene conversion (Mikhailovsky, 1999; Kahn, 2000). Most of the observed spontaneous attachments to telomeres are HeT-A transpositions (Biessmann, 1992a; Kahn, 2000; Golubovsky, 2001), but TART attachments (Sheen, 1994) were also detected (Savitsky, 2006 and references therein).

The spn-E and aub genes, encoding an RNA helicase and a protein of Argonaute family, respectively, are involved in double-stranded RNA (dsRNA)-triggered RNA interference (RNAi) in embryos, in transcriptional silencing of transgenes, and in the control of Drosophila retrotransposon transcript abundance in the germline, especially in ovaries. No effects of RNAi gene mutations on HeT-A and TART expression and telomere structure were observed in somatic tissues (Perrini, 2004). This study shows that increased HeT-A and TART transcript abundance in ovaries, owing to RNAi mutations, is correlated with a high frequency of telomeric element attachments to broken chromosome ends. Addition of HeT-A or TART to a truncated X chromosome, with a break in the upstream regulatory region of yellow, activates yellow expression in aristae, which enables monitoring of the elongation events (Kahn, 2000; Savitsky, 2002). Using this genetic system, the effects of RNAi mutations were studied on the frequency and molecular nature of telomeric attachments. A high frequency of TART but not HeT-A attachments in heterozygous RNAi mutants suggests that TART may be the primary target of the RNAi-based silencing mechanism. These results highlight for the first time the importance of TART, but not the more abundant HeT-A element, in Drosophila telomere maintenance. The disappearance of short TART and HeT-A RNAs was found in spn-E mutant ovaries, strongly suggesting an RNAi-based pathway in the control of telomere maintenance in the Drosophila germline (Savitsky, 2006).

An RNAi-based mechanism has been proposed to evolve in order to immobilize transposable elements and was found to control expression of endogenous transposable elements and their mobility in different species. Drosophila telomeres are maintained by successive transpositions of specialized telomeric retroelements HeT-A and TART. This study shows that transposition of both telomeric elements is under the control of the spn-E and aub genes, known to be related to the RNAi machinery. Hence, an RNAi-based mechanism may be considered not only as a defense against retrotransposon expansion, but also as a regulatory system responsible for proper telomere length maintenance in Drosophila (Savitsky, 2006).

spn-E is required for appropriate localization of mRNA and proteins involved in the establishment of axis formation in the embryo and encodes a member of the DEAD/DE-H protein family possessing RNA-binding and RNA helicase activity. aub encodes a protein of the Argonaute family that was shown to be a component of the RNAi effector complex RISC. aub and spn-E mutations strongly diminished effects of the injected dsRNA into mature oocytes. Both genes are implicated in small interfering RNA (siRNA)-dependent silencing of testis-expressed Stellate genes. Thus, spn-E and aub are components of RNAi-based silencing pathways in Drosophila. Mutations in these genes result in the derepression of a wide spectrum of retrotransposons in the germline, including the HeT-A telomeric element (Aravin, 2001; Stapleton, 2001; Kogan, 2003). This study demonstrates that spn-E and aub mutations increase the frequency of telomeric element retrotranspositions to broken chromosome termini, suggesting that the RNAi machinery controls telomere length in Drosophila (Savitsky, 2006).

Both telomeric elements are shown to be the targets of RNAi. The present results emphasize the differences in the response of HeT-A and TART elements to RNAi mutations. Surprisingly, two different spn-E mutant alleles and an aub mutation in the heterozygous state increase considerably TART mobility, whereas attachments of HeT-A to broken chromosome ends were detected much more rarely in spn-E1/+ ovaries and are not observed in ovaries of spn-Ehls3987/+ and aubQC42/+ flies. One copy of a spn-E mutation is sufficient to increase TART transcript abundance. Strong accumulation of HeT-A transcripts is found only in homozygous mutants, correlating with a high frequency of HeT-A attachments to the broken chromosome ends in the developing oocytes. This observation argues that TART is a primary target of the RNAi machinery in ovaries (Savitsky, 2006).

TART and HeT-A, in spite of sharing the region of integration, are dissimilar in their structure and expression strategy. While both sense and antisense TART transcription has been demonstrated, antisense transcripts are more abundant. In situ RNA analysis detected sense and antisense TART transcripts in the cytoplasm of nurse cells in the late-stage egg chambers, suggesting a possibility of dsRNA formation. However, it was found that the level of antisense TART transcripts is not affected in RNAi mutants. Only sense HeT-A transcription was observed by Northern or by in situ RNA analyses. Nevertheless, HeT-A- and TART-specific siRNAs were revealed among the cloned short RNA species in Drosophila, and short RNAs corresponding to both HeT-A and TART elements are detected by Northern analysis. Antisense HeT-A RNA is probably transcribed at a low level from an unidentified promoter, possibly, from the HeT-A internal region. Actually, a low level of antisense activity of the HeT-A 3' end has been observed . While TART transcripts were observed only in the nurse cells, HeT-A transcripts were detected both in the growing oocyte and nurse cells. It is proposed that TART is a primary target of the RNAi controlling system, since one dose of an RNAi mutation causes preferential TART, but not HeT-A, attachments to broken chromosome ends in ovaries. In contrast, one dose of a mutant Su(var)205 gene (HP1) considerably increasess the frequency of HeT-A rather than TART attachments to the chromosome ends (Savitsky, 2002). Thus, a specific effect of RNAi components on telomeric element expression is observed . Although TART copies are much less abundant in the genome than HeT-A and no TART elements are detected in some telomeres, TART is a conserved component of telomeres in distant Drosophila species. TART was considered as a source of RT production, thus ensuring retrotranspositions of both TART and HeT-A elements. One may propose that TART supplies an RNAi-regulated template for RT production, thus providing telomere-specific transpositions of both elements (Savitsky, 2006).

Drosophila telomeres contain a multisubunit protein complex forming a chromosome cap protecting chromosomes from DNA repair and end-to-end fusions. However, no HeT-A or TART sequences were detected at the stably maintained broken chromosome end, which is protected from telomere fusions. Thus, a sequence-independent system performs telomere capping functions. The capping complex contains HP1, HOAP (HP1/ORC associated protein), as well as ATM-kinase and DNA repair MRN complex and the Ku70/Ku80 heterodimer. HP1 and the Ku heterodimer act also as negative regulators of telomere elongation by retrotransposition of telomeric elements. Deficiencies that remove either the Ku70 or the Ku80 gene increase the transposition rate of HeT-A and TART elements but exert no effect on the HeT-A expression, suggesting that Ku proteins control the accessibility of the telomere to transposition events. At the same time, mutations in the Su(var)205 gene increase both transcript abundance of HeT-A and TART and the frequency of their attachments to chromosome ends. RNAi affects both telomeric retrotransposon expression and the rate of transposition to the telomere. Probably, this effect is mediated through HP1 recruitment and silencing of HeT-A and/or TART chromatin (Savitsky, 2006).

siRNAs produced from telomeric elements TART and HeT-A belong to the long size class (25-29 nt) in contrast to 21-22-nt RNAs guiding post-transcriptional RNAi. In plants, long siRNAs are associated with RNA-directed DNA methylation and play an essential role in the transcriptional retrotransposon silencing. dsRNA and proteins of the RNAi machinery can direct chromatin alteration to homologous DNA sequences and induce transcriptional silencing. RNAi mutations cause delocalization of HP1 in yeast and Drosophila. Actually, the increase in accessibility of HeT-A chromatin and its enrichment in K9-acetylated H3 histone were revealed in ovaries of spn-E mutants. It is also possible that TART and/or HeT-A short RNAs can be targeted to telomeric repeats in a transcriptional silencing complex (Savitsky, 2006).

RNAi disruption affects neither HeT-A and TART expression, nor telomere fusions in somatic cells. No effect was observed of spn-E mutations on HeT-A expression, even in actively dividing cells of imaginal discs, where HeT-A expression was found. The data indicate a crucial role of the RNAi machinery in the regulation of telomere elongation in germinal cells. The appearance of a cluster of individuals with identical retroelement attachments indicates that dsRNA-mediated control of terminal elongation may occur at premeiotic stages of oogenesis (Savitsky, 2006).

This study has demonstrated that expression and retrotransposition of specific telomeric repeats is under control of an RNAi-based system in the Drosophila germline. In this case, the telomerase-dependent mechanism of telomere stability is substituted by retrotranspositions. Interestingly, telomerase-dependent telomere functioning during meiosis in the yeasts Schizosaccharomyces pombe and Tetrahymena is also under the control of RNAi machinery. These observations and the current data indicate that dsRNA-mediated regulation of telomere dynamics in the germline may be a general phenomenon independent of a mode of telomere maintenance (Savitsky, 2006).

A distinct small RNA pathway silences selfish genetic elements in the germline: Requirement for Aubergine

In the Drosophila germline, repeat-associated small interfering RNAs (rasiRNAs) ensure genomic stability by silencing endogenous selfish genetic elements such as retrotransposons and repetitive sequences. Whereas small interfering RNAs (siRNAs) derive from both the sense and antisense strands of their double-stranded RNA precursors, rasiRNAs arise mainly from the antisense strand. rasiRNA production appears not to require Dicer-1, which makes microRNAs (miRNAs), or Dicer-2, which makes siRNAs, and rasiRNAs lack the 2',3' hydroxy termini characteristic of animal siRNA and miRNA. Unlike siRNAs and miRNAs, rasiRNAs function through the Piwi, rather than the Ago, Argonaute protein subfamily. These data suggest that rasiRNAs protect the fly germline through a silencing mechanism distinct from both the miRNA and RNA interference pathways (Vagin, 2006).

In plants and animals, RNA silencing pathways defend against viruses, regulate endogenous gene expression, and protect the genome against selfish genetic elements such as retrotransposons and repetitive sequences. Common to all RNA silencing pathways are RNAs 19 to 30 nucleotides (nt) long that specify the target RNAs to be repressed. In RNA interference (RNAi), siRNAs are produced from long exogenous double-stranded RNA (dsRNA). In contrast, ~22-nt miRNAs are endonucleolytically processed from endogenous RNA polymerase II transcripts. Dicer ribonuclease III (RNase III) enzymes produce both siRNAs and miRNAs. In flies, Dicer-2 (Dcr-2) generates siRNAs, whereas the Dicer-1 (Dcr-1)–Loquacious (Loqs) complex produces miRNAs. After their production, small silencing RNAs bind Argonaute proteins to form the functional RNA silencing effector complexes believed to mediate all RNA silencing processes (Vagin, 2006 and references therein).

In Drosophila, processive dicing of long dsRNA and the accumulation of sense and antisense siRNAs without reference to the orientation of the target mRNA are hallmarks of RNAi in vitro. Total small RNA was prepared from the heads of adult males expressing a dsRNA hairpin that silences the white gene via the RNAi pathway. white silencing requires Dcr-2, R2D2, and Ago2. siRNAs were detected with a microarray containing TM (melting temperature)–normalized probes, 22 nt long, for all sense and antisense siRNAs that theoretically can be produced by dicing the white exon 3 hairpin. Both sense and antisense white siRNAs were detected in wild-type flies but not in dcr-2L811fsX homozygous mutant flies. The Dcr-2–dependent siRNAs were produced with a periodicity of ~22 nt, consistent with the phased processing of the dsRNA hairpin from the end formed by the 6-nt loop predicted to remain after splicing of its intron-containing primary transcript (Vagin, 2006).

Drosophila repeat-associated small interfering RNAs (rasiRNAs) can be distinguished from siRNAs by their longer length, 24 to 29 nt. rasiRNAs have been proposed to be diced from long dsRNA triggers, such as the ~50 copies of the bidirectionally transcribed Suppressor of Stellate [Su(Ste)] locus on the Y chromosome that in testes silence the ~200 copies of the protein-coding gene Stellate (Ste) found on the X chromosome (Vagin, 2006).

Microarray analysis of total small RNA isolated from fly testes revealed that Su(Ste) rasiRNAs detectably accumulate only from the antisense strand, with little or no phasing. As expected, Su(Ste) rasiRNAs were not detected in testes from males lacking the Su(Ste) loci (cry1Y). Su(Ste) rasiRNAs were also absent from armitage (armi) mutant testes, which fail to silence Ste and do not support RNAi in vitro. armi encodes a non–DEAD-box helicase homologous to the Arabidopsis thaliana protein SDE3, which is required for RNA silencing triggered by transgenes and some viruses, and depletion by RNAi of the mammalian Armi homolog Mov10 blocks siRNA-directed RNAi in cultured human cells. Normal accumulation of Su(Ste) rasiRNA and robust Ste silencing also require the putative helicase Spindle-E (Spn-E), a member of the DExH family of adenosine triphosphatases (Vagin, 2006).

The accumulation in vivo of only antisense rasiRNAs from Su(Ste) implies that sense Su(Ste) rasiRNAs either are not produced or are selectively destroyed. Either process would make Ste silencing mechanistically different from RNAi. In support of this view, mutations in the central components of the Drosophila RNAi pathway—dcr-2, r2d2, and ago2—did not diminish Su(Ste) rasiRNA accumulation. Deletion of the Su(Ste) silencing trigger (cry1Y) caused a factor of ~65 increase in Ste mRNA, but null or strong hypomorphic mutations in the three key RNAi proteins did not (Vagin, 2006).

Fly Argonaute proteins can be subdivided into the Ago (Ago1 and Ago2) and Piwi [Aubergine (Aub), Piwi, and Ago3] subfamilies. Unlike ago1 and ago2, the aub, piwi, and ago3 mRNAs are enriched in the germline. Aub is required for Ste silencing and Su(Ste) rasiRNA accumulation. In aubHN2/aubQC42 trans-heterozygous mutants, Su(Ste) rasiRNAs were not detected by microarray or Northern analysis, and Su(Ste)-triggered silencing of Ste mRNA was lost completely. Even aubHN2/+ heterozygotes accumulated less of the most abundant Su(Ste) rasiRNA than did the wild type. That the Ago subfamily protein Ago2 is not required for Ste silencing, whereas the Piwi subfamily protein Aub is essential for it, supports the view that Ste is silenced by a pathway distinct from RNAi. Intriguingly, Su(Ste) rasiRNAs hyperaccumulated in piwi mutant testes, where Ste is silenced normally (Vagin, 2006).

Mutations in aub also cause an increase in sense, but not antisense, Su(Ste) RNA; these results suggest that antisense Su(Ste) rasiRNAs can silence both Ste mRNA and sense Su(Ste) RNA, but that no Su(Ste) rasiRNAs exist that can target the antisense Su(Ste) transcript. The finding that Su(Ste) rasiRNAs are predominantly or exclusively antisense is essentially in agreement with the results of small RNA cloning experiments, in which four of five Su(Ste) rasiRNAs sequenced were in the antisense orientation, but is at odds with earlier reports detecting both sense and antisense Su(Ste) rasiRNAs by non-quantitative Northern hybridization (Vagin, 2006).

Is germline RNA silencing of selfish genetic elements generally distinct from the RNAi and miRNA pathways? The expression of a panel of germline-expressed selfish genetic elementswas examined in mutants defective for eight RNA silencing proteins: three long terminal repeat (LTR)-containing retrotransposons (roo, mdg1, and gypsy); two non-LTR retrotransposons (I-element and HeT-A, a component of the Drosophila telomere), and a repetitive locus (mst40). All selfish genetic elements tested behaved like Ste: Loss of the RNAi proteins Dcr-2, R2D2, or Ago2 had little or no effect on retrotransposon or repetitive element silencing. Instead, silencing required the putative helicases Spn-E and Armi plus one or both of the Piwi subfamily Argonaute proteins, Aub and Piwi. Silencing did not require Loqs, the dsRNA-binding protein required to produce miRNAs (Vagin, 2006).

The null allele dcr-1Q1147X is homozygous lethal, making it impossible to procure dcr-1 mutant ovaries from dcr-1Q1147X/dcr-1Q1147X adult females. Therefore, clones of dcr-1Q1147X/dcr-1Q1147X cells were generated in the ovary by mitotic recombination in flies heterozygous for the dominant female-sterile mutation ovoD1. RNA levels, relative to rp49 mRNA, were measured for three retrotransposons (roo, HeT-A, and mdg1) and one repetitive sequence (mst40) in dcr-1/dcr-1 recombinant ovary clones and in ovoD1/TM3 and dcr-1/ovoD1 nonrecombinant ovaries. The ovoD1 mutation blocks oogenesis at stage 4, after the onset of HeT-A and roo rasiRNA production. Retrotransposon or repetitive sequence transcript abundance was unaltered or decreased in dcr-1/dcr-1 relative to ovoD1/TM3 and dcr-1/ovoD1 controls. It is concluded that Dcr-1 is dispensable for silencing these selfish genetic elements in the Drosophila female germline (Vagin, 2006).

roo is the most abundant LTR retrotransposon in flies. roo silencing was analyzed in the female germline with the use of microarrays containing 30-nt probes, tiled at 5-nt resolution, for all ~18,000 possible roo rasiRNAs; the data was corroborated at 1-nt resolution for those rasiRNAs derived from LTR sequences. As observed for Su(Ste) but not for white RNAi, roo rasiRNAs were nonhomogeneously distributed along the roo sequence and accumulated primarily from the antisense strand. In fact, the most abundant sense rasiRNA peak corresponded to a set of probes containing 16 contiguous uracil residues, which suggests that these probes nonspecifically detected fragments of the mRNA polyadenylate [poly(A)] tail. Most of the remaining sense peaks were unaltered in armi mutant ovaries, in which roo expression is increased; this result implies that they do not contribute to roo silencing. No phasing was detected in the distribution of roo rasiRNAs (Vagin, 2006).

As for Su(Ste), wild-type accumulation of antisense roo rasiRNA required the putative helicases Armi and Spn-E and the Piwi subfamily Argonaute proteins Piwi and Aub, but not the RNAi proteins Dcr-2, R2D2, and Ago2. Moreover, accumulation of roo rasiRNA was not measurably altered in loqs f00791, an allele that strongly disrupts miRNA production in the female germline (Vagin, 2006).

Loss of Dcr-2 or Dcr-1 did not increase retrotransposon or repetitive element expression, which suggests that neither enzyme acts in rasiRNA-directed silencing. Moreover, loss of Dcr-2 had no detectable effect on Su(Ste) rasiRNA in testes or roo rasiRNA in ovaries. The amount of roo rasiRNA and miR-311 was measured in dcr-1/dcr-1 ovary clones generated by mitotic recombination. Comparison of recombinant (dcr-1/dcr-1) and nonrecombinant (ovoD1/TM3 and dcr-1/ovoD1) ovaries by Northern analysis revealed that roo rasiRNA accumulation was unperturbed by the null dcr-1Q1147X mutation. Pre–miR-311 increased and miR-311 declined by a factor of ~3 in the dcr-1/dcr-1 clones, consistent with about two-thirds of the tissue corresponding to mitotic dcr-1/dcr-1 recombinant cells. Yet, although most of the tissue lacked dcr-1 function, improved, rather than diminished, silencing was observed for the four selfish genetic elements examined. Moreover, the dsRNA-binding protein Loqs, which acts with Dcr-1 to produce miRNAs, was also dispensable for roo rasiRNA production and selfish genetic element silencing. Although the possibility that dcr-1 and dcr-2 can fully substitute for each other in the production of rasiRNA in the ovary cannot be excluded, biochemical evidence suggests that none of the three RNase III enzymes in flies—Dcr-1, Dcr-2, and Drosha—can cleave long dsRNA into small RNAs 24 to 30 nt long (Vagin, 2006).

Animal siRNA and miRNA contain 5' phosphate and 2',3' hydroxy termini. Enzymatic and chemical probing was used to infer the terminal structure of roo and Su(Ste) rasiRNAs. RNA from ovaries or testes was treated with calf intestinal phosphatase (CIP) or CIP followed by polynucleotide kinase plus ATP. CIP treatment caused roo and Su(Ste) rasiRNA to migrate more slowly in polyacrylamide gel electrophoresis, consistent with the loss of one or more terminal phosphate groups. Subsequent incubation with polynucleotide kinase and ATP restored the original gel mobility of the rasiRNAs, indicating that they contained a single 5' or 3' phosphate before CIP treatment. The roo rasiRNA served as a substrate for ligation of a 23-nt 5' RNA adapter by T4 RNA ligase, a process that requires a 5' phosphate; pretreatment with CIP blocked ligation, thus establishing that the monophosphate lies at the 5' end. The rasiRNA must also contain at least one terminal hydroxyl group, because it could be joined by T4 RNA ligase to a preadenylated 17-nt 3' RNA adapter. Notably, the 3' ligation reaction was less efficient for the roo rasiRNA than for a miRNA in the same reaction (Vagin, 2006).

RNA from ovaries or testes was reacted with NaIO4, then subjected to ß-elimination, to determine whether the rasiRNA had either a single 2' or 3' terminal hydroxy group or had terminal hydroxy groups at both the 2' and 3' positions, as do animal siRNA and miRNA. Only RNAs containing both 2' and 3' hydroxy groups react with NaIO4; ß-elimination shortens NaIO4-reacted RNA by one nucleotide, leaving a 3' monophosphate terminus, which adds one negative charge. Consequently, NaIO4-reacted, ß-eliminated RNAs migrate faster in polyacrylamide gel electrophoresis than does the original unreacted RNA. Both roo and Su(Ste) rasiRNA lack either a 2' or a 3' hydroxyl group, because they failed to react with NaIO4; miRNAs in the same samples reacted with NaIO4. Together, these results show that rasiRNAs contain one modified and one unmodified hydroxyl. Because T4 RNA ligase can make both 3'-5' and 2'-5' bonds, the blocked position cannot currently be determined. Some plant small silencing RNAs contain a 2'-O-methyl modification at their 3' terminus (Vagin, 2006).

Drosophila and mammalian siRNA and miRNA function through members of the Ago subfamily of Argonaute proteins, but Su(Ste) and roo rasiRNAs require at least one member of the Piwi subfamily for their function and accumulation. To determine whether roo rasiRNAs physically associate with Piwi and Aub, ovary lysate were prepared from wildtype flies or transgenic flies expressing either myc-tagged Piwi or green fluorescent protein (GFP)–tagged Aub protein; they were immunoprecipitated with monoclonal antibodies (mAbs) to myc, GFP, or Ago1; and then the supernatant and antibody-bound small RNAs were analyzed by Northern blotting. Six different roo rasiRNAs were analyzed. All were associated with Piwi but not with Ago1, the Drosophila Argonaute protein typically associated with miRNAs; miR-8, miR-311, and bantam immunoprecipitated with Ago1 mAb. No rasiRNAs immunoprecipitated with the myc mAb when lysate was used from flies lacking the myc-Piwi transgene (Vagin, 2006).

Although aub mutant ovaries silenced roo mRNA normally, they showed reduced accumulation of roo rasiRNA relative to aub/+ heterozygotes, which suggests that roo rasiRNAs associate with both Piwi and Aub. The supernatant and antibody-bound small RNAs were analyzed after GFP mAb immunoprecipitation of ovary lysate from GFP-Aub transgenic flies and flies lacking the transgene. roo rasiRNA was recovered only when the immunoprecipitation was performed with the GFP mAb in ovary lysate from GFP-Aub transgenic flies. The simplest interpretation of these data is that roo rasiRNAs physically associate with both Piwi and Aub, although it remains possible that the roo rasiRNAs are loaded only into Piwi and that Aub associates with Piwi in a stable complex. The association of roo rasiRNA with both Piwi and Aub suggests that piwi and aub are partially redundant, as does the modest reduction in roo silencing in piwi but not in aub mutants. Alternatively, roo silencing might proceed through Piwi alone, but the two proteins could function in the same pathway to silence selfish genetic elements (Vagin, 2006).

These data suggest that in flies, rasiRNAs are produced by a mechanism that requires neither Dcr-1 nor Dcr-2, yet the patterns of rasiRNAs that direct roo and Ste silencing are as stereotyped as the distinctive siRNA population generated from the white hairpin by Dcr-2 or the unique miRNA species made from each pre-miRNA by Dcr-1. A key challenge for the future will be to determine what enzyme makes rasiRNAs and what sequence or structural features of the unknown rasiRNA precursor lead to the accumulation of a stereotyped pattern of predominantly antisense rasiRNAs (Vagin, 2006).

Drosophila rasiRNA pathway mutations disrupt embryonic axis specification through activation of an ATR/Chk2 DNA damage response

Small repeat-associated siRNAs (rasiRNAs) mediate silencing of retrotransposons and the Stellate locus. Mutations in the Drosophila rasiRNA pathway genes armitage and aubergine disrupt embryonic axis specification, triggering defects in microtubule polarization as well as asymmetric localization of mRNA and protein determinants in the developing oocyte. Mutations in the ATR/Chk2 DNA damage signal transduction pathway dramatically suppress these axis specification defects, but do not restore retrotransposon or Stellate silencing. Furthermore, rasiRNA pathway mutations lead to germline-specific accumulation of γ-H2Av foci characteristic of DNA damage. It is concluded that rasiRNA-based gene silencing is not required for axis specification, and that the critical developmental function for this pathway is to suppress DNA damage signaling in the germline (Klattenhoff, 2007).

Mutations in the Drosophila armi, aub, and spn-E genes disrupt oocyte microtubule organization and asymmetric localization of mRNAs and proteins that specify the posterior apole and dorsal-ventral axis of the oocyte and embryo. Mutations in these genes block homology-dependent RNA cleavage and RISC assembly in ovary lysates, RNAi-based gene silencing during early embryogenesis, rasiRNA production, and retrotransposon and Stellate silencing. Mutations in dcr-2 and ago-2 genes, by contrast, block siRNA function, but they do not disrupt the rasiRNA pathway or embryonic axis specification. The rasiRNA pathway thus appears to be required for embryonic axis specification. However, the function of rasiRNAs in the axis specification pathway has not been previously established (Klattenhoff, 2007).

Cytoskeletal polarization, morphogen localization, and eggshell patterning defects associated with armi and aub are efficiently suppressed by mnk and mei-41, which respectively encode Chk2 and ATR kinase components of the DNA damage signaling pathway. In addition, armi and aub mutants accumulate γ-H2Av foci characteristic of DNA DSBs and trigger Chk2-dependent phosphorylation of Vas, an RNA helicase required for posterior and dorsal-ventral specification. Mutations in spn-E also disrupt the rasiRNA pathway, trigger axis specification defects, and lead to germline-specific accumulation of γ-H2Av foci. Significantly, the mnk and mei-41 mutations do not suppress Stellate or HeT-A overexpression, indicating that axis specification does not directly require rasiRNA-dependent gene silencing. Based on these findings, it is concluded that the rasiRNA pathway suppresses DNA damage signaling in the female germline, and that mutations in this pathway disrupt axis specification by activating an ATR/Chk2 kinase pathway that blocks microtubule polarization and morphogen localization in the oocyte (Klattenhoff, 2007).

The cause of DNA damage signaling in armi, aub, and spn-E mutants remains to be established. In wild-type ovaries, γ-H2Av foci begin to accumulate in region 2 of the germarium, when the Spo11 nuclease (encoded by the mei-W68 gene) initiates meiotic breaks. The axis specification defects associated with DNA DSB repair mutations are efficiently suppressed by mei-W68 mutations, indicating that meiotic breaks are the source of DNA damage in these mutants. The axis specification defects and γ-H2Av focus formation associated with armi, by contrast, are not suppressed by mei-W68. mei-W68 double mutants with aub or spn-E have not been analyzed, but this observation strongly suggests that meiotic DSBs are not the source of DNA damage in rasiRNA pathway mutations. Retrotransposon silencing is disrupted in armi, aub, and spn-E mutants, and transcription of LINE retrotransposons in mammalian cells leads to DNA damage and DNA damage signaling. Loss of retrotransposon silencing could therefore directly induce the DSBs in rasiRNA pathway mutants. However, DNA damage can also lead to loss of retrotransposon silencing. Mutations in the rasiRNA pathway could therefore disrupt DNA repair and thus induce DNA damage, which, in turn, induces loss of retrotransposon silencing. Finally, the HeT-A retrotransposon is associated with telomeres, and overexpression of this element could reflect a loss of telomere protection and could damage signaling by chromosome ends in the rasiRNA pathway mutants. The available data do not distinguish between these alternatives (Klattenhoff, 2007).

In mouse, the piwi-related Argonauts Miwi and Mili bind piRNAs, 30 nt RNAs derived primarily from a single strand that appear to be related to rasiRNAs. Mutations in these genes disrupt spermatogenesis and lead to germline apoptosis, which can be induced by DNA damage signaling. Mammalian piRNAs and Drosophila rasiRNAs may therefore serve similar functions in suppressing a germline-specific DNA damage response (Klattenhoff, 2007).

cutoff and aubergine mutations result in retrotransposon upregulation and checkpoint activation in Drosophila

Gametogenesis is a highly regulated process in all organisms. In Drosophila, a meiotic checkpoint which monitors double-stranded DNA breaks and involves Drosophila ATR and Chk2 coordinates the meiotic cell cycle with signaling events that establish the axis of the egg and embryo. Checkpoint activity regulates translation of the transforming growth-factor-alpha-like Gurken signaling molecule which induces dorsal cell fates in the follicle cells. Mutations in the Drosophila gene cutoff (cuff) affect germline cyst development and result in ventralized eggs as a result of reduced Grk protein expression. Surprisingly, cuff mutations lead to a marked increase in the transcript levels of two retrotransposable elements, Het-A and Tart. Small interfering RNAs against the roo element are still produced in cuff mutant ovaries. These results indicate that Cuff is involved in the rasiRNA pathway and most likely acts downstream of siRNA biogenesis. The eggshell and egg-laying defects of cuff mutants are suppressed by a mutation in chk2. Mutations in aubergine (aub), another gene implicated in the rasiRNA pathway, are significantly suppressed by chk2 mutation. These results indicate that mutants in rasiRNA pathways lead to elevated transposition incidents in the germline, and that this elevation activates a checkpoint that causes a loss of germ cells and a reduction of Gurken protein in the remaining egg chambers (Chen, 2007).

cutoff (cuff) mutations were isolated in a large-scale female-sterile screen of Drosophila, and one additional allele was identified in a screen for P element insertions. Females transheterozygous for cuff alleles lay eggs with various degrees of ventralization. The dorsoventral polarity of the egg and embryo depends on the levels of the Gurken (Grk) ligand, which is produced and secreted by the germline and activates the EGF receptor (Egfr) in the overlying follicle cells. To determine whether Grk-Egfr signaling was affected, the grk expression pattern was analyzed in a strong cuff mutant background. In wild-type egg chambers at stage 9 of oogenesis, grk RNA becomes restricted to the future dorsal-anterior side of the oocyte and forms a cap around the oocyte nucleus. Grk protein is translated from the tightly localized RNA and is also spatially restricted to the membrane overlying the oocyte nucleus. cuff mutants do not significantly disrupt grk RNA localization. However, in many mid-stage egg chambers, the Grk protein level is greatly reduced, such that between 10% and 40% of the egg chambers contain no detectable Gurken protein at all, consistent with defects in grk translation. In wild-type egg chambers by stage 3 of oogenesis, the oocyte nucleus forms a compact structure termed the karyosome. In cuff mutants, karyosome formation is affected in 10%–20% of the egg chambers, in which the DNA assumes various shapes and is often found in separate clumps (Chen, 2007).

Genomic database searches identified the yeast gene Rai1 as a homolog of cuff. This gene has been shown to interact with a nuclear 5′–3′ exoribonuclease (Rat1) that is involved in rRNA processing and transcriptional termination. A cytoplasmic homolog of Rat1, Xrn1, has also been described in yeast and vertebrates and has been implicated in mRNA regulation that is localized to cytoplasmic processing bodies. An HA-tagged Drosophila Rat1 (CG10354) construct was generated and overexpressed with a fully functional FLAG-tagged Cuff in the ovary. Using immunoprecipitation (IP), no any interaction between the exoribonuclease and Cuff was detected. It is therefore possible that Drosophila Rat1 is not the correct partner for Cuff. This is also supported by the observation that overexpressed Rat1, as expected, localizes to the nucleus, whereas overexpressed Cuff localizes to the cytoplasm. It was not possible to to detect endogenous Cuff protein with an anti-Cuff antibody, presumably because of low levels of protein expression. However, overexpressed HA-tagged Cuff partially colocalizes with perinuclear puncta in the nurse cells in younger egg chambers. A similar localization pattern has been described for the helicase Vasa, and it was found that Cuff partially colocalizes with Vasa in the cytoplasm. The perinuclear localization pattern, also designated as nuage in the germ cells and related to mammalian P bodies, has been described for components of the RNAi machinery and for genes involved in RNA degradation (Chen, 2007).

Given the eggshell ventralization and the karyosome defect, cuff has mutant phenotypes similar to those of a group of mutants known as the spindle-class genes. Several members of this group encode DNA-repair genes, for instance, spindle(spn) B (XRCC3) and okra (DmRad54). In these mutants, the DSBs that are created during recombination persist and thus activate Chk2 through the Drosophila ATR homolog mei-41. The activity of these kinases negatively regulates the translation of Grk, possibly through a posttranslational modification of Vasa; this modification in turn leads to ventralization of the eggs laid by mutant females. Inactivation of the checkpoint, for instance through mutations in chk2 or mei-41, suppresses the eggshell defects of the spindle-class DNA-repair mutants. In addition, in double mutants of the DNA-repair genes and the genes required for initiating the DSBs, such as c(3)g, mei-W68, or mei-P22, DSBs are not generated; therefore, the checkpoint is not activated, and the eggshell morphology is normal, even in the presence of the repair mutants. To check whether Cuff is involved in the repair of DSBs initiated in prophase of meiosis I, mei41;cuff and cuff;c(3)g double mutants were generated. Although both mutations suppress the eggshell defect of spnB or okra to wild-type morphology, neither suppresses the eggshell defect of cuff, indicating that Cuff does not function in the meiotic repair pathway. Surprisingly, however, a mutation in chk2 partially suppresses the eggshell defect of cuff as well as the defects in cyst development. chk2 cuff double mutants lay mostly wild-type-looking eggs, and have cysts with highly branched fusomes in the germaria, and the females lay more eggs than cuff single mutants, although the rescue is not 100%. In certain allelic combinations, it was possible to observe a dominant effect in the chk2 suppression of the cuff eggshell defect (Chen, 2007).

Previous work has suggested the DNA-repair checkpoint, upon activation, regulates Grk translation through a posttranslational modification of Vas, and that this modification results in slower Vas electrophoretic mobility. To address whether the checkpoint acts in the same manner in cuff mutants, Vas mobility was assayed in cuff mutant combinations. In cuff mutants, Vas migrates slightly more slowly than wild-type control, consistent with the modification seen in the DNA-repair mutants. The mobility is not changed in mei41;cuff double-mutant background, which is consistent with the fact that mei41 mutants do not significantly suppress the eggshell phenotype of cuff. However, Vasa mobility is restored to wild-type in the chk2 cuff double mutant. This suggests that although the checkpoint is activated through a different sensing mechanism in cuff mutants, upon activation the checkpoint involves Chk2 and acts through similar pathways to affect Gurken translation in the egg chambers that escape the early arrest (Chen, 2007).

Several of the spindle-class genes, such as spnE and aub, have been shown to be essential components of the RNAi machinery. Because overexpressed Cuff has a perinuclear localization, whether Cuff might also be required in RNAi pathways was tested. Recently, a specific branch of the RNAi pathways, that involving the repeat-associated small interfering RNA (rasiRNA), has been implicated in the control of retrotransposable elements in the Drosophila germline. Using qRT-PCR, the level of Het-A and Tart, two of the retrotransposable elements responsible for maintaining the telomere in Drosophila, was studied. Previously, it has been shown that in spnE and aub mutants, Het-A and Tart transcripts are derepressed and that this derepression results in a marked elevation in the transcripts level. Compared with heterozygous controls, spnE homozygous mutant females have Het-A and Tart transcript levels that are upregulated by approximately 10-fold, whereas in aub mutants only Het-A is significantly upregulated. In cuff mutant females, the elevation for both transcripts is even more pronounced. Compared with Het-A levels in the heterozygous control, those in cuff mutants are elevated more than 800-fold, and Tart transcript levels increase by more than 20-fold. Transposable elements are normally silenced in the Drosophila germline by the rasiRNA pathway; this silencing process appears to be strongly impaired in the cuff mutants. Whether the upregulation of the transposable elements in cuff mutants could be due to a reduction in the level of rasiRNAs was further tested. However, it was found that the levels of the 25-nt-long roo interfering RNA are not reduced in cuff mutant ovaries, in contrast to ovaries mutant for aub. This indicates that Cuff is not involved in the biogenesis of the rasiRNAs and points to a function for Cuff in the actual silencing process. Because high transcript levels of the retrotransposable elements in the germline are correlated with elevated transposition incidents, which in turn lead to decreased chromosomal integrity, it is possible that such chromosomal defects activate the checkpoint involving chk2. In addition, because transposable elements are involved in the regulation of chromatin structure, the existence of a chromatin checkpoint that involves Chk2 activity is also possible. Once Chk2 is activated, either by the mutants in DNA-repair pathways or by RNAi components such as Cuff and Aub, Chk2 activity leads to posttranslational Vas modification and a negative regulation of Grk translation. However, unlike DNA repair mutations, cuff and aub mutations are not suppressed to wild-type morphology and fecundity by mutations in mei41, suggesting that they activate the checkpoint through a different, or additional, sensing mechanism. Furthermore, most of the mutants in DNA-repair pathways do not cause defects in cyst development or germline stem cell maintenance. These additional defects seen in cuff mutants could be due to the timing of checkpoint activation. DNA-repair mutants activate the meiotic checkpoint during meiotic prophase, which initiates after the formation of the 16 cell cyst, whereas cuff and aub mutants appear to act earlier in oogenesis, given that they already have effects during the mitotic cycles preceding the onset of meiosis. The transposon-activated checkpoint leads not only to translational arrest of Grk, but also to mitotic cell-cycle arrest. Many of the arrested germline cells and cysts eventually undergo apoptosis, leading to gradual loss of both germline stem cells and developing cysts in cuff mutants. However, germ cells that escape the early arrest encounter the second checkpoint effect, which leads to a reduction in Gurken translation (Chen, 2007).

It was recently discovered that there are a large number of different small RNAs generated in the germline of both mammals and flies. Many of them are associated with Piwi family proteins, and most have no known functions. Because the germline represents a special cell type that will pass its DNA on to future progeny, it is possible that selfish elements have developed a high propensity to remobilize in the germline. Furthermore, it is very plausible that in most organisms the germline has evolved sophisticated mechanisms to defend itself against such transposable elements. Many of the small RNAs found in the germline may be involved in the defense against transposable elements, as well as in the regulation of transcription and translation. When the machinery to generate these small silencing RNAs or the effector complexes that are responsible for transcript degradation are disrupted, chromosomal integrity might be at risk. This study has found that a checkpoint involving the conserved Chk2 kinase monitors the RNAi-mediated events in the Drosophila germline and ensures the genomic integrity of the progeny. Chk2 therefore acts as a surveillance factor for both transposon-generated problems as well as DNA-repair problems in the germline. Whether Chk2 has a similar role in the mammalian germline will be interesting to investigate in the future (Chen, 2007).

Ectopic expression of germline genes drives malignant brain tumor growth in Drosophila

Model organisms such as the fruit fly Drosophila melanogaster can help to elucidate the molecular basis of complex diseases such as cancer. Mutations in the Drosophila gene lethal (3) malignant brain tumor cause malignant growth in the larval brain. This study shows that l(3)mbt tumors exhibited a soma-to-germline transformation through the ectopic expression of genes normally required for germline stemness, fitness, or longevity. Orthologs of some of these genes were also expressed in human somatic tumors. In addition, inactivation of any of the germline genes nanos, vasa, piwi, or aubergine suppressed l(3)mbt malignant growth. These results demonstrate that germline traits are necessary for tumor growth in this Drosophila model and suggest that inactivation of germline genes might have tumor-suppressing effects in other species (Janic, 2010).

The Drosophila tumor-suppressor gene l(3)mbt was identified as a temperature-sensitive mutation that caused malignant growth in the larval brain. Other l(3)mbt mutant alleles obtained later show the same temperature-sensitive phenotype. L(3)mbt's closest homologs, Drosophila Scm (Sex comb on midleg) and Sfmbt (Scm-related gene containing four mbt domains), encode Polycomb Group proteins. L3MBTL1, the human homolog of Drosophila L(3)MBT, is a transcriptional repressor that is found in a complex with core histones, heterochromatin protein 1γ (HP1γ), and RB (Retinoblastoma protein) and can compact nucleosomes. Drosophila L(3)MBT is a substoichiometric component of the dREAM-MMB complex, which includes the two Drosophila Retinoblastoma-family proteins and the Myb-MuvB (MMB) complex. Depletion of components of the dREAM/MMB complex in Drosophila Kc cells by RNA interference results in genome-wide changes in gene expression. These data strongly suggest that l(3)mbt function might contribute to establishing and maintaining certain differentiated states through the stable silencing of specific genes (Janic, 2010).

To identify the genes whose misexpression might account for the growth of l(3)mbt tumors (henceforth referred to as mbt tumors), genome-wide gene expression profiling was carried out of l(3)mbtE2 and l(3)mbtts1 homozygous and transheterozygous larval brains raised at restrictive temperature (29°C). l(3)mbtts1 tumors were also analyzed at the 1st, 5th, and 10th rounds of allograft culture in adult flies (T1, T5, and T10, respectively). Brains from homozygous white1118 (w1118), l(3)mbtE2, or l(3)mbtts1 larvae raised at permissive temperature (17°C) were used as controls. For comparison, larval brain malignant neoplasms caused by mutation in brain tumor (brat) as well as allograft cultures at T1,T5, and T10 of tumors caused by mutants in brat, lethal giant larvae (lgl), miranda (mira), prospero (pros), and partner of inscuteable (pins), were also profiled (Janic, 2010).

Hierarchical clustering plots of these data reveal three distinct clusters that include control larval brains, mbt larval brain tumors, and cultured l(3)mbtts1 tumors, respectively. From these data, 151 genes were identified that were either overexpressed or underexpressed in all three larval mbt tumor types compared to all three controls. From this list, those genes were removed that were also up- or down-regulated) in larval brat neoplasms and, hence, likely to encode functions generally required for larval brain tumor growth. The expression levels of the remaining 102 up-regulated genes are referred to as as the mbt signature (MBTS). MBTS is notably enhanced in cultured mbt tumors and can be used unequivocally to distinguish mbt tumors from other cultured malignant brain neoplasms like lgl, mira, pros, pins, or brat. Individual MBTS genes, however, are also up-regulated in some of these tumors (Janic, 2010).

The function of most MBTS genes remains unknown. However, a quarter of them (26 of 102) are genes required in the germ line. For instance, nanos (nos), female sterile(1)Yb (fs(1)Yb), and zero population growth (zpg) function in the establishment of the pole plasm in the egg and cystoblasts differentiation. The gonad-specific thioredoxins ThioredoxinT (TrxT) and deadhead (dhd), giant nuclei (gnu), corona (cona), hold'em (hdm), matotopetli (topi), and the female germline-specific γTUB37C isoform function during oocyte differentiation, meiosis, and syncytial embryo development. Also piwi, aubergine (aub), krimper (krimp), and tejas (tej) are involved in the biogenesis of Piwi-interacting RNAs (piRNAs) that protect germline cells against transposable elements and viruses. Some of these genes also have functions that are not germline related. For instance, some piwi alleles display synthetic lethality), and nos is required during nervous system development (Janic, 2010).

Driven by the high percentage of MBTS genes that have germline functions, other germline-related genes were sought that do not meet the stringent criteria applied to select the 102 MBTS genes, but are overexpressed in mbt tumors. Among these, the genes were found that encode the synaptonemal complex protein Crossover suppressor on 3 of Gowen [C(3)G] and the cell cycle kinase Pan gu (PNG), which interact with the proteins encoded by the MBTS genes cona and gnu, respectively. The same applies to Squash (SQU), Spindle-E (SPN-E), Maelstrom (MAEL), and AGO3, components of the piRNA machinery, which colocalize with other MBTS proteins in nuage (Janic, 2010).

To determine whether the mRNAs found ectopically expressed in mbt tumors are translated, protein expression was examined with a selected number of currently available antibodies. Given the key role of VASA in the assembly of the pole plasm and germline development, it was included in this study, even though vasa mRNA levels are not significantly increased in mbt tumors. By Western blot, it was confirmed that PIWI, AUB, and VASA are ectopically expressed in mbt tumors. Immunofluorescence studies also revealed the ectopic expression in l(3)mbtts1 brains raised at 29°C of C(3)G, SQU, and VASA. These results show that some of the germline genes ectopically expressed in mbt tumors are translated. However, it has not been possible to confirm the expression of other proteins, including MAEL, ORB, BAM, GNU, and TOPI, which suggests that, possible technical problems aside, either the corresponding mRNAs are not translated or these proteins might be unstable in such an ectopic environment. The expression of VASA, by contrast, suggests that other mRNAs whose levels are not appreciably increased in mbt tumors might actually be ectopically translated (Janic, 2010).

Prompted by the expression in l(3)mbtts1 brains of several genes involved in the biogenesis and regulation of piRNAs, 23- to 30-nucleotide RNAs were sequenced from l(3)mbtts1 larval brain tumors and from wild-type brains and ovaries. 117 known piRNAs and microRNAs (miRNAs) were detected in l(3)mbtts1 larval brain tumor samples. Of these, 31 are either not expressed in wild-type brains or are expressed there at less than 10% their level in larval brain tumors. Most of them are highly expressed in wild-type ovaries, thus substantiating further the ectopic acquisition of germline traits that characterizes mbt tumors (Janic, 2010).

It is not known which, if any, of the germline genes that are up-regulated in mbt tumors are direct targets of l(3)mbt or if their ectopic expression is a downstream consequence of intermediate events. The putative direct targets of l(3)mbt are many. The dREAM-MMB complex, of which L(3)MBT is a substoichiometric component, has been found to be promoter-proximal to 32% of Drosophila genes, and MMB factors are known to regulate transcription of a wide range of genes in Drosophila Kc cells. In addition, there is no estimate for the number of proteins like VASA that, despite their low mRNA expression levels, might be up-regulated in mbt tumors. Indeed, many of these genes, as well as the piRNAs and miRNAs expressed in mbt tumors, might themselves regulate the basal transcription and translation machineries, adding a further layer of gene expression modulation (Janic, 2010).

The extent to which ectopic expression of germline genes contributes to mbt tumor growth was determined. To this end, larval brain growth was quantified in individuals that were mutant for l(3)mbtts1 alone, or double mutant for l(3)mbtts1 and one of several of the germline genes that are ectopically expressed in mbt tumors. Measured as the total amount of protein, the average brain size in l(3)mbtts1 is about seven times as large as that in control w1118 larvae, a difference that is not significantly reduced by the additional loss of zpg, Pxt, or AGO3. However, brain overgrowth is reduced to a size similar to that of the control in l(3)mbtts1 larvae that are also mutant for either piwi, vasa, aub, or nos. The loss of piwi does not prevent brain overgrowth in brat k06028 mutant larvae. Then tumor growth was quantified after allograft in adult flies. The frequency with which l(3)mbtts1 homozygous larval brain tissue develops tumors in this assay is not significantly reduced by the additional loss of zpg or AGO3 and is only moderately reduced by the loss of Pxt, but it is markedly reduced by the additional loss of piwi, vasa, aub), or nos. The frequency of brat k06028 tumor formation is not affected by the loss of piwi or nos. These results demonstrate that the ectopic expression of germline genes, particularly piwi, vasa, nos, and aub, significantly contributes to mbt tumor growth (Janic, 2010).

A closely reminiscent soma-to-germline transformation observed in mutants in the Caenorhabditis elegans Rb homolog LIN-35, as well as in long-lived C. elegans strains, has led some to propose that the acquisition of germline characteristics by somatic cells might contribute to increased fitness and survival, a mechanism that could contribute to the transformation of mammalian cells. Also in humans, some genes that are predominantly expressed in germline cells and have little or no expression in somatic adult tissues become aberrantly activated in various malignancies, including melanoma and several types of carcinomas. These are known as cancer-testis (CT) genes or cancer-germline (CG) genes. A subset of these CG genes encode antigens that are immunogenic in cancer patients and are being pursued as biomarkers and as targets for therapeutic cancer vaccines (Janic, 2010 and references therein).

Human CG genes are suspected to contribute to oncogenesis germline traits like immortality, invasiveness, and hypomethylation, but their actual role in cancer remains unknown. The current results demonstrate that ectopic germline traits are necessary for tumor growth in Drosophila mbt tumors, suggesting that their inactivation might have tumor-suppressing effects in other species. Some germline genes up-regulated in mbt tumors are orthologs of human CG genes like PIWIL1/piwi, NANOS1/nanos, and SYCP1 /c(3)G. The list of genes up-regulated in mbt tumors includes many other germline genes that might also be relevant in human cancer (Janic, 2010).

Mutations to the piRNA pathway component aubergine enhance meiotic drive of segregation distorter in Drosophila melanogaster

Diploid sexual reproduction involves segregation of allelic pairs, ensuring equal representation of genotypes in the gamete pool. Some genes, however, are able to 'cheat' the system by promoting their own transmission. The Segregation distorter (Sd) locus in Drosophila melanogaster males is one of the best-studied examples of this type of phenomenon. In this system the presence of Sd on one copy of chromosome 2 results in dysfunction of the non-Sd-bearing (Sd+) sperm and almost exclusive transmission of Sd to the next generation. The mechanism by which Sd wreaks such selective havoc has remained elusive. However, its effect requires a target locus on chromosome 2 known as Responder (Rsp). The Rsp locus comprises repeated copies of a satellite DNA sequence and Rsp copy number correlates with sensitivity to Sd. Under distorting conditions during spermatogenesis, nuclei with chromosomes containing greater than several hundred Rsp repeats fail to condense chromatin and are eliminated. Recently, Rsp sequences were found as small RNAs in association with Argonaute family proteins Aubergine (Aub) and Argonaute3 (AGO3). These proteins are involved in a germline-specific RNAi mechanism known as the Piwi-interacting RNA (piRNA) pathway, which specifically suppresses transposon activation in the germline. This study evaluated the role of piRNAs in segregation distortion by testing the effects of mutations to piRNA pathway components on distortion. Further, mutations to the aub locus of a Segregation Distorter (SD) chromosome were specifically targeted using ends-out homologous recombination. The data in this study demonstrate that mutations to piRNA pathway components act as enhancers of SD (Gell, 2013).

PIWI proteins are essential for early Drosophila embryogenesis

PIWI proteins, a subfamily of the ARGONAUTE/PIWI protein family, have been implicated in transcriptional and posttranscriptional gene regulation and transposon silencing mediated by small non-coding RNAs, especially piRNAs. Although these proteins are known to be required for germline development, their somatic function remains elusive. This study examined the maternal function of all three PIWI proteins in Drosophila; Piwi, Aubergine (Aub) and Argonaute3 (Ago3) during early embryogenesis. In syncytial embryos, Piwi displays an embryonic stage-dependent localization pattern. Piwi is localized in the cytoplasm during mitotic cycles 1-10. Between cycles 11 and 14, Piwi remains in the cytoplasm during mitosis but moves into the somatic nucleus during interphase. Beyond cycle 14, it stays in the nucleus. Aub and Ago3 are diffusely cytoplasmic from cycle 1 to 14. Embryos maternally depleted of any one of the three PIWI proteins display severe mitotic defects, including abnormal chromosome and nuclear morphology, cell cycle arrest, asynchronous nuclear division and aberrant nuclear migration. Furthermore, all three PIWI proteins are required for the assembly of mitotic machinery and progression through mitosis. Embryos depleted of maternal PIWI proteins also exhibit chromatin organization abnormalities. These observations indicate that maternal Piwi, Aub and Ago3 play a critical role in the maintenance of chromatin structure and cell cycle progression during early embryogenesis, with compromised chromatin integrity as a possible cause of the observed mitotic defects. This study demonstrates the essential function of PIWI proteins in the first phase of somatic development (Mani, 2013).

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

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


Search PubMed for articles about Drosophila aubergine

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