InteractiveFly: GeneBrief

Argonaute 3: Biological Overview | References

BIOLOGICAL OVERVIEW

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 (Engels, 1979). 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 (Bregliano, 1980; Bucheton, 1984). The number of I copies builds during subsequent crosses of surviving female progeny until it reaches an average of 1015 per genome (Pelisson, 1987). 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 (Robert, 2001). 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 (Misra, 1990). 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 (Biemont, 1990). 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 (Biemont, 1990; Ronsseray, 1991). Additionally, several groups isolated insertions of incomplete P elements in this same cytological location that acted as dominant transposition suppressors (Marin, 2000; Stuart, 2002). 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 (Pal-Bhadra, 1997). 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 (Hamilton, 1999; Pal-Bhadra, 2002). Moreover, mutations in RNAi pathway genes impact transposon mobility in flies (Kalmykova, 2005; Sarot, 2004; Savitsky, 2006) and C.elegans. Finally, small RNAs (rasiRNAs) corresponding to transposons and repeats have been isolated from flies and zebrafish (Aravin, 2001, 2003; Chen, 2005; 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 (Sarot, 2004), and aubergine mutations impact TART (Savitsky, 2006) and P elements (Reiss, 2004). Finally, both Piwi and Aubergine bind rasiRNAs (Saito, 2006; Vagin, 2006) 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 (Aravin, 2003), and their presence was correlated with suppression of hybrid dysgenesis (Blumenstiel, 2005) 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 (Karpen, 1992), 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).

Slicer-mediated mechanism for repeat-associated siRNA 5' end formation in Drosophila

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 (Williams, 2002), 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).

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).

Krimper enforces an antisense bias on piRNA pools by binding AGO3 in the Drosophila germline

Piwi-interacting RNAs (piRNAs) suppress transposon activity in animal germ cells. In the Drosophila ovary, primary Aubergine (Aub)-bound antisense piRNAs initiate the ping-pong cycle to produce secondary AGO3-bound sense piRNAs. This increases the number of secondary Aub-bound antisense piRNAs that can act to destroy transposon mRNAs. This study shows that Krimper (Krimp), a Tudor-domain protein, directly interacts with piRNA-free AGO3 to promote symmetrical dimethylarginine (sDMA) modification, ensuring sense piRNA-loading onto sDMA-modified AGO3. In aub mutant ovaries, AGO3 associates with ping-pong signature piRNAs, suggesting AGO3's compatibility with primary piRNA loading. Krimp sequesters ectopically expressed AGO3 within Krimp bodies in cultured ovarian somatic cells (OSCs), in which only the primary piRNA pathway operates. Upon krimp-RNAi in OSCs, AGO3 loads with piRNAs, further showing the capacity of AGO3 for primary piRNA loading. It is proposed that Krimp enforces an antisense bias on piRNA pools by binding AGO3 and blocking its access to primary piRNAs (Sato, 2015).

The data suggest that Krimp regulates sDMA modification of AGO3 through direct binding to the N-terminal portion of the protein, thereby controlling interactions of other Tudor-domain proteins, such as Tud (see Krimp Enforces an Antisense Bias on piRNA Pools by Assembling AGO3 in the Ping-Pong Cycle) (Sato, 2015).

A recent study showed that a lack of Qin, another Tudor domain protein, disrupts the interaction between AGO3 and Aub and triggers Aub-Aub homotypic ping-pong in fly ovaries, resulting in an increase in the abundance of sense piRNAs while the overall abundance of piRNAs is preserved. Therefore, Qin seems to act to suppress Aub-Aub homotypic ping-pong by promoting the Aub-AGO3 interaction, which leads to an increase in heterotypic Aub-AGO3 ping-pong. A considerable amount of Aub-Aub homotypic ping-pong occurs even in controls, so Aub-Aub homotypic ping-pong is probably a default pathway for piRNA biogenesis; however, this pathway increases the proportion of sense piRNAs in the piRNA pool and so does not efficiently repress TEs. Therefore, there should be a system(s) that promotes heterotypic Aub-AGO3 ping-pong and that also increases the proportion of antisense piRNAs. Although Aub-Aub homotypic ping-pong also prevails in krimp mutant ovaries, the overall abundance of piRNAs in germline cells is dramatically decreased. This profile of piRNAs in krimp mutant ovaries is very similar to that in ago3 mutant ovaries in which the ping-pong cycle collapses. The increase in Aub-Aub homotypic ping-pong observed in krimp mutant ovaries may largely reflect the loss of AGO3-Aub heterotypic ping-pong, rather than a direct effect on promoting Aub-Aub homotypic ping-pong. Therefore, Krimp plays a very different role to Qin in the ping-pong cycle (Sato, 2015).

The ping-pong cycle is mediated by the Slicer-dependent mutual cleavage of sense and antisense transcripts of a TE, which should result in equal amounts of sense and antisense piRNAs. Thus, an important question is this: what makes sense piRNAs less abundant and disproportionately bound to AGO3 in the ping-pong cycle, enforcing the characteristic antisense bias of Aub-bound piRNAs? In other words, why is Aub-Aub homotypic ping-pong not sufficient to produce the characteristic antisense bias of Aub-bound piRNAs? In krimp mutant ovaries, AGO3 is no longer sDMA modified and is free from piRNAs. Therefore, Krimp not only masks the N-terminal portion of AGO3 where sDMA-modifiable arginine residues reside, but also mediates the sDMA modification that leads to interactions of AGO3 with other Tudor domain proteins, including Tud, and to its nuage localization. In this way, Krimp promotes the ping-pong cycle. Because sDMA modification of Aub is not affected in krimp mutant ovaries, complex formation between Aub loaded with primary piRNAs and Tudor domain proteins may occur before sDMA-modified AGO3 joins the complexes to ensure Aub-AGO3 heterotypic ping-pong. Qin may act in this step to suppress Aub-Aub homotypic ping-pong to further promote Aub-AGO3 heterotypic ping-pong (Sato, 2015).

AGO3 is clearly compatible with the primary piRNA pathway in OSCs when Krimp is depleted. Therefore, Krimp may not only promote the Aub-AGO3 heterotypic ping-pong cycle but also actively prevent AGO3 from becoming loaded with primary piRNAs in germline cells. In contrast, in aub mutant ovaries where AGO3 no longer accumulates at nuage but is sequestered into Krimp bodies, AGO3 is still loaded with reduced levels of piRNAs that have characteristics of primary piRNAs. These results suggest that complete blockage of primary piRNA loading onto AGO3 in ovarian germline cells requires an additional factor(s). An alternative possibility may be that AGO3 does not selectively accept primary piRNAs when aub is mutated but is perhaps loaded in a rather non-specific manner. Thus, together, these findings suggest that the characteristic antisense bias of Aub-bound piRNAs is created by the sum of piRNAs produced in the ping-pong cycle and the continual flow of primary piRNAs onto Aub. Aub is much more abundant than AGO3 in fly ovaries, further suggesting that the continuous flow of primary piRNAs to Aub should contribute to the antisense bias of Aub-bound piRNAs. Alternatively, it is also conceivable that AGO3-sense piRNA complexes may be catalytically more active than Aub-antisense piRNA complexes, potentially resulting in the antisense bias of Aub-bound piRNAs. However, in OSCs, AGO3 is loaded with primary piRNAs when Krimp is depleted while AGO3 in germline cells is not loaded with piRNAs. This suggests that in addition to Krimp, a second/backup system may also operate to further prevent AGO3 from associating with primary piRNAs in germline cells (Sato, 2015).

In aub mutant ovaries, Krimp is no longer accumulated at the nuage but forms Krimp bodies (Lim, 2007, Nagao, 2011), suggesting that the nuage localization of Krimp is Aub dependent. However, Krimp does not directly interact with Aub, and Krimp in OSCs forms Krimp bodies when Aub is ectopically expressed. Therefore, there must be a germline-specific factor(s) that links Aub and Krimp. The N-terminal coiled-coil domains of Krimp are required for Krimp body formation. Thus, a factor(s) may exist to mask the coiled-coil domains to prevent Krimp from forming the aggregates in germline cells. The function and/or stability of this putative factor could be Aub dependent in germline cells. The next key challenge will be to identify such a factor that regulates Krimp, thereby contributing to the operation of the ping-pong cycle (Sato, 2015).

Both AGO3 and Aub have the potential to load sense and antisense piRNAs, and the loading of these two proteins likely relies on the functions of interacting proteins, such as Krimp. It was therefore hypothesized that AGO3 and Aub are functionally very similar and that their interacting partners play essential roles in determining the behaviors of the two PIWI proteins, such that they effectively participate in ping-pong amplification. If both Aub and AGO3 are compatible with the primary piRNA pathway, why then are primary piRNAs loaded only onto Aub and Piwi? Perhaps this is a system to ensure a heterotypic ping-pong between Aub and AGO3 and some other as yet unknown mechanism to amplify antisense piRNAs. During Drosophila germline development, Piwi and Aub, but not AGO3, are directly deposited from mother to offspring through germline transmission. Antisense piRNAs that can act to repress the activation of TEs and initiate the ping-pong cycle are loaded onto Piwi and Aub. The maternal loading of antisense piRNAs, together with the functions of Tudor domain proteins such as Krimp and Qin, could establish an antisense bias in the ping-pong amplification loop (Sato, 2015).

There is no apparent homolog of Krimp in mammals. This may be because the ping-pong amplification mechanism in mammals is different from that in Drosophila. For example, the mouse PIWI protein, MILI, loads sense rather than antisense primary piRNAs. However, heterotypic ping-pong with ping-pong partner MIWI2 may not operate because, once loaded with secondary piRNAs, MIWI2 is imported into the nucleus to direct specific DNA methylation of transposon loci. It is likely that a Krimp-like protein exists in mammals to enforce a piRNA strand bias (Sato, 2015).

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).

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 2-3 h 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 (mnk^P6) 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).

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).

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).

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 4^th chromosomes move toward the poles. In OregonR, distinct 4^th 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 4^th 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).

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 (αβ_c) 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-2^L811fsX mutant flies. In parallel, trans-heterozygous aub (aub^HN2/aub^QC42) and ago3 heads (ago3^t2/ago3^t3) and trans-heterozygous armitage heads (armi¹/armi^72.1) 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).

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).

The cochaperone shutdown defines a group of biogenesis factors essential for all piRNA populations in Drosophila

In animal gonads, PIWI proteins and their bound 23-30 nt piRNAs guard genome integrity by the sequence specific silencing of transposons. Two branches of piRNA biogenesis, namely primary processing and ping-pong amplification, have been proposed. Despite an overall conceptual understanding of piRNA biogenesis, identity and/or function of the involved players are largely unknown. This study demonstrates an essential role for the female sterility gene shutdown in piRNA biology. Shutdown, an evolutionarily conserved cochaperone collaborates with Hsp90 during piRNA biogenesis, potentially at the loading step of RNAs into PIWI proteins. Shutdown is shown to be essential for both primary and secondary piRNA populations in Drosophila. An extension of this study to previously described piRNA pathway members revealed three distinct groups of biogenesis factors. Together with data on how PIWI proteins are wired into primary and secondary processing, a unified model for piRNA biogenesis is proposed (Olivieri, 2012).

PIWI interacting RNAs (piRNAs) are a class of animal small RNAs. They are bound by PIWI family proteins and guide the sequence specific silencing of selfish genetic elements such as transposable elements (TEs). Defects in the piRNA pathway lead to TE derepression, genomic instability and ultimately sterility (Olivieri, 2012).

In Drosophila, most piRNAs are generated from two sources; on the one hand, these are piRNA cluster transcripts that originate from discrete genomic loci and serve as reservoirs of TE sequences; on the other hand, these are RNAs derived from active TEs that engage - together with cluster transcripts - in a piRNA amplification loop called the ping-pong cycle (Olivieri, 2012).

Two modes of piRNA biogenesis exist: (1) during primary piRNA biogenesis, a single stranded RNA is processed into pre-piRNAs, which are loaded onto PIWI proteins and are subsequently 3' trimmed and methylated, yielding mature piRNA induced silencing complexes (piRISCs). (2) piRISCs with active slicer activity can trigger secondary piRNA biogenesis, where a new piRNA is formed out of the sliced target RNA. In the presence of corresponding sense and antisense precursor RNAs, secondary piRNA biogenesis acts as the ping-pong amplification loop. The two piRNAs engaged in ping-pong have opposite orientation and exhibit a characteristic ten nucleotide 5' overlap (ping-pong signature) (Olivieri, 2012).

Primary and secondary piRNA biogenesis co-occur in germline cells, complicating the genetic and mechanistic dissection of these processes. However, somatic cells of the gonad also harbor a piRNA pathway and this is based exclusively on primary piRNA biogenesis. The Drosophila ovary is therefore ideally suited to identify and characterize factors required for either primary or secondary piRNA biogenesis or both (Olivieri, 2012).

Somatic support cells of the Drosophila ovary express Piwi as the only PIWI family protein. Primary piRNA biogenesis is thought to take place in peri-nuclear Yb-bodies, where the RNA helicases Armitage (Armi) and Yb as well as the TUDOR domain protein Vreteno (Vret) accumulate. In addition to these three factors, the putative mitochondria-localized nuclease Zucchini (Zuc) and the RNA helicase Sister of Yb (SoYb) are essential for piRNA biogenesis in the soma. Formation of mature Piwi-RISC triggers its nuclear import, while failure in piRNA biogenesis results in destabilization of presumably unloaded Piwi. Mature Piwi-RISC triggers TE silencing by an unknown mechanism that requires Piwi's nuclear localization but not its slicer activity (Olivieri, 2012).

With the exception of Yb, the above mentioned biogenesis factors are also essential in germline cells for the formation of Piwi-RISC. Germline cells, however, express two additional PIWI proteins, Aubergine (Aub) and Argonaute 3 (AGO3), which localize to the cytoplasm and are enriched in peri-nuclear nuage. Aub and AGO3 are the main players in the ping-pong cycle. Several factors with essential or modulatory roles in the ping-pong cycle have been identified. These are the RNA helicases Spindle-E and Vasa and the TUDOR domain proteins Krimper, Tejas (Tej), Qin and Tudor (Olivieri, 2012).

The analysis of piRNA populations from wild-type and piRNA pathway mutant ovaries indicated that Piwi is mainly a recipient of primary piRNAs, while Aub and AGO3 are predominantly or exclusively recipients of secondary piRNA biogenesis. Given this, three major questions arise: (1) Are primary and secondary piRNA biogenesis processes genetically and mechanistically separate or do common factors act in both processes? (2) In which processing step do identified piRNA biogenesis factors act? (3) How are the three PIWI family proteins wired into piRNA biogenesis? In other words, are certain PIWI proteins only receiving primary or only secondary piRNAs (Olivieri, 2012)?

This study shows that the female sterility gene shutdown encodes a piRNA biogenesis factor. Shu is required for all piRNA populations in ovaries and it acts downstream of known piRNA biogenesis factors. A comparison of Shu to several other pathway factors led to the definition of three major groups of piRNA biogenesis factors. In combination with data on how PIWI proteins are wired into piRNA biogenesis, a model is proposed that accounts for the distinct association of piRNA subpopulations with specific PIWI proteins in Drosophila (Olivieri, 2012).

The outcome of this work is threefold: (1) The cochaperone Shutdown is essential for the biogenesis of all Drosophila piRNA populations. (2) Three major groups of piRNA biogenesis factors can be distinguished. (3) Piwi and Aub but not AGO3 are loaded with primary piRNAs, explaining how the cell maintains highly specific piRNA populations in the three PIWI proteins (Olivieri, 2012).

A remarkable feature of the shu mutant phenotype is that piRNA populations for every TE collapse. This already points to a common piRNA biogenesis step downstream of the primary and secondary branches. Epistatic analysis in somatic follicle cells is consistent with Shu acting at a late step in piRNA biogenesis: Shu is not required for the localization of any known biogenesis factor to Yb-bodies. On the other hand, Shu's localization to Yb-bodies depends on all other biogenesis factors and even on Piwi, arguing that unloaded Piwi recruits Shu to the Yb-body. Similarly, Shu colocalizes with nonloadable AGO3 in OSCs as well as in ovaries defective of ping-pong in discrete foci that also contain and are dependent on Krimp. Thus, in wild-type and in biogenesis mutants, Shu appears to colocalize with unloaded PIWI proteins (Olivieri, 2012).

Shu's C-terminal TPR domain falls into the class of Hsp90 binders and Hsp90 is important for small RNA loading into Argonaute proteins (Iki, 2010; Iwasaki, 2010; Miyoshi, 2010). In addition, the plant cochaperone Cyp40 interacts with Hsp90 via its TPR domain and is a critical cofactor for small RNA loading into AGO1. The genetic and localization data support an analogous role for Shu and Hsp90 during small RNA loading into PIWI proteins. Clearly, in vitro assays will be crucial to dissect the precise order of events and the molecular role of Shu, especially its PPIase domain (Olivieri, 2012).

A major challenge in the field is to assemble piRNA biogenesis factors into pathways that explain the stereotypic populations of piRNAs in vivo. Advantage was taken of efficient germline specific knockdowns to study the impact of several factors on piRNA populations. Based on levels and localization of PIWI proteins as well as on piRNA populations obtained from several pathway factor knockdowns, three major groups of piRNA biogenesis factors are proposed (Olivieri, 2012).

Group I factors are required for primary piRNA biogenesis but dispensable for secondary biogenesis. In fact, piRNAs that initiated ping-pong in group I knockdowns were amplified and ping-pong signatures of such TEs were strongly increased, presumably as primary piRNAs that do not feed into ping-pong were absent (Olivieri, 2012).

Group II factors are specific for ping-pong, as primary piRNA biogenesis feeding into Piwi was unaffected. An alternative explanation that cannot be excluded is that some or all group II genes are required specifically for Aub biology (primary and secondary) per se. This would similarly leave Piwi bound piRNAs intact and would lead to a collapse in ping-pong. Given the data on Aub loading in OSCs, a model is favored however where the primary biogenesis machineries that feed Aub and Piwi are very similar (Olivieri, 2012).

Finally, group III factors are required for the biogenesis of Piwi/Aub/AGO3 bound piRNAs. The prototypic member of this group is Shu. Loss of Shu affects essentially all piRNA populations to the same extent. It is noted that analysis of piRNA populations from vret mutants indicated a role for this group III factor in primary biogenesis but not ping-pong (Handler, 2011; Zamparini, 2011). The distorted tissue composition of vret mutant ovaries coupled with perdurance of maternal Vret protein or RNA may underlie this discrepancy. The existence of group III factors predicts that primary and secondary piRNA biogenesis feed into a final piRISC maturation step that requires a set of common factors for all PIWI proteins. Given that piRNA biogenesis -- irrespective of the source of the precursor RNA -- requires an RNA loading step as well as a 3' trimming step, the existence of group III factors suggests itself (Olivieri, 2012).

The three proposed groups serve as a rough classification of biogenesis factors. Clearly, at a molecular level, the precise role of each factor within the biogenesis process will vary considerably. Of note, the classification of group I and group II genes extends to the mouse piRNA pathway. The Armi and Zuc orthologs MOV10L1 and PLD6 are required for primary piRNA biogenesis, whereas mouse VAS and TDRD9 (mouse Spn-E) were reported to be dispensable for primary biogenesis but are required for secondary biogenesis pathway (Olivieri, 2012).

The data indicate that Aub is not only loaded via ping-pong, but also via primary piRNA biogenesis. It is also postulate that Aub and Piwi proteins are wired into primary piRNA biogenesis processes in a very similar manner, meaning that they require the same or highly overlapping core factors (e.g., Armi or Zuc). In agreement with this, ectopically expressed Aub is loaded in OSCs that harbor a fully functional primary pathway but lack critical ping-pong factors such as Vas. The genetic requirements for Aub loading in OSCs are identical to those for Piwi. It is extrapolated from this that the core primary biogenesis machinery that loads Piwi in the soma also loads Piwi and Aub in the germline. Analyses of piRNA populations from armi versus piwi or aub-GLKDs support a model where Armi and Zuc are required for the biogenesis of both Piwi and Aub bound primary piRNAs. The possibility is not excluded that - despite a similar biogenesis machinery - populations of primary piRNAs in Aub and Piwi are different. For example, differences in subcellular localizations of PIWI proteins as well as piRNA precursor RNAs might result in such differences (Olivieri, 2012).

In contrast to Aub, AGO3 was unstable in OSCs. Coexpression of Aub or simultaneous knockdown of krimp had no impact on AGO3 stability. It is therefore concluded that primary piRNA biogenesis is incompatible with AGO3. In fact, also in the germline genetic data indicated that AGO3 depends on secondary piRNA biogenesis for being loaded. Blocking AGO3's access to the primary biogenesis machinery would allow the cell to load AGO3 with a unique class of piRNAs if it couples AGO3 loading to a precursor RNA originating from Aub-slicer mediated cleavage of an active TE. This would explain the remarkable bias of AGO3 bound piRNAs being sense and carrying an Adenosine at position ten (Olivieri, 2012).

Interestingly, on a primary sequence level Aub -- despite its significantly different biology -- is more closely related to Piwi than to AGO3. A critical question emanating from this is to which extent Piwi is participating in ping-pong, and if it does not, why. A weak, yet statistically significant, ping-pong signature has been observed between Piwi and AGO3 bound piRNAs. This could mean that there is indeed low level of Piwi-AGO3 ping-pong. An alternative explanation is that the Piwi-AGO3 signal is a misleading computational signal: If Piwi and Aub are loaded via the same primary biogenesis machinery, initiator piRNAs for ping-pong that end up in Aub also end up in Piwi. As primary piRNA biogenesis appears to be nonrandom and preferentially processed piRNAs likely trigger ping-pong more robustly, an 'artificial' AGO3/Piwi ping-pong signature might result (Olivieri, 2012).

What could be the molecular basis of why Piwi does not or only moderately participate in ping-pong? Either, specific features of Aub (e.g., symmetric Arginine methylation) are funneling this protein into ping-pong and similar features are absent on Piwi. Or, the mere sequestration of Piwi into the nucleus prevents Piwi from participating in ping-pong. Notably, N-terminally truncated Piwi that is still loaded but that cannot translocate into the nucleus is enriched in nuage the proposed site of secondary piRNA biogenesis. A simple difference in the subcellular localization of Aub and Piwi might thus contribute to the dramatic differences of piRNA populations residing in Aub or Piwi (Olivieri, 2012).

The initial uridine of primary piRNAs does not create the tenth adenine that is the hallmark of secondary piRNAs

PIWI-interacting RNAs (piRNAs) silence transposons in animal germ cells. PIWI proteins bind and amplify piRNAs via the "Ping-Pong" pathway. Because PIWI proteins cleave RNAs between target nucleotides t10 and t11-the nucleotides paired to piRNA guide positions g10 and g11-the first ten nucleotides of piRNAs participating in the Ping-Pong amplification cycle are complementary. Drosophila piRNAs bound to the PIWI protein Aubergine typically begin with uridine (1U), while piRNAs bound to Argonaute3, which are produced by Ping-Pong amplification, often have adenine at position 10 (10A). The Ping-Pong model proposes that the 10A is a consequence of 1U. This study found that 10A is not caused by 1U. Instead, fly Aubergine as well as its homologs, Siwi in silkmoth and MILI in mice, have an intrinsic preference for adenine at the t1 position of their target RNAs; during Ping-Pong amplification, this t1A subsequently becomes the g10A of a piRNA bound to Argonaute3 (Wang, 2014).

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).

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).

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 csul^RM50/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).

Search PubMed for articles about Drosophila Ago3

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date revised: 25 October 2023

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