InteractiveFly: GeneBrief

cutoff: Biological Overview | References

Gene name - cutoff

Synonyms -

Cytological map position - 48C5-48C5

Function - chromatin protein

Keywords - functions in the piRNA pathway in stem- and germ-cell development during Drosophila oogenesis - piRNA cluster regulation, nuage assembly and piRNA production,

Symbol - cuff

FlyBase ID: FBgn0000390

Genetic map position - chr2R:7773572-7775108

Classification - RAI1 like PD-(D/E)XK nuclease

Cellular location - nuclear

NCBI links: EntrezGene

cuff orthologs: Biolitmine
Recent literature
Hur, J.K., Luo, Y., Moon, S., Ninova, M., Marinov, G.K., Chung, Y.D. and Aravin, A.A. (2016). Splicing-independent loading of TREX on nascent RNA is required for efficient expression of dual-strand piRNA clusters in Drosophila. Genes Dev 30: 840-855. PubMed ID: 27036967
The conserved THO/TREX (transcription/export) complex is critical for pre-mRNA processing and mRNA nuclear export. In metazoa, TREX is loaded on nascent RNA transcribed by RNA polymerase II in a splicing-dependent fashion; however, how TREX functions is poorly understood. This study shows that Thoc5 and other TREX components are essential for the biogenesis of piRNA, a distinct class of small noncoding RNAs that control expression of transposable elements (TEs) in the Drosophila germline. Mutations in TREX lead to defects in piRNA biogenesis, resulting in derepression of multiple TE families, gametogenesis defects, and sterility. TREX components are enriched on piRNA precursors transcribed from dual-strand piRNA clusters and colocalize in distinct nuclear foci that overlap with sites of piRNA transcription. The localization of TREX in nuclear foci and its loading on piRNA precursor transcripts depend on Cutoff, a protein associated with chromatin of piRNA clusters. Finally, it was shown that TREX is required for accumulation of nascent piRNA precursors. These data reveal a novel splicing-independent mechanism for TREX loading on nascent RNA and its importance in piRNA biogenesis. 

Chen, Y. A., Stuwe, E., Luo, Y., Ninova, M., Le Thomas, A., Rozhavskaya, E., Li, S., Vempati, S., Laver, J. D., Patel, D. J., Smibert, C. A., Lipshitz, H. D., Fejes Toth, K. and Aravin, A. A. (2016). Cutoff suppresses RNA Polymerase II termination to ensure expression of piRNA precursors. Mol Cell [Epub ahead of print]. PubMed ID: 27292797
Small non-coding RNAs called piRNAs serve as guides for an adaptable immune system that represses transposable elements in germ cells of Metazoa. In Drosophila the RDC complex, composed of Rhino, Deadlock and Cutoff (Cuff) bind chromatin of dual-strand piRNA clusters, special genomic regions, which encode piRNA precursors. The RDC complex is required for transcription of piRNA precursors, though the mechanism by which it licenses transcription remained unknown. This study shows that Cuff prevents premature termination of RNA polymerase II. Cuff prevents cleavage of nascent RNA at poly(A) sites by interfering with recruitment of the cleavage and polyadenylation specificity factor (CPSF) complex. Cuff also protects processed transcripts from degradation by the exonuclease Rat1. This work reveals a conceptually different mechanism of transcriptional enhancement. In contrast to other factors that regulate termination by binding to specific signals on nascent RNA, the RDC complex inhibits termination in a chromatin-dependent and sequence-independent manner.
Hermant, C., Boivin, A., Teysset, L., Delmarre, V., Asif-Laidin, A., Beek, M. V., Antoniewski, C. and Ronsseray, S. (2015). Paramutation in Drosophila requires both nuclear and cytoplasmic actors of the piRNA pathway and induces cis-spreading of piRNA production. Genetics [Epub ahead of print]. PubMed ID: 26482790
Transposable element (TE) activity is repressed in the germline in animals by PIWI-Interacting RNAs (piRNAs), a class of small RNAs produced by genomic loci mostly composed of TE sequences. The mechanism of induction of piRNA production by these loci is still enigmatic. In Drosophila, a cluster of tandemly repeated P-lacZ-white transgenes can be activated for piRNA production by maternal inheritance of a cytoplasm containing homologous piRNAs. This activated state is stably transmitted over generations and allows trans-silencing of a homologous transgenic target in the female germline. Such an epigenetic conversion displays the functional characteristics of a paramutation, i.e., a heritable epigenetic modification of one allele by the other. This paper reports that piRNA production and trans-silencing capacities of the paramutated cluster depend on the function of the rhino, cutoff and zucchini genes involved in primary piRNA biogenesis in the germline, as well as on that of the aubergine gene implicated in the ping-pong piRNA amplification step. The 21nt RNAs which are produced by the paramutated cluster in addition to 23-28nt piRNAs are not necessary for paramutation to occur. Production of these 21nt RNAs requires Dicer-2 but also all the piRNA genes tested. Moreover, cytoplasmic transmission of piRNAs homologous to only a subregion of the transgenic locus can generate a strong paramutated locus which produces piRNAs along the whole length of the transgenes. Finally, it was observed that maternally-inherited transgenic small RNAs can also impact transgene expression in the soma. In conclusion, paramutation involves both nuclear (Rhino, Cutoff) and cytoplasmic (Aubergine, Zucchini) actors of the piRNA pathway. In addition, since it is observed between non-fully homologous loci located on different chromosomes, paramutation may play a crucial role in epigenome shaping in Drosophila natural populations.
Pritykin, Y., Brito, T., Schupbach, T., Singh, M. and Pane, A. (2017). Integrative analysis unveils new functions for the Drosophila Cutoff protein in non-coding RNA biogenesis and gene regulation. RNA [Epub ahead of print]. PubMed ID: 28420675
Piwi-Interacting RNAs (piRNAs) are central components of the piRNA pathway, which directs transposon silencing and guarantees genome integrity in the germ cells of several metazoans. In Drosophila, piRNAs are produced from discrete regions of the genome termed piRNA clusters, whose expression relies on the RDC complex comprised of the core proteins Rhino, Deadlock and Cutoff. To date, the RDC complex has been exclusively implicated in the regulation of the piRNA loci. This study further elucidates the function of Cutoff and the RDC complex by performing genome-wide ChIP-seq and RNA-seq assays in the Drosophila germline and analyzing these data together with other publicly available data sets. In agreement with previous studies, it was confirmed that Cutoff is involved in the transcriptional regulation of piRNA clusters and in the repression of transposable elements in germ cells. Surprisingly, however, it was found that Cutoff is enriched at and affects the expression of other non-coding RNAs, including spliceosomal RNAs (snRNAs) and small nucleolar RNAs (snoRNAs). At least in some instances, Cutoff appears to act at a transcriptional level in concert with Rhino and perhaps Deadlock. Finally, mutations in Cutoff result in the deregulation of hundreds of protein-coding genes in germ cells.

Parhad, S. S., Yu, T., Zhang, G., Rice, N. P., Weng, Z. and Theurkauf, W. E. (2020). Adaptive Evolution Targets a piRNA Precursor Transcription Network. Cell Rep 30(8): 2672-2685. PubMed ID: 32101744
In Drosophila, transposon-silencing piRNAs are derived from heterochromatic clusters and a subset of euchromatic transposon insertions, which are bound by the Rhino-Deadlock-Cutoff complex. The HP1 homolog Rhino binds to Deadlock, which recruits TRF2 to promote non-canonical transcription from both genomic strands. Cuff function is less well understood, but this Rai1 homolog shows hallmarks of adaptive evolution, which can remodel functional interactions within host defense systems. Supporting this hypothesis, Drosophila simulans Cutoff is a dominant-negative allele when expressed in Drosophila melanogaster, in which it traps Deadlock, TRF2, and the conserved transcriptional co-repressor CtBP in stable complexes. Cutoff functions with Rhino and Deadlock to drive non-canonical transcription. In contrast, CtBP suppresses canonical transcription of transposons and promoters flanking the major germline clusters, and canonical transcription interferes with downstream non-canonical transcription and piRNA production. Adaptive evolution thus targets interactions among Cutoff, TRF2, and CtBP that balance canonical and non-canonical piRNA precursor transcription.


In a broad range of organisms, Piwi-interacting RNAs (piRNAs) have emerged as core components of a surveillance system that protects the genome by silencing transposable and repetitive elements. A vast proportion of piRNAs is produced from discrete genomic loci, termed piRNA clusters, which are generally embedded in heterochromatic regions. The molecular mechanisms and the factors that govern their expression are largely unknown. This study shows that Cutoff (Cuff), a Drosophila protein related to the yeast transcription termination factor Rai1, is essential for piRNA production in germline tissues. Cuff accumulates at centromeric/pericentromeric positions in germ-cell nuclei and strongly colocalizes with the major heterochromatic domains. Remarkably, Cuff is enriched at the dual-strand piRNA cluster 1/42AB and is likely to be involved in regulation of transcript levels of similar loci dispersed in the genome. Consistent with this observation, Cuff physically interacts with the Heterochromatin Protein 1 (HP1) variant Rhino (Rhi). These results reveal a link between Cuff activity, heterochromatin assembly and piRNA cluster expression, which is critical for stem-cell and germ-cell development in Drosophila (Pane, 2011).

A significant fraction of eukaryotic genomes is made up of repetitive sequences, such as transposable elements (TEs) and tandem repeats, whose deregulation has been linked to DNA damage and sterility. Germline tissues, which are responsible for transferring the genetic information to the progeny, appear to be the most sensitive targets of TE deregulation. A specialized RNAi pathway, centered on small non-coding RNAs known as Piwi-interacting RNAs (piRNAs), guarantees the repression of transposable and repetitive elements and ensures the maintenance of genomic stability during germ-cell division and differentiation. piRNAs are 23-30-nt-long non-coding RNAs and mainly correspond to sequences in transposable and repetitive elements dispersed in the genome (Aravin, 2003). In Drosophila, the production of these molecules relies on the activity of the Argonaute family members Piwi, Aubergine (Aub) and Argonaute-3 (Ago3) (Saito, 2006; Brennecke, 2007; Gunawardane, 2007; Yin, 2007). Deep-sequencing analyses of the small RNAs associated with these proteins revealed that Piwi and Aub complexes are enriched in antisense piRNAs, while Ago3 is mostly bound by sense piRNAs. Furthermore, a subset of piRNAs displays a 10-nt overlap at their 5' end (Brennecke, 2007; Gunawardane, 2007). These observations were captured in a model, whereby antisense piRNAs bound to Aub/Piwi can pair with transposon transcripts and catalyse the production of sense piRNA molecules through cleavage of the transposon transcripts. The latter are then loaded into an Ago3 complex, which in turn triggers the production of antisense piRNAs by cleaving cluster-derived antisense transcripts. This cycle is the core of the so-called 'Ping-pong' model, a feed-forward loop that amplifies the piRNA population and is thought to reinforce the repression of active transposons dispersed in the genome (Brennecke, 2007; Gunawardane, 2007). piRNA processing requires additional activities, including the eIF-4A-like translation factor Vasa (Vas), the Heterochromatin Protein 1 (HP1) variant Rhino (Rhi), the helicases Spindle-E and Armitage, the putative nucleases Maelstrom, Zucchini and Squash and the Tudor-domain proteins Tejas and Krimper (Khurana, 2010; Saito, 2010; Senti, 2010). The precise biochemical function of many of these proteins is, however, still largely unclear (Pane, 2011).

In Drosophila, primary sources of piRNAs are discrete genomic regions termed piRNA clusters (Brennecke, 2007). These genomic loci are composed of repetitive sequences, TEs and inactive transposon remnants. The transcripts produced from these loci are processed by cytoplasmic activities to produce the mature piRNAs. The major sources of piRNAs in Drosophila are the cluster 1/42AB located on chromosome 2R in pericentromeric position and clusters 2 and flamenco/COM (flam) located on chromosome X. Cluster 1/42AB and the majority of the piRNA clusters in Drosophila are transcribed from both DNA strands (dual-strand clusters). Conversely, a few loci, including cluster 2 and flam, produce piRNAs only from one genomic DNA strand (uni-strand clusters) (Brennecke, 2007; Malone, 2009). To date, flam represents the best-characterized piRNA cluster. The flam cluster harbours fragments of the Zam, gypsy and idefix retro-transposons and is required for the silencing of these retroviral elements in the follicle cells of the Drosophila ovaries. Interestingly, the vast majority of the transposon remnants that populate the flam locus are oriented towards the centromere of the X chromosome (Brennecke, 2007). Genetic and molecular analyses strongly suggest that the transcription of this cluster occurs from an external promoter on the opposite strand and is likely to produce a long transcript encompassing the entire locus (Brennecke, 2007; Mevel-Ninio, 2007). The structure of the cluster and the strand bias of the transcriptional events thus generate mostly antisense piRNAs, which do not engage a ping-pong amplification loop. piRNA clusters in zebrafish, mouse and human also display a clear strand bias similar to flam, whereby piRNAs map only to one DNA strand. The mechanism that regulates the expression of the piRNA clusters and the proteins involved in this nuclear process remain largely elusive. It has recently been shown that a germline-specific HP1 variant encoded by the rhino (rhi) gene is essential for the regulation of the dual-strand clusters (Volpe, 2001; Klattenhoff, 2009). Accordingly, mutations in rhi abolish the expression of these loci, which results in a general depletion of the corresponding piRNA population (Klattenhoff, 2009). The activity of Rhi seems to be limited to the dual-strand clusters, since mutations in this protein do not affect the production of piRNAs from the uni-strand clusters. Surprisingly, the expression of the piRNA loci appears to also rely on the activity of the Piwi proteins Aub and Piwi. In mutants of these factors, heterochromatic states spread over the clusters causing their transcriptional downregulation. These findings, therefore, implicate the production of piRNAs in the maintenance of active transcription from the piRNA clusters (Pane, 2011).

It has been reported that the protein product of the cutoff (cuff) gene is essential for transposon silencing and germline development (Chen, 2007). Mutations in cuff impair the establishment of dorsal–ventral polarity during oogenesis and cause a significant loss of germline cells, possibly due to defects in stem-cell maintenance and/or division. Cuff shows similarity to the yeast transcription termination factor Rai1 (Xue, 2000; Kim, 2004). In yeast, Rai1 is found in a complex with the 5'- 3' exoribonuclease Rat1/Xrn2 and promotes the termination of transcriptional events initiated by the PolI and PolII RNA polymerases (Kim, 2004; El Hage, 2008). Different from Rai1, however, Cuff does not display a Dom3Z domain, which is critical for Rai1 activity. Given the observation that an additional Rai1 homologue exists in Drosophila which contains the Rai1 catalytic domain, it is hypothesized that Cuff might be a germline-specific Rai1 variant, which exerts a novel function in germline cells (Pane, 2011).

This study shows that Cuff localizes to discrete foci in germline nuclei and accumulates in proximity of and within heterochromatic domains. Cuff is essential for the expression of the piRNA clusters embedded in heterochromatic regions, including the major dual-strand cluster 1/42AB, but not for the flam locus, which is primarily expressed in the soma. In agreement with these observations, it was found that Cuff colocalizes and physically interacts with Rhi. It is proposed that these proteins assemble into a complex, which regulates the production of piRNAs from the dual-strand clusters at a transcriptional level. Mutations in Cuff cause a severe disruption of the nuage, which in turn might contribute to the depletion of piRNAs not only from the dual-strand, but also from some uni-strand clusters, including the major cluster 2. These results uncover a role for Cuff in piRNA cluster regulation, nuage assembly and piRNA production and highlight a critical function for the piRNA pathway in stem- and germ-cell development during Drosophila oogenesis (Pane, 2011).

In Drosophila, piRNAs are mostly produced from discrete genomic loci, termed piRNA clusters, which are composed of repetitive sequences and inactive transposon remnants (Brennecke, 2007). The major piRNA clusters, including the dual-strand cluster 1/42AB and the uni-strand cluster 2 and flam, are located at the border between euchromatic and heterochromatic regions or within the major heterochromatic domains (Brennecke, 2007). Heterochromatic states have been generally associated with transcriptionally repressed genomic loci. The expression of the piRNA clusters, therefore, needs to be tightly controlled to ensure piRNA production, while the heterochromatin is being assembled. This study reports the functional characterization of Cuff, a Drosophila protein with similarity to the yeast transcription termination factor Rai1, and this protein is shown to control piRNA cluster expression and piRNA production during Drosophila germline development. Cuff is critical for the correct levels of transcripts produced from the major dual-strand cluster 1/42AB in the pericentromere of chromosome II. piRNAs originating from this locus and from virtually all the dual-strand clusters dispersed in the genome are substantially reduced or depleted in the cuff mutant. Consistent with a germline-specific function of Cuff, mutations in this protein do not impact the flam locus, which is known to be active only in the somatic follicle cells. The data suggest that Cuff regulates the piRNA master loci presumably acting in a complex with the HP1 variant Rhi. Both Cuff and Rhi are enriched at the cluster 1/42AB, while they do not interact with sequences in the cluster 2 and flam. These observations strongly suggest that a Cuff/Rhi complex regulates the transcription of the dual-strand piRNA clusters. The activity of this complex appears to be crucial for the expression of the dual-strand clusters and might be critical to overcome the complexity of these genomic loci, where bidirectional transcription of the different elements can potentially hinder the production of the proper piRNA population. Different from Rhi, cuff also affects the production of piRNAs from some uni-strand clusters, including the major cluster 2. ChIP assays, however, failed to reveal a significant enrichment of Cuff at this locus. It is, therefore, likely that the loss of piRNAs from cluster 2 is not directly caused by mutations in the Cuff protein, but it might rather ensue from the disruption of the nuage observed in the cuff ovaries and the mislocalization of factors required for piRNA production. Nevertheless, the comparison of cuff and rhi piRNA libraries revealed some interesting differences between the two mutants. For instance, mutations in cuff appear to have a more prominent impact on the sense piRNA population compared with the rhi mutant, thus suggesting that these proteins might display partially different functions in piRNA cluster expression and piRNA production. Intriguingly, the expression levels of the piRNA clusters in the cuff mutant do not precisely mirror the profiles of the corresponding piRNA population. One would predict that the depletion of a specific piRNA set would ensue from a general downregulation of the corresponding cluster-derived transcripts. While some regions within cluster 1/42AB are clearly downregulated in the cuff mutant, other regions are not affected and only one, among those analysed, appears to be upregulated. These data point to a role for Cuff in the transcriptional control of dual-strand cluster expression, whereby this protein may be required to activate/permit the transcription of these loci. In the absence of Cuff, cluster-derived piRNA precursor transcripts do not accumulate, thus leading to a general collapse of the piRNA population. It is noteworthy that some regions in the cluster 1/42AB are not affected in the cuff mutant. This observation suggests that the dual-strand clusters might not produce single transcripts spanning the entire locus, as it is the case for the flam locus (Brennecke, 2007), but they might be rather transcribed by multiple internal and external promoters (Pane, 2011).

It has been recently reported that mutations in the Argonaute proteins Piwi and Aub reduce the expression levels of genomic regions located within or next to the piRNA clusters (Moshkovich, 2010). In these mutants, HP1 spreading triggers a significant repression of the piRNA loci, which led to the proposal that in Drosophila, piRNAs ensure the transcription of the clusters by counteracting the spreading of heterochromatic states (Moshkovich, 2010). Remarkably, the recruitment of Cuff and Rhi to nuclear foci early in oogenesis seems to parallel the assembly of the major heterochromatic blocks. The levels of Cuff, Rhi and HP1 are low in the stem cells and in the early mitotic cyst, while these proteins appear to accumulate in the late mitotic cyst, where they extensively colocalize in specific regions of the nuclei. It is, therefore, tempting to speculate that Cuff and Rhino might protect the clusters from the repressive effects associated with the assembly of the major heterochromatic blocks, which takes place early in oogenesis while the germ cells undergo the mitotic divisions (Pane, 2011).

Mutations in cuff negate the production of both sense and antisense piRNAs corresponding to 46 transposon families. For a significant number of transposons only piRNAs matching one DNA strand seem to be affected in cuff, while piRNAs corresponding to a few transposon families are apparently unaltered. It is noteworthy that the HeT-A and TART retro-transposons, which are involved in the maintenance of telomere integrity, display contrasting piRNA profiles despite the fact that the transcript levels of both these elements are upregulated in cuff ovaries (Chen, 2007). Virtually, all the piRNAs corresponding to HeT-A sequences are depleted in the cuff mutant, which might account for the severe deregulation of this retro-transposon in the absence of Cuff. Conversely, only a limited number of piRNAs matching TART sequences could be identified in wild-type ovaries, while a surprising variety of reads corresponding to this element are produced in cuff mutant ovaries. Similar to the piRNA clusters, also the different classes of transposons appear to undergo differential regulation, and it will be a challenge for the future to uncover the underlying rules (Pane, 2011).

The introduction of a sensor transgene based on the Zam retro-transposon in the germline of cuff, rhi, vas and aub flies led to further insight into the temporal requirement of these proteins during Drosophila oogenesis. Zam is an LTR retro-transposon, which is specifically repressed in the ovarian follicle cells by the piRNAs produced from the flam locus. While the sensor transgene is consistently repressed in wild-type germ cells, it is significantly deregulated in cuff, rhi, vas and aub germline. The silencing of the reporter appears to be very sensitive to mutations in cuff and rhi, which produce a strong GFP signal in stem and germ cells as well as in fully formed egg chambers. Mutations in vas and aub instead seem to have a milder impact on the sensor construct, whereby a detectable GFP signal is mostly observed in the dividing or late mitotic cysts. The mechanism by which the sensor construct is silenced in wild-type ovaries is unlikely to mirror the piRNA-dependent repression of the Zam retro-transposon in somatic cells, since mutations in cuff do not impact the production of piRNAs from the flam locus. Similarly, flam piRNA levels are unaltered in rhi, aub and vas mutant backgrounds. These observations reveal that Zam sequences can be targeted for silencing also in germline tissues in a flam-independent fashion. Interestingly, no piRNAs corresponding to the env fragment were detected either in wild-type or in the cuff mutant libraries. While it is not possible to exclude that the expression of the sensor transgene induced by the Act5C-Gal4 driver in the wild-type germline might trigger the production of piRNAs directed against the reporter, it is possible that the silencing of the pGFP-ZenvAS transgene might not proceed through a piRNA-based mechanism. Since nuclear Piwi is dramatically lost in the germaria of all these mutants, it is also unlikely that the strong deregulation of the reporter observed in cuff and rhi mutant stem and germ cells is primarily caused by Piwi mislocalization. Instead, these results suggest that Cuff and Rhi might act upstream of Piwi to regulate the expression of the piRNA clusters and ensure piRNA production early in oogenesis. The mature piRNAs are then loaded into Piwi and serve to drive the nuclear accumulation of this protein. It is conceivable that the activity of Cuff and Rhi protects the piRNA clusters from the repressing effects associated with heterochromatin formation. In later stages, Vas, Aub and most likely other piRNA pathway components might further contribute to the maintenance of active transcription at these loci (Pane, 2011).

Mutations in cuff cause a progressive loss of germ cells, including stem cells, over time (Chen, 2007). In addition, previous studies have implicated germline Piwi in the control of germ-cell division rate, while somatic Piwi is required for stem-cell self-renewal. The current data strongly suggest that stem- and germ-cell loss in the cuff mutant is caused by a failure to express the piRNA master loci. In the absence of primary piRNAs, the entire piRNA machinery is dispersed and Piwi fails to accumulate in the stem- and germ-cell nuclei, thus further contributing to the cuff phenotype (Pane, 2011).

piRNA-based mechanisms appear to be conserved across the phyla and homologues of the Piwi-clade Argonaute proteins are present in organisms as distant as flies and mouse. Similarly, members of the Rai1 superfamily can be found both in unicellular organisms and in higher Eukaryotes. Therefore, it is conceivable that specific Rai1 and HP1 variants might exist in other organisms and, similar to Drosophila, their interaction might ensure the maintenance of genome integrity during stem- and germ-cell division (Pane, 2011).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Transcription termination by nuclear RNA polymerases

Argonaute proteins of the PIWI clade are central to transposon silencing in animal gonads. Their target specificity is defined by 23-30 nt PIWI interacting RNAs (piRNAs), which mostly originate from discrete genomic loci termed piRNA clusters. This study shows that a complex composed of Rhino, Deadlock, and Cutoff (RDC) defines dual-strand piRNA clusters genome-wide in Drosophila ovaries. The RDC is anchored to H3K9me3-marked chromatin in part via Rhino's chromodomain. Depletion of Piwi results in loss of the RDC and small RNAs at a subset of piRNA clusters, demonstrating a feedback loop between Piwi and piRNA source loci. Intriguingly, profiles of RNA polymerase II occupancy, nascent transcription, and steady-state RNA levels reveal that the RDC licenses noncanonical transcription of dual-strand piRNA clusters. Likely, this process involves 5' end protection of nascent RNAs and suppression of transcription termination. These data provide key insight into the regulation and evolution of piRNA clusters (Mohn, 2014).

The data indicate that the Rhi-Del-Cuff complex acts as the central licensing factor for dual-strand piRNA cluster transcription in Drosophila. This discussion focuses on two aspects that emerge from this work. (1) The molecular function of the RDC, and (2) the role of Piwi in specifying piRNA source loci and resulting implications for maternally deposited Piwi (Mohn, 2014).

Rhi, Del, and Cuff form an interdependent protein complex that occupies hundreds of mostly heterochromatic loci in ovarian germline cells. Loss of any individual RDC factor results in loss of transcription at dual-strand piRNA source loci and in downstream distortions of piRNA populations and TE silencing (Mohn, 2014).

At the molecular level, Rhi appears to be an adaptor that anchors Del and Cuff to H3K9 methylated chromatin via its chromodomain. While required, it is unlikely that H3K9me3 is sufficient for RDC recruitment as only a subset of H3K9me3-enriched domains are occupied by the RDC. Further, Su(var)205 is highly expressed in germline cells and occupies H3K9me3 domains genome-wide, suggesting that additional factors or chromatin marks exist that allow Rhi to compete against Su(var)205 (Mohn, 2014).

Rhi's chromo-shadow domain interacts with Del, which-due to lack of any recognizable domains-might act as a flexible linker between Rhi and Cuff. Intriguingly, Cuff is related to the Rai1/Dom3Z family of proteins. This is highly interesting in light of the transcriptional patterns at piRNA source loci: the data strongly point toward a central role of the RDC in preventing termination of RNA Pol II, either from flanking transcription units or from atypical promoters. Termination of Pol II transcription occurs in a poorly understood process but is typically linked to endonucleolytic cleavage of the nascent RNA at poly(A) signals by the cleavage and poly-adenylation specificity factor (CPSF) complex (reviewed in Richard, 2009). While RNA Pol II continues transcription, the cleaved and 5' phosphorylated end of the nascent RNA is targeted by the Rat1/Xrn2 5'-to-3' exonuclease. Upon reaching the elongating Pol II, Xrn2 has been postulated to elicit transcription termination . A central step in preventing termination would therefore be the protection of the nascent RNA's 5' end against degradation. It is speculated that Cuff is centrally involved in this process as it resembles a catalytically inactive Dom3Z/Rai1 paralog. Cuff's binding to the nascent RNA 5' end would prevent RNA degradation and would uncouple transcription from the CBC. This in turn would result in noncanonical Pol II transcription, consistent with RDC licensed transcription ignoring poly(A) sites and splice sites (Mohn, 2014).

While the Pol II scavenging model predicts that Cuff stabilizes 5' phosphorylated transcripts, it is noted that Cuff likely also acts on nascent RNA 5' ends without prior CPSF-mediated cleavage, such as would be the case at the promoters of cluster 38C or the Hsp70A genes. It is speculated that Cuff competes with the capping enzyme or with the CBC for the nascent RNA 5' end. Furthermore, it is also conceivable that Cuff protects 5' ends of spurious noncanonical initiation events within RD-SL. In particular, at large heterochromatic piRNA-SL such as 42AB, which encompass nearly 250 kb, internal initiation events would ensure steady piRNA generation over the entire length (Mohn, 2014).

Taken together, dual-strand piRNA source loci are not hardwired transcription units but are dynamic features of heterochromatin that depend on their heterochromatic nature and the transcriptional status of nearby loci. Considering this, dual-stranded transcription of RD-SL appears to be a consequence of, rather than a requirement for, RDC binding (Mohn, 2014).

The data indicate that Piwi is involved in the definition of piRNA source loci, likely by guiding H3K9 methylation that is required for RDC recruitment. This was particularly evident for stand-alone euchromatic TE insertions. In contrast, most RD-SL within pericentromeric heterochromatin and the major dual-strand piRNA clusters were not or only mildly affected upon Piwi depletion. This resembles findings from S. pombe where concepts of heterochromatin initiation and maintenance have been established and where loci also depend differentially on the RITS complex. Probably, more robust maintenance pathways for chromatin patterns exist within heterochromatin versus euchromatin. Of note, mutations in the Drosophila H3K9 methyl-transferase SETDB1 result in widespread defects in piRNA cluster transcription (Mohn, 2014).

The fact that Piwi can impact RDC recruitment indicates that evolution of piRNA source loci is much more dynamic than previously anticipated. Any TE can act as a piRNA source as long as it inserts at a favorable position and is transcribed to provide a piRNA target. Piwi therefore has a dual function in the germline: First, it guides transcriptional silencing of TE promoters. Second, it permits low-level transcription of TE sequences and specification of emerging transcripts as piRNA precursors via the RDC (Mohn, 2014).

Piwi-piRNA complexes are maternally deposited into the developing egg and are abundant at the posterior pole where future primordial germ cells will form. Given its role in RDC guidance, maternal Piwi might in fact be the central factor to specify piRNA source loci during early development. Subsequently, heterochromatin-and thus RDC occupancy-at most sites is probably maintained independent of Piwi (Mohn, 2014).

Hybrid dysgenesis and paramutation, two epigenetic TE repression phenomena, have been linked to maternally deposited piRNA populations in Drosophila. The findings that Piwi can specify genomic piRNA sources are probably central for the further understanding of the molecular events underlying these phenomena (Mohn, 2014).

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

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

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

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

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

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

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

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

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

Andersen, P. R., Tirian, L., Vunjak, M. and Brennecke, J. (2017). A heterochromatin-dependent transcription machinery drives piRNA expression. Nature 549(7670): 54-59. PubMed ID: 28847004

Mohn, F., Sienski, G., Handler, D. and Brennecke, J. (2014). The rhino-deadlock-cutoff complex licenses noncanonical transcription of dual-strand piRNA clusters in Drosophila. Cell 157(6): 1364-1379. PubMed ID: 24906153

Parhad, S. S., Tu, S., Weng, Z. and Theurkauf, W. E. (2017). Adaptive evolution leads to cross-species incompatibility in the piRNA transposon silencing machinery. Dev Cell 43(1): 60-70 e65. PubMed ID: 28919205

Parhad, S. S., Yu, T., Zhang, G., Rice, N. P., Weng, Z. and Theurkauf, W. E. (2020). Adaptive evolution targets a piRNA precursor transcription network. Cell Rep 30(8): 2672-2685. PubMed ID: 32101744

Adaptive evolution targets a piRNA precursor transcription network

In Drosophila, transposon-silencing piRNAs are derived from heterochromatic clusters and a subset of euchromatic transposon insertions, which are bound by the Rhino-Deadlock-Cutoff complex. The HP1 homolog Rhino binds to Deadlock, which recruits TRF2 to promote non-canonical transcription from both genomic strands. Cuff function is less well understood, but this Rai1 homolog shows hallmarks of adaptive evolution, which can remodel functional interactions within host defense systems. Supporting this hypothesis, Drosophila simulans Cutoff is a dominant-negative allele when expressed in Drosophila melanogaster, in which it traps Deadlock, TRF2, and the conserved transcriptional co-repressor CtBP in stable complexes. Cutoff functions with Rhino and Deadlock to drive non-canonical transcription. In contrast, CtBP suppresses canonical transcription of transposons and promoters flanking the major germline clusters, and canonical transcription interferes with downstream non-canonical transcription and piRNA production. Adaptive evolution thus targets interactions among Cutoff, TRF2, and CtBP that balance canonical and non-canonical piRNA precursor transcription (Parhad, 2020).

Transposable elements (TEs) are major genome components that can induce mutations and facilitate ectopic recombination, but transposons have also been co-opted for normal cellular functions, and transposon mobilization has rewired transcriptional networks to drive evolution. Species survival may therefore depend on a balance of transposon silencing and activation. The Piwi interacting RNA (piRNA) pathway transcriptionally and post-transcriptionally silences transposons in the germline. However, how this pathway is regulated is not completely understood (Parhad, 2020).

In Drosophila, piRNAs are produced from heterochromatic clusters composed of complex arrays of nested transposon fragments, which appear to provide genetic memory of past genome invaders. Adaptation to new genome invaders is proposed to involve transposition into a cluster, which leads to sequence incorporation into precursors that are processed into trans-silencing anti-sense piRNAs. However, a subset of isolated transposon insertions also produce sense and anti-sense piRNAs, providing an independent adaptation mechanism and epigenetic memory of genome invaders. Expression of piRNAs from these loci is disrupted by piwi mutations, but Piwi-bound piRNAs map to all insertions, not just the subset that function in piRNA biogenesis. The mechanism that defines these 'mini-cluster' thus remains to be determined (Parhad, 2020).

In Drosophila, germline piRNA clusters and transposon mini-clusters are bound by the RDC complex, which is composed of the HP1 homolog Rhino (Rhi), which co-localizes with the linker protein Deadlock (Del) and the Rai1 homolog Cutoff (Cuff). The three components of the RDC are co-dependent for localization to clusters and essential to germline piRNA production. Rhi is composed of chromo, hinge, and shadow domains. The chromo domain binds to H3K9me3-modified histones, and the shadow domain binds Del, which recruits Moonshiner (Moon) and TATA box binding protein-related factor 2 (TRF2), promoting 'non-canonical' transcription from both genomic strands (Parhad, 2020).

The third RDC component, Cuff, was identified in a screen for female sterile mutations and found to encode a homolog of the decapping exonuclease Rai1 required for transposon silencing and piRNA biogenesis. Critical residues in the catalytic pocket of Rai1 are not conserved in Cuff, but sidechains that bind the RNA backbone are retained, suggesting that Cuff may be an RNA 5' end-binding protein. Intriguingly, germline piRNAs in Drosophila are preferentially produced from unspliced transcripts, and cuff mutations significantly increase piRNA precursor splicing, and 5' cap binding by the nuclear cap binding complex (CBC) promotes splicing. Together, these findings suggest that that Cuff competes with the CBC for binding to capped cluster transcripts, suppressing splicing and promoting piRNA biogenesis. However, tethering Cuff to a reporter transcript increases read-through transcription, consistent with suppression of transcription termination. The molecular function for Cuff in piRNA biogenesis thus remains enigmatic (Parhad, 2020).

All three RDC genes are rapidly evolving under positive selection, suggesting that adaptive evolution of the complex is driven by a genetic conflict with the transposons the piRNA pathway silences, but other mechanisms are possible. Previous work found that rapid evolution has modified the Rhi-Del interface, producing orthologs that function as mutant alleles when moved across species. Analysis of these cross-species incompatibilities defined an interaction between the Rhi shadow domain and Del that prevents ectopic assembly of piRNA cluster chromatin. Crosses between Drosophila melanogaster and Drosophila simulans, which are sibling species, lead to hybrid sterility and are important model for genetic control of reproductive isolation. Significantly, sterile hybrids between these species phenocopy piRNA pathway mutations. Adaptive evolution of piRNA pathway genes may therefore contribute to reproductive isolation and speciation (Parhad, 2020).

These findings also suggest that cross-species analysis of rapidly evolving genes may provide a powerful genetic approach to structure-function analysis. This study applies this approach to cuff. These studies indicate that adaptive evolution has targeted direct or indirect interactions among Cuff, the Del-TRF2 non-canonical transcriptional complex, and the transcriptional co-repressor C-terminal binding protein (CtBP). CtBP was first identified as a host binding partner of Adenovirus E1A and subsequently implicated in diverse developmental pathways and cancer. This study shows that Drosophila CtBP suppresses canonical transcription from promoters in transposon terminal repeats and from promoters flanking two major germline piRNA clusters. Significantly, in both contexts, activation of canonical transcription interferes with downstream non-canonical transcription and piRNA production. Adaptive evolution has therefore targeted interactions between Cuff and two transcription regulators, which coordinately control germline piRNA expression (Parhad, 2020).

Adaptive evolution is a hallmark of genes engaged in a genetic conflict, which typically leads to co-evolution of host-pathogen gene pairs that encode interacting proteins. However, pathogens can also produce mimics that target interactions within host defense systems, raising the possibility that adaptation can also remodel interaction between host proteins. Supporting the possibility, adaptive evolution has remodeled an interface between the Rhi and Del, which are core components of the host transposon defense machinery. These adaptive changes prevent gene function across closely relates species and define an interaction that is required to restrict the RDC to piRNA clusters, which defines the specificity of the transposon silencing machinery. These findings suggest that adaptive evolution targets important functional domains, which can be functionally analyzed using cross-species complementation. This approach is applied to the third RDC component, cuff and shows that adaptive evolution targets interactions between this Rai1 homolog and proteins that coordinate canonical and non-canonical piRNA cluster transcription and piRNA biogenesis (Parhad, 2020).

Transposon silencing piRNAs are derived from heterochromatic clusters and a subset of euchromatic transposon insertions, and Cuff co-localizes with Rhi and Del at these piRNA source loci. Rhi binds to H3K9me3 marks and recruits Del. Del, in turn, binds Moon, which recruits TRF2 to initiate non-canonical transcription from both genomic strands. In contrast, the current data suggest that Cuff coordinates canonical and non-canonical cluster expression. D. simulans cuff ortholog fails to rescue D. melanogaster cuff mutations and leads to dominant sterility when overexpressed in wild-type flies. Significantly, these phenotypes are associated with stable binding to Del, TRF2, and CtBP. As noted above, Del and TRF2 function in non-canonical transcription of piRNA clusters. CtBP is a conserved transcriptional co-repressor, first identified as a host factor that binds to Adenovirus E1a, and subsequently shown to function in numerous developmental pathways. CtBP does not directly interact with DNA, but binds sequence specific transcription factors and recruits histone-modifying enzymes. This study shows that CtBP-kd activates canonical promoters linked to piRNA source loci. Adaptive evolution has therefore remodeled interactions between Cuff and factors that control both canonical and non-canonical transcription of piRNA precursors loci (Parhad, 2020).

Dominant phenotypes can result from mutations that produce new interactions or functions (neomorphic mutations) and assembly of complexes that are not formed by wild-type proteins. However, the current findings, with previous studies, suggest that substitutions in sim-Cuff stabilize normally transient complexes with both TRF2 and CtBP. In D. melanogaster, Cuff and Del do not co-precipitate, but the proteins co-localize to nuclear foci, interact in two-hybrid assays, and are co-dependent for association with piRNA clusters (Mohn, 2014). Del, in turn, co-precipitates with TRF2 and Moon, and all three proteins are required for non-canonical cluster transcription (Andersen, 2017), but TRF2 does not normally accumulate at clusters. In contrast, overexpression of sim-Cuff drives TRF2 co-localization with the RDC. Similarly, ChIP-seq shows that Cuff and Del localize to canonical promoters that are suppressed by CtBP, but CtBP does not accumulate at these promoters. Substitutions in the sim-Cuff ortholog thus appear to stabilize normally transient associations with Del and TRF2 and with CtBP (Parhad, 2020).

The majority of Drosophila germline clusters are transcribed from internal non-canonical initiation sites and do not have flanking canonical promoters. CtBP-kd does not significantly alter long RNA or piRNA expression from these loci. However, canonical promoters flank the right side of the 42AB cluster and both ends of the 38C cluster, and CtBP-kd increases transcription from these canonical promoters, which is associated with reduced transcription and piRNA production from downstream regions]. It has not been possible directly assay non-canonical transcription at most transposon insertions that produce piRNAs, as the inserted sequences are repeated, but CtBP-kd increases canonical transcription of transposons and is linked to collapse of piRNAs mapping to sequences flanking these insertions. In addition, deletion of the promoters flanking 42AB and 38C leads to spreading of piRNA production into flanking domains (Andersen, 2017). Together, these findings indicate that canonical transcription directly or indirectly represses non-canonical transcription and piRNA production (Parhad, 2020).

On the basis of these findings, it is proposed that Cuff coordinates canonical and non-canonical piRNA precursor transcription. By stabilizing Rhi, Del, Moon and TRF2, Cuff promotes non-canonical transcription. By contrast, Cuff appears to function with CtBP to control canonical transcription. Rescue of cuff mutants with sim-Cuff, which shows enhanced binding to CtBP, is phenocopied by CtBP-kd: both lead to increased canonical transcription. Formation of stable complexes with sim-Cuff thus appears to inhibit CtBP, activating canonical transcription and reducing downstream non-canonical transcription. Normally, the interaction between Cuff and CtBP is weak and free CtBP suppresses canonical promoters, while Cuff functions with Del-TRF2 to drive of non-canonical transcription. It is speculated that this balance may be altered in response to stress or environmental signals, which can activate transposons. Intriguingly, CtBP is also an NADH/NAD binding protein, suggesting that the balance between canonical and non-canonical piRNA precursor transcriptions may be regulated in response to metabolic state (Parhad, 2020).

The RDC proteins Moon and TRF2 are required for piRNA precursor transcription, and all of these factors are rapidly evolving. By contrast, CtBP is conserved from flies to humans, and a putative human oncogene. The data presented here, with earlier analysis of Rhi and Del (Parhad, 2017), indicate that rapid evolution has modified multiple interactions between rapidly evolving proteins in the piRNA biogenesis, and association of these proteins with a highly conserved transcriptional co-repressor. Rapidly evolving genes with specialized functions are frequently the most accessible to phenotype-based forward genetic approaches in model systems, and linking these specialized genes to conserved pathways can be a challenge. The studies reported in this study indicate that cross-species studies can help define these links, bridging the gap between genetically tractable model organisms and human biology (Parhad, 2020).

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

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

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

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

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

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

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

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


Search PubMed for articles about Drosophila Cutoff

Aravin, A. A., et al. (2003). The small RNA profile during Drosophila melanogaster development. Dev Cell 5: 337-350. PubMed ID: 12919683

Brennecke. J., Aravin, A. A., Stark, A., Dus, M., Kellis, M., Sachidanandam, R. and Hannon, G. J. (2007). Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila. Cell 128(6): 1089-103. PubMed ID: 17346786

Chen, Y., Pane, A. and Schupbach, T. (2007). cutoff and aubergine mutations result in retrotransposon upregulation and checkpoint activation in Drosophila. Curr. Biol. 17(7): 637-42. PubMed ID: 17363252

de Vanssay, A., Bouge, A. L., Boivin, A., Hermant, C., Teysset, L., Delmarre, V., Antoniewski, C. and Ronsseray, S. (2012). Paramutation in Drosophila linked to emergence of a piRNA-producing locus. Nature 490: 112-115. PubMed ID: 22922650

El Hage, A., Koper, M., Kufel, J. and Tollervey, D. (2008). Efficient termination of transcription by RNA polymerase I requires the 5' exonuclease Rat1 in yeast. Genes Dev. 22: 1069-1081. PubMed ID: 18413717

Gunawardane, L. S., Saito, K., Nishida, K. M., Miyoshi, K., Kawamura, Y., Nagami, T., Siomi, H. and Siomi, M. C. (2007). A slicer-mediated mechanism for repeat-associated siRNA 5' end formation in Drosophila. Science. 315(5818): 1587-90. PubMed ID: 17322028

Khurana, J. S. and Theurkauf, W. (2010). piRNAs, transposon silencing, and Drosophila germline development. J. Cell Biol. 191: 905-913. PubMed ID: 21115802

Kim, M., et al. (2004). The yeast Rat1 exonuclease promotes transcription termination by RNA polymerase II. Nature 432: 517-522. PubMed ID: 15565157

Klattenhoff, C., et al. (2009). The Drosophila HP1 homolog Rhino is required for transposon silencing and piRNA production by dual-strand clusters. Cell 138: 1137-1149. PubMed ID: 19732946

Le Thomas, A., Stuwe, E., Li, S., Du, J., Marinov, G., Rozhkov, N., Chen, Y. C., Luo, Y., Sachidanandam, R., Toth, K. F., Patel, D., Aravin, A. A. (2014) Transgenerationally inherited piRNAs trigger piRNA biogenesis by changing the chromatin of piRNA clusters and inducing precursor processing. Genes Dev 28: 1667-1680. PubMed ID: 25085419

Malone, C. D., et al. (2009). Specialized piRNA pathways act in germline and somatic tissues of the Drosophila ovary. Cell 137: 522-535. PubMed ID: 19395010

Mevel-Ninio, M., et al. (2007). The flamenco locus controls the gypsy and ZAM retroviruses and is required for Drosophila oogenesis. Genetics 175: 1615-1624. PubMed ID: 17277359

Mohn, F., Sienski, G., Handler, D. and Brennecke, J. (2014). The Rhino-Deadlock-Cutoff complex licenses noncanonical transcription of dual-strand piRNA clusters in Drosophila. Cell 157: 1364-1379. PubMed ID: 24906153

Moshkovich, N. and Lei, E. P. (2010). HP1 recruitment in the absence of argonaute proteins in Drosophila. PLoS Genet 6: e1000880. PubMed ID: 20300658

Pane, A., Jiang, P., Zhao, D. Y., Singh, M. and Schüpbach, T. (2011). The Cutoff protein regulates piRNA cluster expression and piRNA production in the Drosophila germline. EMBO J. 30(22): 4601-15. PubMed ID: 21952049

Richard, P. and Manley, J. L. (2009). Transcription termination by nuclear RNA polymerases. Genes Dev 23: 1247-1269. PubMed ID: 19487567

Saito, K., et al. (2006). Specific association of Piwi with rasiRNAs derived from retrotransposon and heterochromatic regions in the Drosophila genome. Genes Dev 20: 2214-2222. PubMed ID: 16882972

Saito, K. and Siomi, M. C. (2010). Small RNA-mediated quiescence of transposable elements in animals. Dev. Cell 19: 687-697. PubMed ID: 21074719

Senti, K. A. and Brennecke, J. (2010). The piRNA pathway: a fly's perspective on the guardian of the genome. Trends Genet. 26: 499-509. PubMed ID: 20934772

Volpe, A. M., et al. (2001). Drosophila rhino encodes a female-specific chromo-domain protein that affects chromosome structure and egg polarity. Genetics 159: 1117-1134. PubMed ID: 11729157

Xue, Y., et al. (2000). Saccharomyces cerevisiae RAI1 (YGL246c) is homologous to human DOM3Z and encodes a protein that binds the nuclear exoribonuclease Rat1p. Mol. Cell Biol. 20: 4006-4015. PubMed ID: 10805743

Yin, H. and Lin, H. (2007) An epigenetic activation role of Piwi and a Piwi-associated piRNA in Drosophila melanogaster. Nature 450: 304-308. PubMed ID: 17952056

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date revised: 23 August 2014

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