female sterile (1) Yb: Biological Overview | References
Gene name - female sterile (1) Yb
Synonyms - Yb
Cytological map position - 3B3-3B3
Keywords - tudor domain RNA helicase, post-transcriptional gene regulation, regulates transposon silencing via the piRNA pathway, component of the Yb body, a site for Piwi-associated RNA biogenesis, regulates Piwi transport to the nucleus
Symbol - fs(1)Yb
FlyBase ID: FBgn0000928
Genetic map position - chrX:2720468-2724158
Classification - Tudor domain
Cellular location - cytoplasmic
Primary piRNAs in Drosophila ovarian somatic cells arise from piRNA cluster transcripts and the 3' UTRs of a subset of mRNAs, including Traffic jam (Tj) mRNA. However, it is unclear how these RNAs are determined as primary piRNA sources. This study identified a cis-acting 100-nt fragment in the Tj 3' UTR that is sufficient for producing artificial piRNAs from unintegrated DNA. These artificial piRNAs were effective in endogenous gene transcriptional silencing. The Tudor domain RNA helicase Yb, a core component of primary piRNA biogenesis center Yb bodies, directly binds the Tj-cis element. Disruption of this interaction markedly reduces piRNA production. Thus, Yb is the trans-acting partner of the Tj-cis element. Yb-CLIP revealed that Yb binding correlates with somatic piRNA production but Tj-cis element downstream sequences produced few artificial piRNAs. It is thus proposed that Yb determines primary piRNA sources through two modes of action: primary binding to cis elements to specify substrates and secondary binding to downstream regions to increase diversity in piRNA populations (Ishizu, 2015).
PIWI-interacting RNAs (piRNAs) interact with PIWI proteins to form piRNA-induced silencing complexes (piRISCs), which repress target genes, mostly transposons, either transcriptionally or at the post-transcriptional level by cleaving transcripts in the cytoplasm. Interestingly, not all cells in the gonads use both mechanisms. Follicle cells in Drosophila ovaries use transcriptional silencing but lack piRISC-mediated post-transcriptional silencing, while germ cells possess both transcriptional and post-transcriptional piRISC machineries. In Bombyx ovaries, only posttranscriptional silencing occurs. This variation largely depends on which PIWI proteins are expressed in a given cell type; transcriptional silencing requires nuclear PIWI proteins while post-transcriptional silencing requires cytoplasmic PIWI proteins (Ishizu, 2015).
Primary piRNAs are produced from single-stranded long noncoding RNAs transcribed from piRNA clusters in a Dicer-independent manner. The Drosophila genome contains 142 piRNA clusters (Brennecke, 2007), whose expression is regulated differently in different cell types. flamenco (flam), a representative of unidirectional piRNA clusters, is expressed only in follicle cells, whereas the bidirectional cluster 42AB is expressed specifically in nurse cells (Brennecke, 2007). The types of transposon fragments inserted in individual piRNA clusters also vary; therefore, piRNA populations differ among cell types. piRNAs in nurse cells are rather complex because primary piRNAs are amplified through the amplification loop, yielding secondary piRNAs (Ishizu, 2012). Recent studies showed that secondary piRNAs further produce phased trailer piRNAs (Han, 2015; Mohn, 2015). Follicle cells do not use this amplification system and thus only contain primary piRNAs (Ishizu, 2015).
The biogenesis of somatic primary piRNAs has been studied using ovaries and an ovarian somatic cell (OSC). A current model suggests that upon transcription flam-piRNA precursors are localized to perinuclear Flam bodies (Murota, 2014) and processed at adjacent Yb bodies (Olivieri, 2010; Saito, 2010). Yb bodies contain many piRNA factors besides Yb. Zucchini (Zuc), an endonuclease required for processing piRNA intermediates into mature piRNAs, is localized on the surface of mitochondria. Yb bodies tend to be observed in inter-mitochondrial regions. This arrangement of organelles appears crucial for accelerating piRNA processing because it centralizes all the necessary factors in the cytoplasm. Upon maturation, piRNAs associate with Piwi, a Drosophila PIWI protein, to form piRISCs, which are then translocated to the nucleus to implement nuclear transposon silencing through chromatin modifications on target transposon loci with support from co-factors such as GTSF1/ Asterix and Maelstrom (Ishizu, 2015).
flam is the major source of primary piRNAs in OSCs and follicle cells in the ovaries. flam is largely occupied by transposon remnants, whose orientation predominantly opposes that of the parental transposons; thus, most primary piRNAs arising from the piRNA cluster act as antisense oligos to repress parental transposons. Some protein-coding genes such as Traffic jam (Tj) also act as primary piRNA sources, and genic piRNA sources express proteins in OSCs and follicle cells. The TJ protein, encoded by Tj, is a large Maf transcriptional factor necessary for controlling gonad morphogenesis (Li, 2003). Loss of Tj function abolishes Piwi expression in follicle cells. However, Piwi expression in nurse cells is not influenced by TJ loss. Thus, the dependence of Piwi expression on TJ differs between follicle cells and germ cells (Ishizu, 2015).
Only a limited number of transcripts serve as somatic primary piRNA precursors. However, the mechanism underlying the recognition and selection of these transcripts as piRNA precursors is poorly understood. To better understand the mechanism, the Tj 3' UTR was used as representative of somatic primary piRNA sources to identify a cis element and its trans-acting partner necessary for producing primary piRNAs in OSCs (Ishizu, 2015).
Yb bodies and Flam bodies in OSCs are considered to be the centers for primary piRNA maturation/piRISC formation and piRNA intermediate storage, respectively, and exist in close proximity (Murota, 2014; Olivieri, 2010; Saito, 2010). The formation of both bodies depends on the Yb protein, particularly its RNA-binding activity (Murota, 2014). In the absence of this, piRNA processing fails, resulting in piRNA loss, although piRNA intermediates and processing factors are present in the cytosol. Thus, Yb binding to piRNA sources centralizes all necessary ingredients for piRNA biogenesis, which is crucial for primary piRNA production (Murota, 2014). This study discovered that the direct association of Yb with a specific ~100-nt element (i.e., cis element) within the piRNA precursors provokes somatic primary piRNA biogenesis from downstream regions. Insertion of the Yb-binding element within RNA molecules that do not otherwise serve as piRNA precursors converts the RNA transcripts into piRNA sources. Artificial primary piRNAs were mapped only downstream, but not upstream, of regions of the Yb-binding element. Previous studies demonstrated that natural genic piRNAs mostly arise from 3' UTRs rather than mRNA CDS or 5' UTRs. The present study also showed that few Tj-piRNAs mapped to the Tj CDS, and that few Yb-CLIP tags were also found in the Tj CDS. Thus, Yb determines not only substrate specificity but also processing directionality in the somatic primary piRNA biogenesis pathway. This may occur through the Yb- controlled recruitment of other piRNA factors, such as another putative RNA helicase Armi and endonuclease Zuc, only to downstream sequences (Ishizu, 2015).
Yb-CLIP tags greatly overlap with primary piRNA-producing loci in the genome. This strongly supports the idea that Yb is the central player in determining substrates in the piRNA pathway. An unexpected but intriguing observation in this study is that Tj-R1 and Tj-R2 in the Tj 3' UTR show strong Yb-binding marks, as does the Tj-cis element, but provoked very little artificial piRNA production in contrast to the Tj-cis element. Yb-CLIP experiments showed that Yb binding to Tj-R1 and Tj-R2 within the Drosophila genome largely depends on Yb binding to its upstream Tj-cis element. Therefore, a model is proposed in which Yb determines primary piRNA sources by two sequential modes of action: primary binding to cis elements that represents selection of piRNA precursors among cellular RNAs, then secondary binding to downstream regions, representing the defining domains to be processed by precursors. This complexity in determining piRNA precursors could ensure the high diversity in piRNA populations, which is a unique feature of piRNAs (Ishizu, 2015).
The RNA-binding activity of Yb is required for primary piRNA production in OSC. Yb mutants carrying a point mutation within the DEAD box showed little RNA binding activity (Murota, 2014). When these Yb mutants were expressed individually in OSC lacking endogenous Yb, piRNA precursors were not accumulated in Flam bodies, and few piRNAs were produced. As a consequence, transposons were de-silenced. Therefore, there is little doubt that the RNA-binding activity of Yb through the DEAD-box is indispensable for primary piRNA production. HITS-CLIP experiments clarified direct interaction of Yb with piRNA sources, including Tj mRNA. Insertion of a particular Yb-bound RNA element within Tj mRNA, i.e., the Tj-cis element, upstream of any given RNA molecule enables the arbitrary sequences to produce artificial piRNAs. Deletion of the Tj-cis element from the Drosophila genome significantly abolished piRNA production from its downstream region spanning at least ~200 nt. These observations strongly support the proposed model, in which Yb is the trans-acting factor that recognizes and binds cis elements within piRNA precursors to provoke primary piRNA biogenesis in ovarian somatic cells. However, it does not exclude the possibility that Yb collaboratively achieves this task with unknown factors. Moreover, it is not certain if Yb is the uppermost factor in the cytoplasmic phase of the biogenesis pathway upon nuclear transport of piRNA precursors (Ishizu, 2015).
PIWI-interacting RNAs (piRNAs) direct Piwi to repress transposons and maintain genome integrity in Drosophila ovarian somatic cells. piRNA maturation and association with Piwi occur at perinuclear Yb bodies, the centers of piRNA biogenesis. This study shows that piRNA intermediates arising from the piRNA cluster flamenco (flam) localize to perinuclear foci adjacent to Yb bodies, termed Flam bodies. RNAi-based screening of piRNA factors revealed that Flam body formation depends on Yb, the core component of Yb bodies, while Piwi and another Yb body component, Armitage, are dispensable for formation. Abolishing the RNA-binding activity of Yb disrupts both Flam bodies and Yb bodies. Yb directly binds flam, but not transcripts from neighboring protein-coding genes. Thus, Yb integrates piRNA intermediates and piRNA processing factors selectively into Flam bodies and Yb bodies, respectively. It is suggested that Yb is a key upstream factor in the cytoplasmic phase of the piRNA pathway in ovarian somatic cells (Murota, 2014).
This study visualized flam-piRNA intermediates in OSCs and follicle cells using RNA-FISH and EM-ISH and revealed that they concentrate at perinuclear Flam bodies. Flam bodies locate in very close proximity to Yb bodies, the sites of piRNA maturation and piRISC formation. It is postulated that flam signals might also be detectable within Yb bodies. However, this was not the case. The simplest explanation for this observation is that piRNA processing at Yb bodies occurs so quickly, and the processed piRNAs localize to the nucleus as piRISCs so immediately, that the flam signal at Yb bodies was below the level of detection at Yb bodies (Murota, 2014).
In Zuc-depleted cells, flam transcripts were detected predominantly as flam-piRNA intermediates, being several hundred to 4,000 nt in length, while the full transcriptional unit of flam is estimated to be over 180 kb. Both Yb body and Flam body formation require Yb, or more precisely, its RNA-binding activity through its NTD. Yb binds flam-piRNA intermediates directly. Based on these findings, a new model is proposed for primary piRNA biogenesis in ovarian soma, in which the association of Yb with piRNA intermediates, which most likely occurs in the cytoplasm because Yb is a cytoplasmic protein (Olivieri, 2010; Saito, 2010), is the initiation point of the cytoplasmic phase of piRNA biogenesis. This follows the nuclear phase of piRNA biogenesis: flam transcription and nuclear export of flam transcripts through the nuclear pores. flam transcription is initiated by RNA polymerase II and requires the transcriptional factor Cubitus interruptus. However, it is not known by which export factors and in what lengths the flam transcripts are exported from the nucleus. Further investigation will be required for a detailed understanding of the nuclear phase of piRNA biogenesis (Murota, 2014).
The locations of the genomic flam loci in the nucleus and Flam bodies do not seem to be arranged to be close to each other, meaning that the flam transcripts move a long distance to arrive at Flam bodies. Do the flam transcripts move within the nucleus to get closer to Flam bodies before export to the cytoplasm? Alternatively, does nuclear export occur first and then flam transcripts are localized to Flam bodies? Yb localization in the cytoplasm seems to be so dynamic that a point mutation in Yb that disrupts the RNA-binding capacity of Yb drastically changes the subcellular localization of Yb, causing it to be scattered evenly in the cytosol. Thus, the latter scenario appears more likely, in which Yb plays a crucial role; upon nuclear export, Yb captures flam transcripts through direct binding and localizes them, as flam-piRNA intermediates, to Flam bodies. Flam body formation depends on the RNA-binding activity of Yb, a cytoplasmic protein (Olivieri, 2010; Saito, 2010); this notion further supports the idea that Flam bodies are cytoplasmic structures (Murota, 2014).
Unlike flam transcripts, DIP1 mRNAs were virtually undetectable in Yb-CLIP tags, although the DIP1 protein-coding gene and the flam piRNA cluster are neighbors on chromosome X and DIP1 is expressed in OSCs. The sequences of Yb-CLIP tags were examined closely, but no obvious consensus sequences were found. Yb may recognize binding substrates owing to higher-order structures. Immunoelectron microscopy using an anti-Yb antibody showed that Yb bodies are often attached to mitochondria, to which Zuc endoribonuclease, the piRNA intermediate processor, anchors on the surface to face into cytoplasmic Yb bodies. This peculiar spatial arrangement of Zuc and Yb bodies, along with Flam bodies, integrates all the ingredients necessary for primary piRNA production locally, enhancing the rates of piRISC assembly. Another virtue of this perinuclear arrangement is that it enables assembled piRISCs to be immediately imported into the nucleus, where the RNP complex (i.e., the PIWI-piRNA complex) exerts its nuclear-specific function of silencing transposon transcription. How does Yb decide where within the perinuclear region to integrate all the materials necessary for primary piRNA biogenesis? Reconstitution of the whole machinery in, for instance, nongonadal somatic Schneider2 cells, in which no primary piRNAs are otherwise expressed, might address this fundamental question (Murota, 2014).
The piRNA pathway protects genomes by silencing mobile elements. Despite advances in understanding the processing events that generate piRNAs for silencing, little is known about how primary transcripts are transported from their genomic clusters to their processing centers. Using a model of the Drosophila COM/flamenco locus in ovarian somatic cells, this study identified a prominent nuclear structure called Dot COM, which is enriched in long transcripts from piRNA clusters but located far from their transcription sites. Remarkably, transcripts from multiple clusters accumulate at Dot COM, which is often juxtaposed with Yb-bodies, the cytoplasmic processing centers for cluster transcripts. Genetic evidence suggests that the accumulation of precursor transcripts at Dot COM represents one of the most upstream events in the piRNA pathway. These results provide new insights into the initial steps of the piRNA pathway, and open up a new research area important for a complete understanding of this conserved pathway (Dennis, 2013).
Despite exciting progress in understanding the Piwi-interacting RNA (piRNA) pathway in the germ line, less is known about this pathway in somatic cells. Previous work has shown that Piwi, a key component of the piRNA pathway in Drosophila, is regulated in somatic cells by Yb, a novel protein containing an RNA helicase-like motif and a Tudor-like domain. Yb is specifically expressed in gonadal somatic cells and regulates piwi in somatic niche cells to control germ line and somatic stem cell self-renewal. However, the molecular basis of the regulation remains elusive. This study reports that Yb recruits Armitage (Armi), a putative RNA helicase involved in the piRNA pathway, to the Yb body, a cytoplasmic sphere to which Yb is exclusively localized. Moreover, co-immunoprecipitation experiments show that Yb forms a complex with Armi. In Yb mutants, Armi is dispersed throughout the cytoplasm, and Piwi fails to enter the nucleus and is rarely detectable in the cytoplasm. Furthermore, somatic piRNAs are drastically diminished, and soma-expressing transposons are desilenced. These observations indicate a crucial role of Yb and the Yb body in piRNA biogenesis, possibly by regulating the activity of Armi that controls the entry of Piwi into the nucleus for its function. Finally, this study has discovered putative endo-siRNAs in the flamenco locus and the Yb dependence of their expression. These observations further implicate a role for Yb in transposon silencing via both the piRNA and endo-siRNA pathways (Qi, 2011).
This study reports that Yb is a novel component of the somatic piRNA pathway. Because Yb is localized only in the Yb body in somatic cells, the results further implicate the Yb body as a key site in the cytoplasm for piRNA biogenesis in ovarian somatic cells. How is Yb involved in the piRNA pathway? Previous studys have shown that Yb genetically acts upstream of Piwi to regulate its expression in somatic cells, yet physically, Armi is the only known piRNA pathway component that colocalizes and physically interacts with Yb. While this manuscript was in preparation and under consideration, Olivieri (2010) and Saito (2010) also reported the Armi-Yb interaction and their role in the piRNA pathway (Qi, 2011).
In addition, Haase (2010) reported the role of Armi in Piwi and piRNA expression. These studies verify the current observations. It is likely that Yb regulates Piwi via Armi, a RNA helicase. It is possible that Yb first recruits Armi to the Yb body, where the Armi-Yb complex, possibly involving other factors, might then serve as a site for the biogenesis and/or loading piRNAs and/or other factors to Piwi. The resulting Piwi-piRNA complex then enters the nucleus to achieve epigenetic regulatory functions. Such epigenetic regulation then leads to transposon silencing in somatic cells and niche cell function. Without Yb, Armi might fail to facilitate the biogenesis and/or loading of piRNA to Piwi. The unloaded Piwi might then fail to enter the nucleus and is subject to degradation. Of course, other possibilities exist, such as Armi-Yb interaction leading to the regulation of transcription or translation of Piwi. In any case, such regulation is specific to Piwi because Aub and Ago3 are not expressed in somatic cells in the ovary, and Yb mutations do not affect the expression or localization of Aub and Ago3 (Qi, 2011).
Although Yb has long been regarded as a protein required in somatic niche cells to regulate germ line and follicle stem cell division, its underlying molecular mechanism remains elusive. The current study reveals a molecular mechanism through which Yb functions in the niche cells: it regulates Piwi activity, possibly via Armi. The activated Piwi then enters the nucleus to epigenetically regulate the niche cell genome, which ensures the genome integrity and defines the niche signaling function toward stem cells (Qi, 2011).
In addition to its involvement in the piRNA pathway, Yb appears to be also involved in the endo-siRNA pathway. The gypsy6 endo-siRNA, which is decreased by 8-fold in the Yb mutant, is from the flamenco cluster. The flamenco cluster generates both endo-siRNAs and piRNAs. It is possible that the precursor transcripts of the piRNA pathway also serve as precursors of the endo-siRNA pathway, either in their original forms or as processed intermediates. Alternatively, piRNAs can target the single-stranded precursor of endo-siRNAs to form mature double-stranded precursors to produce endo-siRNAs. In either case, the dual role of Yb is reminiscent of the possible involvement of Piwi in the miRNA pathway. These observations together point to an 'inconvenient' fact, i.e. the specificity of Ago and Piwi proteins with respect to the siRNA/miRNA pathway versus piRNA pathway is only in a relative sense, just like the specificity of Drosophila Ago1 and Ago2 for the siRNA versus miRNA pathway is also a relative sense. In fact, the data suggest that Yb is required for transposon silencing, likely via both the piRNA and endo-siRNA pathways. Further investigation of the Yb-mediated mechanism should reveal a new dimension of the biogenesis and regulatory function of the piRNA pathway (Qi, 2011).
PIWI-interacting RNAs (piRNAs) protect genome integrity from transposons. In Drosophila ovarian somas, primary piRNAs are produced and loaded onto Piwi. This study describes roles for the cytoplasmic Yb body components Armitage and Yb in somatic primary piRNA biogenesis. Armitage binds to Piwi and is required for localizing Piwi into Yb bodies. Without Armitage or Yb, Piwi is freed from the piRNAs and does not enter the nucleus. Thus, piRNA loading is required for Piwi nuclear entry. It is proposed that a functional Piwi-piRNA complex is formed and inspected in Yb bodies before its nuclear entry to exert transposon silencing (Saito, 2010).
In Drosophila, three sets of endogenous small RNAs have been identified so far: microRNAs (miRNAs), endogenous siRNAs (endo-siRNAs/esiRNAs), and PIWI-interacting RNAs (piRNAs). Of these, piRNAs are considered unique because of their germline-specific expression and specific interaction with germline-specific Argonaute proteins, PIWI proteins. The identification of the piRNAs associated with three PIWI proteins (Aubergine [Aub], Argonaute 3 [AGO3], and Piwi) has revealed distinct features of piRNAs associated with each PIWI and has led to two models for piRNA biogenesis: the primary processing pathway and the amplification loop pathway. In the amplification loop model, the Slicer (endonuclease) activity of Aub and AGO3 determines the formation of the 5' end of piRNAs. Zucchini (Zuc), a putative cytoplasmic nuclease, is involved in the primary processing pathway; however, its precise molecular function remains unclear. Furthermore, the factors other than zuc required for primary piRNA biogenesis are unknown (Saito, 2010).
The ovarian somatic cell (OSC) line consists of ovarian somas only. The expression of Aub and AGO3 is not detectable in OSCs because both proteins are germ cell-specific. This implies that the amplification loop does not operate in OSCs. However, OSCs express piRNAs and are loaded onto Piwi, indicating that the piRNAs in OSCs are generated specifically through the primary processing pathway. Thus, OSCs are an ideal tool to elucidate the molecular mechanisms of primary piRNA processing and Piwi function. Loss of zuc function drastically reduced the level of primary piRNAs in the ovaries. This was recapitulated in OSCs: Zuc depletion by RNAi caused a severe reduction in the piRNA level in OSCs. This result prompted a screen for other factors necessary for primary piRNA production using RNAi in OSCs (Saito, 2010).
To identify the genes required for somatic primary piRNA biogenesis, RNAi-based screening was performed in OSCs. The genes screened included armitage (armi), spindle-E (spn-E), and maelstrome (mael), all of which are implicated in piRNA biogenesis. However, their roles in somatic primary piRNA production remain unknown. Depletion of Armi reduced the piRNA levels to an extent very similar to that of Piwi and Zuc depletion, indicating that Armi is necessary for primary piRNA biogenesis in OSCs. Depletion of Mael and Spn-E showed little or no effect on piRNA accumulation in OSCs. Mutations in both genes have been shown to significantly reduce the piRNA levels in ovaries. Thus, spn-E and mael are factors functioning in the amplification loop. Depletion of Dicer1 and Dicer2 had little or no effect on the piRNA levels, confirming that neither protein is necessary for piRNA production (Saito, 2010).
Armi is the Drosophila ortholog of Arabidopsis Silencing-Defective 3 (SDE3) and mammalian Moloney leukemia virus 10 (MOV10). These orthologs contain a conserved ATP-dependent RNA helicase domain at their C termini and have been implicated in small RNA-mediated gene silencing. However, their precise functions remain unknown. To gain further insight into the function of Armi in somatic primary piRNA processing, a monoclonal antibody was produced against Armi. Western blotting showed a discrete band in both ovary and cultured Schneider2 (S2) cell lysates, indicating that Armi expression is not germline-specific. The ~150-kDa protein immunopurified from S2 cells with the anti-Armi antibody was confirmed to be Armi by mass spectrometry (Saito, 2010).
Immunostaining of OSCs and ovaries with the anti-Armi antibody confirmed an earlier observation that Armi is a cytoplasmic protein. The Armi signals were detected in both somatic and germ cells of ovaries. The somatic signal was considered a background signal because it did not disappear even in armi homozygous mutant egg chambers. In the present study, the cytoplasmic signal in OSCs mostly disappeared when Armi was depleted by RNAi. Thus, it is concluded that Armi is expressed in both somatic and germ cells in ovaries (Saito, 2010).
The subcellular localization of Armi in the armi trans-heterozygous mutants appeared very similar to that in the homozygous mutants. In addition, Western blotting revealed a band corresponding to Armi in the armi ovaries. By what mechanisms Armi is expressed in the mutant somas remains unclear. The simplest explanation is that the armi gene uses two distinct genomic elements as promoters in ovarian somas. In fact, armi homozygous mutants weakly express a shorter armi transcript than that expressed in the wild-type strain (Saito, 2010).
The Armi signal in germ cells was rather weak, and only a small proportion of Armi accumulated at, or near, the nuage, an electron-dense structure associated with nurse cell nuclei. Thus, Armi might not be a component of the nuage per se. This correlates well with the fact that armi mutations barely affected the ability of the ovaries to amplify endogenous piRNAs. In ovarian somas, Armi accumulated strongly at discrete cytoplasmic foci. Each somatic cell contained one or several foci. Interestingly, the Armi-positive foci were often located near the nucleus in both ovaries and OSCs (Saito, 2010)
Piwi is required for the silencing of transposons in gonads. In fact, Piwi depletion in OSCs caused derepression of transposons, as with Armi, Yb, and Zuc depletion. Under conditions where endogenous Piwi was depleted, expression of myc-Piwi-r, which was designed to be RNAi-insensitive, rescued transposon silencing. However, myc-Piwi-δN, which lacks 72 amino acids at the N terminus of Piwi and thus does not localize to the nucleus, did not rescue transposon silencing, although it does associate with piRNAs to the same extent as does the wild-type Piwi. myc-Piwi-δN13, which lacks 13 amino acids at the N terminus, behaved similarly. On the other hand, myc-Piwi-DDAA-r, a Slicer mutant of Piwi, could bind to mature piRNAs in OSCs, as does the wild-type Piwi, and rescued transposon silencing. These results might suggest that Piwi must be localized in the nucleus to silence the transposable elements, and that Piwi Slicer activity is unnecessary for its function. It is assumed that this system has evolved to prevent nascent Piwi, not loaded with piRNAs, from being imported into the nucleus. In other words, only the functional Piwi-piRNA complex (piRISC) formed at Yb bodies could be transported to the nucleus. At present, the mechanisms of this control system remain unclear. In the nongonadal somatic S2 cell line, where the expression of piRNAs is undetectable, transfected Piwi is localized to the nucleus, indicating that 'empty' Piwi can be transported to the nucleus. It seems that the machineries necessary for the nuclear transport of Piwi might recognize different features of Piwi in different cell types (Saito, 2010).
How is piRNA-free Piwi restrained in the cytoplasm in OSCs? One possibility is that some unknown protein binds the N-terminal end of Piwi, where its NLS (nuclear localization signal) resides, and interferes with the nuclear import machinery's ability to recognize Piwi as a cargo. The nuclear localization inhibitory factors may be retained on Piwi until a functional Piwi-piRNA complex is formed at Yb bodies. Once the complex is formed, a conformational change in Piwi would be induced, which would release the regulatory factors and reveal the Piwi NLS for recognition by the nuclear import machinery. It would be very interesting to determine the proteins that are associated with Piwi in OSCs under conditions of Armi or Zuc depletion, thus identifying the protein factors that restrain Piwi in the cytoplasm until it is loaded with mature piRNAs at Yb bodies (Saito, 2010).
In Drosophila, PIWI proteins and bound PIWI-interacting RNAs (piRNAs) form the core of a small RNA-mediated defense system against selfish genetic elements. Within germline cells, piRNAs are processed from piRNA clusters and transposons to be loaded into Piwi/Aubergine/AGO3 and a subset of piRNAs undergoes target-dependent amplification. In contrast, gonadal somatic support cells express only Piwi, lack signs of piRNA amplification and exhibit primary piRNA biogenesis from piRNA clusters. Neither piRNA processing/loading nor Piwi-mediated target silencing is understood at the genetic, cellular or molecular level. This study developed an in vivo RNAi assay for the somatic piRNA pathway and identified the RNA helicase Armitage, the Tudor domain containing RNA helicase Yb and the putative nuclease Zucchini as essential factors for primary piRNA biogenesis. Lack of any of these proteins leads to transposon de-silencing, to a collapse in piRNA levels and to a failure in Piwi-nuclear accumulation. Armitage and Yb interact physically and co-localize in cytoplasmic Yb bodies, which flank P bodies. Loss of Zucchini leads to an accumulation of Piwi and Armitage in Yb bodies, indicating that Yb bodies are sites of primary piRNA biogenesis (Olivieri, 2010).
This paper describes a robust in vivo assay that allows the identification of genes with a critical involvement in the somatic piRNA pathway. It takes advantage of the recently constructed genome-wide RNAi library at the VDRC and, therefore, will allow the conduction of a genome-wide RNAi screen. The somatic support cells of the Drosophila ovary are the only described cells containing a piRNA pathway without ping-pong cycle. Elucidation of this pathway will, therefore, not only uncover the basic concepts behind the piRNA biogenesis and silencing machineries. It will also simplify the genetic and functional characterization of identified candidates, aided by the availability of the OSC culture system. Similar to any screen system, this assay has drawbacks such as availability of an RNAi line, potential off-target effects or inefficient knockdown. However, an RNAi screen has the major advantage that knockdown of genes essential for other vital functions in the cell might still allow the development of ovaries suitable for analysis, whereas EMS generated null alleles would often prevent this (Olivieri, 2010).
This assay was used to assign essential functions to the piRNA pathway proteins Armi and Zuc within the somatic pathway and identified Yb as a novel piRNA pathway gene. Detailed cell biological analyses in conjunction with protein interaction studies and piRNA sequencing efforts place all three factors upstream of the active Piwi–piRNA complex, either in piRNA biogenesis or in piRNA loading into Piwi. All three proteins are required for piRNA accumulation and for the nuclear accumulation of Piwi. The cytoplasmic localization of Armi, Yb and Zuc suggests that piRNA biogenesis/loading occurs in the cytoplasm. This is supported by the observation that N-terminally truncated Piwi that cannot translocate into the nucleus is still loaded with piRNAs. In addition, piRNAs derived from cellular mRNAs typically map to the 3′UTR, indicating that ribosomal association with those mRNAs did precede their funnelling into piRNA biogenesis (Olivieri, 2010).
The data indicate that unloaded Piwi does not translocate into the nucleus. This is highly reminiscent to the situation in mouse and Tetrahymena. The mouse PIWI family protein MIWI2 as well as the Tetrahymena Twi1 protein accumulate in the cytoplasm under conditions that do not allow piRNA biogenesis or loading into these factors. Furthermore, unloaded Piwi appears to be unstable in vivo. This effect is most apparent in ovaries lacking Armi and Zuc, two factors that are essential for Piwi–piRNA biogenesis/loading in soma and germline (Olivieri, 2010).
The results further indicate that aspects of piRNA biogenesis and/or piRNA loading take place in discrete peri-nuclear foci that have been previously termed Yb bodies (Szakmary, 2009). The two RNA helicases Armi and Yb localize both to Yb bodies and interact physically. Both proteins are required for their reciprocal localization to cytoplasmic foci and potentially for Yb-body formation per se. A strong argument for the importance of Yb bodies in the piRNA pathway stems from the observation that fs(1)Yb mutant ovaries also show Piwi loss from the nucleus. In this allele, a conserved Arginine residue in the Tudor domain is mutated. This still allows Yb protein expression, but blocks Yb-body formation (Szakmary, 2009). Ultra-structurally, Yb bodies were described as electron dense cytoplasmic spheres that are directly adjacent to an RNA containing electron dense structure (Szakmary, 2009). Localization studies with DCP1 indicate that these previously identified neighbouring structures are likely a subset of cellular P bodies. The functional significance of this is unclear, as no defects are detected in gypsy silencing upon knockdown of two central P-body components LSM1 and Me31b, which have been reported to lead to a dispersal of P bodies in Drosophila S2 cells. Nevertheless, the physical neighbourhood of these two bodies is intriguing and is reminiscent of the neighbouring and/or coinciding localization of a set of piRNA pathway proteins and P-body components in mouse germ cells and Drosophila nurse cells (Olivieri, 2010).
Of the three identified proteins, Yb is the only factor that seems specific for the Piwi pathway in somatic support cells. This is consistent with genetic analyses showing that germline-specific mutants for fs(1)Yb are fertile (King, 2001). The function of Yb in the somatic support cells is required for maintenance of somatic and indirectly also for germline stem cells (King, 2001). In this, fs(1)Yb mutants strongly resemble piwi mutants and indicate once again that the Piwi-dependent piRNA pathway in the somatic support cells is essential for germline stem cell niche maintenance. The specificity of Yb for the somatic cells raises the question, whether there is a related protein with equivalent functions in germline cells. Protein BLAST analysis indeed identified two proteins (CG31755 and CG11133) with similarity to Yb over most of their sequence (Szakmary, 2009). Similar to Yb, CG31755 and CG11133 also contain a recognizable Tudor domain downstream of their helicase domain. Both proteins are selectively expressed in ovaries and testes. These observations indicate that CG31755 and CG11133 could serve Yb's function within the germline. It is noted that knockdown of these proteins individually does not impact the somatic piRNA pathway (not shown). CG31755 and CG11133 share considerable similarity to mouse TDRD12, a gene with testis and oocyte-specific expression (Olivieri, 2010).
The function of Armi as an essential factor in primary piRNA biogenesis is conserved in mouse. In the absence of the germline-specific orthologue MOV10L1, spermatogenesis is blocked at a similar stage as in MILI mutants and MILI and MIWI2, which is thought to function downstream of MILI in the mouse ping-pong cycle are lacking bound piRNAs. In Drosophila, Armi is per se not required for the ping-pong cycle, indicating that the function of this RNA helicase is restricted to primary piRNA processing or Piwi loading. Two Armi isoforms are expressed in ovaries with germline cells expressing only the larger one. Although this might suggest functional specialization, it is noted that the two isoforms show an identical distribution in a glycerol gradient, interact both with Piwi and show identical subcellular localization in follicle cells based on GFP fusions (Olivieri, 2010).
Similar to Armi, Zuc is well conserved in vertebrates. The mouse orthologue PLD6 is expressed specifically in testes consistent with a function in the piRNA pathway. Zuc contains a single phospho-lipase D domain, which is predicted to confer endo-nucleolytic activity. The three important residues for catalysis are conserved in Zuc and a point mutation in the active site causes a similar de-localization of Piwi from the nucleus as the null mutant (not shown). These data indicate that Zuc function (potentially endo-nucleolytic generation of piRNA 5' or 3' ends) is required to release Piwi and Armi from Yb bodies. This study further showed that Zuc is essential for Piwi-piRNA biogenesis. This casts doubts on the rather weak effects of zuc mutations on flamenco piRNA biogenesis reported previously. Genotyping of trans-heterozygous zuc mutants from stocks balanced over CyO is complicated by a dominant wing phenotype of the zuc[Def] allele interfering with confident selection against the CyO balancer. It is suspected that this led to a mix of homozygous and heterozygous ovaries analysed in that study. It is unclear, however, how the defects in Piwi localization went unnoticed in such a situation (Olivieri, 2010).
The phenotype of armi and zuc mutant ovaries indicates that Piwi biology is similar in germline and somatic support cells. It is noted that the analysis of piRNAs bound by Piwi and AGO3 also showed a participation of Piwi in the germline-specific ping-pong cycle. However, in armi germline mutants, the ping-pong cycle is still active, yet Piwi is not loaded effectively (Malone, 2009). This suggests that primary piRNA biogenesis through Zuc/Armi is the major pathway feeding into Piwi in germline cells (Olivieri, 2010).
In summary, this study identified the RNA helicases Armi and Yb and the predicted nuclease Zuc as essential components of primary piRNA biogenesis. Peri-nuclear Yb bodies were linked to piRNA biogenesis, and it was shown that Piwi biology in germline and soma follows a common logic. The described genetic assay system and the availability of the cultured OSC line will be important resources to work towards a mechanistic understanding of piRNA biogenesis and the silencing mechanism, two of the most mysterious open questions in the small RNA field (Olivieri, 2010).
Yb regulates the proliferation of both germline and somatic stem cells in the Drosophila melanogaster ovary by activating piwi and hh expression in niche cells. This study shows that Yb protein is localized as discrete cytoplasmic spots exclusively in the somatic cells of the ovary and testis. These spots, which are different from all known cytoplasmic structures in D. melanogaster, are evenly electron-dense spheres 1.5 microm in diameter (herein termed the Yb body). The Yb body is frequently associated with mitochondria and a less electron-dense sphere of similar size that appears to be RNA rich. There are one to two Yb bodies/cell, often located close to germline cells. The N-terminal region of Yb is required for hh expression in niche cells, whereas the C-terminal region is required for localization to Yb bodies. The entire Yb protein is necessary for piwi expression in niche cells. A double mutant of Yb and a novel locus show male germline loss, revealing a function for Yb in male germline stem cell maintenance (Szakmary, 2000).
Stem cell research has been focused on niche signaling and epigenetic programming of stem cells. However, epigenetic programming of niche cells remains unexplored. Previous studies have shown that Piwi plays a crucial role in Piwi-interacting RNA-mediated epigenetic regulation and functions in the niche cells to maintain germline stem cells (GSCs) in the Drosophila ovary. To investigate the epigenetic programming of niche cells by Piwi, mutations in the Polycomb and trithorax group genes, and an enhancer of Polycomb and trithorax called corto, were screened for their potential genetic interaction with piwi. corto encodes a chromatin protein. corto mutations restored GSC division in mutants of piwi and fs(1)Yb (Yb), a gene that regulates piwi expression in niche cells to maintain GSCs. Consistent with this, corto appears to be expressed in the niche cells and is not required in the germline. Furthermore, in corto-suppressed Yb mutants, the expression of hedgehog (hh) is restored in niche cells, which is likely responsible for corto suppression of the GSC and somatic stem cell defects of Yb mutants. These results reveal a novel epigenetic mechanism involving Corto and Piwi that defines the fate and signaling function of niche cells in maintaining GSCs (Smulders-Srinivasan, 2010).
The coordinated division of distinctive types of stem cells within an organ is crucial for organogenesis and homeostasis. This study shows genetic interactions among fs(1)Yb (Yb), piwi, and hedgehog (hh) that regulate the division of both germline stem cells (GSCs) and somatic stem cells (SSCs), the two constituent stem cell populations of the Drosophila ovary. Yb is required for both GSC and SSC divisions; loss of Yb function eliminates GSCs and reduces SSC division, while Yb overexpression increases GSC number and causes SSC overproliferation. Yb acts via the piwi- and hh-mediated signaling pathways that emanate from the same signaling cells to control GSC and SSC division, respectively. hh signaling also has a minor effect in GSC division (King, 2001).
A screen was performed for female sterile mutations on the X chromosome of Drosophila and new loci were identified that are required for developmental events in oogenesis: new alleles of previously described genes were identified as well. The screen has identified genes that are involved in cell cycle control, intracellular transport, cell migration, maintenance of cell membranes, epithelial monolayer integrity and cell survival or apoptosis. New roles are described for the genes dunce, brainiac and fs(1)Yb, and new alleles of Sex lethal, ovarian tumor, sans filles, fs(1)K10, singed, and defective chorion-1 have been identified (Swan, 2001).
Drosophila oogenesis starts when a germline stem cell divides asymmetrically to generate a daughter germline stem cell and a cystoblast that will develop into a mature egg. This study shows that the fs(1)Yb gene is essential for the maintenance of germline stem cells during oogenesis. fs(1)Yb was delineated within a 6.4 kb genomic region by transgenic rescue experiments. fs(1)Yb encodes a 4.1 kb RNA that is present in the third instar larval, pupal and adult stages, consistent with its role in regulating germline stem cells during oogenesis. Germline clonal analysis shows that all fs(1)Yb mutations are soma-dependent. In the adult ovary, fs(1)Yb is specifically expressed in the terminal filament cells, suggesting that fs(1)Yb acts in these signaling cells to maintain germline stem cells. fs(1)Yb encodes a novel hydrophilic protein with no potential signal peptide or transmembrane domains, suggesting that this protein is not itself a signal but a key component of the signaling machinery for germline stem cell maintenance (King, 1999).
Phenotypic and genetic analyses demonstrate that fs(1)Yb activity is required in the soma for the development of a subset of ovarian follicle cells and to support later stages of egg maturation. Mutations in fs(1)Yb cause a range of ovarian phenotypes, from the improper segregation of egg chambers to abnormal dorsal appendage formation. The mutant phenotypes associated with fs(1)Yb are very similar to the ovarian aberrations produced by temperature-sensitive alleles of Notch and Delta. Possible functional or regulatory interactions between fs(1)Yb and Notch are suggested by genetic studies. A duplication of the Notch locus partially suppresses the female-sterility caused by fs(1)Yb mutations, while reducing Notch dosage makes the fs(1)Yb mutant phenotype more severe. In addition, fs(1)Yb alleles also interact with genes that are known to act with or regulate Notch activity, including Delta, daughterless, and mastermind. However, differences between the mutant ovarian phenotype of ffs(1)Yb and that of Notch or Delta indicate that the genes do not have completely overlapping functions in the ovary. It is proposed that fs(1)Yb acts as an ovary-specific factor that determines follicle cell fate (Johnson, 1995).
Search PubMed for articles about Drosophila Yb
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: 1089-1103. PubMed ID: 17346786
Dennis, C., Zanni, V., Brasset, E., Eymery, A., Zhang, L., Mteirek, R., Jensen, S., Rong, Y. S. and Vaury, C. (2013). 'Dot COM', a nuclear transit center for the primary piRNA pathway in Drosophila. PLoS One 8: e72752. PubMed ID: 24039799
Haase, A. D., Fenoglio, S., Muerdter, F., Guzzardo, P. M., Czech, B., Pappin, D. J., Chen, C., Gordon, A. and Hannon, G. J. (2010). Probing the initiation and effector phases of the somatic piRNA pathway in Drosophila. Genes Dev 24: 2499-2504. PubMed ID: 20966049
Han, B. W., Wang, W., Li, C., Weng, Z. and Zamore, P. D. (2015). Noncoding RNA. piRNA-guided transposon cleavage initiates Zucchini-dependent, phased piRNA production. Science 348: 817-821. PubMed ID: 25977554
Ishizu, H., Siomi, H. and Siomi, M. C. (2012). Biology of PIWI-interacting RNAs: new insights into biogenesis and function inside and outside of germlines. Genes Dev 26: 2361-2373. PubMed ID: 23124062
Ishizu, H., Iwasaki, Y. W., Hirakata, S., Ozaki, H., Iwasaki, W., Siomi, H. and Siomi, M. C. (2015). Somatic primary piRNA biogenesis driven by cis-acting RNA elements and trans-acting Yb. Cell Rep 12: 429-440. PubMed ID: 26166564
Johnson, E., Wayne, S. and Nagoshi, R. (1995). fs(1)Yb is required for ovary follicle cell differentiation in Drosophila melanogaster and has genetic interactions with the Notch group of neurogenic genes. Genetics 140: 207-217. PubMed ID: 7635286
King, F. J. and Lin, H. (1999). Somatic signaling mediated by fs(1)Yb is essential for germline stem cell maintenance during Drosophila oogenesis. Development 126: 1833-1844. PubMed ID: 10101118
King, F. J., Szakmary, A., Cox, D. N. and Lin, H. (2001). Yb modulates the divisions of both germline and somatic stem cells through piwi- and hh-mediated mechanisms in the Drosophila ovary. Mol Cell 7: 497-508. PubMed ID: 11463375
Li, M. A., Alls, J. D., Avancini, R. M., Koo, K. and Godt, D. (2003). The large Maf factor Traffic Jam controls gonad morphogenesis in Drosophila. Nat Cell Biol 5: 994-1000. PubMed ID: 14578908
Mohn, F., Handler, D. and Brennecke, J. (2015). Noncoding RNA. piRNA-guided slicing specifies transcripts for Zucchini-dependent, phased piRNA biogenesis. Science 348: 812-817. PubMed ID: 25977553
Murota, Y., Ishizu, H., Nakagawa, S., Iwasaki, Y. W., Shibata, S., Kamatani, M. K., Saito, K., Okano, H., Siomi, H. and Siomi, M. C. (2014). Yb integrates piRNA intermediates and processing factors into perinuclear bodies to enhance piRISC assembly. Cell Rep 8: 103-113. PubMed ID: 24953657
Olivieri, D., Sykora, M. M., Sachidanandam, R., Mechtler, K. and Brennecke, J. (2010). An in vivo RNAi assay identifies major genetic and cellular requirements for primary piRNA biogenesis in Drosophila. EMBO J 29: 3301-3317. PubMed ID: 20818334
Qi, H., Watanabe, T., Ku, H. Y., Liu, N., Zhong, M. and Lin, H. (2011). The Yb body, a major site for Piwi-associated RNA biogenesis and a gateway for Piwi expression and transport to the nucleus in somatic cells. J Biol Chem 286: 3789-3797. PubMed ID: 21106531
Saito, K., Ishizu, H., Komai, M., Kotani, H., Kawamura, Y., Nishida, K. M., Siomi, H. and Siomi, M. C. (2010). Roles for the Yb body components Armitage and Yb in primary piRNA biogenesis in Drosophila. Genes Dev 24: 2493-2498. PubMed ID: 20966047
Smulders-Srinivasan, T. K., Szakmary, A. and Lin, H. (2010). A Drosophila chromatin factor interacts with the Piwi-interacting RNA mechanism in niche cells to regulate germline stem cell self-renewal. Genetics 186: 573-583. PubMed ID: 20647505
Swan, A., Hijal, S., Hilfiker, A. and Suter, B. (2001). Identification of new X-chromosomal genes required for Drosophila oogenesis and novel roles for fs(1)Yb, brainiac and dunce. Genome Res 11: 67-77. PubMed ID: 11156616
Szakmary, A., Reedy, M., Qi, H. and Lin, H. (2009). The Yb protein defines a novel organelle and regulates male germline stem cell self-renewal in Drosophila melanogaster. J Cell Biol 185: 613-627. PubMed ID: 19433453
date revised: 9 September 2015
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