Yb Bodies Research Articles

PiRNA Biogenesis and Function in Drosophila

PIWI‐interacting RNAs (piRNAs) repress transposons to protect the germline genome from their harmful invasion. To this end, piRNAs stoichiometrically associate with members of the PIWI protein family to form piRNA‐induced silencing complexes (piRISCs) and target them to their RNA substrates via Watson‐Crick (RNA‐RNA) base pairing. Drosophila expresses three PIWI members, Piwi, Aubergine (Aub), and Argonaute3 (Ago3); the lack of any of these members causes transposon derepression, leading to severe defects in gonadal development and infertility, showing their functional non‐redundancy. In Drosophila ovarian somatic cells (OSCs), transposon‐repressing piRNAs originate from a soma‐specific piRNA cluster flamenco (flam) on the X chromosome. The flam locus is rich in transposon remnants, whose directions of insertion at the locus are mostly opposite to the direction of flam transcription. Therefore, by their nature, flam‐derived piRNAs are created to act as antisense oligonucleotides to silence their parental transposons. OSCs express Piwi but no other PIWI proteins. The Piwi‐piRISC assembly takes place in the cytoplasm. The complex is then imported into the nucleus and represses transposons at the transcriptional level. In the current model, Armitage (Armi) brings the Piwi‐piRISC precursor (pre‐piRISC) to mitochondria, where Zucchini‐dependent piRISC maturation occurs. We recently found that Armi is necessary for Piwi‐pre‐piRISC formation at Yb bodies and that Armi triggers the exit of Piwi‐pre‐piRISC from Yb bodies prior to translocation to mitochondria. Piwi‐pre‐piRISC resists leaving Yb bodies until Armi binds Piwi‐pre‐piRISC through piRNA precursor. Lack of Armi N‐terminus also blocks the Piwi‐pre‐piRISC exit from Yb bodies. Thus, Armi determines Piwi‐piRISC processing, in a multilayered manner, from precursor formation and quality control to inter‐organelle translocation for maturation.

Armitage determines Piwi-piRISC processing from precursor formation and quality control to inter-organelle translocation.

Piwi and piRNA form the piRNA-induced silencing complex (piRISC) to repress transposons. In the current model, Armitage (Armi) brings the Piwi-piRISC precursor (pre-piRISC) to mitochondria, where Zucchini-dependent piRISC maturation occurs. Here, we show that Armi is necessary for Piwi-pre-piRISC formation at Yb bodies and that Armi triggers the exit of Piwi-pre-piRISC from Yb bodies and the translocation to mitochondria. Piwi-pre-piRISC resist leaving Yb bodies until Armi binds Piwi-pre-piRISC through the piRNA precursors. The lack of the Armi N-terminus also blocks the Piwi-pre-piRISC exit from Yb bodies. Thus, Armi determines Piwi-piRISC processing, in a multilayered manner, from precursor formation and quality control to inter-organelle translocation for maturation.

Requirements for multivalent Yb body assembly in transposon silencing in Drosophila.

Female sterile (1) Yb (Yb) is a primary component of Yb bodies, perinuclear foci considered to be the site of PIWI-interacting RNA (piRNA) biogenesis in Drosophila ovarian somatic cells (OSCs). Yb consists of three domains: Helicase C-terminal (Hel-C), RNA helicase, and extended Tudor (eTud) domains. We previously showed that the RNA helicase domain is necessary for Yb-RNA interaction, Yb body formation, and piRNA biogenesis. Here, we investigate the functions of Hel-C and eTud and reveal that Hel-C is dedicated to Yb-Yb homotypic interaction, while eTud is necessary for Yb-RNA association, as is the RNA helicase domain. All of these domains are indispensable for Yb body formation and transposon-repressing piRNA production. Strikingly, however, genic piRNAs unrelated to transposon silencing are produced in OSCs where Yb bodies are disassembled. We also reveal that Yb bodies are liquid-like multivalent condensates whose assembly depends on Yb-Yb homotypic interaction and Yb binding particularly with flamenco RNA transcripts, the source of transposon-repressing piRNAs. New insights into Yb body assembly and biological relevance of Yb bodies in transposon silencing have emerged.

Yb body assembly on the flamenco piRNA precursor transcripts reduces genic piRNA production.

In Drosophila ovarian somatic cells, PIWI-interacting small RNAs (piRNAs) against transposable elements are mainly produced from the ∼180-kb flamenco (flam) locus. flam transcripts are gathered into foci, located close to the nuclear envelope, and processed into piRNAs in the cytoplasmic Yb bodies. The mechanism of Yb body formation remains unknown. Using RNA fluorescence in situ hybridization, we found that in the follicle cells of ovaries the 5′-ends of flam transcripts are usually located in close proximity to the nuclear envelope and outside of Yb bodies, whereas their extended downstream regions mostly overlap with Yb bodies. In flamKG mutant ovaries, flam transcripts containing the first and, partially, second exons but lacking downstream regions are gathered into foci at the nuclear envelope, but Yb bodies are not assembled. Strikingly, piRNAs from the protein-coding gene transcripts accumulate at higher levels in flamKG ovaries indicating that piRNA biogenesis may occur without Yb bodies. We propose that normally in follicle cells, flam downstream transcript regions function not only as a substrate for generation of piRNAs but also as a scaffold for Yb body assembly, which competitively decreases piRNA production from the protein-coding gene transcripts. By contrast, in ovarian somatic cap and escort cells Yb body assembly does not require flam transcription.

Recruitment of Armitage and Yb to a transcript triggers its phased processing into primary piRNAs in Drosophila ovaries.

Small RNAs called PIWI -interacting RNAs (piRNAs) are essential for transposon control and fertility in animals. Primary processing is the small RNA biogenesis pathway that uses long single-stranded RNA precursors to generate millions of individual piRNAs, but the molecular mechanisms that identify a transcript as a precursor are poorly understood. Here we demonstrate that artificial tethering of the piRNA biogenesis factor, Armi, to a transcript is sufficient to direct it into primary processing in Drosophila ovaries and in an ovarian cell culture model. In the fly ovarian somatic follicle cells, the transcript becomes cleaved in a stepwise manner, with a 5′→3′ directionality, liberating U1-containing ~24 nt piRNAs that are loaded into Piwi. Although uridines are preferred for generation of piRNA 5′ ends, processing takes place even in their absence, albeit at a lower efficiency. We show that recombinant Armi has 5′→3′ helicase activity, and mutations that abolish this activity also reduce piRNA processing in vivo. Another somatic piRNA pathway factor Yb, an interactor of Armi, is also able to trigger piRNA biogenesis when tethered to a transcript. Tethering-mediated primary piRNA biogenesis is also functional in the fly ovarian germline and loads all the three PIWI proteins present in this environment. Our study finds a broad correlation between piRNA processing and localization of the tethered factors to the cytoplasmic perinuclear ribonucleoprotein granules called germline nuage or somatic Yb bodies. We conclude that transcripts bound by Armi and Yb are identified as piRNA precursors, resulting in localization to cytoplasmic processing granules and their subsequent engagement by the resident piRNA biogenesis machinery.

Characterization of Armitage and Yb containing granules and their relationship to nuage in ovary-derived cultured silkworm cell

PIWI-interacting RNAs (piRNAs) are a class of endogenous small non-coding RNAs, which are mostly 24–32 nucleotides in length and interact specifically with PIWI subfamily of argonaute proteins. Despite the significant research progress in germ line piRNA pathway, its role in somatic cell is not well known. In Drosophila ovarian somatic cell, maturation of primary piRNA and its loading onto Piwi occurs at perinuclear Yb body. The Armitage (Armi) and Yb proteins are the major components of Yb body and specially expressed in ovarian somatic cell. Based on the reports, here we studied the BmArmi and BmYb in Bombyx mori ovary-derived BmN4 cells expressing BmVasa. In this study, we show that BmArmi and BmYb co-localized with BmVasa at nuage. The helicase domains of BmArmi and BmYb are important for nuage localization. Moreover, RNAi of piRNA components reveal that BmArmi depend on BmAgo3 for nuage localization, and BmArmi and BmYb form cytoplasmic granules independently in the absence of BmVasa. Our results provide evidence that the BmArmi and BmYb coexist with BmVasa and play an important role in perinuclear nuage granules formation in ovary-derived BmN4 cell.

Somatic Primary piRNA Biogenesis Driven by cis-Acting RNA Elements and trans-Acting Yb.

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. Here, we identify 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. Yb, a core component of primary piRNA biogenesis center Yb bodies, directly bound the Tj-cis element. Disruption of this interaction markedly reduced piRNA production. Thus, Yb is the trans-acting partner of the Tj-cis element. Yb-CLIP revealed that Yb binding correlated with somatic piRNA production but Tj-cis element downstream sequences produced few artificial piRNAs. We thus propose 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.

Yb Integrates piRNA Intermediates and Processing Factors into Perinuclear Bodies to Enhance piRISC Assembly

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. Here, we show 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. We suggest that Yb is a key upstream factor in the cytoplasmic phase of the piRNA pathway in ovarian somatic cells.

A systematic analysis ofDrosophilaTUDOR domain-containing proteins identifies Vreteno and the Tdrd12 family as essential primary piRNA pathway factors

PIWI proteins and their bound PIWI-interacting RNAs (piRNAs) form the core of a gonad-specific small RNA silencing pathway that protects the animal genome against the deleterious activity of transposable elements. Recent studies linked the piRNA pathway to TUDOR biology as TUDOR domains of various proteins bind symmetrically methylated Arginine residues in PIWI proteins. We systematically analysed the Drosophila TUDOR protein family and identified four previously not characterized TUDOR domain-containing proteins (CG4771, CG14303, CG11133 and CG31755) as essential piRNA pathway factors. We characterized CG4771 (Vreteno) in detail and demonstrate a critical role for this protein in primary piRNA biogenesis. Vreteno physically and/or genetically interacts with the primary pathway components Piwi, Armitage, Yb and Zucchini. Vreteno also interacts with the Tdrd12 orthologues CG11133 (Brother of Yb) and CG31755 (Sister of Yb), which are essential for the primary piRNA pathway in the germline and probably replace the function of the related but soma-specific factor Yb.

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

Despite exciting progress in understanding the Piwi-interacting RNA (piRNA) pathway in the germ line, less is known about this pathway in somatic cells. We showed previously 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. Here, we report 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, we 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.

P41. Primary piRNA biogenesis occurs through Yb bodies in the Drosophila ovarian somatic cells

PIWI proteins, a subset of Argonaute proteins that are the key factors in small RNA-mediated gene silencing, are expressed predominantly in the germline and are associated specifically with PIWI-interacting RNAs (piRNAs), small RNAs expressed predominantly in the germline. The PIWI-piRNA complex silences transposable elements and other genes that show complementarities to the piRNA sequences. Without functions of PIWI proteins and piRNAs, transposable elements become active, which has high potential to injure the genome. Thus, both PIWI proteins and piRNAs are essential for maintenance of genomic integrity and germline development. piRNA in Drosophila ovarian somatic cells (OSCs) are generated only through the primary processing pathway and are loaded onto Piwi, a member of PIWI proteins. However, factors required for the primary piRNA processing remain elusive. We focused on elucidating the mechanism of the primary processing pathway using the OSC line, which we have established from the female germline stem cell/ovarian somatic sheet (fGS/OSS) line, and found that primary piRNA biogenesis occur through Yb bodies, cytoplasmic structures that have been previously implicated in RNA processing. armitage (armi), a gene encoding an ATP-dependent RNA helicase, is necessary for primary piRNA biogenesis in OSCs. Armi localizes Piwi to Yb bodies by associating with Piwi and Yb, the main component of Yb bodies. piRNA intermediates are loaded onto the Armi-Piwi complex and processed into mature piRNAs. Loss of Yb caused depletion of Yb bodies and resulted in a reduction in overall piRNA levels. Yb bodies also impacted piRNA intermediates production, suggesting that they play important roles in primary piRNA biogenesis. zucchini (zuc), a gene encoding a putative cytoplasmic nuclease, is involved in processing piRNA intermediates into mature piRNAs. Without Armi, Yb or Zuc, Piwi is not loaded with piRNAs and does not localize to nucleus. A Piwi mutant that lacks the nuclear localization signal and therefore is not localized in the nucleus failed to silence transposons. These data suggest that Armi, Yb and Zuc are required for primary piRNA biogenesis and Piwi function. We further identified components of Yb bodies other than Armi and Yb and cloned piRNA intermediates. We will discuss in detail primary piRNA biogenesis based on our analyses.

Roles for the Yb body components Armitage and Yb in primary piRNA biogenesis in Drosophila

PIWI-interacting RNAs (piRNAs) protect genome integrity from transposons. In Drosophila ovarian somas, primary piRNAs are produced and loaded onto Piwi. Here, we describe 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. We propose that a functional Piwi-piRNA complex is formed and inspected in Yb bodies before its nuclear entry to exert transposon silencing.

An in vivo RNAi assay identifies major genetic and cellular requirements for primary piRNA biogenesis in Drosophila

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. We 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. We show that 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.

The Yb protein defines a novel organelle and regulates male germline stem cell self-renewal in Drosophila melanogaster

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. In this study, we show 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 µm 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.