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.

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PIWI-interacting RNAs (piRNAs) repress transposons to maintain germline genome integrity. Previous studies showed that artificial tethering of Armitage (Armi) to reporter RNAs induced piRNA biogenesis. However, the lack of female sterile (1) Yb (Yb) in Drosophila ovarian somatic cells (OSCs) impaired the production of transposon-targeting piRNAs, even in the presence of Armi. Here, we show that the specific interaction of Armi with RNA transcripts of the flamenco piRNA cluster, the primary source of transposon-targeting piRNAs in OSCs, is strictly regulated by Yb. The lack of Yb allowed Armi to bind RNAs promiscuously, leading to the production of piRNAs unrelated to transposon silencing. The ATP hydrolysis-defective mutants of Armi failed to unwind RNAs and were retained on them, abolishing piRNA production. These findings shed light on distinct and collaborative requirements of Yb and Armi in transposon-targeting piRNA biogenesis. We also provide evidence supporting the direct involvement of Armi but not Yb in Zucchini-dependent piRNA phasing.

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.

PIWI-interacting RNAs (piRNAs) are small non-coding RNAs that repress transposons in animal germlines. This protects the genome from the invasive DNA elements. piRNA pathway failures lead to DNA damage, gonadal development defects, and infertility. Thus, the piRNA pathway is indispensable for the continuation of animal life. piRNA-mediated transposon silencing occurs in both the nucleus and cytoplasm while piRNA biogenesis is a solely cytoplasmic event. piRNA production requires a number of proteins, the majority of which localize to non-membranous organelles that specifically appear in the gonads. Other piRNA factors are localized on outer mitochondrial membranes. In situ RNA hybridization experiments show that piRNA precursors are compartmentalized into other non-membranous organelles. In this review, we summarize recent findings about the function of these organelles in the Drosophila piRNA pathway by focusing on their assembly and function.