Requirements for multivalent Yb body assembly in transposon silencing in Drosophila

Abstract

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.

Armitage determines Piwi−piRISC processing from precursor formation and quality control to inter‐organelle translocation

“…In OSCs, transposon-repressing piRISC formation starts with Ybbody formation, which is initiated by multivalent cis-trans association between Yb and flam RNAs [28]. Other Yb-body components are subsequently incorporated into the bodies in a hierarchical manner: Armi goes in first and then the heterodimer composed of SoYb and Vret joins the body structures.…”

“…To this end, we expressed Armi-Flag N756A mutant in OSCs where Zuc and Armi were depleted by RNAi and then performed immunoprecipitation. These factors probably depend markedly on Yb-Armi protein-protein interaction [28]. Immunofluorescence showed that this mutant does not accumulate to mitochondria and localizes to Yb bodies in Zuc-and Armi-lacking OSCs (Figs 5B and EV5C and D).…”

“…The fly extract was prepared as shown previously [28]. The fly extract was prepared as shown previously [28].…”

“…OSCs were cultured at 26°C in Shields and Sang M3 medium (USBiological) supplemented with 10% fetal bovine serum (Funakoshi), 0.6 mg/ml glutathione, 10 mU/ml insulin, and 10% fly extract. The fly extract was prepared as shown previously [28]. S2 cells were cultured at 26°C in Schneider's Drosophila medium (Gibco) supplemented with 10% fetal bovine serum (Equitech-Bio) and 1× peni-cillinÀstreptomycinÀglutamine (Gibco) [31].…”

Assembly and Function of Gonad-Specific Non-Membranous Organelles in Drosophila piRNA Biogenesis

“…Therefore, Yb is considered the core component of Yb body assembly. The hierarchy of the other components in Yb body assembly is Armi to the SoYb-Vret heterodimer [61][62][63][64][65]92]. SoYb and Vret interact with and stabilize each other in vivo [92].…”