Requirements for multivalent Yb body assembly in transposon silencing in Drosophila. 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 known as the site of PIWI-interacting RNA (piRNA) biogenesis in Drosophila ovarian somatic cells. Yb consists of 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 investigated the functions of the two other domains and found that Hel-C and eTud are necessary for Yb to self-associate and to interact with RNAs and other Yb body components, respectively. Both domains were essential for Yb body formation and transposon silencing. Without eTud, piRNA production was completely impaired. Loss of Hel-C severely reduced the levels of transposon-targeting piRNAs, although genic piRNAs unrelated to transposon silencing were still produced. Similar phenotypes were observed when mutations in flamenco, the primary source of transposon-targeting piRNAs, led to the expression of attenuated RNAs. Yb bodies are liquid-like multivalent condensates whose assembly depends on Yb self-association and widespread Yb−flamenco RNA binding. Overall design: Four piRNA-seq libraries: piRNA libraries were prepared from RNA associated with Flag-Piwi immunoprecipitates (with size selection at 23-30 nt).

雌性不育(1) Yb（Yb）是Yb小体的核心组分，Yb小体是一类核周聚集点，为果蝇卵巢体细胞中PIWI互作RNA（piRNA）的生物发生位点。Yb蛋白包含解旋酶C端（Hel-C）、RNA解旋酶以及延伸Tudor（eTud）结构域。我们既往研究表明，RNA解旋酶结构域对于Yb与RNA的结合、Yb小体形成以及piRNA生物发生均不可或缺。本研究针对其余两个结构域的功能展开探究，结果发现Hel-C结构域与eTud结构域分别是Yb自身聚集、Yb与RNA及其他Yb小体组分相互结合的必要条件。二者均为Yb小体形成与转座子沉默所必需。缺失eTud结构域会完全阻断piRNA的生成；而Hel-C结构域的缺失则会显著降低靶向转座子的piRNA水平，但此时与转座子沉默无关的基因源性piRNA仍可正常产生。当flamenco（靶向转座子piRNA的主要来源位点）发生突变并表达弱化RNA时，可观察到类似表型。Yb小体属于类液态多价凝聚体，其组装依赖于Yb的自身聚集以及Yb与flamenco RNA的广泛结合。总体实验设计：共构建4个piRNA测序文库：文库均制备自Flag-Piwi免疫沉淀所结合的RNA（片段大小筛选范围为23~30 nt）。