Maelstrom functions in the production of Siwi-piRISC capable of regulating transposons in Bombyx germ cells. Maelstrom functions in the production of Siwi-piRISC capable of regulating transposons in Bombyx germ cells

PIWI-interacting RNAs (piRNAs) bind to PIWI proteins to assemble the piRISC, which represses germline transposons. Maelstrom (Mael) is necessary for piRISC biogenesis in germ cells, but its function remains unclear. Here, we show that Mael interconnects Spindle-E (Spn-E), a key piRISC biogenesis factor, with unloaded Siwi, one of two silkworm PIWI members. Mael also assembles a subset of nuage, a non-membranous organelle involved in piRISC biogenesis. Loss of Mael abrogated the Spn-E–Siwi interaction and Ago3-piRISC biogenesis, but Siwi-piRISC was produced. Bioinformatic analysis showed that Siwi-bound piRNAs in Mael-lacking cells were rich in transposon-targeting piRNAs as in normal cells but were biased toward transposons that are marginally controlled by Siwi-piRISC. This explains the impairment in Ago3-piRISC production because transposon mRNAs cleaved by Siwi are the origin of Ago3-loaded piRNAs. We argue that Mael plays a role in the production of primary Siwi-piRISC capable of regulating transposon expression in germ cells. Overall design: Six smRNA-seq libraries: PIWI-associated small RNA libraries were prepared from small RNAs associated with Flag-Siwi immunoprecipitates with size selection at 22-35nt. Six RNA-seq libraries: RNA libraries were prepared from total RNAs with poly-A selection.

PIWI互作RNA（PIWI-interacting RNAs，piRNAs）与PIWI蛋白结合，组装形成piRNA诱导沉默复合物（piRISC），后者可抑制生殖系转座子（germline transposons）。漩涡蛋白（Maelstrom，Mael）对生殖细胞中piRISC的生物发生至关重要，但其具体功能仍未阐明。本研究发现，Mael可将关键piRISC生物发生因子纺锤体-E蛋白（Spindle-E，Spn-E）与家蚕PIWI家族的两个成员之一的空载Siwi蛋白连接起来；同时，Mael还能组装一类参与piRISC生物发生的非膜包被细胞器Nuage颗粒（nuage）。缺失Mael会破坏Spn-E与Siwi的相互作用以及Ago3-piRNA诱导沉默复合物（Ago3-piRISC）的生物发生，但Siwi-piRNA诱导沉默复合物（Siwi-piRISC）的生成不受影响。生物信息学分析显示，在缺失Mael的细胞中，与Siwi结合的piRNAs与正常细胞一样，富含靶向转座子的piRNAs，但这些piRNAs更倾向于靶向那些仅受Siwi-piRISC微弱调控的转座子。这一结果解释了Ago3-piRISC生成受损的原因：被Siwi切割的转座子mRNA是装载至Ago3的piRNAs的来源。本研究认为，Mael的作用是促进能够调控生殖细胞中转座子表达的原发性Siwi-piRISC的生成。实验整体设计：构建6个小分子RNA测序（small RNA sequencing，smRNA-seq）文库：以22-35nt为长度筛选范围，从Flag标记的Siwi免疫沉淀复合物结合的小分子RNA中制备PIWI相关小分子RNA文库。同时构建6个RNA测序（RNA-seq）文库：从经过polyA尾富集（poly-A selection）的总RNA中制备RNA测序文库。