Silkworm ovarian germ cells produce the Siwi‐piRNA‐induced silencing complex (piRISC) through two consecutive mechanisms, the primary pathway and the secondary ping‐pong cycle. Primary Siwi‐piRISC production occurs on the outer mitochondrial membrane in an Ago3‐independent manner, where Tudor domain‐containing Papi binds unloaded Siwi via its symmetrical dimethylarginines (sDMAs). Here, we now show that secondary Siwi‐piRISC production occurs at the Ago3‐positive nuage Ago3 bodies, in an Ago3‐dependent manner, where Vreteno (Vret), another Tudor protein, interconnects unloaded Siwi and Ago3‐piRISC through their sDMAs. Upon Siwi depletion, Ago3 is phosphorylated and insolubilized in its piRISC form with cleaved RNAs and Vret, suggesting that the complex is stalled in the intermediate state. The Ago3 bodies are also enlarged. The aberrant morphology is restored upon Siwi re‐expression without Ago3‐piRISC supply. Thus, Siwi depletion aggregates the Ago3 bodies to protect the piRNA intermediates from degradation until the normal cellular environment returns to re‐initiate the ping‐pong cycle. Overall, these findings reveal a unique regulatory mechanism controlling piRNA biogenesis.

中文翻译：

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Siwi水平通过影响家蚕中的Ago3身体形态可逆地调节继发piRISC生物发生。

蚕卵巢生殖细胞通过两个连续的机制（主要途径和次要乒乓循环）产生Siwi-piRNA诱导的沉默复合体（piRISC）。最初的Siwi-piRISC生产以不依赖Ago3的方式发生在线粒体外膜上，其中Tudor域包含的Papi通过其对称的二甲基精氨酸（sDMAs）结合了未加载的Siwi。在这里，我们现在表明，次生Siwi-piRISC生产以依赖Ago3的方式发生在Ago3阳性nugo Ago3体上，其中另一种Tudor蛋白Vreteno（Vret）通过其sDMA连接卸载的Siwi和Ago3-piRISC。Siwi耗尽后，Ago3被piRISC形式磷酸化，并被裂解的RNA和Vret溶解，表明该复合物停滞在中间状态。Ago3体也被放大。Siwi重新表达后，无需Ago3-piRISC即可恢复异常形态。因此，Siwi耗竭聚集了Ago3体，以保护piRNA中间体免于降解，直到正常细胞环境恢复以重新启动乒乓循环。总体而言，这些发现揭示了控制piRNA生物发生的独特调控机制。