Siwi levels reversibly regulate secondary piRISC biogenesis by affecting Ago3 body morphology in Bombyx mori.

Abstract

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.