Siwi levels reversibly regulate secondary pi RISC 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 domaincontaining 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.

DEAD‐box polypeptide 43 facilitates piRNA amplification by actively liberating RNA from Ago3‐piRISC

“…Both Vret and DDX43 interconnect Ago3‐piRISC and piRNA‐unbound Siwi in BmN4 cells (Nishida et al , 2020 and this study). We speculated that Vret and DDX43 physically interact with each other through the ping‐pong cycle although such interaction would be transient.…”

“…Our recent investigations showed that the Tudor domain‐containing protein, Vreteno (Vret), facilitates Ago3‐dependent Siwi‐piRISC production in the ping‐pong cycle (Nishida et al , 2020). Our study also showed that a subset of nuage (germ cell‐specific granules), which we termed Ago3 bodies, are central hubs in the Siwi‐piRISC production.…”

“…We speculated that Vret and DDX43 physically interact with each other through the ping-pong cycle although such interaction would be transient. Probing the DDX43 complex components with an anti-Vret antibody showed that Vret, and particularly its longer isoform (Nishida et al, 2020) . This suggested the intriguing idea that Ago3 and DDX43 interact in the cytosol and co-localize to Ago3 bodies.…”

“…Monoclonal antibodies for Vasa, Siwi, and Ago3 detection were produced as described previously (Nishida et al , 2015). The anti‐Vret monoclonal antibody was raised in our laboratory (Nishida et al , 2020). We produced the anti‐DDX43 monoclonal antibody by immunizing mice with the purified GST‐tagged N‐terminal region (amino acids 1–200) of DDX43.…”

“…Vret monoclonal antibody was raised in our laboratory (Nishida et al, 2020). We produced the anti-DDX43 monoclonal antibody by immunizing mice with the purified GST-tagged N-terminal region (amino acids 1-200) of DDX43.…”