RESUMO
Piwi-interacting RNAs (piRNAs) direct PIWI proteins to transposons to silence them, thereby preserving genome integrity and fertility. The piRNA population can be expanded in the ping-pong amplification loop. Within this process, piRNA-associated PIWI proteins (piRISC) enter a membraneless organelle called nuage to cleave their target RNA, which is stimulated by Gtsf proteins. The resulting cleavage product gets loaded into an empty PIWI protein to form a new piRISC complex. However, for piRNA amplification to occur, the new RNA substrates, Gtsf-piRISC, and empty PIWI proteins have to be in physical proximity. In this study, we show that in silkworm cells, the Gtsf1 homolog BmGtsf1L binds to piRNA-loaded BmAgo3 and localizes to granules positive for BmAgo3 and BmVreteno. Biochemical assays further revealed that conserved residues within the unstructured tail of BmGtsf1L directly interact with BmVreteno. Using a combination of AlphaFold modeling, atomistic molecular dynamics simulations, and in vitro assays, we identified a novel binding interface on the BmVreteno-eTudor domain, which is required for BmGtsf1L binding. Our study reveals that a single eTudor domain within BmVreteno provides two binding interfaces and thereby interconnects piRNA-loaded BmAgo3 and BmGtsf1L.
Assuntos
Bombyx , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Bombyx/genética , Bombyx/metabolismo , RNA de Interação com Piwi , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Domínio TudorRESUMO
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.