An extended Tudor domain within Vreteno interconnects Gtsf1L and Ago3 in Bombyx mori

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 so-called ping-pong amplification loop. Within this process, piRNA-associated PIWI proteins (piRISC) enter the nuage to cleave target RNA, a process that 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, it is required that new RNA substrates, Gtsf-piRISC and empty PIWI proteins are all in physical proximity. A recent study in the silkworm has shown that BmVreteno can act as a scaffolding protein for BmAgo3-dependent piRISC-Siwi assembly within the nuage. However, how BmAgo3-catalyzed target RNA cleavage is restricted to a nuage environment remains unknown. In this study we show that BmGtsf1L binds to piRNA-loaded BmAgo3 and co-localizes to BmAgo3-BmVreteno positive granules, which mark the nuage. In addition, biochemical assays revealed that conserved residues within the unstructured tail of BmGtsf1L directly interact with BmVreteno. Next, using a combination of AlphaFold modeling, atomistic molecular dynamics simulations and in vitro assays we identified a novel binding interface on a BmVreteno-eTudor domain, which is required for BmGtsf1L binding. Interestinly, this is the same eTudor domain that also harbors an aromatic cage that is required for binding to piRNA-loaded, methylated BmAgo3. Our study revealed that a single eTudor domain within BmVreteno can form a dual binding interface and interconnects loaded BmAgo3 and BmGtsf1L, possibly restricting BmAgo3 slicing to an environment where empty Siwi can accept a target-cleavage product for de novo piRISC assembly.