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

The piRNA amplification pathway in Bombyx is operated by Ago3 and Siwi in their piRISC form. The DEAD-box protein, Vasa, facilitates Ago3-piRISC production by liberating cleaved RNAs from Siwi-piRISC in an ATP hydrolysis-dependent manner. However, the Vasa-like factor facilitating Siwi-piRISC production along this pathway remains unknown. Here, we identify DEAD-box polypeptide 43 (DDX43) as the Vasa-like protein functioning in Siwi-piRISC production. DDX43 belongs to the helicase superfamily II along with Vasa, and it contains a similar helicase core. DDX43 also contains a K-homology (KH) domain, a prevalent RNA-binding domain, within its N-terminal region. Biochemical analyses show that the helicase core is responsible for Ago3-piRISC interaction and ATP hydrolysis, while the KH domain enhances the ATPase activity of the helicase core. This enhancement is independent of the RNA-binding activity of the KH domain. For maximal DDX43 RNA-binding activity, both the KH domain and helicase core are required. This study not only provides new insight into the piRNA amplification mechanism but also reveals unique collaborations between the two domains supporting DDX43 function within the pathway.