PIWI proteins associate with Piwi-interacting RNAs (piRNAs) that are mainly derived from intergenic repetitive elements on the genome and function in maintaining the integrity of the germline genome by silencing selfish transposable elements. piRNA biogenesis consists of two sequential steps; primary piRNA processing and the Ping-Pong cycle that depends on reciprocal Slicer-mediated RNA cleavage by PIWI proteins. The entire process requires many factors; however, their molecular functions remain elusive. We aimed to elucidate functions of factors in piRNA pathway.

A Bombyx mori germ cell line, BmN4, is an excellent tool to investigate the molecular pathways of piRNA biogenesis.We show that RNAs cleaved by a Bombyx mori PIWI, Siwi, remain bound to the protein upon cleavage, but are released by a DEAD-box protein BmVasa. BmVasa co-purifies with Siwi, but not with another PIWI BmAgo3. A lack of BmVasa does not affect primary piRNA processing but abolishes the Ping-Pong cycle. Siwi also forms a complex with two Tudor proteins, BmSpn-E and BmQin. This complex is physically separable from the Siwi/BmVasa complex. BmSpn-E, unlike BmVasa, is necessary for primary piRNA production. We propose a new model for piRNA biogenesis, where the BmSpn-E/BmQin dimer binds Siwi to function in primary piRNA processing, whereas BmVasa, by associating with Siwi, ensures target RNA release from the protein upon cleavage, to facilitate the Ping-Pong cycle.