Proof-of-concept for the ping-pong amplification cycle of PIWI-interacting RNAs

PIWI-interacting RNAs (piRNAs) play essential roles in the defense system against selfish elements in animal germ line cells by cooperating with PIWI proteins. A subset of piRNAs are supposed to be generated via the ping-pong amplification cycle, which is controlled mainly by two different PIWI proteins. At present, however, this ping-pong cycle is just a proposed model based only on bioinformatic analyses of piRNA sequences. Here, we establish a cell-based artificial piRNA production system using silkworm ovarian cultured cells, which are believed to possess a complete piRNA pathway. We took advantage of a unique, silkworm sex-determining one-to-one ping-pong piRNA pair, which enables us to monitor the behavior of an individual artificial piRNA. With this novel strategy, we successfully generated artificial piRNAs against endogenous silkworm genes via the expected back-and-forth mechanism, showing for the first time the proof-of-concept evidence for the ping-pong amplification cycle of piRNAs.