Artificial ``ping-pong'' cascade of PIWI-interacting RNA in silkworm cells

Abstract

o̧pyright 2016 Shoji et al.PIWI-interacting RNAs (piRNAs) play essential roles in the defense system against selfish elements in animal germline cells by cooperating with PIWI proteins. A subset of piRNAs is predicted to be generated via the ping-pong'' cascade, which is mainly controlled by two different PIWI proteins. Here we established a cell-based artificial piRNA production system using a silkworm ovarian cultured cell line that is believed to possess a complete piRNA pathway. In addition, we took advantage of a unique silkworm sex-determining one-to-one ping-pong piRNA pair, which enabled us to precisely monitor the behavior of individual artificial piRNAs. With this novel strategy, we successfully generated artificial piRNAs against endogenous proteincoding genes via the expected back-and-forth traveling mechanism. Furthermore, we detected primary’’ piRNAs from the upstream region of the artificial ping-pong'' site in the endogenous gene. This artificial piRNA production system experimentally confirms the existence of the ping-pong’’ cascade of piRNAs. Also, this system will enable us to identify the factors involved in both, or each, of the ping'' and pong’’ cascades and the sequence features that are required for efficient piRNA production.