Piwi and piRNAs act upstream of an endogenous siRNA pathway to suppress Tc3 transposon mobility in the Caenorhabditis elegans germline

Abstract

Several hundred endogenous small RNAs, namely microRNAs (miRNAs), repeat- associated small interfering RNAs (rasiRNAs), small interfering RNAs (siRNAs) have been discovered in diverse organisms. These small RNAs are derived from different sources and they cause transcriptional gene silencing, translational repression and mRNA cleavage through effector complexes. Several functionally distinct RNA silencing effector complexes have already been isolated from different organisms. Effector complexes are composed of members of the Argonaute (AGO) protein family and single-stranded small RNAs, along with other associated proteins. Members of the Piwi subfamily of Argonaute proteins have conserved roles in germline development and stem cell maintenance. In C. elegans, PRG-1 and PRG-2 are the two members of Piwi sub-family of Argonaute proteins. In order to study the role of PRG-1 and PRG-2 in C. elegans germline development, we generated prg-1 and prg-2 single mutants as well as prg-1; prg-2 double mutant worms, which show severe germline defects. I identify a class of 21 nucleotide RNAs, previously named 21U-RNAs, as the Piwi-interacting RNAs in C. elegans. Piwi and piRNA expression is restricted to the male and female germline and piRNA biogenesis is independent of many other proteins involved in small RNA pathways including DCR-1. I show that Piwi is specifically required for suppression of Tc3 transposons. The excision rate of Tc3 is 100-fold higher in piwi mutants compared to wild-type. There is no evidence for a ping-pong mechanism in C. elegans for amplification of piRNAs. Finally, I demonstrate that a Piwi-piRNA complex acts upstream of the endogenous siRNA pathway for Tc3 silencing and that might suggest a link between the function of endogenous siRNAs and piRNAs.