PIWI Associated siRNAs and piRNAs Specifically Require the Caenorhabditis elegans HEN1 Ortholog henn-1

Abstract

Small RNAs—including piRNAs, miRNAs, and endogenous siRNAs—bind Argonaute proteins to form RNA silencing complexes that target coding genes, transposons, and aberrant RNAs. To assess the requirements for endogenous siRNA formation and activity in Caenorhabditis elegans, we developed a GFP-based sensor for the endogenous siRNA 22G siR-1, one of a set of abundant siRNAs processed from a precursor RNA mapping to the X chromosome, the X-cluster. Silencing of the sensor is also dependent on the partially complementary, unlinked 26G siR-O7 siRNA. We show that 26G siR-O7 acts in trans to initiate 22G siRNA formation from the X-cluster. The presence of several mispairs between 26G siR-O7 and the X-cluster mRNA, as well as mutagenesis of the siRNA sensor, indicates that siRNA target recognition is permissive to a degree of mispairing. From a candidate reverse genetic screen, we identified several factors required for 22G siR-1 activity, including the chromatin factors mes-4 and gfl-1, the Argonaute ergo-1, and the 3′ methyltransferase henn-1. Quantitative RT–PCR of small RNAs in a henn-1 mutant and deep sequencing of methylated small RNAs indicate that siRNAs and piRNAs that associate with PIWI clade Argonautes are methylated by HENN-1, while siRNAs and miRNAs that associate with non-PIWI clade Argonautes are not. Thus, PIWI-class Argonaute proteins are specifically adapted to associate with methylated small RNAs in C. elegans.

Author Summary

RNA interference (RNAi) is the process in which endogenous small RNA pathways are exploited by researchers to direct RNA silencing of particular genes. Plants and animals use endogenous RNA silencing pathways for protection against viruses and transposable elements and to regulate genes during development. The features that route genes into specific RNA silencing pathways are poorly understood. Furthermore, it is not clear how small RNAs identify target mRNAs and how they repress their activity. Here, we show that a single siRNA target site is sufficient to trigger gene silencing in C. elegans without requiring perfect complementarity for target recognition. We also discovered an endogenous siRNA that acts in trans to initiate siRNA amplification. Finally, we show that siRNAs and PIWI-interacting RNAs (piRNAs) that bind specifically to PIWI clade Argonautes are methylated by the C. elegans HEN1 ortholog HENN-1.