RNA interference (RNAi)–related pathways affect gene activity by sequence-specific recruitment of Ago proteins to mRNA target molecules. The sequence specificity of this process stems from small RNA (sRNA) co-factors bound by the Ago protein. Stability of sRNA molecules in some pathways is in part regulated by Hen1-mediated methylation of their 3′ ends. Here we describe the effects of the Caenorhabditis elegans HEN1 RNA–methyl-transferase homolog, HENN-1, on the different RNAi pathways in this nematode. We reveal differential effects of HENN-1 on the two pathways that are known to employ methylated sRNA molecules: the 26G and 21U pathways. Surprisingly, in the germline, stability of 21U RNAs, the C. elegans piRNAs, is only mildly affected by loss of methylation; and introduction of artificial 21U target RNA does not further destabilize non-methylated 21U RNAs. In contrast, most 26G RNAs display reduced stability and respond to loss of HENN-1 by displaying increased 3′-uridylation frequencies. Within the 26G RNA class, we find that specifically ERGO-1–bound 26G RNAs are modified by HENN-1, while ALG-3/ALG-4–bound 26G RNAs are not. Global gene expression analysis of henn-1 mutants reveals mild effects, including down-regulation of many germline-expressed genes. Our data suggest that, apart from direct effects of reduced 26G RNA levels of henn-1 on gene expression, most effects on global gene expression are indirect. These studies further refine our understanding of endogenous RNAi in C. elegans and the roles for Hen1 like enzymes in these pathways.
Small RNAs (sRNAs) have been shown to be potent regulators of gene expression in many different systems. They act by providing sequence specificity to Argonaute (Ago) proteins that in turn affect the expression and/or stability of mRNAs, or affect chromatin structures through recognition of nascent transcripts. Stability of sRNAs can be regulated by methylation of their 3′ end. This modification prevents addition of uridine residues that can destabilize the sRNA. The enzyme that catalyzes the methylation of sRNAs has been identified in Arabidopsis: HEN1. We describe studies on the C. elegans homolog of Hen1, henn-1. Our findings show that HENN-1 protein does not stably associate with the Ago proteins binding methylated sRNAs, but that HENN-1 does localize to subcellular regions known to host these factors. We find that the two known methylated sRNA species in C. elegans (21U and 26G) respond differently to loss of henn-1. While HENN-1 is required for 26G RNA stability in the germline, it has limited impact on 21U RNAs. In addition, we demonstrate that only ERGO-1–bound 26G RNAs are methylated, while those bound by ALG-3/4, are not. Our findings further refine the general understanding of 21U and 26G RNA pathways and identify two separable effects of HENN-1 on these RNAi–related mechanisms.