Abstract
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.
Highlights
Small RNAs 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
Our findings show that HENN-1 protein does not stably associate with the Ago proteins binding methylated small RNA (sRNA), but that HENN-1 does localize to subcellular regions known to host these factors
We have addressed the C. elegans HEN1 ortholog HENN-1 in terms of its activity and its sub-cellular localization and have described its effect on the various endogenous RNA interference (RNAi) pathways of this nematode
Summary
Stability of sRNA molecules in some pathways is in part regulated by Hen1-mediated methylation of their 39 ends. We reveal differential effects of HENN-1 on the two pathways that are known to employ methylated sRNA molecules: the 26G and 21U pathways. Global gene expression analysis of henn-1 mutants reveals mild effects, including down-regulation of many germline-expressed genes. 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 Hen like enzymes in these pathways.
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