Maturation of some classes of small RNAs includes 2'-O-methylation at the 3'-terminal nucleotide by S-adenosylmethionine-dependent RNA methyltransferase protein HEN1. Fly, zebrafish, and plant studies indicate that methylation protects small RNAs from terminal polyuridylation/polyadenylation, which induces exonucleolytic degradation (1-3). Thus, methylation by HEN1 increases the perdurance of a small RNA, enabling persistence of target silencing beyond the period of biogenesis. In C. elegans, methylation of several classes of small RNAs has been reported (4), but the methyltransferase catalyzing this modification has not yet been identified. C02F5.6 encodes the sole C. elegans HEN1 ortholog. Here, we report that this protein, which we refer to as HEN1-like methyltransferase (HLM-1), terminally methylates both 21U RNAs (worm piRNAs) and the ERGO-1 class of primary 26G endo-siRNAs, which are enriched in female germline and embryo. ALG-3/4 class primary 26G endo-siRNAs, enriched in spermatogenic cells (5,6), are not methylated, suggesting that HLM-1-mediated methylation may selectively stabilize female germline-derived 26G endo-siRNAs to direct their inheritance. While 2'-O-methylation has not been convincingly linked to small RNA stability in some animal models (7), accumulation of methylated worm small RNAs is impaired in the
hlm-1 mutant. Consequently, loss of HLM-1 results in some defects in secondary siRNA production and target silencing downstream of methylated primary endo-siRNAs. Because loss of HLM-1 impairs function of worm piRNA and ERGO-1 class endo-siRNA pathways, the
hlm-1 mutant exhibits phenotypes that approach those observed in mutants lacking these small RNAs. Due presumably to decreased 21U RNA levels, the
hlm-1 mutant shows a modest fertility defect at 25 deg C. Similarly, the
hlm-1 mutant exhibits enhanced sensitivity to somatic RNAi, mirroring the enhanced RNAi phenotype observed in mutants defective for production of ERGO-1 class 26G RNAs. Together, these data provide the first example of germline-specific methylation of a subclass of endo-siRNAs and may further our understanding of a germline small RNA regulatory mechanism that is widespread in higher eukaryotic organisms. N.B. C02F5.6 has recently been named
rem-1. 1. Ameres, S. L. et al., Science 328, 1534 (2010). 2. Kamminga, L. M. et al., EMBO J 29, 3688 (2010). 3. Li, J. et al., Curr Biol 15, 1501 (2005). 4. Ruby, J. G. et al., Cell 127, 1193 (2006). 5. Conine, C. C. et al., PNAS 107, 3588 (2010). 6. Han, T. et al., PNAS 106, 18674 (2009). 7. Saito, K. et al., Genes Dev 21, 1603 (2007).