Plant microRNAs (miRNAs) and small interfering RNAs (siRNAs) bear a 2'-O-methyl group on the 3'-terminal nucleotide. This methyl group is post-synthetically added by the methyltransferase protein HEN1 and protects small RNAs from enzymatic activities that target the 3'-OH. A mutagenesis screen for suppressors of the partial loss-of-function hen1-2 allele in Arabidopsis identified second-site mutations that restore miRNA methylation. These mutations affect two subunits of the DNA-dependent RNA polymerase IV (Pol IV), which is essential for the biogenesis of 24 nt endogenous siRNAs. A mutation in RNA-dependent RNA polymerase 2, another essential gene for the biogenesis of endogenous 24-nt siRNAs, also rescued the defects in miRNA methylation of hen1-2, revealing a previously unsuspected, negative influence of siRNAs on HEN1-mediated miRNA methylation. In addition, our findings imply the existence of a negative modifier of HEN1 activity in the Columbia genetic background.
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