Methylation protects miRNAs and siRNAs from a 3′-end uridylation activity in Arabidopsis

Small RNAs of 21-25 nucleotides (nt), including small interfering RNAs (siRNAs) and microRNAs (miRNAs), act as guide RNAs to silence target-gene expression in a sequence-specific manner [1]. In addition to a Dicer homolog, DCL1, the biogenesis of miRNAs in Arabidopsis requires another protein, HEN1 [2, 3]. miRNAs are reduced in abundance and increased in size in hen1 mutants [2, 4-7]. We found that HEN1 is a miRNA methyltransferase that adds a methyl group to the 3′-most nucleotide of miRNAs [8], but the role of miRNA methylation was unknown. Here, we show that siRNAs from sense transgenes, hairpin transgenes, and transposons or repeat sequences, as well as a new class of siRNAs known as trans-acting siRNAs, are also methylated in vivo by HEN1. In addition, we show that the size increase of small RNAs in the hen1-1 mutant is due to the addition of one to five U residues to the 3′ ends of the small RNAs. Therefore, a novel uridylation activity targets the 3′ ends of unmethylated miRNAs and siRNAs in hen1 mutants. We conclude that 3′-end methylation is a common step in miRNA and siRNA metabolism and likely protects the 3′ ends of the small RNAs from the uridylation activity.