Small RNAs ∼20–30 nucleotides (nt) in length regulate gene expression at the transcriptional and post-transcriptional levels. In the plant Arabidopsis, all small RNAs are 3′-terminal 2′-O-methylated by HEN1, whereas only a subset of small RNAs carry this modification in metazoans. This methylation is known to stabilize small RNAs, but its biological significance remains unclear. In the ciliated protozoan Tetrahymena thermophila, two classes of small RNAs have been identified: RNAs ∼28–29 nt long (scnRNAs) that are expressed only during sexual reproduction, and constitutively expressed ∼23–24 nt siRNAs. In this study, we demonstrate that scnRNAs, but not siRNAs, are 2′-O-methylated at their 3′ ends. The Tetrahymena HEN1 homolog Hen1p is responsible for scnRNA 2′-O-methylation. Loss of Hen1p causes a gradual reduction in the level and length of scnRNAs, defects in programmed DNA elimination, and inefficient production of sexual progeny. Therefore, Hen1p-mediated 2′-O-methylation stabilizes scnRNA and ensures DNA elimination in Tetrahymena. This study clearly shows that 3′-terminal 2′-O-methylation on a selected class of small RNAs regulates the function of a specific RNAi pathway
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