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 39-terminal 29-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 29-O-methylated at their 39 ends. The Tetrahymena HEN1 homolog Hen1p is responsible for scnRNA 29-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 29-O-methylation stabilizes scnRNA and ensures DNA elimination in Tetrahymena. This study clearly shows that 39-terminal 29-O-methylation on a selected class of small RNAs regulates the function of a specific RNAi pathway.

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Summary Emerging evidence suggests that RNAi-related processes act both in the cytoplasm and in the nucleus. However, the process by which the RNAi machinery is transported into the nucleus remains poorly understood. The Tetrahymena Argonaute protein Twi1p localizes to the nucleus and is crucial for small RNA-directed programmed DNA elimination. In this study, we identify Giw1p, which binds to Twi1p and is required for its nuclear localization. Further, the endoribonuclease (Slicer)-activity of Twi1p plays a vital role in the removal of one of the two strands of Twi1p-associated siRNAs, leading to a functionally mature Twi1p-siRNA complex. Slicer activity is also shown to be required for nuclear localization of Twi1p and for its association with Giw1p. These results suggest that Giw1p senses the state of Twi1p-associated siRNAs and selectively transports the mature Twi1p-siRNA complex into the nucleus.