Terminal Modification, Sequence, and Length Determine Small RNA Stability in Animals

In animals, piRNAs, siRNAs, and miRNAs silence transposons, fight viral infections, and regulate gene expression. piRNA biogenesis concludes with 3′ terminal trimming and 2′- O -methylation. Both trimming and methylation influence piRNA stability. Here, we report that trimming and methylation protect mouse piRNAs from different decay mechanisms. In the absence of 2′- O -methylation, mouse piRNAs with extensive complementarity to long RNAs become unstable. In flies, 2′- O -methylation similarly protects both piRNAs and siRNAs from complementarity-dependent destabilization. Animal miRNAs are unmethylated, and complementarity-dependent destabilization helps explain differences in miRNA decay rates in both mice and flies. In contrast, trimming protects mouse piRNAs from a separate degradation pathway unaffected by target complementarity but sensitive to the 3′ terminal, untrimmed sequence. Because distinct sets of mouse piRNAs are protected by each of these mechanisms, loss of both trimming and 2′- O -methylation causes the piRNA pathway to collapse, demonstrating that these two small RNA modifications collaborate to stabilize piRNAs. Highlights ### Competing Interest Statement The authors have declared no competing interest.