The RNA phosphatase PIR-1 regulates endogenous small RNA pathways in C. elegans

Eukaryotic cells regulate 5’ triphosphorylated (ppp) RNAs to promote cellular functions and to prevent recognition by viral RNA sensors. For example, the triphosphatase domain of RNA capping enzymes removes the γ phosphate of ppp-RNAs in RNA capping reactions. Members of a related RNA polyphosphotase family including human PIR1 remove both the β and γ phosphates from ppp-RNAs. Here we demonstrate that C. elegans PIR-1, previously identified as a Dicer-interacting protein, dephosphorylates single and double-stranded ppp-RNAs, and is required for the maturation of 26G-RNAs, which regulate thousands of genes during spermatogenesis and embryogenesis. We find that 26G-RNAs are generated in a unique semi-phasing manner in which primary 26G-RNAs are required for generating secondary 26G-RNAs and PIR-1 is required for removing a diphosphate group from each triphosphorylated precursor, which only generates one 26G-RNA. Our findings also implicate PIR-1 in regulating ppp-RNAs in Dicer-independent pathways, supporting PIR-1 as a master phosphatase regulating ppp-RNAs. This study examines how PIR-1, a novel RNA polyphosphatase, regulates small RNA biogenesis in C. elegans. We generated pir-1 mutants and compared the small RNA profiles between the mutants and the WT control using high-throughput sequencing followed by bioinformatics. We also utilized biochemistry to characterize the in vitro activity of PIR-1 in C. elegans.