biostudies-literatureDowling MRepublic and Canton of GenevaSwiss National Science FoundationSwiss National Science Foundation, Switzerland308-316https://www.ebi.ac.uk/biostudies/studies/S-EPMC9945443biostudies-literatureUnknown29(3)Argonautes are small RNA-binding proteins, with some having small RNA-guided endonuclease (slicer) activity that cleaves target nucleic acids. One cardinal rule that is structurally defined is the inability of slicers to cleave target RNAs when nucleotide mismatches exist between the paired small RNA and the target at the cleavage site. Animal-specific PIWI clade Argonautes associate with PIWI-interacting RNAs (piRNAs) to silence transposable elements in the gonads, and this is essential for fertility. We previously demonstrated that purified endogenous mouse MIWI fails to cleave mismatched targets in vitro. Surprisingly, here we find using knock-in mouse models that target sites with cleavage-site mismatches at the 10th and 11th piRNA nucleotides are precisely sliced in vivo. This is idenRNA (New York, N.Y.)In vivo PIWI slicing in mouse testes deviates from rules established in vitro.PMC994544316692351NF40_182880CRETP3_166923310030_207468Raad NPillai RSDowling MGos PPandey RRHomolka DfalseIn vivo PIWI slicing in mouse testes deviates from rules established in vitro.Argonautes are small RNA-binding proteins, with some having small RNA-guided endonuclease (slicer) activity that cleaves target nucleic acids. One cardinal rule that is structurally defined is the inability of slicers to cleave target RNAs when nucleotide mismatches exist between the paired small RNA and the target at the cleavage site. Animal-specific PIWI clade Argonautes associate with PIWI-interacting RNAs (piRNAs) to silence transposable elements in the gonads, and this is essential for fertility. We previously demonstrated that purified endogenous mouse MIWI fails to cleave mismatched targets in vitro. Surprisingly, here we find using knock-in mouse models that target sites with cleavage-site mismatches at the 10th and 11th piRNA nucleotides are precisely sliced in vivo. This is iden2023-01-01T00:00:00Z2023 Mar2026-05-28T17:57:47.946Z2025-04-04T14:30:58.337ZS-EPMC99454433661765810.1261/rna.079349.122