Structure of the MIWI endoribonuclease bound to pachytene piRNAs from mouse testes

Animal germlines express small RNAs called PIWI-interacting RNAs (piRNAs) that guide their associated PIWI endoribonucleases to destroy transposon transcripts, ensuring animal fertility. Mouse MIWI binds millions of sequences called pachytene piRNAs that use partial complementarity to target transposons and cellular mRNAs. Here we report the first cryo-EM structure of the MIWI-pachytene piRNA complex isolated from mouse testes. It shows a positively charged channel where the piRNA is held, with specific recognition of the signature first nucleotide uridine being mediated by amino acids within the MID and PIWI domains. The first six nucleotides of the guide RNA are visible and take up the A-form conformation to facilitate pairing with the target. Importantly, part of the channel accommodating the seed sequence is wider than that for insect PIWI, likely explaining the reported tolerance of MIWI to seed sequence mismatches. The catalytic site in the PIWI endonuclease domain presents an inactive state, with the loop containing one of the catalytic residues (E671) requiring structural re-orientation to adopt the catalytically competent state. Finally, we identify a conserved pre-formed pocket on the PIWI domain that may serve to accommodate a conserved tryptophan from the interacting factor GTSF1. Modelling shows how this interaction with GTSF1 can allosterically enforce reorganization of the catalytic pocket to promote small RNA-guided endoribonuclease activity. Our structure informs how MIWI functions using relaxed target engagement rules and associates with its interacting partner.