In vivo PIWI slicing in mouse testes deviates from rules established in vitro

Purified endogenous mouse MIWI fails to cleave mismatched targets in vitro. Surprisingly, here we find using knock-in mouse models that piRNA target sites with cleavage-site mismatches are precisely sliced in vivo. This is identical to the slicing outcome in knock-in mice where targets are identified by perfect complementarity base-pairing with the piRNA. Additionally, we find that such pachytene piRNA-guided MIWI/MILI slicing in vivo failed to initiate phased piRNA production from the specific target mRNA we studied. Instead, the two slicer cleavage fragments were retained in PIWI proteins as a pre-piRNA and 17-19 nt by-product fragments. Our results indicate that PIWI slicing rules established in vitro are not respected in vivo, and that all targets of PIWI slicing are not substrates for piRNA biogenesis. Overall design: We designed four knock-in mouse lines: two lines with one or ten sites perfectly complementary to the pachytene piRNA piR-A (1xPerf, 10xPerf) and two lines with one or ten sites having mismatches at positions 10-11 (1xBulge,10xBulge). The piRNA targeted sites were inserted into 3'UTR of Ythdc2. We immunoprecipitated and sequenced the MILI and MIWI associated small RNAs to search for piRNAs generated by piR-A cleavage and used RNA-seq to analyze the gene expression changes. Two biological replicas were analyzed.