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. 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.纯化的内源性小鼠MIWI蛋白在体外无法切割错配靶标。令人意外的是，本研究通过敲入小鼠模型发现，带有切割位点错配的PIWI互作RNA（piRNA）靶位点在体内可被精准切割，这与靶标与piRNA完全互补配对的敲入小鼠体内的切割结果一致。此外，本研究发现，此类粗线期piRNA引导的MIWI/MILI体内切割无法从我们研究的特定靶标信使RNA（mRNA）起始阶段性piRNA生成；相反，两种切割酶切割产生的片段以piRNA前体和17-19 nt副产物的形式滞留在PIWI蛋白中。本研究结果表明，体外建立的PIWI切割规则在体内并未被遵循，且并非所有PIWI切割的靶标均可作为piRNA生物发生的底物。我们构建了四种敲入小鼠品系：两种分别带有1个或10个与粗线期piRNA piR-A完全互补的靶位点（分别记为1xPerf、10xPerf），另外两种分别带有1个或10个在10-11位存在错配的靶位点（分别记为1xBulge、10xBulge）。上述piRNA靶位点被插入至Ythdc2基因的3'非翻译区（3'UTR）。我们通过免疫沉淀获取与MILI和MIWI结合的小RNA并进行测序，以搜寻piR-A切割产生的piRNA；同时利用RNA测序（RNA-seq）分析基因表达变化。本研究对两个生物学重复样本进行了分析。