The long term goal of this project is to elucidate the functions of MOV10L1 in piRNA biogenesis. Control of transposable elements in the germline is of paramount significance in protecting transgenerational genome integrity. Piwi-interacting RNAs (piRNAs) are small non-coding RNAs that associate with P-element induced wimpy testes (PIWI) proteins to silence transposable elements in the germline. The biogenesis of piRNAs is a highly conserved process required for transposon silencing, germ cell development, and fertility in many species. During piRNA biogenesis, piRNA precursors are processed into piRNA intermediates, loaded onto PIWI proteins, and trimmed and modified into mature piRNAs. However, the mechanisms underlying piRNA biogenesis remain unclear. The Wang lab previously identified Moloney leukemia virus 10-like 1 (MOV10L1) as a testis-specific putative RNA helicase that is required for piRNA biogenesis in mammals. It is the only known master regulator of mammalian piRNA biogenesis. MOV10L1 associates with all three mouse PIWI proteins. The disruption of Mov10l1 results in the depletion of piRNAs and an accumulation of piRNA precursors. I hypothesize that MOV10L1 functions in the processing of piRNA precursors and piRNA intermediates during piRNA biogenesis. In Specific Aim 1, I will determine the role of MOV10L1 RNA helicase activity in the processing of piRNA precursors. I have generated two Mov10l1 knock-in mouse models in which MOV10L1 RNA helicase activity is disrupted. The testes of homozygous Mov10l1 knock-in mice show defects in the localization of piRNA pathway proteins and transposon silencing. Male homozygous Mov10l1 knock-in mice also exhibit meiotic arrest. I plan to generate conditional Mov10l1 knock-in mouse models in which MOV10L1 RNA helicase activity is disrupted post-natally. I will characterize the phenotype of conditional Mov10l1 knock-in mice and piRNA processing in both the testes of homozygous Mov10l1 knock-in mice and conditional Mov10l1 knock-in mice. In Specific Aim 2, I will characterize the role of MOV10L1 in the loading and trimming of piRNA intermediates into piRNAs in vitro using a novel biochemical assay I plan to develop. This biochemicl assay will use a cell-free testicular lysate system, and I will use testicular lysate from Mov10l1 mutant mice. These studies will reveal conserved mechanisms in piRNA biogenesis and provide insights into the etiology of human diseases including male infertility.