Piwi-interacting RNAs (piRNAs) are essential for silencing of transposable elements in the germline, but their biogenesis is poorly understood. Here we demonstrate that MOV10L1, a germ cell–specific putative RNA helicase, is associated with Piwi proteins. Genetic disruption of the MOV10L1 RNA helicase domain in mice renders both MILI and MIWI2 devoid of piRNAs. Absence of a functional piRNA pathway in Mov10l1 mutant testes causes loss of DNA methylation and subsequent derepression of retrotransposons in germ cells. The Mov10l1 mutant males are sterile owing to complete meiotic arrest. This mouse mutant expresses Piwi proteins but lacks piRNAs, suggesting that MOV10L1 is required for piRNA biogenesis and/or loading to Piwi proteins.

Background: Life-long production of spermatozoa depends on spermatogonial stem cells. Spermatogonial stem cells exist among the most primitive population of germ cells – undifferentiated spermatogonia. Transplantation experiments have demonstrated the functional heterogeneity of undifferentiated spermatogonia. Although the undifferentiated spermatogonia can be topographically divided into A_s(single), A_pr(paired), and A_al(aligned) spermatogonia, subdivision of this primitive cell population using cytological markers would greatly facilitate characterization of their functions.Results: In the present study, we show that LIN28, a pluripotency factor, is specifically expressed in undifferentiated spermatogonia (A_s, A_pr, and A_al) in mouse.Ngn3also specifically labels undifferentiated spermatogonia. We usedNgn3-GFP knockin mice, in which GFP expression is under the control of allNgn3transcription regulatory elements. Remarkably,Ngn3-GFP is only expressed in ~40% of LIN28-positive A_s(single) cells. The percentage ofNgn3-GFP-positive clusters increases dramatically with the chain length of interconnected spermatogonia.Conclusion: Our study demonstrates that LIN28 specifically marks undifferentiated spermatogonia in mice. These data, together with previous studies, suggest that the LIN28-expressing undifferentiated spermatogonia exist as two subpopulations:Ngn3-GFP-negative (high stem cell potential) andNgn3-GFP-positive (high differentiation commitment). Furthermore,Ngn3-GFP-negative cells are found in chains ofNgn3-GFP-positive spermatogonia, suggesting that cells in the A_alspermatogonia could revert to a more primitive state.

Piwi–piRNA (Piwi-interacting RNA) ribonucleoproteins (piRNPs) enforce retrotransposon silencing, a function critical for preserving the genome integrity of germ cells. The molecular functions of most of the factors that have been genetically implicated in primary piRNA biogenesis are still elusive. Here we show that MOV10L1 exhibits 5′-to-3′ directional RNA-unwinding activity in vitro and that a point mutation that abolishes this activity causes a failure in primary piRNA biogenesis in vivo. We demonstrate that MOV10L1 selectively binds piRNA precursor transcripts and is essential for the generation of intermediate piRNA processing fragments that are subsequently loaded to Piwi proteins. Multiple analyses suggest an intimate coupling of piRNA precursor processing with elements of local secondary structures such as G quadruplexes. Our results support a model in which MOV10L1 RNA helicase activity promotes unwinding and funneling of the single-stranded piRNA precursor transcripts to the endonuclease that catalyzes the first cleavage step of piRNA processing.

Piwi-interacting RNAs are a diverse class of small non-coding RNAs implicated in the silencing of transposable elements and the safeguarding of genome integrity. In mammals, male germ cells express two genetically and developmentally distinct populations of piRNAs at the pre-pachytene and pachytene stages of meiosis, respectively. Pre-pachytene piRNAs are mostly derived from retrotransposons and required for their silencing. In contrast, pachytene piRNAs originate from ∼3,000 genomic clusters, and their biogenesis and function remain enigmatic. Here, we report that conditional inactivation of the putative RNA helicase MOV10L1 in mouse spermatocytes produces a specific loss of pachytene piRNAs, significant accumulation of pachytene piRNA precursor transcripts, and unusual polar conglomeration of Piwi proteins with mitochondria. Pachytene piRNA–deficient spermatocytes progress through meiosis without derepression of LINE1 retrotransposons, but become arrested at the post-meiotic round spermatid stage with massive DNA damage. Our results demonstrate that MOV10L1 acts upstream of Piwi proteins in the primary processing of pachytene piRNAs and suggest that, distinct from pre-pachytene piRNAs, pachytene piRNAs fulfill a unique function in maintaining post-meiotic genome integrity. Small non-coding RNAs play critical roles during development and in disease. The integrity of the germline genome is of paramount importance to the wellbeing of offspring and the survival of species. Piwi-interacting RNAs (piRNAs) are a class of small non-coding RNAs abundantly expressed in the gonad. Compared to microRNAs and small-interfering RNAs (siRNAs), the biogenesis and function of piRNAs remain poorly understood. Here we have identified MOV10L1, a putative RNA helicase, as a master regulator of piRNA biogenesis in mouse. We find that production of pachytene piRNAs requires MOV10L1. Blockade of pachytene piRNAs disrupts germ cell development and results in defects in post-meiotic genome integrity. Therefore, mutations in MOV10L1 and other piRNA pathway components may contribute to male infertility in humans.

Significance: TATA-binding protein (TBP)-associated factor 7l ( Taf7l ), Taf4b , and TBP-related factor 2 ( Trf2 ) were found to be three important atypical testis-specific core promoter recognition factors. However, whether there was any functional cross-talk between them and at which stages of spermatogenesis remained uncharacterized. Here we report that Taf7l ^{− /Y} becomes sterile rather than merely subfertile after backcrossing. Importantly, we found that Taf7l cooperates with Trf2 , but not Taf4b , at late stages to direct the transcription of key spermatogenic genes. Our findings thus provide unique insights into the function of cell-type–specific Tafs and how two core promoter recognition factors mediate transcriptional control of spermiogenesis. The testis-specific role of Taf7l identifies this regulator as a potential target for male contraceptive intervention.