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.T he Piwi clade of Argonaute proteins associates with a class of 26-31-nt germline-specific small RNAs called "piRNAs". Together they participate in suppression of transposable elements in all animals studied (1-4). In mice, the Piwi clade contains three members: Miwi2, Mili, and Miwi. These three Piwi members exhibit distinct developmental expression patterns. Miwi2 is expressed in perinatal male germ cells (5), whereas Mili is more broadly expressed from embryonic germ cells to postnatal round spermatids (6). Miwi expression begins in pachytene spermatocytes and persists in haploid round spermatids (7). The overlapping temporal expression of Mili with Miwi and Miwi2 points to the pivotal role of MILI in the piRNA pathway, as further supported by the fact that MILI is associated with developmental stage-dependent pools of piRNAs: prenatal, prepachytene, and pachytene piRNAs (5,8,9).The mechanisms of piRNA biogenesis are largely unclear (1-4). One feature of piRNAs in all species is their highly clustered genomic origins. Several of these clusters produce piRNAs only from one strand. This leads to a hypothesized primary processing pathway whereby an unknown nuclease cleaves off mature piRNAs from a long single-stranded precursor transcript. On the other hand, some piRNAs in prenatal and prepachytene pools display signatures indicative of a proposed RNA-mediated amplification loop that uses primary piRNAs to generate secondary piRNAs from precursor transcripts (ping-pong mechanism) (10, 11). Apart from the Piwi proteins themselves, factors directly impacting piRNA production are unknown.We previously identified Mov10l1 as a gene specifically expressed in mouse germ cells, which encodes a putative RNA helicase of unknown function (12). Whereas the N-terminal half of MOV10L1 is not homologous to any other mouse proteins, its C-terminal RNA helicase domain exhibits low homology (45% amino acid identity) with MOV10. MOV10, the vertebrate homolog of Drosophila Armi, is ubiquitously expressed. In mammalian cells, MOV10 is associated with Argonaute proteins in the RNA-induced silencing complex (RISC) and is functionally required for RNA interference (13, 14). Here we demonstrate that MOV10L1 is an essential factor in the piRNA pathway. Results MOV10L1 Is Associated with Piwi Proteins.To identi...

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.

BackgroundLife-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 As (single), Apr (paired), and Aal (aligned) spermatogonia, subdivision of this primitive cell population using cytological markers would greatly facilitate characterization of their functions.ResultsIn the present study, we show that LIN28, a pluripotency factor, is specifically expressed in undifferentiated spermatogonia (As, Apr, and Aal) in mouse. Ngn3 also specifically labels undifferentiated spermatogonia. We used Ngn3-GFP knockin mice, in which GFP expression is under the control of all Ngn3 transcription regulatory elements. Remarkably, Ngn3-GFP is only expressed in ~40% of LIN28-positive As (single) cells. The percentage of Ngn3-GFP-positive clusters increases dramatically with the chain length of interconnected spermatogonia.ConclusionOur 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) and Ngn3-GFP-positive (high differentiation commitment). Furthermore, Ngn3-GFP-negative cells are found in chains of Ngn3-GFP-positive spermatogonia, suggesting that cells in the Aal spermatogonia could revert to a more primitive state.

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.

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