vations suggest that the piRNA pathway, specifically MOV10L1 and pachytene piRNAs, play a yet undefined role in maintaining genome integrity in post-meiotic round spermatids. The MOV10L1 RNA Helicase Domain Is Required for Its Interaction with Piwi Proteins We previously found that MOV10L1 interacts with all three Piwi proteins (MILI, MIWI, and MIWI2) [22]. The low abundance of MOV10L1D in both Mov10l12/2 (ubiquitous mutant) testes [22] and adult Mov10l1 conditional mutant testes (Figure S2A) precluded co-immunoprecipitation (IP) experiments to ascertain if deletion of the helicase domain affected interaction of the truncated protein with Piwi proteins in vivo. However, a peak expression of MOV10L1D protein in post-natal day 20 Mov10l1fl/- Neurog3-Cre testes, with a level highly exceeding that of the remaining wild-type MOV10L1 (Figure S2A), allowed us to perform co-immunoprecipitation of day 20 testicular extracts with anti-MILI and anti- MOV10L1 antibodies (Figure 6A). While wild-type MOV10L1 (due to the lack of Neurog3-Cre expression in spermatogonia) could be detected as a very faint band in the MILI immunoprecipitate as expected, the much more abundant MOV10L1D was absent (Figure 6A, Lane 6). Notably, MILI was not detectable in the MOV10L1/MOV10L1D immunoprecipi- tate, and the level of MIWI was extremely low (Figure 6A, Lane 4). These results suggest that, apart from its putative enzymatic activity, the RNA helicase domain of MOV10L1 is also essential for its association with MILI and MIWI, and that piRNA production could be affected by disruption of the MOV10L1-Piwi interactions. Accumulation of Pachytene piRNA Precursors in Mov10l1 Mutant Testes Pachytene piRNAs are derived from only one strand of genomic clusters [11,13–16], prompting the hypothesis that a single long primary piRNA transcript is made from each cluster and is cleaved into intermediate RNAs by an unknown Dicer-independent mechanism [5,20,21]. Due to their large size and low abundance, detection of these precursor transcripts requires RT-PCR analysis, with the exception of a ,10 kb piLR (piRNA like small RNA) transcript that can be visualized on Northern blots of testicular extracts [50]. As the depletion of pachytene piRNAs in Mov10l1fl/- Neurog3-Cre testes may be due to a blockade of pachytene piRNA precursor processing, we examined the abundance of precursors of four pachytene piRNAs (piR1, piR2, piR3, and piLR) by RT- PCR assays (Figure S7). All four precursors accumulated substantially in Mov10l1fl/- Neurog3-Cre testes, at 8 to 20 fold increased levels (Figure 6B, 6C). As expected, abundance of the pre-pachytene piRNA precursor (cluster 10) [10] and the miRNA precursor Pri-let7g remained constant (Figure 6B, 6C). These data suggest that MOV10L1 is required for the primary processing of precursor transcripts and thus plays an essential role in the early steps of the piRNA biogenesis pathway, i.e. primary processing and loading onto Piwi proteins (Figure 6D). Discussion We have identified MOV10L1 as the only factor known to date that is required for the production of all pachytene piRNAs in mouse. As the biogenesis of pachytene piRNAs only involves the primary processing pathway, our conditional Mov10l1 mutants provide a unique opportunity to delineate this enigmatic component of piRNA biogenesis in mammalian species. Presum- ably, long piRNA precursor transcripts are first cleaved into intermediate molecules, and then processed into mature piRNAs Pachytene piRNA Biogenesis Requires MOV10L1 (Figure 6D). Observations that the Drosophila Armi-Piwi-Yb complex is associated with a population of 25–70 nt piRNA intermediate-like (piR-IL) molecules support this hypothesis [20]. Furthermore, recent biochemical studies using silkworm ovarian cell lysate have shown that intermediate piRNA molecules with 59 U are specifically loaded onto Piwi proteins and then trimmed from the 39end to generate mature piRNAs [21]. Here, we show that, in the mouse male germline, postnatal disruption of Mov10l1 does not affect the expression of Piwi proteins (MILI and MIWI) but causes a complete loss of pachytene piRNAs, demonstrating that MOV10L1 functions upstream of Piwi proteins in the piRNA biogenesis pathway. Consistent with its homology to Drosophila Armi [25,26], MOV10L1 is therefore a master regulator of piRNA biogenesis in mouse. This notion is further supported by the dramatic accumulation of pachytene piRNA precursors in the Mov10l1 mutant testes. As MOV10L1 interacts with Piwi proteins, we postulate that MOV10L1 may facilitate the loading of intermediate piRNA molecules onto the Piwi proteins in mouse (Figure 6D). In spermatocytes, proteins of the piRNA pathway such as MILI, MIWI, TDRD1, MAEL, and GASZ, localize to the nuage - inter- mitochondrial cement [7,8,36,37,44,51]. However, the functional significance of the physical association of nuage with mitochondria in germ cells is poorly understood. MitoPLD, a mitochondrial signaling protein, is essential for nuage formation and piRNA production, suggesting an important role for mitochondria in these mechanisms [39,40]. In this study, we find an unusual polar congregation of piRNA pathway proteins (such as MILI, MIWI, TDRD1, and GASZ). Similar to wild-type MOV10L1, truncated MOV10L1D is distributed diffusely through the cytoplasm of pachytene spermatocytes; therefore the polar coalescence of the other piRNA pathway components in MOV10L1-deficient pachytene cells is likely caused by the absence of pachytene piRNAs. However, as the association of Piwi-MOV10L1 is disrupted in the Mov10l1 mutant, it is also possible that the localization of Piwi proteins and their interacting partners has become perturbed as a consequence of this disruption. The unusual polar congregation of piRNA pathway proteins with mitochondria in Mov10l1 mutant spermatocytes suggests that MOV10L1 and/or pachytene piRNAs are essential for nuage formation and proper mitochondria distribution. Consistent with such a role, we find that the chromatoid body, a prominent nuage in spermatids, is fragmented in pachytene piRNA-deficient mutant cells. This previously unknown role in organelle distribution shows that pachytene piRNAs are intricately integrated in the inter- dependent relationships among piRNA production, nuage forma- tion, and mitochondria organization that are essential for male germ cell maturation. A recent study has shown that MIWI is an RNA-guided RNase with slicer activity that directly cleaves transcripts of the LINE1 retrotransposon [18]. Miwi-deficient and MiwiADH (slicer inactive) mutant testes, in which MIWI is either absent or lacks slicer activity, exhibit substantial accumulation of LINE1 transcripts and protein. In Mov10l1fl/- Neurog3-Cre testes, however, LINE1 RNA levels are not affected. One possible explanation for these differential effects on LINE1 abundance could be that, in Mov10l1fl/- Neurog3-Cre testes, MIWI is catalytically intact and may function as a slicer through pachytene piRNA-independent mechanisms. Moreover, MIWI directly binds to spermiogenic mRNAs, independent of piRNAs [17]. Although previous genetic studies of piRNA pathway mutants show that perturbation of pre-pachytene piRNAs causes meiotic arrest and de-repression of LINE1 and IAP retrotransposons, the functions of pachytene piRNAs have remained elusive. Our study PLOS Genetics | www.plosgenetics.org 9 November 2012 | Volume 8 | Issue 11 | e1003038