Blockade of Pachytene piRNA Biogenesis Reveals a Novel Requirement for Maintaining Post-Meiotic Germline Genome Integrity Ke Zheng, P. Jeremy Wang* Department of Animal Biology, Center for Animal Transgenesis and Germ Cell Research, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania, United States of America Abstract 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. Citation: Zheng K, Wang PJ (2012) Blockade of Pachytene piRNA Biogenesis Reveals a Novel Requirement for Maintaining Post-Meiotic Germline Genome Integrity. PLoS Genet 8(11): e1003038. doi:10.1371/journal.pgen.1003038 Editor: John C. Schimenti, Cornell University, United States of America Received March 16, 2012; Accepted September 5, 2012; Published November 15, 2012 Copyright: 2012 Zheng, Wang. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institutes of Health/National Institute of Child Health and Human Development grant R01HD069592. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: pwang@vet.upenn.edu Introduction Piwi-interacting RNAs (piRNAs) are a diverse class of gonad- specific small interfering RNAs that bind to members of the Piwi subfamily of Argonaute proteins. One common function of piRNAs in all species studied so far is the silencing of transposable elements, which is essential for the protection of genome integrity during germ cell development [1–3]. Distinct from miRNAs and siRNAs in origin, length, structure, and biogenesis, piRNAs are generated by dicer-independent processing of long precursor transcripts, however, the precise mechanisms of their biogenesis remain largely unclear [4,5]. In mice, the Piwi family has three members: Miwi (Piwil1), Mili (Piwil2), and Miwi2 (Piwil4). These Piwi genes exhibit different developmental expression patterns in testis. While Miwi2 is expressed in fetal and perinatal germ cells [6], the expression of Miwi is restricted to pachytene spermatocytes and round spermatids in adult testes [7]. Mili is expressed from the fetal germ cell stage onwards through the round spermatid stage [8]. Two developmentally distinct populations of piRNAs are expressed in mouse male germ cells at the pre-pachytene and pachytene stages. Pre-pachytene piRNAs are mostly derived from transposable elements and are associated with MILI and MIWI2 in fetal and perinatal male germ cells [6,9,10]. Pachytene piRNAs originate from ,3000 genomic clusters [11] and bind to both MILI and MIWI [12–17]. Interestingly, more than 90% of MILI- and MIWI-bound pachytene piRNAs shared identical 59end sequences [18]. As a result, most MILI- and MIWI-bound pachytene piRNAs map to the same genomic clusters [18]. The biogenesis of piRNAs involves primary and secondary processing mechanisms [1,2]. Pre-pachytene piRNAs derive from precursor transcripts that are cleaved into putative primary piRNA intermediate molecules by a yet unknown primary processing mechanism, followed by loading onto MILI for further processing. In embryonic germ cells, the endonuclease (slicer) activity of MILI is required for the secondary piRNA processing mechanism, which amplifies MILI-bound piRNAs through an intra-MILI ping-pong loop and generates all MIWI2-bound secondary piRNAs [19]. In this feed-forward ping-pong model, Piwi proteins with piRNAs complimentary to retroelement- derived transcripts drive transcript cleavage and piRNA amplifi- cation [6,9,10,19]. In contrast, the biogenesis of pachytene piRNAs only engages the primary processing mechanism, i.e. the presumptive cleavage by an unknown nuclease and eventual processing of the precursor transcript into mature piRNAs [5,17,20,21]. Therefore, pachytene piRNAs provide a simple and ideal system for dissecting the mysterious primary processing mechanism in mammals [11,13–16]. We and others previously demonstrated that MOV10L1, a putative RNA helicase, interacts with all mouse Piwi proteins and is required for biogenesis of pre-pachytene piRNAs [22,23]. MOV10L1 homologues are evolutionarily conserved among insects (Armi in Drosophila melanogaster), plants (SDE3 in Arabidopsis PLOS Genetics | www.plosgenetics.org 1 November 2012 | Volume 8 | Issue 11 | e1003038