Abstract
Germ cells implement elaborate mechanisms to protect their genetic material and to regulate gene expression during differentiation. Piwi proteins bind Piwi-interacting RNAs (piRNAs), small germline RNAs whose biogenesis and functions are still largely elusive. We used high-throughput sequencing after cross-linking and immunoprecipitation (HITS-CLIP) coupled with RNA–sequencing (RNA-seq) to characterize the genome-wide target RNA repertoire of Mili (Piwil2) and Miwi (Piwil1), two Piwi proteins expressed in mouse postnatal testis. We report the in vivo pathway of primary piRNA biogenesis and implicate distinct nucleolytic activities that process Piwi-bound precursor transcripts. Our studies indicate that pachytene piRNAs are the end products of RNA processing. HITS-CLIP demonstrated that Miwi binds spermiogenic mRNAs directly, without using piRNAs as guides, and independent biochemical analyses of testis mRNA ribonucleoproteins (mRNPs) established that Miwi functions in the formation of mRNP complexes that stabilize mRNAs essential for spermiogenesis.
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Acknowledgements
We are grateful to J. Schug for assistance with Illumina analysis approaches in the early phase of this project and to S. Fayngerts, S. Rafail and members of the Mourelatos laboratory for technical help and lively discussions. The antibody to Tnp2 was a gift from W.S. Kistler (University of South Carolina, Columbia). This work was supported by the US National Institute of General Medical Sciences of the National Institutes of Health (R01GM0720777) and in part by Penn Institute for Regenerative Medicine and Perelman School of Medicine, University of Pennsylvania grants to Z.M. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Work in the Gregory lab was supported in part by grant #IRG-78-002-30 from the American Cancer Society as well as the Penn Genome Frontiers Institute and a grant with the Pennsylvania Department of Health. The Department of Health specifically disclaims responsibility for any analyses, interpretations or conclusions.
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A.V. and Z.M. conceived and directed experiments. A.V. performed HITS-CLIP, RNA-seq and Nycodenz experiments; Y.K. performed overexpression of Mili and Miwi and in vitro cross-linking experiments. Q.Z., M.M., P.A., A.V., B.D.G., Z.M. analyzed data and A.V. wrote the manuscript with input and editing from Q.Z., B.D.G. and Z.M.
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Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–7, Supplementary Note (PDF 5290 kb)
Supplementary Table 1
Number of raw and mapped deep sequencing reads of HITS-CLIP libraries. (XLSX 31 kb)
Supplementary Table 2
Comparison of CLIP and standard IP piRNA populations. (XLSX 28 kb)
Supplementary Table 3
Number of raw and mapped deep sequencing reads of RNA-seq libraries. (XLSX 34 kb)
Supplementary Table 4
Genomic locations of Intergenic piRNA hotspots. (XLSX 114 kb)
Supplementary Table 5
CLIP tag abundance and fold difference (glog-odds) of Miwi covered genes. (XLSX 64 kb)
Supplementary Table 6
Gene Ontology terms enriched in Miwi-covered genes. (XLSX 20 kb)
Supplementary Table 7
Mass spectrometry analysis of eluates from oligo(dT) pull-down experiments. (XLSX 10 kb)
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Vourekas, A., Zheng, Q., Alexiou, P. et al. Mili and Miwi target RNA repertoire reveals piRNA biogenesis and function of Miwi in spermiogenesis. Nat Struct Mol Biol 19, 773–781 (2012). https://doi.org/10.1038/nsmb.2347
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DOI: https://doi.org/10.1038/nsmb.2347
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