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Mili and Miwi target RNA repertoire reveals piRNA biogenesis and function of Miwi in spermiogenesis

Nature Structural & Molecular Biology volume 19, pages 773–781 (2012)Cite this article

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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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Figure 1: Mili and Miwi HITS-CLIP.
Figure 2: Size distribution, nucleotide preference and genomic classification of mapped CLIP reads.
Figure 3: Processing of intergenic piRNA precursors.
Figure 4: Nonrepeat piRNAs lack complementary RNA targets.
Figure 5: Absence of piRNA processing in Miwi-bound mRNAs essential for spermiogenesis.
Figure 6: Miwi, devoid of piRNAs, binds and stabilizes spermiogenic mRNAs in repressed mRNPs.
Figure 7: Model for primary piRNA biogenesis, and functions of Mili and Miwi piRNPs during mouse male germ cell differentiation.

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References

  1. Cox, D.N. et al. A novel class of evolutionarily conserved genes defined by piwi are essential for stem cell self-renewal. Genes Dev. 12, 3715–3727 (1998).

    Article  CAS  Google Scholar 

  2. Deng, W. & Lin, H. miwi, a murine homolog of piwi, encodes a cytoplasmic protein essential for spermatogenesis. Dev. Cell 2, 819–830 (2002).

    Article  CAS  Google Scholar 

  3. Kuramochi-Miyagawa, S. et al. Mili, a mammalian member of piwi family gene, is essential for spermatogenesis. Development 131, 839–849 (2004).

    Article  CAS  Google Scholar 

  4. Boswell, R.E. & Mahowald, A.P. tudor, a gene required for assembly of the germ plasm in Drosophila melanogaster. Cell 43, 97–104 (1985).

    Article  CAS  Google Scholar 

  5. Chen, C., Nott, T.J., Jin, J. & Pawson, T. Deciphering arginine methylation: Tudor tells the tale. Nat. Rev. Mol. Cell Biol. 12, 629–642 (2011).

    Article  CAS  Google Scholar 

  6. Kirino, Y. et al. Arginine methylation of Piwi proteins catalysed by dPRMT5 is required for Ago3 and Aub stability. Nat. Cell Biol. 11, 652–658 (2009).

    Article  CAS  Google Scholar 

  7. Nishida, K.M. et al. Functional involvement of Tudor and dPRMT5 in the piRNA processing pathway in Drosophila germlines. EMBO J. 28, 3820–3831 (2009).

    Article  CAS  Google Scholar 

  8. Vagin, V.V. et al. Proteomic analysis of murine Piwi proteins reveals a role for arginine methylation in specifying interaction with Tudor family members. Genes Dev. 23, 1749–1762 (2009).

    Article  CAS  Google Scholar 

  9. Kirino, Y. et al. Arginine methylation of Aubergine mediates Tudor binding and germ plasm localization. RNA 16, 70–78 (2010).

    Article  CAS  Google Scholar 

  10. Yokota, S. Historical survey on chromatoid body research. Acta Histochem. Cytochem. 41, 65–82 (2008).

    Article  CAS  Google Scholar 

  11. Lau, N.C. et al. Characterization of the piRNA complex from rat testes. Science 313, 363–367 (2006).

    Article  CAS  Google Scholar 

  12. Aravin, A. et al. A novel class of small RNAs bind to MILI protein in mouse testes. Nature 442, 203–207 (2006).

    Article  CAS  Google Scholar 

  13. Girard, A., Sachidanandam, R., Hannon, G.J. & Carmell, M.A. A germline-specific class of small RNAs binds mammalian Piwi proteins. Nature 442, 199–202 (2006).

    Article  Google Scholar 

  14. Grivna, S.T., Beyret, E., Wang, Z. & Lin, H. A novel class of small RNAs in mouse spermatogenic cells. Genes Dev. 20, 1709–1714 (2006).

    Article  CAS  Google Scholar 

  15. Siomi, M.C., Sato, K., Pezic, D. & Aravin, A.A. PIWI-interacting small RNAs: the vanguard of genome defence. Nat. Rev. Mol. Cell Biol. 12, 246–258 (2011).

    Article  CAS  Google Scholar 

  16. Kawaoka, S., Izumi, N., Katsuma, S. & Tomari, Y. 3′ End formation of PIWI-interacting RNAs in vitro. Mol. Cell 43, 1015–1022 (2011).

    Article  CAS  Google Scholar 

  17. Pillai, R.S. & Chuma, S. piRNAs and their involvement in male germline development in mice. Dev. Growth Differ. 54, 78–92 (2012).

    Article  CAS  Google Scholar 

  18. Besse, F. & Ephrussi, A. Translational control of localized mRNAs: restricting protein synthesis in space and time. Nat. Rev. Mol. Cell Biol. 9, 971–980 (2008).

    Article  CAS  Google Scholar 

  19. Heidaran, M.A., Showman, R.M. & Kistler, W.S. A cytochemical study of the transcriptional and translational regulation of nuclear transition protein 1 (TP1), a major chromosomal protein of mammalian spermatids. J. Cell Biol. 106, 1427–1433 (1988).

    Article  CAS  Google Scholar 

  20. Kleene, K.C. Patterns of translational regulation in the mammalian testis. Mol. Reprod. Dev. 43, 268–281 (1996).

    Article  CAS  Google Scholar 

  21. Carmell, M.A. et al. MIWI2 is essential for spermatogenesis and repression of transposons in the mouse male germline. Dev. Cell 12, 503–514 (2007).

    Article  CAS  Google Scholar 

  22. Reuter, M. et al. Loss of the Mili-interacting Tudor domain-containing protein-1 activates transposons and alters the Mili-associated small RNA profile. Nat. Struct. Mol. Biol. 16, 639–646 (2009).

    Article  CAS  Google Scholar 

  23. Shoji, M. et al. The TDRD9-MIWI2 complex is essential for piRNA-mediated retrotransposon silencing in the mouse male germline. Dev. Cell 17, 775–787 (2009).

    Article  CAS  Google Scholar 

  24. Kuramochi-Miyagawa, S. et al. MVH in piRNA processing and gene silencing of retrotransposons. Genes Dev. 24, 887–892 (2010).

    Article  CAS  Google Scholar 

  25. Frost, R.J. et al. MOV10L1 is necessary for protection of spermatocytes against retrotransposons by Piwi-interacting RNAs. Proc. Natl. Acad. Sci. USA 107, 11847–11852 (2010).

    Article  CAS  Google Scholar 

  26. Zheng, K. et al. Mouse MOV10L1 associates with Piwi proteins and is an essential component of the Piwi-interacting RNA (piRNA) pathway. Proc. Natl. Acad. Sci. USA 107, 11841–11846 (2010).

    Article  CAS  Google Scholar 

  27. De Fazio, S. et al. The endonuclease activity of Mili fuels piRNA amplification that silences LINE1 elements. Nature 480, 259–263 (2011).

    Article  CAS  Google Scholar 

  28. Tanaka, T. et al. Tudor domain containing 7 (Tdrd7) is essential for dynamic ribonucleoprotein (RNP) remodeling of chromatoid bodies during spermatogenesis. Proc. Natl. Acad. Sci. USA 108, 10579–10584 (2011).

    Article  CAS  Google Scholar 

  29. Vasileva, A., Tiedau, D., Firooznia, A., Muller-Reichert, T. & Jessberger, R. Tdrd6 is required for spermiogenesis, chromatoid body architecture, and regulation of miRNA expression. Curr. Biol. 19, 630–639 (2009).

    Article  CAS  Google Scholar 

  30. Chi, S.W., Zang, J.B., Mele, A. & Darnell, R.B. Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps. Nature 460, 479–486 (2009).

    Article  CAS  Google Scholar 

  31. Robine, N. et al. A broadly conserved pathway generates 3′UTR-directed primary piRNAs. Curr. Biol. 19, 2066–2076 (2009).

    Article  CAS  Google Scholar 

  32. Gunawardane, L.S. et al. A slicer-mediated mechanism for repeat-associated siRNA 5′ end formation in Drosophila. Science 315, 1587–1590 (2007).

    Article  CAS  Google Scholar 

  33. Horwich, M.D. et al. The Drosophila RNA methyltransferase, DmHen1, modifies germline piRNAs and single-stranded siRNAs in RISC. Curr. Biol. 17, 1265–1272 (2007).

    Article  CAS  Google Scholar 

  34. Kirino, Y. & Mourelatos, Z. The mouse homolog of HEN1 is a potential methylase for Piwi-interacting RNAs. RNA 13, 1397–1401 (2007).

    Article  CAS  Google Scholar 

  35. Simon, B. et al. Recognition of 2′-O-methylated 3′-end of piRNA by the PAZ domain of a Piwi protein. Structure 19, 172–180 (2011).

    Article  CAS  Google Scholar 

  36. Tian, Y., Simanshu, D.K., Ma, J.B. & Patel, D.J. Structural basis for piRNA 2′-O-methylated 3′-end recognition by Piwi PAZ (Piwi/Argonaute/Zwille) domains. Proc. Natl. Acad. Sci. USA 108, 903–910 (2011).

    Article  CAS  Google Scholar 

  37. Kirino, Y. & Mourelatos, Z. Site-specific crosslinking of human microRNPs to RNA targets. RNA 14, 2254–2259 (2008).

    Article  CAS  Google Scholar 

  38. Reuter, M. et al. Miwi catalysis is required for piRNA amplification-independent LINE1 transposon silencing. Nature 480, 264–267 (2011).

    Article  CAS  Google Scholar 

  39. Zhang, C. & Darnell, R.B. Mapping in vivo protein-RNA interactions at single-nucleotide resolution from HITS-CLIP data. Nat. Biotechnol. 29, 607–614 (2011).

    Article  CAS  Google Scholar 

  40. Herbert, T.P. & Hecht, N.B. The mouse Y-box protein, MSY2, is associated with a kinase on non-polysomal mouse testicular mRNAs. Nucleic Acids Res. 27, 1747–1753 (1999).

    Article  CAS  Google Scholar 

  41. Bagarova, J., Chowdhury, T.A., Kimura, M. & Kleene, K.C. Identification of elements in the Smcp 5′ and 3′ UTR that repress translation and promote the formation of heavy inactive mRNPs in spermatids by analysis of mutations in transgenic mice. Reproduction 140, 853–864 (2010).

    Article  CAS  Google Scholar 

  42. Kleene, K.C., Bagarova, J., Hawthorne, S.K. & Catado, L.M. Quantitative analysis of mRNA translation in mammalian spermatogenic cells with sucrose and Nycodenz gradients. Reproductive biology and endocrinology 8, 155 (2010).

    Article  CAS  Google Scholar 

  43. Giorgini, F., Davies, H.G. & Braun, R.E. Translational repression by MSY4 inhibits spermatid differentiation in mice. Development 129, 3669–3679 (2002).

    CAS  PubMed  Google Scholar 

  44. Kimura, M., Ishida, K., Kashiwabara, S. & Baba, T. Characterization of two cytoplasmic poly(A)-binding proteins, PABPC1 and PABPC2, in mouse spermatogenic cells. Biol. Reprod. 80, 545–554 (2009).

    Article  CAS  Google Scholar 

  45. Yang, J., Medvedev, S., Reddi, P.P., Schultz, R.M. & Hecht, N.B. The DNA/RNA-binding protein MSY2 marks specific transcripts for cytoplasmic storage in mouse male germ cells. Proc. Natl. Acad. Sci. USA 102, 1513–1518 (2005).

    Article  CAS  Google Scholar 

  46. Yang, J. et al. Absence of the DNA-/RNA-binding protein MSY2 results in male and female infertility. Proc. Natl. Acad. Sci. USA 102, 5755–5760 (2005).

    Article  CAS  Google Scholar 

  47. Huang, H. et al. piRNA-associated germline nuage formation and spermatogenesis require MitoPLD profusogenic mitochondrial-surface lipid signaling. Dev. Cell 20, 376–387 (2011).

    Article  CAS  Google Scholar 

  48. Watanabe, T. et al. MITOPLD is a mitochondrial protein essential for nuage formation and piRNA biogenesis in the mouse germline. Dev. Cell 20, 364–375 (2011).

    Article  CAS  Google Scholar 

  49. Hermo, L., Pelletier, R.M., Cyr, D.G. & Smith, C.E. Surfing the wave, cycle, life history, and genes/proteins expressed by testicular germ cells. Part 2: changes in spermatid organelles associated with development of spermatozoa. Microsc. Res. Tech. 73, 279–319 (2010).

    Article  CAS  Google Scholar 

  50. Haraguchi, C.M. et al. Chromatoid bodies: aggresome-like characteristics and degradation sites for organelles of spermiogenic cells. J. Histochem. Cytochem. 53, 455–465 (2005).

    Article  CAS  Google Scholar 

  51. Austin, C.R. & Sapsford, C.S. The development of the rat spermatid. J. R. Microsc. Soc. 71, 397–406 (1951).

    Article  CAS  Google Scholar 

  52. Dostie, J. & Dreyfuss, G. Translation is required to remove Y14 from mRNAs in the cytoplasm. Curr. Biol. 12, 1060–1067 (2002).

    Article  CAS  Google Scholar 

  53. Faehnle, C.R. & Joshua-Tor, L. Argonaute MID domain takes centre stage. EMBO Rep. 11, 564–565 (2010).

    Article  CAS  Google Scholar 

  54. Liu, X., Jin, D.Y., McManus, M.T. & Mourelatos, Z. Precursor microRNA-programmed silencing complex assembly pathways in mammals. Mol. Cell 46, 507–517 (2012).

    Article  CAS  Google Scholar 

  55. Oko, R.J., Jando, V., Wagner, C.L., Kistler, W.S. & Hermo, L.S. Chromatin reorganization in rat spermatids during the disappearance of testis-specific histone, H1t, and the appearance of transition proteins TP1 and TP2. Biol. Reprod. 54, 1141–1157 (1996).

    Article  CAS  Google Scholar 

  56. Alfonso, P.J. & Kistler, W.S. Immunohistochemical localization of spermatid nuclear transition protein 2 in the testes of rats and mice. Biol. Reprod. 48, 522–529 (1993).

    Article  CAS  Google Scholar 

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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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Author notes

  1. Panagiotis Alexiou and Manolis Maragkakis: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Pathology and Laboratory Medicine, Division of Neuropathology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Anastassios Vourekas, Panagiotis Alexiou, Manolis Maragkakis & Zissimos Mourelatos

  2. Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Qi Zheng & Brian D Gregory

  3. PENN Genome Frontiers Institute, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Qi Zheng, Brian D Gregory & Zissimos Mourelatos

  4. Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, California, USA

    Yohei Kirino

  5. Genomics and Computational Biology Graduate Program, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Brian D Gregory

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  1. Anastassios Vourekas
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Contributions

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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Correspondence to Zissimos Mourelatos.

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The authors declare no competing financial interests.

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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