Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

A novel class of small RNAs bind to MILI protein in mouse testes

Nature volume 442, pages 203–207 (2006)Cite this article

Abstract

Small RNAs bound to Argonaute proteins recognize partially or fully complementary nucleic acid targets in diverse gene-silencing processes1,2,3,4. A subgroup of the Argonaute proteins—known as the ‘Piwi family’5—is required for germ- and stem-cell development in invertebrates6,7, and two Piwi members—MILI and MIWI—are essential for spermatogenesis in mouse8,9. Here we describe a new class of small RNAs that bind to MILI in mouse male germ cells, where they accumulate at the onset of meiosis. The sequences of the over 1,000 identified unique molecules share a strong preference for a 5′ uridine, but otherwise cannot be readily classified into sequence families. Genomic mapping of these small RNAs reveals a limited number of clusters, suggesting that these RNAs are processed from long primary transcripts. The small RNAs are 26–31 nucleotides (nt) in length—clearly distinct from the 21–23 nt of microRNAs (miRNAs) or short interfering RNAs (siRNAs)—and we refer to them as ‘Piwi-interacting RNAs’ or piRNAs. Orthologous human chromosomal regions also give rise to small RNAs with the characteristics of piRNAs, but the cloned sequences are distinct. The identification of this new class of small RNAs provides an important starting point to determine the molecular function of Piwi proteins in mammalian spermatogenesis.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: MILI interacts with 26–28-nt RNAs.
Figure 2: MILI-interacting piRNAs are encoded in clustered genomic loci.
Figure 3: Temporal expression of piRNAs during mouse spermatogenesis.
Figure 4: Predominant mouse piRNA cluster and its orthologous cluster in human.

Similar content being viewed by others

References

  1. Meister, G. & Tuschl, T. Mechanisms of gene silencing by double-stranded RNA. Nature 431, 343–349 (2004)

    Article  ADS  CAS  Google Scholar 

  2. Mello, C. C. & Conte, D. Jr. Revealing the world of RNA interference. Nature 431, 338–342 (2004)

    Article  ADS  CAS  Google Scholar 

  3. Matzke, M. A. & Birchler, J. A. RNAi-mediated pathways in the nucleus. Nature Rev. Genet. 6, 24–35 (2005)

    Article  CAS  Google Scholar 

  4. Zamore, P. D. & Haley, B. Ribo-gnome: the big world of small RNAs. Science 309, 1519–1524 (2005)

    Article  ADS  CAS  Google Scholar 

  5. Carmell, M. A., Xuan, Z., Zhang, M. Q. & Hannon, G. J. The Argonaute family: tentacles that reach into RNAi, developmental control, stem cell maintenance, and tumorigenesis. Genes Dev. 16, 2733–2742 (2002)

    Article  CAS  Google Scholar 

  6. 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 

  7. Reddien, P. W., Oviedo, N. J., Jennings, J. R., Jenkin, J. C. & Sanchez Alvarado, A. SMEDWI-2 is a PIWI-like protein that regulates planarian stem cells. Science 310, 1327–1330 (2005)

    Article  ADS  CAS  Google Scholar 

  8. 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 

  9. 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 

  10. Aravin, A. A. et al. The small RNA profile during Drosophila melanogaster development. Dev. Cell 5, 337–350 (2003)

    Article  CAS  Google Scholar 

  11. Bartel, D. P. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297 (2004)

    Article  CAS  Google Scholar 

  12. Chen, P. Y. et al. The developmental miRNA profiles of zebrafish as determined by small RNA cloning. Genes Dev. 19, 1288–1293 (2005)

    Article  CAS  Google Scholar 

  13. Bellve, A. R. et al. Spermatogenic cells of the prepuberal mouse. Isolation and morphological characterization. J. Cell Biol. 74, 68–85 (1977)

    Article  CAS  Google Scholar 

  14. Siepel, A. et al. Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes. Genome Res. 15, 1034–1050 (2005)

    Article  CAS  Google Scholar 

  15. Mochizuki, K. & Gorovsky, M. A. Small RNAs in genome rearrangement in Tetrahymena. Curr. Opin. Genet. Dev. 14, 181–187 (2004)

    Article  CAS  Google Scholar 

  16. Grewal, S. I. & Rice, J. C. Regulation of heterochromatin by histone methylation and small RNAs. Curr. Opin. Cell Biol. 16, 230–238 (2004)

    Article  CAS  Google Scholar 

  17. Jenab, S. & Morris, P. L. Testicular leukemia inhibitory factor (LIF) and LIF receptor mediate phosphorylation of signal transducers and activators of transcription (STAT)-3 and STAT-1 and induce c-fos transcription and activator protein-1 activation in rat Sertoli but not germ cells. Endocrinology 139, 1883–1890 (1998)

    Article  CAS  Google Scholar 

  18. Pfeffer, S. et al. Identification of microRNAs of the herpesvirus family. Nature Methods 2, 269–276 (2005)

    Article  CAS  Google Scholar 

  19. Pfeffer, S., Lagos-Quintana, M. & Tuschl, T. in Current Protocols in Molecular Biology (eds Ausubel, F. M. et al.) 26.4.1–26.4.18 (John Wiley and Sons, New York, 2003)

    Google Scholar 

  20. Lagos-Quintana, M. et al. Identification of tissue-specific microRNAs from mouse. Curr. Biol. 12, 735–739 (2002)

    Article  CAS  Google Scholar 

  21. Pfeffer, S. et al. Identification of virus-encoded microRNAs. Science 304, 734–736 (2004)

    Article  ADS  CAS  Google Scholar 

  22. Lim, L. P., Glasner, M. E., Yekta, S., Burge, C. B. & Bartel, D. P. Vertebrate microRNA genes. Science 299, 1540 (2003)

    Article  CAS  Google Scholar 

  23. Berezikov, E. et al. Phylogenetic shadowing and computational identification of human microRNA genes. Cell 120, 21–24 (2005)

    Article  CAS  Google Scholar 

  24. Legendre, M., Lambert, A. & Gautheret, D. Profile-based detection of microRNA precursors in animal genomes. Bioinformatics 21, 841–845 (2005)

    Article  CAS  Google Scholar 

  25. Pruitt, K. D., Tatusova, T. & Maglott, D. R. NCBI Reference Sequence (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins. Nucleic Acids Res. 33, D501–D504 (2005)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank J. Brennecke, M. Landthaler, Y. Pei, K. Fejes Toth and A. Sewer for discussion and help with the manuscript. We also thank L. Mitchell and K. Hwang for expert assistance in germ-cell purification, M. Poy and M. Stoffel for providing mouse tissues, and D. Weir and R. Choksi for assistance with sequencing. This work was supported by a FRAXA Research Foundation postdoctoral fellowship to A.A., an NIH grant to P.M., NIH grants to T.T., and an SNF grant to M.Z. Author Contributions A.A., S.P. and M.L.-Q. prepared the mouse, and P.L. and N.I. the human, testes small RNA libraries. A.A. recognized the presence of piRNAs, performed the MILI IPs, prepared the MILI-interacting small RNA library, and performed, together with N.I., the northern blotting analysis. S.K.-M. and T.N. produced, characterized and purified the MILI antibody. T.T. developed the concept of cloning from Ago/Piwi IPs. P.M. isolated germline cells. M.C., J.J.R. and J.J. performed the large-scale sequencing. The bioinformatic analyses of piRNAs were designed and carried out by D.G. and M.Z. with input from A.A. and T.T. The database of RNAs with known function was compiled by M.Z., R.S., P.L. and S.P., the software used for small RNA annotation was developed by M.Z., R.S. and C.S., and the manuscript was written by A.A., M.Z. and T.T.

Author information

Author notes

  1. Alexei Aravin

    Present address: Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, New York, 11724, USA

  2. Sébastien Pfeffer

    Present address: CNRS-UPR 2357, IBMP, 12 rue du Général Zimmer, 67084, Strasbourg Cedex, France

  3. Alexei Aravin and Dimos Gaidatzis: *These authors contributed equally to this work

Authors and Affiliations

  1. Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, New York, 1230 York Avenue, Box 186, New York, 10021, USA

    Alexei Aravin, Sébastien Pfeffer, Mariana Lagos-Quintana, Pablo Landgraf, Nicola Iovino & Thomas Tuschl

  2. Biozentrum, Universität Basel, Basel, Klingelbergstr 50-70, CH-4056, Switzerland

    Dimos Gaidatzis & Mihaela Zavolan

  3. Population Council, The Rockefeller University, New York, 1230 York Avenue, New York, 10021, USA

    Patricia Morris

  4. J. Craig Venter Institute, Functional Genomics, Rockville, 9704 Medical Center Drive, Maryland, 20850, USA

    Michael J. Brownstein

  5. Department of Pathology, Medical School, Graduate School of Frontier Biosciences, Osaka University, Osaka, Yamada-oka 2-2 Suita, 565-0871, Japan

    Satomi Kuramochi-Miyagawa & Toru Nakano

  6. Columbia Genome Center, Russ Berrie Pavilion, New York, 1150 St. Nicholas Avenue, New York, 10032, USA

    Minchen Chien, James J. Russo & Jingyue Ju

  7. Department of Chemical Engineering, Columbia University, New York, 500 West 120 Street, New York, 10027, USA

    Jingyue Ju

  8. Computational Biology Center, Memorial Sloan-Kettering Cancer Center, New York, New York, 10021, USA

    Robert Sheridan & Chris Sander

  9. Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, New York, 11724, USA

    Sébastien Pfeffer

  10. CNRS-UPR 2357, IBMP, 12 rue du Général Zimmer, 67084, Strasbourg Cedex, France

    Alexei Aravin

Authors
  1. Alexei Aravin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dimos Gaidatzis
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sébastien Pfeffer
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mariana Lagos-Quintana
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Pablo Landgraf
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Nicola Iovino
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Patricia Morris
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Michael J. Brownstein
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Satomi Kuramochi-Miyagawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Toru Nakano
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Minchen Chien
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. James J. Russo
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Jingyue Ju
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Robert Sheridan
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Chris Sander
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Mihaela Zavolan
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Thomas Tuschl
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Mihaela Zavolan or Thomas Tuschl.

Ethics declarations

Competing interests

Sequences of the piRNAs determined in this paper are given in Supplementary Tables 4 and 9. Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Supplementary information

Supplementary Notes

This file contains Supplementary Methods, Supplementary Tables 1–10 (excluding 3, 4, 6–9) and their legends, and Supplementary Figure Legends. (DOC 168 kb)

Supplementary Table 3

Summary of the mouse piRNA clusters. (XLS 28 kb)

Supplementary Table 4

List of mouse piRNA clones. (XLS 1039 kb)

Supplementary Table 6

Mouse piRNA minicluster alignments. (DOC 270 kb)

Supplementary Table 7

Human piRNA minicluster alignments. (DOC 30 kb)

Supplementary Table 8

Summary of the human piRNA clusters. (XLS 21 kb)

Supplementary Table 9

Supplementary Table 9 nature04916-s7.xls List of human piRNA clones. (XLS 194 kb)

Supplementary Figures

This file contains Supplementary Figures 1–6. (PDF 5095 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Aravin, A., Gaidatzis, D., Pfeffer, S. et al. A novel class of small RNAs bind to MILI protein in mouse testes. Nature 442, 203–207 (2006). https://doi.org/10.1038/nature04916

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature04916

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing