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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.

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References

  1. 1

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

  2. 2

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

  3. 3

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

  4. 4

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

  5. 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)

  6. 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)

  7. 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)

  8. 8

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

  9. 9

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

  10. 10

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

  11. 11

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

  12. 12

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

  13. 13

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

  14. 14

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

  15. 15

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

  16. 16

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

  17. 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)

  18. 18

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

  19. 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)

  20. 20

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

  21. 21

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

  22. 22

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

  23. 23

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

  24. 24

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

  25. 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)

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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

    • Alexei Aravin

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

    • Sébastien Pfeffer

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

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

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

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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.

Corresponding authors

Correspondence to Mihaela Zavolan or Thomas Tuschl.

Supplementary information

  1. 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)

  2. Supplementary Table 3

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

  3. Supplementary Table 4

    List of mouse piRNA clones. (XLS 1039 kb)

  4. Supplementary Table 6

    Mouse piRNA minicluster alignments. (DOC 270 kb)

  5. Supplementary Table 7

    Human piRNA minicluster alignments. (DOC 30 kb)

  6. Supplementary Table 8

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

  7. Supplementary Table 9

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

  8. Supplementary Figures

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

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DOI

https://doi.org/10.1038/nature04916

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