Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes

Article metrics

Abstract

RNA interference (RNAi) is a mechanism by which double-stranded RNAs (dsRNAs) suppress specific transcripts in a sequence-dependent manner. dsRNAs are processed by Dicer to 21–24-nucleotide small interfering RNAs (siRNAs) and then incorporated into the argonaute (Ago) proteins1,2,3,4. Gene regulation by endogenous siRNAs has been observed only in organisms possessing RNA-dependent RNA polymerase (RdRP)5,6,7,8,9,10. In mammals, where no RdRP activity has been found, biogenesis and function of endogenous siRNAs remain largely unknown. Here we show, using mouse oocytes, that endogenous siRNAs are derived from naturally occurring dsRNAs and have roles in the regulation of gene expression. By means of deep sequencing, we identify a large number of both 25–27-nucleotide Piwi-interacting RNAs (piRNAs) and 21-nucleotide siRNAs corresponding to messenger RNAs or retrotransposons in growing oocytes. piRNAs are bound to Mili and have a role in the regulation of retrotransposons. siRNAs are exclusively mapped to retrotransposons or other genomic regions that produce transcripts capable of forming dsRNA structures. Inverted repeat structures, bidirectional transcription and antisense transcripts from various loci are sources of the dsRNAs. Some precursor transcripts of siRNAs are derived from expressed pseudogenes, indicating that one role of pseudogenes is to adjust the level of the founding source mRNA through RNAi. Loss of Dicer or Ago2 results in decreased levels of siRNAs and increased levels of retrotransposon and protein-coding transcripts complementary to the siRNAs. Thus, the RNAi pathway regulates both protein-coding transcripts and retrotransposons in mouse oocytes. Our results reveal a role for endogenous siRNAs in mammalian oocytes and show that organisms lacking RdRP activity can produce functional endogenous siRNAs from naturally occurring dsRNAs.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Small RNA profile and expression of small RNA pathway components in mouse oocytes.
Figure 2: Structure of the hp-siRNA cluster at the Au76 locus.
Figure 3: Structure of the cis-nat-siRNA cluster at the Pdzd11/Kif4 locus.
Figure 4: Structure of the trans-nat-siRNA cluster pair at the loci of Ppp4r1 and its processed pseudogene.

Accession codes

Primary accessions

GenBank/EMBL/DDBJ

Data deposits

The GenBank accession numbers of the small RNAs mapped to the genome in clusters and piRNAs co-immunoprecipitated with Mili are AB334800–AB349184 and AB349185–AB353040, respectively.

References

  1. 1

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

  2. 2

    Du, T. & Zamore, P. D. microPrimer: the biogenesis and function of microRNA. Development 132, 4645–4652 (2005)

  3. 3

    Filipowicz, W., Jaskiewicz, L., Kolb, F. A. & Pillai, R. S. Post-transcriptional gene silencing by siRNAs and miRNAs. Curr. Opin. Struct. Biol. 15, 331–341 (2005)

  4. 4

    Ambros, V. The functions of animal microRNAs. Nature 431, 350–355 (2004)

  5. 5

    Vaucheret, H. Post-transcriptional small RNA pathways in plants: mechanisms and regulations. Genes Dev. 20, 759–771 (2006)

  6. 6

    Ambros, V. & Chen, X. The regulation of genes and genomes by small RNAs. Development 134, 1635–1641 (2007)

  7. 7

    Pak, J. & Fire, A. Distinct populations of primary and secondary effectors during RNAi in C. elegans . Science 315, 241–244 (2007)

  8. 8

    Ruby, J. G. et al. Large-scale sequencing reveals 21U-RNAs and additional microRNAs and endogenous siRNAs in C. elegans . Cell 127, 1193–1207 (2006)

  9. 9

    Sijen, T., Steiner, F. A., Thijssen, K. L. & Plasterk, R. H. Secondary siRNAs result from unprimed RNA synthesis and form a distinct class. Science 315, 244–247 (2007)

  10. 10

    Yigit, E. et al. Analysis of the C. elegans Argonaute family reveals that distinct Argonautes act sequentially during RNAi. Cell 127, 747–757 (2006)

  11. 11

    Okazaki, Y. et al. Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs. Nature 420, 563–573 (2002)

  12. 12

    Yelin, R. et al. Widespread occurrence of antisense transcription in the human genome. Nature Biotechnol. 21, 379–386 (2003)

  13. 13

    Chen, J. et al. Over 20% of human transcripts might form sense-antisense pairs. Nucleic Acids Res. 32, 4812–4820 (2004)

  14. 14

    Carninci, P. et al. The transcriptional landscape of the mammalian genome. Science 309, 1559–1563 (2005)

  15. 15

    Lavorgna, G. et al. In search of antisense. Trends Biochem. Sci. 29, 88–94 (2004)

  16. 16

    Watanabe, T. et al. Identification and characterization of two novel classes of small RNAs in the mouse germline: retrotransposon-derived siRNAs in oocytes and germline small RNAs in testes. Genes Dev. 20, 1732–1743 (2006)

  17. 17

    Yang, N. & Kazazian, H. H. L1 retrotransposition is suppressed by endogenously encoded small interfering RNAs in human cultured cells. Nature Struct. Mol. Biol. 13, 763–771 (2006)

  18. 18

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

  19. 19

    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)

  20. 20

    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)

  21. 21

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

  22. 22

    Saito, K. et al. Specific association of Piwi with rasiRNAs derived from retrotransposon and heterochromatic regions in the Drosophila genome. Genes Dev. 20, 2214–2222 (2006)

  23. 23

    Vagin, V. V. et al. A distinct small RNA pathway silences selfish genetic elements in the germline. Science 313, 320–324 (2006)

  24. 24

    Hutvagner, G. et al. A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science 293, 834–838 (2001)

  25. 25

    Gray, T. A., Wilson, A., Fortin, P. J. & Nicholls, R. D. The putatively functional Mkrn1-p1 pseudogene is neither expressed nor imprinted, nor does it regulate its source gene in trans. Proc. Natl Acad. Sci. USA 103, 12039–12044 (2006)

  26. 26

    Hirotsune, S. et al. An expressed pseudogene regulates the messenger-RNA stability of its homologous coding gene. Nature 423, 91–96 (2003)

  27. 27

    Peaston, A. E. et al. Retrotransposons regulate host genes in mouse oocytes and preimplantation embryos. Dev. Cell 7, 597–606 (2004)

  28. 28

    Tam, O. H. et al. Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes. Nature doi: 10.1038/nature06904 (this issue)

  29. 29

    Tang, F. et al. Maternal microRNAs are essential for mouse zygotic development. Genes Dev. 21, 644–648 (2007)

  30. 30

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

Download references

Acknowledgements

We thank A. Tarakhovsky and D. O’Carroll for mice with the Dicer and Ago2 conditional alleles; G. J. Hannon and A. Girard for the anti-AGO2 antibody; T. Sado, K. Hata and H. Furuumi for scientific and technical advice; Y. Kurihara, A. Takeda and K. Ichiyanagi for comments on the manuscript; N. Minami and Y. Hoki for expertise in mouse oocytes; and K. Takada and M. Kiyooka for technical assistance. We thank RIKEN for the Super Combined Cluster (RSCC) computational resources. We also thank members of the Sasaki laboratory for discussion and encouragement. T.W. is a research fellow of the Japan Society for the Promotion of Science. This work was supported in part by Grants-in-Aid for Scientific Research on Priority Area from the Ministry of Education, Culture, Sports, Science, and Technology of Japan to H.S.

Author Contributions T.W. performed experiments and interpreted results; Y.T. generated computational programs and analysed data with T.W.; A.T., Y.S. and Y.K. were involved in small RNA sequencing; Y.O., H.C. and T.K. were involved in oocyte collection; M.K. and M.A.S. prepared the samples from conditional Dicer and Ago2 knockout mice; S.K.-M. and T.N. provided samples from Mili knockout mouse; H.S. and T.W. designed the study and wrote the manuscript.

Author information

Correspondence to Toshiaki Watanabe or Hiroyuki Sasaki.

Supplementary information

Supplementary information

The file contains Supplementary Methods, Supplementary Figures 1-10 with Legends and Supplementary Tables 1-3. (PDF 2763 kb)

Supplementary information

The file contains Supplementary Table 4. (XLS 2536 kb)

Rights and permissions

Reprints and Permissions

About this article

Further reading

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.