Letter | Published:

RNAi triggered by specialized machinery silences developmental genes and retrotransposons

Nature volume 493, pages 557560 (24 January 2013) | Download Citation

Abstract

RNA interference (RNAi) is a conserved mechanism in which small interfering RNAs (siRNAs) guide the degradation of cognate RNAs, but also promote heterochromatin assembly at repetitive DNA elements such as centromeric repeats1,2. However, the full extent of RNAi functions and its endogenous targets have not been explored. Here we show that, in the fission yeast Schizosaccharomyces pombe, RNAi and heterochromatin factors cooperate to silence diverse loci, including sexual differentiation genes, genes encoding transmembrane proteins, and retrotransposons that are also targeted by the exosome RNA degradation machinery. In the absence of the exosome, transcripts are processed preferentially by the RNAi machinery, revealing siRNA clusters and a corresponding increase in heterochromatin modifications across large domains containing genes and retrotransposons. We show that the generation of siRNAs and heterochromatin assembly by RNAi is triggered by a mechanism involving the canonical poly(A) polymerase Pla1 and an associated RNA surveillance factor Red1, which also activate the exosome. Notably, siRNA production and heterochromatin modifications at these target loci are regulated by environmental growth conditions, and by developmental signals that induce gene expression during sexual differentiation. Our analyses uncover an interaction between RNAi and the exosome that is conserved in Drosophila, and show that differentiation signals modulate RNAi silencing to regulate developmental genes.

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Primary accessions

Gene Expression Omnibus

Data deposits

Microarray and sequencing data are available at the NCBI Gene Expression Omnibus (GEO) repository under the accession number GSE41643.

References

  1. 1.

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

  2. 2.

    & Different means, same end-heterochromatin formation by RNAi and RNAi-independent RNA processing factors in fission yeast. Curr. Opin. Genet. Dev. 22, 156–163 (2012)

  3. 3.

    et al. Stc1: a critical link between RNAi and chromatin modification required for heterochromatin integrity. Cell 140, 666–677 (2010)

  4. 4.

    , , & Roles of the Clr4 methyltransferase complex in nucleation, spreading and maintenance of heterochromatin. Nature Struct. Mol. Biol. 15, 381–388 (2008)

  5. 5.

    et al. Comprehensive analysis of heterochromatin- and RNAi-mediated epigenetic control of the fission yeast genome. Nature Genet. 37, 809–819 (2005)

  6. 6.

    et al. Analysis of small RNA in fission yeast; centromeric siRNAs are potentially generated through a structured RNA. EMBO J. 28, 3832–3844 (2009)

  7. 7.

    et al. Clr4/Suv39 and RNA quality control factors cooperate to trigger RNAi and suppress antisense RNA. Science 331, 1624–1627 (2011)

  8. 8.

    et al. Ribonuclease activity of Dis3 is required for mitotic progression and provides a possible link between heterochromatin and kinetochore function. PLoS ONE 2, e317 (2007)

  9. 9.

    , , & RNAi-dependent and -independent RNA turnover mechanisms contribute to heterochromatic gene silencing. Cell 129, 707–721 (2007)

  10. 10.

    , , , & Defects in RNA quality control factors reveal RNAi-independent nucleation of heterochromatin. Nature Struct. Mol. Biol. 18, 1132–1138 (2011)

  11. 11.

    et al. Dynamic transcriptome of Schizosaccharomyces pombe shown by RNA-DNA hybrid mapping. Nature Genet. 40, 977–986 (2008)

  12. 12.

    et al. Dynamic repertoire of a eukaryotic transcriptome surveyed at single-nucleotide resolution. Nature 453, 1239–1243 (2008)

  13. 13.

    & Cohesin complex promotes transcriptional termination between convergent genes in S. pombe. Cell 132, 983–995 (2008)

  14. 14.

    et al. RNA elimination machinery targeting meiotic mRNAs promotes facultative heterochromatin formation. Science 335, 96–100 (2012)

  15. 15.

    et al. Comparative functional genomics of the fission yeasts. Science 332, 930–936 (2011)

  16. 16.

    et al. Programmed fluctuations in sense/antisense transcript ratios drive sexual differentiation in S. pombe. Mol. Syst. Biol. 7, 559 (2011)

  17. 17.

    & Small RNAs as guardians of the genome. Cell 136, 656–668 (2009)

  18. 18.

    , , , & Host genome surveillance for retrotransposons by transposon-derived proteins. Nature 451, 431–436 (2008)

  19. 19.

    et al. The fission yeast HIRA histone chaperone is required for promoter silencing and the suppression of cryptic antisense transcripts. Mol. Cell. Biol. 29, 5158–5167 (2009)

  20. 20.

    et al. Global effects on gene expression in fission yeast by silencing and RNA interference machineries. Mol. Cell. Biol. 25, 590–601 (2005)

  21. 21.

    , , & Dicer associates with chromatin to repress genome activity in Schizosaccharomyces pombe. Nature Struct. Mol. Biol. 18, 94–99 (2011)

  22. 22.

    et al. Establishment and maintenance of a heterochromatin domain. Science 297, 2232–2237 (2002)

  23. 23.

    , , , & cis-acting DNA from fission yeast centromeres mediates histone H3 methylation and recruitment of silencing factors and cohesin to an ectopic site. Curr. Biol. 12, 1652–1660 (2002)

  24. 24.

    & Red1 promotes the elimination of meiosis-specific mRNAs in vegetatively growing fission yeast. EMBO J. 30, 1027–1039 (2011)

  25. 25.

    et al. The nuclear poly(A)-binding protein interacts with the exosome to promote synthesis of noncoding small nucleolar RNAs. Mol. Cell 37, 34–45 (2010)

  26. 26.

    , , , & Importance of polyadenylation in the selective elimination of meiotic mRNAs in growing S. pombe cells. EMBO J. 29, 2173–2181 (2010)

  27. 27.

    et al. Mmi1 RNA surveillance machinery directs RNAi complex RITS to specific meiotic genes in fission yeast. EMBO J. 31, 2296–2308 (2012)

  28. 28.

    & Optimization of enzymatic reaction conditions for generating representative pools of cDNA from small RNA. RNA 16, 2537–2552 (2010)

  29. 29.

    et al. Histone H2A.Z cooperates with RNAi and heterochromatin factors to suppress antisense RNAs. Nature 461, 419–422 (2009)

  30. 30.

    & Improved northern blot method for enhanced detection of small RNA. Nature Protocols 3, 1077–1084 (2008)

  31. 31.

    et al. Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi. Science 297, 1833–1837 (2002)

  32. 32.

    & Using RepeatMasker to identify repetitive elements in genomic sequences. Curr. Protoc. Bioinformatics 25, 4.10.1–4.10.14 (2009)

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Acknowledgements

We thank M. Zofall and K. Zhang for contributions, J. Barrowman for editing the manuscript, M. Yamamoto for the pla1-37 strain, E. Lei for the Drosophila rrp6 mutant, B. Walker and M. Pineda for their help in sequencing, and Grewal laboratory members for discussions. This study used the Helix Systems and the Biowulf Linux cluster at the National Institutes of Health. This work was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute.

Author information

Affiliations

  1. Laboratory of Biochemistry and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA

    • Soichiro Yamanaka
    • , Sameet Mehta
    • , Francisca E. Reyes-Turcu
    • , Yikang Rong
    •  & Shiv I. S. Grewal
  2. New England Biolabs, Ipswich, Massachusetts 01938, USA

    • Fanglei Zhuang
    • , Ryan T. Fuchs
    •  & Gregory B. Robb

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Contributions

S.Y. and S.I.S.G. designed the experiments. F.Z., R.T.F., S.Y. and G.B.R. prepared the library of small RNA for deep sequencing. S.M. processed the deep-sequencing data. S.Y., S.M. and S.I.S.G. analysed the data. F.E.R.-T. and S.Y. carried out ChIP-chip. Y.R. performed the Drosophila genetic crosses. S.Y. performed all other experiments, if not stated. S.I.S.G. wrote the paper with input from all of the authors.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Shiv I. S. Grewal.

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    Supplementary Information

    This file contains Supplementary Tables 1-4 and Supplementary Figures 1-14.

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https://doi.org/10.1038/nature11716

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