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RITS acts in cis to promote RNA interference–mediated transcriptional and post-transcriptional silencing

Nature Genetics volume 36, pages 1174–1180 (2004)Cite this article

Abstract

RNA interference is a conserved mechanism by which double-stranded RNA is processed into short interfering RNAs (siRNAs) that can trigger both post-transcriptional and transcriptional gene silencing. In fission yeast, the RNA-induced initiation of transcriptional gene silencing (RITS) complex contains Dicer-generated siRNAs and is required for heterochromatic silencing. Here we show that RITS components, including Argonaute protein, bind to all known heterochromatic loci. At the mating-type region, RITS is recruited to the centromere-homologous repeat cenH in a Dicer-dependent manner, whereas the spreading of RITS across the entire 20-kb silenced domain, as well as its subsequent maintenance, requires heterochromatin machinery including Swi6 and occurs even in the absence of Dicer. Furthermore, our analyses suggest that RNA interference machinery operates in cis as a stable component of heterochromatic domains with RITS tethered to silenced loci by methylation of histone H3 at Lys9. This tethering promotes the processing of transcripts and generation of additional siRNAs for heterochromatin maintenance.

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Figure 1: Stable association of RITS with constitutive heterochromatic domains.
Figure 2: RITS cooperates with ATF-CREB family proteins to nucleate heterochromatin assembly at the mat locus.
Figure 3: Mapping of RITS components at the mat region.
Figure 4: Localization of Chp1 and Tas3 at the mat region in dcr1Δ and dcr1Δ swi6Δ mutant cells.
Figure 5: Bidirectional transcription of the cenH element and accumulation of the transcripts in RITS and other RNAi mutants.
Figure 6: RITS localization correlates with H3-Lys9 methylation in clr4 mutants at the mat locus.
Figure 7: H3-Lys9 methylation–dependent stable association of RITS with silenced domains is essential for generation and recruitment of siRNAs into the complex and heterochromatin maintenance.
Figure 8: Schematic model showing the role of RITS and RNAi in transcriptional and post-transcriptional silencing.

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Acknowledgements

We thank I. Hall, J. Rice and B. Paterson for critical reading of the manuscript, members of the laboratory of S.I.S.G. for discussions and Y. Tsukamoto for technical assistance. Research in the laboratories of S.I.S.G. and D.M. is supported by the National Cancer Institute and the National Institutes of Health, respectively.

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Authors and Affiliations

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

    Ken-ichi Noma, Tomoyasu Sugiyama, Hugh Cam, Martin Zofall, Songtao Jia & Shiv I S Grewal

  2. Department of Cell Biology, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Andre Verdel & Danesh Moazed

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Supplementary information

Supplementary Fig. 1

The effect of clr4Δ on the localization of RITS components at centromeres. (PDF 5 kb)

Supplementary Fig. 2

ChIP analyses of H3-K9 methylation and Chp1 localization in wild type, clr4W31G, clr4G486Δ and clr4Δ strains at the mating-type region. (PDF 15 kb)

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Noma, Ki., Sugiyama, T., Cam, H. et al. RITS acts in cis to promote RNA interference–mediated transcriptional and post-transcriptional silencing. Nat Genet 36, 1174–1180 (2004). https://doi.org/10.1038/ng1452

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