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Small regulatory RNAs inhibit RNA polymerase II during the elongation phase of transcription

Abstract

Eukaryotic cells express a wide variety of endogenous small regulatory RNAs that regulate heterochromatin formation, developmental timing, defence against parasitic nucleic acids and genome rearrangement. Many small regulatory RNAs are thought to function in nuclei1,2. For instance, in plants and fungi, short interfering RNA (siRNAs) associate with nascent transcripts and direct chromatin and/or DNA modifications1,2. To understand further the biological roles of small regulatory RNAs, we conducted a genetic screen to identify factors required for RNA interference (RNAi) in Caenorhabditis elegans nuclei3. Here we show that the gene nuclear RNAi defective-2 (nrde-2) encodes an evolutionarily conserved protein that is required for siRNA-mediated silencing in nuclei. NRDE-2 associates with the Argonaute protein NRDE-3 within nuclei and is recruited by NRDE-3/siRNA complexes to nascent transcripts that have been targeted by RNAi. We find that nuclear-localized siRNAs direct an NRDE-2-dependent silencing of pre-messenger RNAs (pre-mRNAs) 3′ to sites of RNAi, an NRDE-2-dependent accumulation of RNA polymerase (RNAP) II at genomic loci targeted by RNAi, and NRDE-2-dependent decreases in RNAP II occupancy and RNAP II transcriptional activity 3′ to sites of RNAi. These results define NRDE-2 as a component of the nuclear RNAi machinery and demonstrate that metazoan siRNAs can silence nuclear-localized RNAs co-transcriptionally. In addition, these results establish a novel mode of RNAP II regulation: siRNA-directed recruitment of NRDE factors that inhibit RNAP II during the elongation phase of transcription.

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Figure 1: The gene nrde-2 encodes a conserved and nuclear-localized protein that is required for nuclear RNAi.
Figure 2: NRDE-2 is recruited by NRDE-3/siRNA complexes to pre-mRNAs that have been targeted by RNAi.
Figure 3: C. elegans siRNAs direct an NRDE-2/3-dependent co-transcriptional gene silencing program.
Figure 4: siRNAs direct an NRDE-2/3-dependent inhibition of RNAP II during the elongation phase of transcription.

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Acknowledgements

We thank P. Anderson, members of the Anderson laboratory, D. Wassarman and D. Brow for comments, and the Caenorhabditis Genetics Center for strains. This work was supported by grants from the Pew and Shaw scholars programmes, the National Institutes of Health and the American Heart Association.

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Contributions

S.G. performed genetic screening, generated constructs and contributed to Figs 1a, 2b, c, 3b, e, 4a and Supplementary Figs 2, 6, 7 and 9–12. A.F.B. mapped nrde-2, generated transgenic lines and contributed to Fig. 1c, d and Supplementary Figs 3 and 5. K.B.B. contributed to Fig. 1b and Supplementary Figs 3 and 8. N.B. contributed to Fig. 3a and Supplementary Fig. 8. D.M.P. contributed to Fig. 2a. S.K. wrote the paper and contributed to Figs 2d, 3c, d, 4b–d and Supplementary Figs 1, 2, 13 and 14.

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Correspondence to Scott Kennedy.

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The authors declare no competing financial interests.

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Guang, S., Bochner, A., Burkhart, K. et al. Small regulatory RNAs inhibit RNA polymerase II during the elongation phase of transcription. Nature 465, 1097–1101 (2010). https://doi.org/10.1038/nature09095

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