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

Journal name:
Nature
Volume:
465,
Pages:
1097–1101
Date published:
DOI:
doi:10.1038/nature09095
Received
Accepted
Published online

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.

At a glance

Figures

  1. The gene nrde-2 encodes a conserved and nuclear-localized protein that is required for nuclear RNAi.
    Figure 1: The gene nrde-2 encodes a conserved and nuclear-localized protein that is required for nuclear RNAi.

    a, Light microscopy of embryos of about six cells with or without GFP RNAi subjected to in situ hybridization detecting pes-10::gfp RNA. b, nrde-2(−) animals fail to silence the lin-15b/lin15a and lir-1/lin-26 nuclear-localized RNAs (n = 4; bars, s.d.). An eri-1(−) genetic background was used for this analysis. c, Predicted domain structure of NRDE-2. Yellow, serine/arginine domain; green, DUF1740; red, potential HAT-like repeats. d, Fluorescent microscopy of an embryo of about 200 cells expressing a rescuing GFP::NRDE-2 fusion protein.

  2. NRDE-2 is recruited by NRDE-3/siRNA complexes to pre-mRNAs that have been targeted by RNAi.
    Figure 2: NRDE-2 is recruited by NRDE-3/siRNA complexes to pre-mRNAs that have been targeted by RNAi.

    a, Animals were exposed to unc-15 dsRNA and scored for uncoordinated phenotypes (Unc) (n = 3; bars, s.d.). rde-1(ne219) animals are defective for RNAi12. b, Top panels: fluorescent microscopy of a seam cell expressing GFP::NRDE-3. Arrows indicate nuclei. eri-1(mg366) animals fail to express endo-siRNAs and consequently NRDE-3 is mislocalized to the cytoplasm3, 13, 14. Bottom panels: FLAG::NRDE-3 co-precipitating RNAs radiolabelled with 32P and analysed by polyacrylamide gel electrophoresis. c, NRDE-2 co-precipitates with nuclear-localized NRDE-3 (Methods) (n = 3). d, rtPCR quantification of NRDE-2/3 co-precipitating pre-mRNA. Throughout this paper, pre-mRNA levels are quantified with exon–intron or intron–intron primer pairs. Data are expressed as ratios of co-precipitating pre-mRNA with or without lin-15b RNAi (n = 4 for NRDE-2 IP, n = 2 for NRDE-3 IP; bars, s.e.m.). Δ, fold change.

  3. C. elegans siRNAs direct an NRDE-2/3-dependent co-transcriptional gene silencing program.
    Figure 3: C. elegans siRNAs direct an NRDE-2/3-dependent co-transcriptional gene silencing program.

    a, Animals were exposed to one part lir-1 dsRNA expressing bacteria and six parts vector control, nrde-2/3 or rpb-7 dsRNA expressing bacteria. The percentage of animals that failed to exhibit lir-1 RNAi phenotypes is indicated. b, ChIP with H3K9me3 antibody (Upstate, 07-523). Data are expressed as the ratio of H3K9me3 co-precipitating DNA with or without lin-15b RNAi (n = 2; bars = s.d.). c, Total RNA was isolated and lin-15b pre-mRNA was quantified with rtPCR. Data are expressed as ratios with or without lin-15b RNAi. The genetic background was eri-1(−) (bars = s.d.). d, FLAG::NRDE-2/3 co-precipitating dpy-28 pre-mRNA. Data are expressed as ratios with or without dpy-28 RNAi (n = 4; bars, s.d.). e, ChIP of AMA-1/Rpb1 with α-AMA-1 antibody (Covance, 8WG16). Data are expressed as ratios of AMA-1 co-precipitating DNA with or without lin-15b RNAi (n = 3; bars, s.d.). The genetic background was eri-1(−).

  4. siRNAs direct an NRDE-2/3-dependent inhibition of RNAP II during the elongation phase of transcription.
    Figure 4: siRNAs direct an NRDE-2/3-dependent inhibition of RNAP II during the elongation phase of transcription.

    a, AMA-1 co-precipitating pre-mRNA. Data are expressed as ratio with or without lin-15b RNAi (n = 3; bars, s.d.). b, A crude preparation of nuclei was subjected to nuclear run-on analysis (see Methods). Transcription detected from wild-type (WT) nuclei was defined as one (n = 3; bars, s.d.). c, d, RNAi inhibits RNAP II activity 3′ to sites of RNAi. An eri-1(−) genetic background was used for these analyses. Nuclei isolated from animals treated with lin-15b (c) or lir-1 (d) RNAi were subjected to run-on analysis. Data are expressed as ratio of transcription detected in nrde-3(+)/nrde-3(−) nuclei (c) or nrde-2(+)/nrde-2(−) nuclei (d) (n = 5 for c, n = 3 for d; bars, s.e.m.).

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Author information

Affiliations

  1. Laboratory of Genetics, University of Wisconsin, Madison, Wisconsin 53706, USA

    • Shouhong Guang,
    • Aaron F. Bochner,
    • Kirk B. Burkhart,
    • Nick Burton &
    • Scott Kennedy
  2. Department of Pharmacology, University of Wisconsin, Madison, Wisconsin 53706, USA

    • Derek M. Pavelec &
    • Scott Kennedy

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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The authors declare no competing financial interests.

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