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 nuclei. For instance, in plants and fungi, short interfering RNA (siRNAs) associate with nascent transcripts and direct chromatin and/or DNA modifications. 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 nuclei. 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.

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

(a) Light microscopy of ≈6-cell embryos +/- 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, +/- s.d.). An eri-1(-) genetic background was used for this analysis (c) Predicted domain structure of NRDE-2. (Yellow) SR domain. (Green) DUF1740. (Red) potential HAT-like repeats. (d) Fluorescent microscopy of a ≈200 cell embryo expressing a rescuing GFP∷NRDE-2 fusion protein.

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, +/- s.d.). rde-1(ne219) animals are defective for RNAi . (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 cytoplasm ,,. (bottom panels) FLAG∷NRDE-3 co-precipitating RNAs ³²P-radiolabeled and analyzed by PAGE. (c) NRDE-2 co-precipitates with nuclear-localized NRDE-3 (materials and methods) (n=3). (d) qRT-PCR quantification of NRDE-2/3 co-precipitating pre-mRNA. Throughout this manuscript pre-mRNA levels are quantified with exon/intron or intron/intron primer pairs. Data are expressed as ratios of co-precipitating pre-mRNA +/- lin-15b RNAi (n=4 for NRDE-2 IP, n=2 for NRDE-3 IP, +/- s.e.m.). Δ=fold change.

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

(a) Animals were exposed to one part lir-1 dsRNA expressing bacteria and 6 parts vector control, nrde-2/3, or rpb-7 dsRNA expressing bacteria. (%) indicates percentage of animals that failed to exhibit lir-1 RNAi phenotypes. (b) Chromatin IP with H3K9me3 antibody (Upstate, #07-523). Data are expressed as ratio of H3K9me3 co-precipitating DNA +/- lin-15b RNAi, (n=2). (c) Total RNA was isolated and lin-15b pre-mRNA was quantified with qRT-PCR. Data are expressed as ratios +/- lin-15b RNAi. (c) and (e) were done in an eri-1(-) genetic background. (d) FLAG∷NRDE-2/3 co-precipitating dpy-28 pre-mRNA. Data are expressed as ratio +/- dpy-28 RNAi (n=4, +/- s.d.). (e) Chromatin IP of AMA-1/Rpb1 with α-AMA-1 antibody (Covance, 8WG16). Data are expressed as ratio of AMA-1 co-precipitating DNA +/- lin-15b RNAi (n=3, +/- s.d.).

Figure 4. siRNAs direct a 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 +/- lin-15b RNAi (n=3, +/- s.d.). (b) A crude preparation of nuclei was subjected to nuclear run-on analysis (see materials and methods). Transcription detected from wild-type (WT) nuclei was defined as one (n=3, +/- 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, and n=3 for d, +/- s.e.m.).