A pre-mRNA-associating factor links endogenous siRNAs to chromatin regulation

Abstract

In plants and fungi, small RNAs silence gene expression in the nucleus by establishing repressive chromatin states. The role of endogenous small RNAs in metazoan nuclei is largely unknown. Here we show that endogenous small interfering RNAs (endo-siRNAs) direct Histone H3 Lysine 9 methylation (H3K9me) in Caenorhabditis elegans. In addition, we report the identification and characterization of nuclear RNAi defective (nrde)-1 and nrde-4. Endo-siRNA-driven H3K9me requires the nuclear RNAi pathway including the Argonaute (Ago) NRDE-3, the conserved nuclear RNAi factor NRDE-2, as well as NRDE-1 and NRDE-4. Small RNAs direct NRDE-1 to associate with the pre-mRNA and chromatin of genes, which have been targeted by RNAi. NRDE-3 and NRDE-2 are required for the association of NRDE-1 with pre-mRNA and chromatin. NRDE-4 is required for NRDE-1/chromatin association, but not NRDE-1/pre-mRNA association. These data establish that NRDE-1 is a novel pre-mRNA and chromatin-associating factor that links small RNAs to H3K9 methylation. In addition, these results demonstrate that endo-siRNAs direct chromatin modifications via the Nrde pathway in C. elegans.

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

(A) Genetic map positions of genes identified in genetic screen. Number of alleles identified in screen are indicated. (B) RNAi-driven transcriptional inhibition requires nrde-1 and nrde-4. Nuclear Run On (NRO) analysis of transcription from the lin-15b/a gene from nuclei of animals exposed to +/− lin-15b dsRNA or +/− α-amanitin. Data are expressed as a ratio +/− lin-15b dsRNA (normalized to transcription detected from eft-3 gene) or +/− α-amanitin (5 µg/ml). Dotted line indicates a ratio of 1; i.e. no change. The genetic background for this experiment was eri-1(mg366). Control +/− lin-15b dsRNA (n = 3–8, +/− s.e.m.), nrde-1(gg088) (n = 4–6, +/− s.e.m., data point represented by triangle n = 1), nrde-4(gg129)(n = 5, +/− s.e.m.), control +/− α-amanitin (n = 3 +/− s.e.m., data point represented by triangle n = 1). Δ = fold change. Below: diagram of lin-15b/lin-15a gene structure indicating location of primers and trigger dsRNA (magenta). (C) Predicted nrde-1 gene structure. Arrows indicate mutant alleles. (D) NRDE-1 localizes to the nucleus. Fluorescence microscopy of two seam cells in a L4 larval animal expressing gfp::nrde-1. Arrows indicate nuclei. (E) gfp::nrde-1 fusion gene rescues nrde-1 mutant phenotype. Animals of the indicated genotypes were exposed to lir-1 dsRNA. A score of 5 indicates all animals died during larval development and a score of 0 indicates animals did not exhibit any developmental defects. Plates were scored blind and in triplicate for lir-1 RNAi-mediated lethality (a.u. arbitrary units).

Figure 2. NRDE-1 is recruited by NRDE-2/-3 to pre-mRNAs that have been targeted by RNAi.

(A) NRDE-3 retains the ability to bind siRNAs in nrde-1(−) animals. FLAG::NRDE-3 co-precipitating RNAs were radiolabeled with ³²P and analyzed by polyacrylamide gel electrophoresis. (B) NRDE-3 localizes to the nucleus in nrde-1(−) animals. Fluorescence microscopy of NRDE-3::GFP in seam cells from ∼L3 animals. (C) The recruitment of NRDE-3 to pre-mRNAs by RNAi is unaffected in nrde-1(−) animals. FLAG::NRDE-3 co-precipitating RNAs were converted to cDNA and quantified by qRT-PCR using primers that span exon-intron junctions. Throughout the remainder of this manuscript pre-mRNA levels are measured using exon-intron primer pairs. Data are expressed as a ratio of NRDE-3 precipitating pre-mRNA with or without lin-15b RNAi. For wild-type (n = 7,+/− s.e.m.), nrde-1(−) (n = 5, +/− s.e.m.), rrf-1(−) (n = 5, +/− s.e.m.), nrde-4(−) (n = 5, +/− s.e.m.), rde-1(−) (n = 3, +/− s.e.m.). Below, western blot detecting FLAG::NRDE-3 verified similar levels of NRDE-3 were Immuno-Precipitated (IP'ed) from each strain and for each condition. (D) NRDE-1 associates with pre-mRNAs that have been targeted by RNAi. NRDE-1 co-precipitating RNAs were converted to cDNA and quantified by qRT-PCR. Data are expressed as a ratio of NRDE-1 co-precipitating pre-mRNAs with or without indicated RNAi. Samples exposed to dpy-28 RNAi or lin-15b RNAi were probed with primers targeting either dpy-28 pre-mRNA or lin-15b pre-mRNA. For dpy-28 RNAi (n = 3, +/− s.e.m.), lin-15b RNAi (n = 5, +/− s.e.m.). For lin-15b RNAi, one data point is also shown in panel E and is marked with * in both panels. (E) NRDE-1 association with pre-mRNAs requires NRDE-2 and NRDE-3. FLAG::NRDE-1 co-precipitating pre-mRNAs were converted to cDNA and quantified by qRT-PCR. Data are expressed as a ratio of co-precipitating lin-15b pre-mRNA with or without lin-15b RNAi. Control (n = 5, +/− s.e.m., data point represented by triangle n = 1), nrde-2(−) (n = 3, +/− s.e.m.), nrde-3(−) (n = 6, +/− s.e.m.), nrde-4(−) (n = 5, +/− s.e.m.), n = 1 for pooled extracts. This experiment was performed in an nrde-1(gg088) background. Below, western blot of FLAG::NRDE-1 verified similar amounts of NRDE-1 were IP'ed.

Figure 3. NRDE-1 is required for RNAi-directed H3K9 methylation.

Chromatin Immunoprecipitation (ChIP) with anti-H3K9me3 (Upstate, 07-523) was performed on extracts derived from embryos of animals exposed to +/− lin-15b RNAi. Co-precipitating H3K9me3 DNA was quantified with qRT-PCR and data are expressed as ratios of samples exposed to lin-15b RNAi or no RNAi (n = 3 +/− s.d).

Figure 4. NRDE-1 associates with chromatin at genomic loci targeted by RNAi.

(A) FLAG::NRDE co-precipitating DNA was quantified with qRT-PCR. Data are expressed as ratios of NRDE associated DNA with or without lin-15b RNAi. Data was normalized to input. NRDE-1 (n = 4, +/− s.e.m.), NRDE-2 (n = 3, +/− s.e.m.), NRDE-3 (n = 3 +/− s.e.m.) * indicates the same FLAG::NRDE-1 ChIP data are shown in panel A and B. (B) Genetic requirements for RNAi-driven NRDE-1/chromatin association. FLAG::NRDE-1 co-precipitating DNA from indicated genetic backgrounds was quantified with qRT-PCR. Data are expressed as ratios of NRDE-1 co-IPed DNA with or without lin-15b RNAi. Control (n = 4, +/− s.e.m.), for all other strains (n = 3,+/− s.e.m.).

Figure 5. Endo-siRNAs promote H3K9 methylation.

(A) e01g4.5 is an endo-siRNA target. Small RNAs cloned from wild type or eri-1(mg366) L4 larval animals were counted in 100 bp non-overlapping windows across a 30 kb region surrounding e01g4.5. Small RNAs were normalized to total sequenced small RNAs from each sample. (B–C) eri-1(−) and nrde-1(−) animals are depleted for H3K9me3 at e01g4.5. H3K9me3 ChIPs were performed in wild-type (WT), eri-1(mg366), and nrde-1(gg088) animals. Data are represented as ratios of H3K9me3 co-precipitating DNA in WT/eri-1(mg366) (B) or WT/nrde-1(gg088) (C). Data in B and C are normalized to ChIPed eft-3 DNA (n = 3, +/− s.d.). (D) NRDE-1 associates with the pre-mRNA of an endo-siRNA target. Co-precipitating FLAG::NRDE-1 RNAs were isolated from +/− nrde-2 animals, converted to cDNA, and quantified with qRT-PCR. Data are expressed as ratios of NRDE-1 associated e01g4.5 pre-mRNA +/− nrde-2 (n = 2, +/− s.d.). (E) Endo-siRNAs inhibit transcription. NRO transcription analysis from wild-type and nrde-1 mutant animals. Data are represented as a ratio of transcription in nrde-1(−)/WT (n = 4, +/− s.e.m.). The genomic region surrounding e01g4.5 is depicted below the graph. (F) endo-siRNAs direct H3K9me marks at genomic loci homologous to endo-siRNAs. H3K9me3 ChIPs were performed in wild-type (WT) or animals of the indicated genotypes. Data are expressed as ratios of H3K9me co-precipitating DNA in WT/indicated genotype. eri-1(−) (n = 3 +/− s.d.), nrde-1(−) (n = 1) , nrde-2(−) (n = 2, +/− s.d.), nrde-3(−) (n = 2, +/− s.d.), nrde-4(−) (n = 3, +/− s.d.).

Figure 6. Model of NRDE pathway.

siRNAs (from either an exogenous or endogenous source) are bound by the Ago NRDE-3 in the cytoplasm and escorted into the nucleus. Once in the nucleus, NRDE-3/siRNA complexes bind nascent transcripts synthesized by RNAP II. NRDE-3/siRNA complexes recruit NRDE-2, and NRDE-2/3 recruit NRDE-1 to these nascent transcripts. NRDE-1 is deposited on chromatin in a NRDE-4 dependent manner. An unknown histone methyltransferase (HMT) catalyzes the methylation of histone 3 lysine 9 (H3K9me3). H3K9me marks may facilitate recruitment of NRDE-1 to chromatin. Alternatively, NRDE-1 may recruit an H3K9 methyltransferase to sites of RNAi. Together, H3K9me marks and NRDE-1 inhibit RNAP II elongation. Additional small RNAs and Argonaute proteins (Ago X) may engage NRDE-1/2/4 to direct chromatin modifications.