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. 2015 Jul 14;6:7367.
doi: 10.1038/ncomms8367.

RC3H1 Post-Transcriptionally Regulates A20 mRNA and Modulates the Activity of the IKK/NF-κB Pathway

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Free PMC article

RC3H1 Post-Transcriptionally Regulates A20 mRNA and Modulates the Activity of the IKK/NF-κB Pathway

Yasuhiro Murakawa et al. Nat Commun. .
Free PMC article

Abstract

The RNA-binding protein RC3H1 (also known as ROQUIN) promotes TNFα mRNA decay via a 3'UTR constitutive decay element (CDE). Here we applied PAR-CLIP to human RC3H1 to identify ∼ 3,800 mRNA targets with >16,000 binding sites. A large number of sites are distinct from the consensus CDE and revealed a structure-sequence motif with U-rich sequences embedded in hairpins. RC3H1 binds preferentially short-lived and DNA damage-induced mRNAs, indicating a role of this RNA-binding protein in the post-transcriptional regulation of the DNA damage response. Intriguingly, RC3H1 affects expression of the NF-κB pathway regulators such as IκBα and A20. RC3H1 uses ROQ and Zn-finger domains to contact a binding site in the A20 3'UTR, demonstrating a not yet recognized mode of RC3H1 binding. Knockdown of RC3H1 resulted in increased A20 protein expression, thereby interfering with IκB kinase and NF-κB activities, demonstrating that RC3H1 can modulate the activity of the IKK/NF-κB pathway.

Figures

Figure 1
Figure 1. PAR-CLIP identifies thousands of human mRNAs directly bound by RC3H1.
(a) Phosphorimage of SDS–PAGE of radiolabelled FLAG/HA-RC3H1–RNA complexes from 365 nm ultraviolet light crosslinked non-labelled, 6SG- or 4SU-labelled cells. Crosslinked protein–RNA complexes were observed upon metabolic labelling with 4SU or 6SG. The lower panel shows an anti-HA western blot, confirming correct size and equal loading of the IPed protein. The box indicates the region that was cut out for PAR-CLIP library preparation. (b) Specific T to C mismatches in aligned reads demonstrate efficient mRNA-RBP crosslinking. The frequency of nucleotide mismatches in 4SU-2 PAR-CLIP reads aligned to mature mRNAs is shown. Sense mapping is shown in blue and antisense mapping in red. (c) A Venn diagram showing the overlap of target mRNA transcripts between 4SU and 6SG PAR-CLIP experiments. (d) Distribution of binding sites along mRNA transcripts based on consensus RC3H1 PAR-CLIP-binding sites. The majority of binding sites are located in 3′UTRs. CDS, coding sequences.
Figure 2
Figure 2. Identification of U-rich sequences and stem–loop secondary structure as recognition elements of RC3H1.
(a) Log10 frequencies of 7mers occurring in the 41-nucleotide (nt) window around the RC3H1 preferred crosslink sites are shown for 4SU-2 PAR-CLIP and 6SG PAR-CLIP libraries. U-rich sequences are frequently occurring in both 4SU and 6SG libraries. (b) Enrichment of indicated 7mers in the 41-nt window around the RC3H1 (left) or IGF2BP1 (right) preferred crosslink sites compared with all 3′UTR sequences. U-rich sequences with A contents are specifically enriched in RC3H1 3′UTR-binding sites, whereas 7mers containing known IGF2BP1 motif (CAU) are enriched in IGF2BP1 3′UTR-binding sites to the similar degree. (c) A heat map showing the coverage of 7mers, indicated on the left, around the preferred crosslinks in 3′UTR RC3H1 consensus binding sites. U-rich elements with A contents and CDE are indicated. U-rich sequences are found in the close vicinity of crosslink sites, which is indicative of direct association of RC3H1 with U-rich sequences. (d) Enrichment of indicated stem–loop structures in the 41-nt window around the RC3H1 (left) or IGF2BP1 (right) preferred crosslink sites compared with 41 nt sequences randomly selected from the 3′UTRs of target transcripts as a background model. Various stem–loop structures (n-m-n indicates a hairpin structure of n-mer stem and m-mer loop) are enriched in RC3H1 3′UTR-binding sites but not in 3′UTR IGF2BP1-binding sites. (e) The seed alignment and consensus structure of motifs 1 and 2 are shown. ARE, AU-rich elements.
Figure 3
Figure 3. RC3H1 recruits deadenylation complex and destabilizes target mRNAs.
(a) A scatter plot of identified peptide counts in two label-swap replicates. Peptides eluted from immunopurified FLAG/HA-tagged RC3H1 complex are analysed by tandem LC-MS/MS. High dose of RNaseT1/RNaseI are treated before immunoprecipitation (IP) to disrupt the indirect interactions mediated by nascent RNA. Peptides derived from the CCR4-CAF1-CNOT deadenylase complex were detected. (b) RC3H1 interactions were confirmed by co-transfection of myc-RC3H1 expression construct with HA-CNOT1, HA-CNOT8 or HA-QUAKING (QKI) expression constructs. IP was performed using anti-myc antibody. IPed proteins were resolved on SDS–PAGE, blotted and probed with anti-myc and anti-HA antibodies. Protein expression in cellular extract used as input for IP experiments is indicated (IN). (c) A cumulative distribution function (CDF) plot of log2-fold changes of mRNA decay rates of the top 500 normalized RC3H1-bound mRNAs is shown in red and all expressed mRNAs is shown in black. Top RC3H1-bound mRNAs show slower mRNA decay rates compared with all mRNAs upon RC3H1/ RC3H2 knockdown. The mean difference in mRNA decay rates (siRNA/mock) for the top 500 RC3H1 target mRNAs (n=500) and all mRNAs (n=15158) are −1.324 and −0.074, respectively (P value <2.2e–16, Wilcoxon's rank sum test). (d) A CDF plot of log2-fold changes of protein synthesis of consensus RC3H1 target transcripts that have >100 transitions on 3′UTR is shown in red and non-targets is shown in black after siRNA-mediated RC3H1 depletion. Protein synthesis of RC3H1-bound mRNAs was upregulated upon RC3H1 knockdown (P value 0.0031, Wilcoxon's rank sum test). The mean log2-fold changes for RC3H1 targets (n=390) and non-targets (n=1,279) are 0.001 and −0.116, respectively.
Figure 4
Figure 4. RC3H1 target transcripts are enriched for mRNAs induced upon DNA damage, and RC3H1 negatively regulates A20 at the post-transcriptional level.
(a) A scatter plot of mRNA expression levels of untreated cells and cells treated for 4 h with 200 ng ml−1 of neocarzinostatin (NCS). The data was retrieved from Elkon et al.. RC3H1 3′UTR target transcripts are shown in red and non-targets are shown in black. Among the RC3H1 targets, A20 was the most differentially expressed mRNA upon DNA damage. (b) A cumulative distribution function (CDF) plot of log2-fold changes upon DNA damage is shown for RC3H1 3′UTR targets in red and for non-targets in black (P value <2.2e-16, Wilcoxon-rank sum test). (c) RC3H1 induction by doxycycline treatment specifically leads to reduced expression of A20 at each time point. mRNA expression level of A20 and GAPDH (negative control) are measured by qPCR at 0, 4, 9 and 24 h post-DNA damage induced by 250 ng ml−1 of NCS. Averages and s.d.'s (error bar) from three technical replicates are shown. A representative data set out of two independent biological replicates is shown. WT, wild type. (d) Induction of RC3H1 leads to increased A20 mRNA decay. At 4 h post-DNA damage induced by NCS (250 ng ml−1), transcription was blocked with actinomycin D, and mRNA expression levels were measured by qRT–PCR. Percentage of A20 mRNA amount at each time point relative to starting point is shown. Error bars indicate s.d.'s calculated from three replicates. (e) Transfection of antisense LNA oligonucleotide targeting the stem–loop structure in HEK293 cells leads to decreased A20 mRNA decay (red) in comparison with control (Ctr) LNA transfection (black). A representative data from two independent experiments are shown.
Figure 5
Figure 5. RC3H1 binds to a composite structure-sequence motif in the A20 3′UTR mediated by the CCCH-type Zn-finger domain.
(a) Illustration of the RC3H1-binding site in the A20 3′UTR. The binding sites of RC3H1 in the 3′UTR of A20 is shown in red and zoomed in below. T to C transitions for indicated base positions are shown. Bases shown in red are forming a potential stem. Phastcon vertebrate conservation is shown in green. RC3H1-binding site in the A20 3′UTR contains a stem–loop structure flanked by AU-rich sequences. (b) The effect of A20 AU-rich element (ARE)-stem–loop hairpin (37 nucleotide (nt)) was assayed by transiently transfecting HEK293 cells with the d2GFP reporter plasmid, which contains the 37-nt sequence inserted into the 3′UTR of d2GFP. mRNA decay of the reporter transcripts were measured in mock and RC3H1/RC3H2 knockdown cells. Average and s.d.'s (error bar) from three technical replicates are shown. (c) EMSA experiments to examine the binding mode of RC3H1 to the A20 target site. Increasing concentration of recombinant RC3H1-N1 (aa 2–399) or RC3H1-N2 containing an additional CCCH-type Zn-finger domain (aa 2–452) was incubated with radiolabelled ICOS (13 nt), A20 stem–loop (21 nt) and A20 ARE-stem–loop (37 nt), and free RNA was separated from RNA–protein complexes by native PAGE. (d) EMSA experiments to examine the sequence specificity of the A20 stem–loop hairpin. Increasing concentration of recombinant RC3H1-N2 was incubated with radiolabelled wild-type (WT) A20 stem–loop (21 nt), mutated A20 sequences (Mut 1 and Mut 2) as indicated below, or 21 nt control sequence (Mut 3) generated by concatenating three 7mers underrepresented in our 7mer analysis. Mutation in the loop slightly reduces the binding, and the control sequence does not virtually bind to RC3H1-N2. (e) Increasing concentration of antisense LNA oligonucleotide targeting the A20 stem–loop structure impairs the interaction of RC3H1-N2 and 37 nt ARE-stem–loop.
Figure 6
Figure 6. RC3H1 modulates the activation of IKK by TNFα.
(a) A scatter plot of mRNA expression levels of untreated cells and cells treated for 4 h with 10 ng ml−1 of TNFα. The data were retrieved from Grimley et al.. RC3H1 3′UTR target transcripts are shown in red and non-targets are shown in black. Several TNFα-induced mRNAs, such as A20, IκBα and NFKBIZ, are targets of RC3H1. Among the RC3H1 target transcripts, A20 was the most differentially expressed mRNA upon TNFα treatment. (b) RC3H1 induction leads to slightly reduced expression of A20 and IκBα at each time point. mRNA expression levels of A20 (left) and IκBα (right) were measured by qPCR at indicated time points after TNFα treatment. Representative data from two independent experiments are shown. Average and s.e.m. (error bar) are from three technical replicates. (c) Western blot analyses of the NF-κB pathway proteins after TNFα stimulation in cells with doxycyline (Dox)-dependent RC3H1 expression. HEK293 cells were treated with Dox (1 μg/ml for 72 h), to induce HA-RC3H1. Subsequently, cells were treated with TNFα as indicated, and analysed by western blot with the indicated antibodies. RC3H1 upregulation results in decreased A20 expression, leading to increased IKK activation (T-loop phosphorylation, P-IKK) and phosphorylation of p65 (P-p65). Representative data from two independent experiments are shown. (d) EMSA analysis of whole-cell extracts for TNFα-induced NF-κB activity. Cells were treated as in c. (e) Western blot analyses of the NF-κB pathway proteins after TNFα stimulation in mock or RC3H1/2 siRNA-treated HEK293. Cells were treated with TNFα, and analysed by western blot with the indicated antibodies. RC3H1 downregulation results in mildly increased A20 expression, leading to decreased IKK activation and phosphorylation of p65. Representative data from two independent experiments are shown. ‘*' indicates phosphorylated form of IKK. (f) EMSA analysis of whole-cell extracts for TNFα-induced NF-κB activity. Cells were treated as in e. Knockdown of RC3H1 expression reduced the NF-κB activity.

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