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m6A modification controls the innate immune response to infection by targeting type I interferons

Abstract

N6-methyladenosine (m6A) is the most common mRNA modification. Recent studies have revealed that depletion of m6A machinery leads to alterations in the propagation of diverse viruses. These effects were proposed to be mediated through dysregulated methylation of viral RNA. Here we show that following viral infection or stimulation of cells with an inactivated virus, deletion of the m6A ‘writer’ METTL3 or ‘reader’ YTHDF2 led to an increase in the induction of interferon-stimulated genes. Consequently, propagation of different viruses was suppressed in an interferon-signaling-dependent manner. Significantly, the mRNA of IFNB, the gene encoding the main cytokine that drives the type I interferon response, was m6A modified and was stabilized following repression of METTL3 or YTHDF2. Furthermore, we show that m6A-mediated regulation of interferon genes was conserved in mice. Together, our findings uncover the role m6A serves as a negative regulator of interferon response by dictating the fast turnover of interferon mRNAs and consequently facilitating viral propagation.

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Data availability

All RNA-seq data sets generated in this manuscript have been deposited in the GEO under accession number GSE114019. Full images of immunoblots presented in this study have been deposited to Mendeley Data and are available at https://doi.org/10.17632/3zb63b6ssj.1. All other data are available from the corresponding author upon reasonable request.

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Change history

  • 11 January 2019

    In the version of this article initially published, the penultimate sentence of the abstract included a typographical error (‘cxgenes’). The correct word is ‘genes’. The error has been corrected in the HTML and PDF version of the article.

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Acknowledgements

We thank M. Schwartz and the rest of the Stern-Ginossar laboratory members for discussions and critical reading of the manuscript. This research was supported by the European Research Council starting grant (StG-2014-638142), the EU-FP7-PEOPLE Career integration grant, the ICORE (Chromatin and RNA Gene Regulation) and the Israeli Science Foundation (1073/14). N.S.-G. is incumbent of the Skirball career development chair in new scientists.

Author information

R.W., E.G., L.L., J.H.H., S.S. and N.S.-G. conceived experiments and interpreted data. L.L., S.G. and J.H.H. generated and characterized the gene-deficient mice. R.W., E.G., M.S., S.G., C.S., A.N., J.T.-S., N.F. and M.M. executed experiments and analysis. V.T.K.L.-T. and M.T. provided critical reagents and advice. R.W., E.G. and N.S.-G. wrote the manuscript with contribution from all other authors.

Competing interests

The authors declare no competing interests.

Correspondence to Noam Stern-Ginossar.

Integrated supplementary information

Supplementary Figure 1 m6A machinery is elevated along HCMV infection and is important for its propagation.

a, mRNA and translation levels of genes encoding for m6A machinery along HCMV infection, as measured by RNA-seq (red) and ribosome profiling (green)35. b, Immunoblot analysis of m6A machinery proteins in cells expressing sgRNAs targeting control gene (WT) or the various m6A machinery genes (indicated on the left) in fibroblasts. GAPDH was used as a loading control. The gel images were cropped to present only relevant proteins. c, Quantification of m6A machinery proteins levels from the immunoblot analysis in b normalized to the levels of GAPDH. d, Fluorescent microscopy of GFP signal in WT fibroblasts infected with supernatant from infected cells in which m6A machinery genes were depleted (indicated at the bottom). e, Immunoblot analysis of HCMV Immediate-early protein (IE1-pp72) (left panel) and fluorescent microscopy of GFP signal (right panel), at 24 hpi in ALKBH5-depleted and control cells. GAPDH was used as a loading control. The gel images were cropped to present only relevant proteins. f, Quantification of viral protein levels from the immunoblot analysis in Fig.1e normalized to the levels of GAPDH. Data are representative of three (d) or two (e) independent experiments.

Supplementary Figure 2 Differences in ISG expression between METTL3-depleted and control cells is abolished by ruxolitinib and does not stem from changes in their stability.

a, ISG relative expression, as measured by RNA-seq, in METTL3-depleted cells versus control cells at 28 hpi, treated or untreated with ruxolitinib. Expression levels of each transcript were normalized to a scale of 0 to 1. ISGs showing significant difference (FDR < 0.01) between control and METTL3-depleted cells are presented. b-e, METTL3-depleted and control cells were treated with actinomycin D at 22 hpi and collected for RNA-seq at 0, 2 and 4 hours post treatment. The mRNA decay of several ISGs that showed enhanced expression in METTL3-depleted cells are presented (n = 2 for each time point). Values represent the mean of RNA-seq replicates and error bars show s.d. RPKM, reads per kilobase of transcript per million mapped reads. f, Quantification of protein levels from the immunoblot analysis in Fig.4a normalized to the levels of GAPDH.

Supplementary Figure 3 IFNB mRNA is m6A-modified and its levels are higher in YTHDF2-depleted cells than in control cells.

a, Specificity of m6A signal on IFNB transcript in immunoprecipitated (IP) samples compared to input (POI, Peak Over Input) and to median coverage across the gene (POM, Peak Over Median), in METTL3-depleted (n = 3) and control cells (n = 3). Thick line, median; box boundaries, 25% and 75% percentiles; whiskers, 1.5-fold interquartile range. b, IFNB mRNA and c, protein levels in YTHDF2-depleted and control cells at indicated time points post infection, measured by qRT-PCR and ELISA, respectively. 18S ribosomal RNA was used as a normalizing gene in qRT-PCR. Dots, measurements; bars, mean of three technical (b) or cell culture (c) replicates. The P values were calculated using a two-sided Student’s t-test. n.d., not detected. d, Nascent RNA was labeled for 2 h with 5-Ethynyluridine (EU). EU was washed out and RNA was extracted at the indicated time points. The relative remaining EU-labeled mRNA abundance, normalized to GAPDH, was analyzed by qRT-PCR for IFNB and USP42 that was used as control. Values represent the mean of three technical replicates and error bars show s.d. The P values were calculated using a two-sided Student’s t-test. e, IFNB gene (5′UTR, coding sequence and 3′UTR) was cloned into a plasmid in its wild-type (WT) version and in a mutant version (MUT), in which three putative m6A-modified adenosines were mutated to guanosines (labeled in red). f, Immunoblot analysis of METTL3 in THP1 cells expressing sgRNAs targeting control gene (WT) or METTL3. GAPDH was used as a loading control. The gel images were cropped to present only relevant proteins. g, Quantification of METTL3 levels from the immunoblot analysis in f normalized to the levels of GAPDH. Data (b-d) are representative of two independent experiments.

Supplementary Figure 4 m6A-mediated IFN regulation is conserved in mouse.

a, RNA-seq of input RNA and m6A immunoprecipitated RNA from mouse dendritic cells treated with lipopolysaccharide (LPS) for 3 and 6 h is presented for Ifna14. b, Immunoblot analysis of m6A machinery proteins in MEFs expressing sgRNAs targeting control gene (WT) or the various m6A machinery genes (indicated on the left). GAPDH was used as a loading control. The gel images were cropped to present only relevant proteins. c, Quantification of m6A machinery protein levels from the immunoblot analysis in b normalized to the levels of GAPDH.

Supplementary Figure 5 Construction of the Ythdf3–/– mouse.

a, Ythdf3–/– mice were generated via one-cell embryo CRISPR/Cas9 injection. sgRNA targeting Ythdf3 exon3 was used. The mutated Ythdf3 gene contains an out of frame 14bp deletion, which leads to the production of a stop codon. b, Immunoblot analysis of YTHDF3 protein expression in MEFs extracted from Ythdf3+/+, Ythdf3+/- and Ythdf3–/– embryos. HSP90 was used as a loading control. The gel images were cropped to present only relevant proteins.

Supplementary information

Supplementary Figures 1-5

Reporting Summary

Supplementary Table 1

Measurement of cell viability in control cells and cells depleted of m6A machinery proteins, before and 96 h after infection with HCMV

Supplementary Table 2

Dataset of 21 putative m6A sites in HCMV transcripts

Supplementary Table 3

Dataset of 7,093 putative m6A sites in human transcripts, obtained following infection with HCMV

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Further reading

Fig. 1: m6A ‘writers’ and ‘readers’ are upregulated during HCMV infection and required for viral growth.
Fig. 2: Inhibition of HCMV growth in m6A-deficient cells is driven by an enhanced type I interferon response.
Fig. 3: ISG-enhanced expression in METTL3-depleted cells is independent of viral gene expression.
Fig. 4: IFNB mRNA is m6A modified and is more stable in METTL3-depleted cells.
Fig. 5: Depletion of m6A machinery proteins leads to an elevation in IFNB levels following infection with diverse viruses.
Fig. 6: Type I interferon m6A methylation and destabilization are conserved in mouse.
Supplementary Figure 1: m6A machinery is elevated along HCMV infection and is important for its propagation.
Supplementary Figure 2: Differences in ISG expression between METTL3-depleted and control cells is abolished by ruxolitinib and does not stem from changes in their stability.
Supplementary Figure 3: IFNB mRNA is m6A-modified and its levels are higher in YTHDF2-depleted cells than in control cells.
Supplementary Figure 4: m6A-mediated IFN regulation is conserved in mouse.
Supplementary Figure 5: Construction of the Ythdf3–/– mouse.