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. 2018 Dec 1;32(23-24):1472-1484.
doi: 10.1101/gad.319475.118. Epub 2018 Nov 21.

RNA m6 A modification enzymes shape innate responses to DNA by regulating interferon β

Rosa M Rubio  1 , Daniel P Depledge  1 , Christopher Bianco  1 , Letitia Thompson  1 , Ian Mohr  1   2
Affiliations

RNA m6 A modification enzymes shape innate responses to DNA by regulating interferon β

Rosa M Rubio et al. Genes Dev. .

Abstract

Modification of mRNA by N6-adenosine methylation (m6A) on internal bases influences gene expression in eukaryotes. How the dynamic genome-wide landscape of m6A-modified mRNAs impacts virus infection and host immune responses remains poorly understood. Here, we show that type I interferon (IFN) production triggered by dsDNA or human cytomegalovirus (HCMV) is controlled by the cellular m6A methyltrasferase subunit METTL14 and ALKBH5 demethylase. While METTL14 depletion reduced virus reproduction and stimulated dsDNA- or HCMV-induced IFNB1 mRNA accumulation, ALKBH5 depletion had the opposite effect. Depleting METTL14 increased both nascent IFNB1 mRNA production and stability in response to dsDNA. In contrast, ALKBH5 depletion reduced nascent IFNB1 mRNA production without detectably influencing IFN1B mRNA decay. Genome-wide transcriptome profiling following ALKBH5 depletion identified differentially expressed genes regulating antiviral immune responses, while METTL14 depletion altered pathways impacting metabolic reprogramming, stress responses, and aging. Finally, we determined that IFNB1 mRNA was m6A-modified within both the coding sequence and the 3' untranslated region (UTR). This establishes that the host m6A modification machinery controls IFNβ production triggered by HCMV or dsDNA. Moreover, it demonstrates that responses to nonmicrobial dsDNA in uninfected cells, which shape host immunity and contribute to autoimmune disease, are regulated by enzymes controlling m6A epitranscriptomic changes.

Keywords: RNA m6A modification; dsDNA signaling; human cytomegalovirus; innate immunity; virus infection.

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Figures

Figure 1.
Figure 1.
Accumulation of the cellular m6A machinery is induced by human cytomegalovirus (HCMV). (A) Regulation of m6A levels in mRNA by the opposing action of a multisubunit methyltransferase and demethylases. A subset of reader proteins that recognize m6A-modified residues is depicted. (B) Normal human dermal fibroblasts (NHDFs) were mock-infected (uninfected) or infected with HCMV (multiplicity of infection [MOI] = 3). At the indicated hours post-infection (hpi), total protein was collected, fractionated by SDS-PAGE, and analyzed by immunoblotting with the indicated antibodies. Actin represents a host antigen whose levels remain unchanged during infection. (C) As in B except cells were infected with UV-inactivated HCMV, and GAPDH was used as a control host antigen. (D) Total RNA collected from HCMV-infected cells (MOI = 3) at the indicated times (hours) post-infection. For each of the indicated genes, mRNA was analyzed by real-time quantitative PCR (qPCR) and normalized to PPIA mRNA levels. Three biological replicates were performed. Error bars indicate SEM. (E) Uninfected NHDFs or NHDFs infected with HCMV (MOI = 3) were treated with the mechanistic target of rapamycin (mTOR) active site inhibitor PP242 or DMSO. At the indicated times (hours post-infection), total protein was collected, fractionated by SDS-PAGE, and analyzed by immunoblotting with the indicated antisera. GAPDH served as a loading control. The change in 4E-BP1 migration in uninfected cells from a slow-migrating hyperphosphorylated species (hyper) to a faster-migrating hypophosphorylated (hypo) form validates the activity of PP242.
Figure 2.
Figure 2.
Control of HCMV replication by the host RNA m6A METTL3/14 methyltransferase and ALKBH5 demethylase. (A) NHDFs were treated with control nonsilencing siRNA, siRNA specific for the m6A methyltransferase METTL3 or METTL14 subunits, or siRNA specific for the ALKBH5 demethylase. After 72 h, cultures were infected with HCMV (MOI = 0.05), and the infection allowed to proceed for 7 d. Supernatants were harvested, and virus titer (tissue culture infective dose at which 50% of cultures are infected [TCID/50]) was determined using NHDFs. Error bars indicate SEM. (**) P ≤ 0.01; (****) P ≤ 0.0001 by Student's t-test. (B) As in A except total protein was harvested and fractionated by SDS-PAGE, and the accumulation of viral proteins encoded by representative immediate–early (IE1/2), early (UL44), or late (pp28) genes was measured by immunoblotting with the indicated antisera. GAPDH served as a loading control. (C) NHDFs treated with control nonsilencing siRNA (blue) or siRNA specific for METTL14 (orange) or ALKBH5 (purple) were infected with HCMV (MOI = 3). After 6 h, total RNA was harvested, and IFNB1 mRNA levels were quantified by qRT–PCR. Three biological replicates (n = 3) were performed, and error bars represent SEM. (**) P ≤ 0.01; (***) P ≤ 0.001 by Student's t-test. (D) NHDFs were treated with control nonsilencing siRNA or siRNA specific for METTL14. After 72 h, cultures were treated with either 1 µM Janus kinase (JAK) inhibitor (pyridone 6) or DMSO and infected with HCMV (MOI = 0.05). Supernatants were harvested, and virus titer (TCID/50) was determined after 7 d using NHDFs.
Figure 3.
Figure 3.
Induction of type I IFN in response to dsDNA is regulated by RNA m6A METTL3/14 methyltransferase and ALKBH5 demethylase. (A) NHDFs treated with control nonsilencing siRNA or siRNAs specific for METTL14 or ALKBH5 were incubated with H2O or dsDNA. After 9 h, total RNA was collected, and IFNB1 mRNA was measured by qRT–PCR. Error bars indicate SEM. (*) P ≤ 0.05; (**) P ≤ 0.01 by Student's t-test. (B) RNA isolated from NHDFs treated with dsDNA for 6 h was immunoprecipitated with anti-m6A antibody. RNA enriched in the immune complex was analyzed by qRT–PCR using primers specific for the indicated genes. (C) Two regions of the IFNB1 transcript are enriched for m6A peaks. Visualization of m6A sequencing (m6A-seq) results shows regions of enrichment for m6A immunoprecipitation (red) over input (blue) for three biological replicates of NHDFs transfected with dsDNA for 12 h. Specific regions enriched for m6A modifications were identified using ExomePeak (see the Supplemental Material) and are shown as horizontal black lines. m6A DRACH motifs (Linder et al. 2015) are shown as red boxes. No IFNB1 mapping reads were detected in control (no dsDNA) immunoprecipitation or input data sets (data not shown), consistent with the minimal background presence of IFNB1 transcripts in untreated cells. The transcript structure of IFNB1 is denoted in dark blue (3′ UTR and 5′ UTR) and blue (CDS). (D) NHDFs treated as in A were incubated with H2O or dsDNA. After 24 h, supernatants or known concentrations of IFNβ standards were placed on indicator cells, and the amount of IFNβ activity was quantified. Error bars indicate SEM. (*) P ≤ 0.05; (***) P ≤ 0.001 by Student's t-test. (E) NHDFs treated with control nonsilencing siRNA or the indicated siRNAs specific for the demethylase ALKBH5 and/or m6A readers YTHDF1, YTHDF2, and YTHDF3 were exposed to dsDNA. After 9 h, total RNA was collected, and IFNB1 mRNA was quantified by qRT–PCR. Error bars indicate SEM. (*) P ≤ 0.05; (**) P ≤ 0.01 by Student's t-test.
Figure 4.
Figure 4.
Control of genome-wide responses to dsDNA by m6A demethylase ALKBH5 and m6A methylase subunit METTL14. Volcano plots show differentially expressed genes (adjusted P-value < 0.01) identified from RNA-seq of polyadenylated RNA collected from cells transfected with ALKBH5 siRNA (siALKBH5; n = 3 biological replicates) (A) or METTL14 siRNA (siMETTL14; n = 3 biological replicates) (B) and stimulated with dsDNA for 12 h. Genes up-regulated versus a nonsilencing siRNA control (stimulated with dsDNA for 12 h; n = 3 biological replicates) are shown in red, while down-regulated genes are shown in blue. Nonregulated genes are shown in gray. (C) One-thousand-four-hundred-forty-seven significantly regulated genes (P < 0.01) overlap between data sets generated using siALKBH5 and siMETTL14. A scatter plot of these shows genes that are commonly up-regulated or down-regulated following either METTL14 or ALKBH5 silencing (highlighted in red and blue, respectively). Genes regulated in a reciprocal manner are highlighted in green (up-regulated when METTL14 is silenced and down-regulated when ALKBH5 is silenced) and yellow (down-regulated when METTL14 is silenced and up-regulated when ALKBH5 is silenced). The red circle highlights the IFNB1 mRNA. (D) Heat map depicting 349 ISGs, colored according to log2 fold change in expression versus the nonsilencing siRNA control. (E,F) Pathway analyses (gene ontology direct terms) of significantly differentially expressed genes from A and B were conducted using DAVID and filtered according to a Benjamini-Hochberg procedure (<0.05).
Figure 5.
Figure 5.
Control of IFNB1 mRNA biogenesis and decay by ALKBH5 and METTL14. NHDFs were treated with control nonsilencing siRNA or siRNA specific for the METTL14 m6A methyltransferase subunit or the ALKBH5 demethylase for 72 h. (A) NHDFs treated with the indicated siRNAs were transfected with dsDNA in the presence of DMSO or the JAK inhibitor (pyridone 6). At the indicated times, total RNA was harvested, and the abundance of IFNB1 mRNA was quantified by qRT–PCR. Error bars indicate SEM. (*) P ≤ 0.05; (**) P ≤ 0.01; (***) P ≤ 0.001 by Student's t-test. (B) As in A except cultures were pulse-labeled for 30 min with 5-ethyluridine (EU) at either 7 or 10 h after exposure to dsDNA. Immediately following the EU pulse, nuclear RNA was collected, nascent EU-containing RNA was isolated, and the abundance of IFNB1 mRNA was quantified by qRT–PCR. Error bars indicate SEM. (*) P ≤ 0.05; (**) P ≤ 0.01 by Student's t-test. (C, left panel) NHDFs treated with the indicated siRNAs were exposed to dsDNA for 3 h and pulse-labeled for 2 h with EU. Free EU was washed out, and total RNA was harvested at the indicated time points. Following isolation of nascent EU-containing RNA, overall IFNB1 mRNA levels were measured by qRT–PCR and normalized to GAPDH. Error bars indicate SEM. (*) P < 0.04 by Student's t-test. (Right panel) As in the left panel, except cultures were also treated with the JAK inhibitor (pyridone 6). (D) Model illustrating the relationship between IFNB1 mRNA levels, induced in response to dsDNA in uninfected cells or HCMV infection, and m6A levels in mRNA. The balance between methyltransferase METTL14 and demethylase ALKBH5 activities is depicted as a scale. METTL14 depletion (siMETTL14) tips the scale in favor of the ALKBH5 demethylase, which increases IFNB1 mRNA levels and is predicted to reduce m6A levels. In contrast, ALKBH5 depletion (siALKBH5) disrupts the balance in favor of methyltransferase activity, which decreases IFNB1 mRNA and is predicted to increase m6A levels.
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