m6A modifications regulate intestinal immunity and rotavirus infection
- Department of Digestive Disease, The First Affiliated Hospital of University of Science and Technology of China, Division of Life Sciences and Medicine, University of Science and Technology of China, China
- Institute of Immunology, the Chinese Academy of Sciences Key Laboratory of Innate Immunity and Chronic Disease, Division of Life Sciences and Medicine, University of Science and Technology of China, China
- Shanghai Institute of Immunology, Department of Microbiology and Immunology, Shanghai Jiao Tong University School of Medicine (SJTU-SM), China
- Department of Molecular Microbiology, Washington University School of Medicine in St. Louis, United States
- Department of Immunobiology, Yale University School of Medicine, United States
- Howard Hughes Medical Institute, Yale University School of Medicine, United States
- School of Data Science, University of Science and Technology of China, China
- Institute of Health and Medicine, Hefei Comprehensive National Science Center, Hefei, China, China
Figures
Figure 1 with 4 supplements
Rotavirus infection increases the global frequency of m6A modifications, and METTL3 deficiency in intestinal epithelial cells (IECs) results in increased resistance to rotavirus infection.
(a) m6A dot blot analysis of total RNA in ileum tissues from mice of different ages. Methylene blue (MB) staining was used as the loading control. (b) Quantitative analysis of the dot blot analysis shown in (a) (mean ± SEM), statistical significance was determined by Student’s t-test (*p < 0.05, **p < 0.005, ***p < 0.001, NS, not significant). The quantitative m6A signals were normalized against the quantitative MB staining signals. (c) Mass spectrum (MS) analysis of m6A level in ileum tissues from mice of different ages (mean ± SEM). Statistical significance was determined by Student’s t-test (*p < 0.05, NS, not significant). (d) qPCR analysis of the expression of the indicated genes (relative to the expression of the reference gene Hprt) in ileum tissues from mice of different ages (mean ± SEM). Statistical significance was determined by Student’s t-tests between groups (*p < 0.05, ***p < 0.001, NS, not significant). (e) WT and Mettl3ΔIEC mice were infected by rotavirus EW strain at 8 days post birth. m6A dot blot analysis of total RNA in ileum IEC at 2 days post infection (dpi). Methylene blue (MB) staining was used as the loading control. (f) Quantitative analysis of the dot blot analysis shown in (e) (mean ± SEM). Statistical significance was determined by Student’s t-test (*p < 0.05, ***p < 0.001, NS, not significant). The quantitative m6A signals were normalized against the quantitative MB staining signals. (g–h) Mettl3ΔIEC mice and littermate controls were infected by the rotavirus EW strain at 8 days post birth. qPCR analysis of RV viral loads in ileum tissue (g) or fecal samples (h) from Mettl3ΔIEC mice and littermate controls was carried out at 4 dpi (littermate WT n = 4, Mettl3ΔIEC n = 4, mean ± SEM). Statistical significance was determined by Student’s t-tests between genotypes (*p < 0.05). (i) qPCR analysis of the indicated genes in Rhesus rotavirus (RRV)-infected HT-29 cells transduced with Mettl3 single guide RNA (sgRNA) or control sgRNA, at the indicated hours post infection (hpi) (mean ± SEM). Statistical significance was determined by Student’s t-test (*p < 0.05, ***p < 0.001, NS, not significant). Experiments in (a, d-f, and i) were repeated twice, whereas those in (g and h) were are repeated four times.
Figure 1—figure supplement 1
ALKBH5 regulates total RNA m6A modification level in the intestine.
(a) Immunoblotting with antibodies targeting ALKBH5 and TUBULIN in ileum tissues from mice of different ages. (b) Quantitative analysis of the blots shown in (a) (mean ± SEM). Statistical significance was determined by Student’s t-test (*p < 0.05, NS, not significant). (c) Immunoblotting with antibodies targeting ALKBH5 and TUBULIN in MODE-K cells transfected with pSIN-EV or with pSIN-mAlkbh5-3xFlag for 24 hr,, and m6A dot blot analysis of total RNA in the indicated samples. Methylene blue (MB) staining was used as the loading control. Experiments in (a and b) were repeated twice, (c) were repeated three times.
Figure 1—figure supplement 2
Dot blot analysis of total RNA m6A modification level in the ileum of germ-free mice.
(a) m6A dot blot analysis of total RNA in ileum tissues from germ-free mice of different ages. Methylene blue (MB) staining was used as the loading control. (b) Quantitative analysis of the dot blot analysis shown in (a) (mean ± SEM). Statistical significance was determined by Student’s t-test (NS, not significant). The quantitative m6A signals were normalized against the quantitative MB staining signals.
Figure 1—figure supplement 3
Mass spectrum (MS) analysis of total RNA m6A modification level in RV-infected ileum.
(a) WT and Mettl3ΔIEC mice were infected by the rotavirus EW strain at 8 days post birth. m6A level in ileum tissue from mice was analysed by MS at 2 days post infection (dpi) (mean ± SEM). Statistical significance was determined by Student’s t-test (**p < 0.005).
Figure 1—figure supplement 4
Schematic design of the RV infection model.
Figure 2 with 5 supplements
METTL3 deficiency in intestinal epithelial cells (IECs) results in decreased m6A deposition on Irf7, and increased interferon responses.
(a) Gene ontology (GO) analysis of differentially expressed genes in IECs from Mettl3ΔIEC mice vs IECs from littermate wild-type (WT) mice. (b) Heat map of a subset of upregulated IFN-stimulated genes (ISGs) in IECs from Mettl3ΔIEC mice vs IECs from littermate WT mice, as revealed by RNA-seq (normalized data). (c) m6A-RIP-seq analysis of Irf7 and (as a reference) Gapdh mRNA in the ileum of WT mice. (d) Gene regulation network of a subset of upregulated genes including Irf7. (e) Heat map of Interferon regulatory factor genes (Irfs) in IECs from Mettl3ΔIEC mice vs IECs from littermate WT mice, as revealed by RNA-seq (RPKM). (f) Mettl3ΔIEC mice and littermate controls were infected by EW at 8 days post birth. qPCR analysis of Irf7 expression in the ileum and jejunum of Mettl3ΔIEC mice and littermate controls was carried out at 2 days post infection (dpi) (littermate WT n = 4, Mettl3ΔIEC mice n = 3, mean ± SEM). Statistical significance was determined by Student’s t-test (*p < 0.05, **p < 0.005). (g) q-PCR analysis of Irf7 mRNA in METTL3 knockdown HT-29 cells or in control cells at the indicated time points post actinomycin D treatment (n = 3, mean ± SEM). Statistical significance was determined by Student’s t-test (*p < 0.05, ***p < 0.001, NS, not significant). (h) Relative luciferase activity of sgEV and sgMettl3 HEK293T cells transfected with pmirGLO-Irf7-3’UTR (Irf7-WT) or pmirGLO-Irf7-3’UTR containing mutated m6A modification sites (Irf7-MUT). The firefly luciferase activity was normalized to Renilla luciferase activity (n = 3, mean ± SEM). Statistical significance was determined by Student’s t-tests between groups (*p < 0.05, NS, not significant). (i) Mettl3ΔIEC mice and littermate controls and were infected with EW at 8 days post birth. qPCR analysis of selected IFNs and ISGs in ileum tissue was carried out at 2 dpi (littermate WT, n = 4, Mettl3ΔIEC mice, n = 3, mean ± SEM). Statistical significance was determined by Student’s t-tests between genotypes (*p < 0.05, **p < 0.005). Experiments in (f, h and i) were repeated three times, whereas those in (g) were repeated twice.
Figure 2—figure supplement 1
Characterization of m6A modifications on Irf7 mRNA.
(a) Putative m6A sites on the transcript mRNA of mouse Irf7 or human IRF7 genes. (b) Metagene plots of m6A-modified sites. (c) m6A-RIP-qPCR confirms that Irf7 is an m6A-modified gene in IECs. The RNA of sgEV and sgMettl3 MODE-K cells was incubated with an anti-m6A antibody (Sigma Aldrich, ABE572-I). The eluted RNA and input were processed as described in the ‘RT-qPCR’ section, the data were normalized to the input samples (n = 3, mean ± SEM, statistical significance was determined by Student’s t-test (*p < 0.05, **p < 0.005, NS, not significant)). Tlr3 and Rps14 were measured with m6A-site-specific qPCR primers as positive and negative controls, respectively. Irf7 was measured with a predicted m6A-site-specific qPCR primer. (d) Efficiency of METTL3 knockdown in MODE-K cells. (e) Efficiency of METTL3 knockdown in the 293t cells used in the luciferase assay. (f) Western blot analysis of sgEV and sgMettl3 MODE-K cells transfected with 2 µg/ml poly I:C at the indicated hours post transfection, at least three replicate experiments were performed. Experiments in (c–f) were repeated twice.
Figure 2—figure supplement 2
METTL3 knockdown in HT-29 cells results in increased IFN response.
(a) qPCR analysis of the expression levels of IFNs and ISGs in Rhesus rotavirus-infected METTL3 WT and KD HT-29 cells at the indicated hours post infection (hpi) (mean ± SEM); statistical significance was determined by Student’s t-test (*p < 0.05, **p < 0.005, ***p < 0.001, NS, not significant). (b) Efficiency of METTL3 knockdown in HT-29 cells. Experiments in (a and b) were repeated three times.
Figure 2—figure supplement 3
METTL3 deficiency in MA104 cells results in increased resistance to Rhesus rotavirus infection.
(a) qPCR analysis of the expression of viral RNAs, IFNs, and ISGs in Rhesus rotavirus-infected METTL3 WT and KO MA104 cells at 24 hours post infection (hpi) (WT control n = 4, METTL3 ko n = 3, mean ± SEM); statistical significance was determined by Student’s t-test (**p < 0.005, ***p < 0.001, NS, not significant). (b) Efficiency of METTL3 knockout in MA104 cells. Experiments in (a and b) were repeated three times.
Figure 2—figure supplement 4
m6A modification on Irf7 mRNA regulates its expression.
(a) WT and Irf7-/- MEF cells were transduced with lentivirus expressing pLVX-EV-puro, pLVX-Irf7-WT-puro or pLVX-Irf7-Mut-puro containing mutated m6A-modification sites. qPCR analysis was used to measure the expression of genes in different cells at the indicated hours post infection (hpi) (mean ± SEM); statistical significance was determined by Student’s t-tests between groups (*p < 0.05, **p < 0.005, ***p < 0.001, NS, not significant). (b) Western blot analysis of IRF7 expression levels in the different MEF cells. Experiments in (a and b) were repeated three times.
Figure 2—figure supplement 5
Expression of Irf7 and ISGs in the ileum of mice during early-life development.
(a) qPCR analysis of the expression of the indicated genes in the ileum (mean ± SEM); statistical significance was determined by Student’s t-tests between ages (*p < 0.05, **p < 0.005, NS, not significant). Experiments in (a) were repeated twice.
Figure 3
IRF7 deficiency attenuated the increased interferon response and resistance to rotavirus infection in Mettl3ΔIEC mice.
(a–c) The wild-type (WT) control mice, Mettl3ΔIEC mice, Irf7-/- mice and Mettl3ΔIEC Irf7-/- mice are all littermates. They were infected by RV EW at 8 days post birth. The expression of selected interferon (IFN) genes (a), IFN-stimulated genes (ISGs) (b), or Irf7 (c) in ileum from indicated groups of mice was analyzed by qPCR at 2 dpi (littermate WT n = 7, Mettl3ΔIEC n = 5, Irf7-/- n = 3, Mettl3ΔIEC Irf7-/- n = 6, mean ± SEM). Statistical significance was determined by Student’s t-tests between genotypes (*p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001, NS, not significant). (d) Fecal rotaviral shedding by the indicated groups of mice was analyzed by qPCR at 4 dpi (littermate WT n = 5, Mettl3ΔIEC n = 5, Irf7-/- n = 3, Mettl3ΔIEC Irf7-/- n = 4, mean ± SEM). Statistical significance was determined by Student’s t-tests between genotypes (*p < 0.05, NS, not significant). (e–f) Expression of RV proteins (e) or Mettl3 and Irf7 (f) in ileum tissue or IECs from the indicated groups of mice was analyzed by qPCR at 4 dpi (littermate WT n = 7, Mettl3ΔIEC n = 5, Irf7-/- n = 3, Mettl3ΔIEC Irf7-/- n = 6, mean ± SEM). Statistical significance was determined by Student’s t-tests between genotypes (*p < 0.05, **p < 0.005, ***p < 0.001, NS, not significant). Experiments in (a–f) were repeated twice.
Figure 4 with 4 supplements
Rotavirus suppresses ALKBH5 expression through NSP1 to evade immune defense.
(a) WT mice were infected by RV EW at 8 days post birth. Immunoblotting with antibodies targeting ALKBH5, FTO, METTL14 and METTL3 in ileum tissue from mice infected with RV EW or treated with PBS was carried out at 2 days post infection (dpi). (b) Quantitative analysis of the immunoblot shown in (a) (mean ± SEM). Statistical significance was determined by Student’s t-test (*p < 0.05, NS, not significant). (c–e) Alkbh5ΔIEC mice and littermate controls were infected by RV EW at 8 days post birth. The expression of the indicated genes in ileum tissue or IECs (c), viral shedding in feces (d), and the expression of viral proteins in ileum tissues (e), were analyzed for Alkbh5ΔIEC mice or littermate controls at 4 dpi (littermate WT n = 6, Alkbh5ΔIEC n = 5, mean ± SEM). Statistical significance was determined by Student’s t-tests between genotypes (*p < 0.05, NS, not significant). (f) Immunoblotting with antibodies targeting ALKBH5, NSP1, VP6 and GAPDH in HEK293 cells infected by SA11-4F and SA11-NSP1null (MOI = 1) for 24 hr. (g) Graphical abstract illustrating the functions and molecular mechanisms of m6A modification on Irf7 in anti-RV infection. Experiments in (a–e) were repeated three time, whereas those in (f) were repeated twice.
Figure 4—figure supplement 1
A non-redundant role of Alkbh5 in regulating m6A modification in intestinal epithelial cells (IECs).
(a) Relative expression levels of ALKBH5/Alkbh5 and FTO/Fto in human intestinal enteroid, mice IEC and mice IEC cell MODE-K. (b) m6A dot blot analysis of total RNA in different MODE-K cells. Methylene blue (MB) staining was the loading control. Experiments in (b) were repeated twice.
Figure 4—figure supplement 2
ALKBH5 overexpression in MODE-K cells results in enhanced IFN response and increased resistance to Rhesus rotavirus infection.
(a) MODE-K cells were transduced with lentivirus (expressing pLVX-EV-puro or pLVX-mAlkbh5-puro). The expression of virus genes, interferon genes (IFNs) and IFN-stimulated genes (ISGs) in Rhesus rotavirus-infected EV and mAlkbh5 overexpressing cells were analyzed by qPCR at the indicated hours post infection (hpi) (mean ± SEM); statistical significance was determined by Student’s t-test (*p < 0.05, **p < 0.005, NS, not significant). (b) Expression levels of ALKBH5 in EV and mAlkbh5-overexpressing MODE-K cells at the indicated hours post infection. Experiments in (a and b) were repeated twice.
Figure 4—figure supplement 3
m6A-RIP-qPCR analysis of the predicted m6A sites on Rotavirus RNA.
(a) Total RNA was extracted from IECs from 1-week-old WT mice infected by RV for 2 days. Fragmented RNA was incubated with an anti-m6A antibody (Sigma Aldrich ABE572) and an IgG IP Grade Rabbit polyclonal antibody (abcam, lot: 934197). The eluted RNA and input were processed as described in the ‘RT-qPCR’ section, the data were normalized to the input samples (n = 2, mean ± SEM). Rps14 was chosen as a negative control.
Figure 4—figure supplement 4
METTL3 deficiency leads to aberrant double-stranded RNA (dsRNA) formation in isolated IECs.
(a) IECs from 6-week-old Mettl3ΔIEC mice and from WT littermate controls were isolated. dsRNA was labeled by immunostaining with a mouse monoclonal antibody J2 (Scisons), cells nuclei were visualized with 4,6-diamidino-2-phenylindole (DAPI, Invitrogen). All fluorescence images were analyzed via confocal imaging using Zeiss LSM880. Experiments in (a) were repeated twice.
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m6A modifications regulate intestinal immunity and rotavirus infection
eLife 11:e73628.
https://doi.org/10.7554/eLife.73628
