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 global m6A modifications, and METTL3 deficiency in IECs results in increased resistance to rotavirus infection.
(a) m6A dot blot analysis of total RNA in ileum tissues from mice with different ages. Methylene blue (MB) staining was the loading control. (b) Quantitative analysis of (a) (mean ± SEM), statistical significance was determined by Student’s t-test (**p < 0.005, ***p < 0.001, NS., not significant). The quantitative m6A signals were normalized to quantitative MB staining signals. (c) mass spectrum (MS) analysis of m6A level in ileum tissue from mice with different ages. (mean ± SEM), Statistical significance was determined by Student’s t-test (*p < 0.05, NS., not significant). (d) qPCR analysis of indicated genes in ileum tissues from mice with 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 dpi. Methylene blue (MB) staining was the loading control. (f) Quantitative analysis of (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 to quantitative MB staining signals. (g–h) Mettl3ΔIEC mice and littermate controls were infected by 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 at 4 days post infection (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 indicated genes in Rhesus rotavirus (RRV)-infected HT-29 cells transduced with Mettl3 sgRNA or control sgRNA, at 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) are repeated twice, (g and h) are repeated four times.
Figure 1—figure supplement 1
ALKBH5 regulates total RNA m6A modification level in intestine.
(a) Immunoblotting with antibodies target ALKBH5 and TUBULIN in ileum tissues from mice with different ages. (b) Quantitative analysis of (a) (mean ± SEM), Statistical significance was determined by Student’s t-test (*p < 0.05, NS., not significant). (c) Immunoblotting with antibodies target ALKBH5 and TUBULIN in MODE-K cells transfected with pSIN-EV or pSIN-mAlkbh5-3xFlag for 24 hr. m6A dot blot analysis of total RNA in indicated samples. Methylene blue (MB) staining was the loading control. Experiments in (a and b) are repeated twice, (c) are 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 with different ages. Methylene blue (MB) staining was the loading control. (b) Quantitative analysis of (a) (mean ± SEM), statistical significance was determined by Student’s t-test (NS., not significant). The quantitative m6A signals were normalized to 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 rotavirus EW strain at 8 days post birth. MS analysis of m6A level in ileum tissue from mice at two dpi (mean ± SEM). Statistical significance was determined by Student’s t-test (**p < 0.005).
Figure 1—figure supplement 4
Schematic design of RV infection model.
Figure 2 with 5 supplements
METTL3 deficiency in 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 WT mice. (b) Heat map of a subset of upregulated 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 Gapdh mRNA in the ileum of WT mice. (d) Gene regulation network of a subset of up-regulated genes including IRF7. (e) Heat map of Interferon regulatory factors (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 the Irf7 expression in ileum and jejunum from Mettl3ΔIEC mice and littermate control at 2 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 control cells in 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 control and were infected by EW at 8 days post birth. qPCR analysis of selected IFNs and ISGs in ileum tissue at two 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) are repeated three times, (g) are repeated twice.
Figure 2—figure supplement 1
Characterization of m6A modifications on Irf7 mRNA.
(a) Putative m6A sites of mouse or human Irf7/IRF7 on the genomes. (b) Metagene plots of m6A-modified sites. (c) m6A-RIP-qPCR confirms Irf7 as an m6A-modified gene in IECs. 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 ‘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 sites specific qPCR primers as positive control and negative control, Irf7 was measured with predicted m6A sites specific qPCR primer. (d) Knock down efficiency of METTL3 in MODE-K cells. (e) Knock down efficiency of METTL3 in 293t cells used for luciferase assay. (f) Western blot analysis of sgEV and sgMettl3 MODE-K cells transfected with 2 µg/ml poly I:C at indicated hours, at least three replicate experiments were performed. Experiments in (c–f) are repeated twice.
Figure 2—figure supplement 2
METTL3 knockdown in HT-29 cells results in increased IFN response.
(a) qPCR analysis of IFN/ISGs in Rhesus rotavirus-infected METTL3 WT and KD HT-29 cells at 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) Knock down efficiency of METTL3 in HT-29 cells. Experiments in (a and b) are 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 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) Knock out efficiency of METTL3 in MA104 cells. Experiments in (a and b) are repeated three times.
Figure 2—figure supplement 4
m6A modification on Irf7 mRNA regulates its expression.
(a) WT and Irf7-/- MEF cell were transduced with lentivirus expressing pLVX-EV-puro, pLVX-Irf7-WT-puro or pLVX-Irf7-Mut-puro containing mutated m6A modification sites. qPCR analysis of genes in different cells at 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 level in different MEF cells. Experiments in (a and b) are repeated three times.
Figure 2—figure supplement 5
Expression of Irf7 and ISGs in ileum from mice during early life development.
(a) qPCR analysis of 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) are repeated twice.
Figure 3
IRF7 deficiency attenuated the increased interferon response and resistance to rotavirus infection in Mettl3ΔIEC mice.
(a–c) 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. qPCR analysis of selected IFNs (a), ISGs (b), or Irf7 (c) expression in ileum from indicated groups of mice at two 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) qPCR analysis of fecal rotaviral shedding in indicated groups of mice at four 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, **p < 0.005, NS., not significant). (e–f) qPCR analysis of RV proteins expression (e) or Mettl3 and Irf7 (f) in ileum from indicated groups of mice at four 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 and (*p < 0.05, **p < 0.005, ***p < 0.001, NS., not significant). Experiments in (a–f) are 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 target ALKBH5, FTO, METTL14 and METTL3 in ileum tissue from mice infected with RV EW at two dpi or treated with PBS. (b) Quantitative analysis of (a) (mean ± SEM), Statistical significance was determined by Student’s t-test (*p < 0.05). (c–e) Alkbh5ΔIEC mice and littermate controls were infected by RV EW at 8 days post birth. qPCR analysis of indicated genes expression in ileum (c), viral shedding in feces (d), and viral proteins expression in ileum (e), from Alkbh5ΔIEC mice or littermate controls at 4 days post infection (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 target 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 modifications on Irf7 in anti-RV infection. Experiments in (a–e) are repeated three time, (f) are repeated twice.
Figure 4—figure supplement 1
A non-redundant role of Alkbh5 in regulating m6A modification in IECs.
(a) Relative expression level 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) are repeated twice.
Figure 4—figure supplement 2
ALKBH5 over-expression 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). qPCR analysis of virus genes and IFNs/ISGs in Rhesus rotavirus-infected EV and mAlkbh5 over-expressing cells at 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 level of ALKBH5 in EV and mAlkbh5 over-expressed MODE-K cells at indicated hours post infection. Experiments in (a and b) are 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 IgG IP Grade Rabbit polyclonal antibody (abcam, lot: 934197). The eluted RNA and input were processed as described in ‘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 dsRNA formation in isolated IECs.
(a) IECs from 6-week-old Mettl3ΔIEC mice as well as the WT littermate controls were isolated. Double-stranded RNA (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) are repeated twice.
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m6A modifications regulate intestinal immunity and rotavirus infection
eLife 11:e73628.
https://doi.org/10.7554/eLife.73628
