We also validated our results by m6A RIP-qPCR to examine m6A modification sites in mRNA based on our RIP-sequencing data in METTL3 knock-down MODE-K cells and predicted results from the database (http://rna.sysy.edu.cn) (Figure 2figure supplement 1a, Figure 2figure supplement 1c and Figure 2figure supplement 1d). following datasets were generated: Wang A. 2022. Data from: m6A modifications regulate intestinal immunity and rotavirus infection. Dryad Digital Repository. [CrossRef] Wang A. 2022. m6A modifications regulate intestinal immunity and rotavirus infection. NCBI Sequence Read Archive. PRJNA713535 Abstract N6-methyladenosine (m6A) is an abundant mRNA modification and affects many biological processes. However, how m6A levels are regulated during physiological or pathological processes such as virus infections, and the in vivo function of m6A in the intestinal immune defense against virus infections are largely unknown. Here, we uncover a novel antiviral function of m6A modification during rotavirus (RV) infection in small bowel intestinal epithelial cells (IECs). We found that rotavirus infection induced global m6A modifications on mRNA transcripts by down-regulating the SGL5213 m6a eraser ALKBH5. Mice lacking the m6A writer enzymes METTL3 in IECs (mRNA stability and enhanced type I and III IFN expression. Deficiency in IRF7 attenuated the elevated expression of IFNs and ISGs and restored susceptibility to RV infection in decreases the m6A modifications in endogenous retrovirus (ERV) transcripts. The accumulation of ERVs activates pattern recognition receptors (e.g. RIG-I) pathways, resulting in a detrimental interferon response in livers of fetal mice (Gao et al., 2020). The m6A modification of the enterovirus 71 (EV71) RNA genome is important for viral propagation, and EV71 infection increases the expression of m6A writers in vitro (Hao et al., 2019). m6A methyltransferase METTL3 knockdown reduces whereas m6A demethylase FTO knockdown increases EV71 replication (Hao et al., 2019). In addition, human cytomegalovirus can upregulate the expression of m6A-related proteins (Rubio et al., 2018; Winkler et al., 2019). Despite the knowledges about m6A regulation and function during viral infection revealed by these in vitro studies, the regulation of m6A modifications and the specific role of m6A in the antiviral response in vivo, especially in the gastrointestinal tract, remains unclear. Rotavirus (RV), SGL5213 a member of the family mRNA stability and enhanced type I and III IFN expression. Deficiency in IRF7 attenuated the elevated expression of IFNs and ISGs and restored susceptibility to RV infection in expression (Figure 1d, Figure 1figure supplement 1a and Figure 1figure supplement 1b) and development of microbiota SGL5213 might be involved (Figure 1figure supplement 2). Indeed, overexpression of in mouse IEC cell line caused decreased m6A level (Figure 1figure supplement 1c), supporting the role of in regulating global m6A levels in intestine. These data indicating a potential link between the resistance to RV infection and the decline of m6A modification during early life development. Open in a separate window Figure 1. 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 Students 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 Rabbit polyclonal to IQCE (MS) analysis of m6A level in ileum tissue from mice with different ages. (mean SEM), Statistical significance was determined by Students 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 Students t-tests between groups (*p 0.05, ***p 0.001, NS., not significant). (e) WT and sgRNA or control sgRNA, at indicated hours post infection (hpi) (mean SEM), statistical significance was determined by Students 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 1figure supplement 1. Open in a separate window 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 Students 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-m(Figure 2c), which played a key role in.