m6A RNA modification is implicated in multiple cellular responses. However, its function in the innate immune cells is poorly understood. Here, we identified major m6A "writers" as the top candidate genes regulating macrophage activation by LPS in an RNA binding protein focused CRISPR screening. We have confirmed that Mettl3-deficient macrophages exhibited reduced TNF-α production upon LPS stimulation in vitro. Consistently, Mettl3 flox/flox;Lyzm-Cre mice displayed increased susceptibility to bacterial infection and showed faster tumor growth. Mechanistically, the transcripts of the Irakm gene encoding a negative regulator of TLR4 signaling were highly decorated by m6A modification. METTL3 deficiency led to the loss of m6A modification on Irakm mRNA and slowed down its degradation, resulting in a higher level of IRAKM, which ultimately suppressed TLR signaling-mediated macrophage activation. Our findings demonstrate a previously unknown role for METTL3-mediated m6A modification in innate immune responses and implicate the m6A machinery as a potential cancer immunotherapy target.

Colonic mucosal barrier dysfunction is one of the major causes of inflammatory bowel disease (IBD). However, the mechanisms underlying mucosal barrier dysfunction are poorly understood. N6-methyladenosine (m6A) mRNA modification is an important modulator of epitranscriptional regulation of gene expression, participating in multiple physiological and pathological processes. However, the function of m6A modification in colonic epithelial cells and stem cells is unknown. Here, we show that m6A modification is essential for maintaining the homeostatic self-renewal in colonic stem cells. Specific deletion of the methyltransferase 14 (Mettl14) gene in mouse colon resulted in colonic stem cell apoptosis, causing mucosal barrier dysfunction and severe colitis. Mechanistically, we revealed that Mettl14 restricted colonic epithelial cell death by regulating the stability of Nfkbia mRNA and modulating the NF-κB pathway. Our results identified a previously unidentified role for m6A modification in colonic epithelial cells and stem cells, suggesting that m6A modification may be a potential therapeutic target for IBD.

N6-methyladenosine (m6A) modification is dynamically regulated by "writer" and "eraser" enzymes. m6A "writers" have been shown to ensure the homeostasis of CD4+ T cells, but the "erasers" functioning in T cells is poorly understood. Here, we reported that m6A eraser AlkB homolog 5 (ALKBH5), but not FTO, maintains the ability of naïve CD4+ T cells to induce adoptive transfer colitis. In addition, T cell-specific ablation of ALKBH5 confers protection against experimental autoimmune encephalomyelitis. During the induced neuroinflammation, ALKBH5 deficiency increased m6A modification on interferon-γ and C-X-C motif chemokine ligand 2 messenger RNA (mRNA), thus decreasing their mRNA stability and protein expression in CD4+ T cells. These modifications resulted in attenuated CD4+ T cell responses and diminished recruitment of neutrophils into the central nervous system. Our findings reveal an unexpected specific role of ALKBH5 as an m6A eraser in controlling the pathogenicity of CD4+ T cells during autoimmunity.

RNA helicase DHX9 has been extensively characterized as a transcriptional regulator, which is consistent with its mostly nucleic localization. It is also involved in recognizing RNA viruses in the cytoplasm. However, there is no in vivo data to support the antiviral role of DHX9; meanwhile, as a nuclear protein, if and how nucleic DHX9 promotes antiviral immunity remains largely unknown. Here, we generated myeloid-specific and hepatocyte-specific DHX9 knockout mice and confirmed that DHX9 is crucial for host resistance to RNA virus infections in vivo. By additional knockout MAVS or STAT1 in DHX9-deficient mice, we demonstrated that nucleic DHX9 plays a positive role in regulating interferon-stimulated gene (ISG) expression downstream of type I interferon. Mechanistically, upon interferon stimulation, DHX9 is directly bound to STAT1 and recruits Pol II to the ISG promoter region to participate in STAT1-mediated transcription of ISGs. Collectively, these findings uncover an important role for nucleic DHX9 in antiviral immunity.