Molecular Mechanisms Of Innate Immune Regulation Of METTL3 Function

N~6-methyladenosine(also named m~6A)is considered as the most prevalent internal modification in eukaryotic m RNA,which regulates multiple stages throughout the m RNA lifecycle.By determining the m RNA fate at the post-transcriptional level,m~6A is heavily involved in various physiological and pathological processes.In recent years,studies on m~6A methylation during virus infection have been gradually carried out,however,its function in the host antiviral innate immune responses has not been determined.Of note,most current studies have focused on the effects of m~6A modification on antiviral innate immunity,and little is known about the regulatory mechanism by which innate immune signaling regulates m~6A modification.METTL3 is well known as the catalytic core of the m~6A methyltransferase complex,which mediates almost all m~6A modifications in m RNAs.Previous studies have shown that there were multiple phosphorylation and SUMOylation sites on endogenous METTL3 protein,which affected its stability,nuclear localization,and catalytic activity,and contributed to DNA damage repair and tumorigenesis.During virus infection,the expression of METTL3 protein was upregulated and cytoplasmic localization was increased,and the transcriptome-wide m~6A methylome of the host was significantly changed,but the underlying mechanisms have not been clarified.Other studies have shown that METTL3 also promotes m RNA translation independent of its m~6A catalytic activity,and how these two functions work coordinately is still unclear.To identify the interacting proteins with METTL3,we performed mass spectrometry followed by Co-IP assay for validation.Results showed a virus-induced interaction between endogenous proteins of METTL3 and TBK1.Microscale thermophoresis assay further confirmed a direct interaction with their purified proteins.To test whether METTL3 is a substrate of TBK1,we examined the phosphorylation level of endogenous METTL3 protein.Western blot analysis showed that virus infection increased the serine phosphorylation level of METTL3,and a TBK1 inhibitor could antagonize this effect,suggesting that virus infection induced the phosphorylation of METTL3 by TBK1.We then conducted point mutation analysis with several phosphorylation sites of METTL3 identified in our earlier laboratory studies.Among which,S67A mutant METTL3 showed a significantly reduced phosphorylation level and a much weaker synergistic effect with TBK1 to activate IFN-β.S67phosphorylation was subsequently shown to promote the binding of METTL3 to translation-related proteins by mass spectrometry.SUn SET analysis and Tethering assay further confirmed that TBK1 was involved in promoting protein translation through phosphorylation of METTL3 at S67.In addition,in vitro RNA methylation assay suggested that TBK1 could also enhance the catalytic activity of METTL3 in a kinase-independent manner.And Tbk1 knockout nearly abolished the m~6A methylation induced by virus infection.Furthermore,m~6A-IP-q PCR analysis confirmed that TBK1 upregulated IRF3 m RNA methylation level upon virus infection,which was further demonstrated to maintain the m RNA stability and enhanced protein expression by knock-in mutation strategies.Finally,we infected Mettl3 myeloid knockout mice with different viruses,to evaluate the overall effect of m~6A catalyzed by METTL3 on antiviral innate immunity.Besides,the biological functions of m~6A are known to mediated by its specific reader proteins.Among which,YTHDF1 is reported to promote m RNA translation,and was detected in our mass spectrometry to interact with endogenous METTL3 upon VSV infection.To this end,we also constructed Ythdf1 homozygous knockout mice to examine its antiviral function.The results showed that both METTL3 and YTHDF1 positively regulated antiviral innate immune responses in mice.In conclusion,this study revealed a new mechanism by which antiviral innate immune signaling regulated m~6A methylation.In detail,upon virus infection,TBK1 was induced to directly bind to the m~6A-encoded protein METTL3,and enhanced m~6A methylation in a kinase-independent manner.Besides,TBK1 also promoted the phosphorylation of METTL3at S67,which then interacted with translation initiation complex to enhance protein translation.Thus,both functions of METTL3 was regulated by TBK1 and coordinately to enhance type I interferon responses.This study provides new insights into how m~6A machinery interplay with innate immune signaling.