| Innate immune system is the first line of host defense against pathogens or endogenous danger signals and the system recognizes specific pathogen-associated molecular patterns(PAMPs)through multiple pattern recognition receptors(PRRs)to activate different signaling pathways and lead to various antipathogenic responses.Retinoic acid-inducible gene-like receptors(RLRs)in innate antiviral immune response are responsible for recognizing RNA viruses and activating downstream signaling pathways.Mitochondrial antiviral signaling protein(MAVS)is the core signaling adaptor in this signaling pathway.MAVS is recruited and activated after the RLRs recognize the nucleic acid of RNA virus,the activated MAVS induces the production of type I interferons(IFNs)by activating IRF3 to protect the host against virus invasion.It is worth noting that insufficient secretion of IFNs will cause chronic infections,while excessive secretion of IFNs can induce autoimmune or inflammatory diseases.Considering that the production of IFNs is strictly controlled during viral infection,the expression and function of MAVS must be precisely regulated.It has been reported that MAVS is regulated by various post-translational modifications,such as phosphorylation,ubiquitination,and methylation.The spatial structure or protein abundance of modified MAVS will be changed to influence the interaction with other molecules in the RLRs signal.At the same time,the control of its mRNA metabolism is also critical for managing the quantity of MAVS gene expression.For example,miR-3470b promotes bovine ephemeral fever virus(BEFV)replication via directly targeting MAVS mRNA,and miR-27a inhibits MAVS mRNA expression to promote the replication of vesicular stomatitis virus(VSV).Recent studies demonstrated that post-transcriptional regulation of mRNA,such as N6-methyladenosine(m6A)or 5-methylcytosine(m5C)modifications,helps cells respond more rapidly to external signaling at the transcriptional level.However,the molecular regulatory mechanism of post-transcriptional modification on MA VS mRNA remains indistinct.N6-methyladenosine(m6A)is the most common post-transcriptional modification mechanism,which is directly related to mRNA splicing,translation and stability.It is involved in the regulation of a large number of gene expressions and widely participated in the regulation of numerous physiological functions.In this study,we aimed to explore the role of m6A modification in RLRs-mediated type I interferon immune responses.By detecting the level of m6A modification about important molecules in the RLRs-innate immunity,we found that methyltransferase 14(METTL14)promotes the m6A modification of MAVS mRNA to inhibit the production of RLRs-mediated IFNs.In this study,we found that knockdown or deletion of Mettl14 in mouse peritoneal macrophage promoted the Ifnb1,Ifna4 and Il6 mRNA expression induced by RLRs,which also increased the secretion of IFN-β and IL6 in cell supernatant and up-regulated the phosphorylation of IRF3 and TBK1.These results suggested that Mettl14 can inhibit RLRs-induced innate immunity signal transduction.Overexpression assay showed that METTL14 was found to promote VSV replication by inhibiting the secretion of IFN-β and downstream inflammatory factors.To further explore the mechanism of METTL14,we next detected some key molecules in the RLRs signaling pathway after virus infection,which including RIG-I,MDA5,MAVS,TBK1 and IRF3,the results showed that METTL14 could selectively reduce the expression level of MAVS protein.However,we found that METTL14 had no significant effect on the degradation rate of MAVS protein by applying Cycloheximide(CHX)to inhibit protein synthesis,indicating that METTL14 does not regulate MAVS protein stability to inhibit its protein production.By detecting homeostasis MAVS mRNA levels,we found METTL14 reduced the level of MAVS mRNA.METTL14 accelerated the degradation rate of MAVS mRNA and reduced its accumulation in THP-1,BMDM,peritoneal macrophage and HEK293T cells.These results indicate that METTL14 regulates MAVS mRNA and promotes its degradation.Our further mechanism exploration showed that METTL14 increased the m6A modification of MAVS transcripts.The results of whole transcriptome m6A sequencing(MeRIP-seq)showed conspicuous m6A modification of MAVS mRNA after virus infection.Then RNA with m6A modification was purified by RNA immunoprecipitation assay(RIP)and verified by qPCR.The results showed prominent m6A modification of MAVS mRNA in macrophage,and the level of m6A modification decreased with SeV infection.Compared with wild-type cells,the m6A modification level of MAVS mRNA was significantly reduced in the Mettl14 deficient cells.Combining the transcriptome-wide m6A-seq data and a software prediction,we found a putative m6A modification site(131246518 in Chromosome 2).We then generated a luciferase reporter based on that,which verified that the MAVS transcript was directly modified with m6A by METTL14 at 131246518 site in the CDS region,and the modification was dependent on the methyltransferase activity of METTL14.At last,to investigate the role of Metil14 on antiviral immunity in physiological condition,we challenged mice with VSV.Compared with wild-type mice,Mettl14 knockout mice showed higher survival rate,slighter pathological damage in lung,more IFN-β secretion in serum,increased Ifnb1 mRNA and lower VSV titer in organs after VSV infection.These data demonstrated that Mettl14 knockout mice showed a better tolerance to RNA virus infection.In conclusion,we found that METTL14 was able to inhibit RLRs-mediated production of IFNs.METTL14 catalyzes the m6A modification of MAVS mRNA,which reduces the stability of MAVS mRNA to accelerate its degradation,and further reduces the accumulation of MAVS transcripts.We also found a specific m6A modification site catalyzed by METTL14 in the CDS region of MAVS mRNA.This study revealed a new mechanism in detail that METTL14 regulates MAVS-mediated antiviral immune responses,revealing a novel mechanism regulating innate immune signaling at the post-transcriptional level. |
PDF Full Text Request