Roles of m6A mRNA Methylation in Innate Immunity

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PROJECT SUMMARY/ABSTRACT Post-transcriptional modifications of mRNA have emerged as a central regulatory mechanism in genetic information flow. N6-methyladenosine (m6A) is the most abundant post-transcriptional modification in eukaryotic mRNAs. m6A mRNA methylation is reversible and dynamically regulated by writers, erasers and readers. Writers are methyltransferases that install the methyl group on adenosine residues, erasers are demethylases that remove the methyl group, and readers are proteins that recognize and interact with the m6A site. m6A methylation influences all fundamental aspects of mRNA metabolism, including mRNA processing, stability and translation. Despite tremendous progresses, the in vivo roles of m6A mRNA methylation in macrophage biology remains unclear. Sepsis is a major clinical problem and leading cause of death in patients in intensive care units. Sepsis is usually caused by Gram-negative bacterial infection that triggers a fast cytokine storm. Macrophages as the first line of defense are the predominant producer of pro-inflammatory cytokines during infection. Proper resolution of the cytokine response is essential for the host's well-being. The intensity and duration of cytokine storm is delicately regulated by negative feedback regulatory loops, and the SOCS family of proteins are the central players of this feedback regulatory mechanism. We have sought to understand the role of m6A methylation in macrophage biology by genetically targeting METTL14, a core subunit of the m6A methyltransferase (a writer). We found that mice carrying METTL14 deletion in myeloid cells are hypersensitive in both cecal ligation puncture (CLP)- and lipopolysaccharide (LPS)-induced sepsis models. These tissue-specific METTL14-mutant mice produced and maintained much higher levels of serum pro-inflammatory cytokines and suffered much higher mortality than control mice. METTL14-depleted macrophages produced and sustained much higher levels of pro-inflammatory cytokines than the control macrophages, and the underlying cause is that METTL14 deletion impairs SOCS1 induction in macrophages following bacterial infection or LPS challenge. Our data support the hypothesis that m6A methylation plays a critical role in controlling the intensity and resolution of cytokine storm in sepsis by increasing Socs1 mRNA stability and translation. Our data strongly suggest that LPS or bacterial infection activates the NF-κB pathway that stimulates Socs1 mRNA transcription; LPS/bacterial infection further increases Socs1 m6A methylation by promoting FTO (an eraser) mRNA degradation, and then YTHDF1 (a reader) binds to the Socs1 m6A sites to promote Socs1 mRNA stability and increase its translation. In this proposal we will validate that SOCS1 is an essential METTL14 target to control macrophage activation in septic response using in vivo and in vitro models (Aim 1), validate that YTHDF1 is a critical reader to promote Socs1 mRNA stability and translation in septic response (Aim 2), and validate that FTO is a critical eraser whose mRNA degradation promotes Socs1 m6A methylation and greatly contributes to negative feedback control of macrophage activation (Aim 3).