Roles of m6A mRNA Methylation in Innate Immunity

项目摘要/摘要 基因转录后修饰是基因表达的一种重要调控机制 信息流。N6-甲基腺苷(M6A)是生物体内含量最丰富的转录后修饰 真核生物mRNAs。M6A mRNA甲基化是可逆的，并受编写器、擦除器和 读者。写入者是在腺苷残基上安装甲基的甲基转移酶，擦除器是 去除甲基的去甲基酶和阅读器是识别M6A并与其相互作用的蛋白质 地点。M6A甲基化影响信使核糖核酸代谢的所有基本方面，包括信使核糖核酸的处理， 稳定性和平移性。尽管取得了巨大的进展，但m6A mRNA甲基化在体内的作用 巨噬细胞的生物学尚不清楚。脓毒症是一种主要的临床问题，也是导致患者死亡的主要原因。 在重症监护室。败血症通常是由革兰氏阴性细菌感染引起的，这种感染会引发快速 细胞因子风暴。巨噬细胞作为第一道防线是促炎因子的主要产生细胞。 感染过程中的细胞因子。细胞因子反应的正确解决对宿主的健康至关重要。这个 细胞因子风暴的强度和持续时间由负反馈调节环路微妙地调节，并且 SOCS家族蛋白是这一反馈调控机制的核心角色。我们一直在努力 通过基因靶向核心基因METTL14了解m6A甲基化在巨噬细胞生物学中的作用 M6A甲基转移酶的亚单位(写入者)。我们发现携带髓系METTL14缺失的小鼠 盲肠结扎穿刺术(CLP)和脂多糖(LPS)诱导的脓毒症中细胞都是超敏的 模特们。这些组织特异性的METTL14突变小鼠产生并维持了更高的血清水平 致炎细胞因子，死亡率明显高于对照组小鼠。METTL14-耗尽 巨噬细胞产生并维持比对照组高得多的促炎细胞因子水平 其根本原因是METTL14缺失削弱了巨噬细胞SOCS1的诱导 细菌感染或内毒素攻击后。我们的数据支持这样一种假设，即m6A甲基化在 上调Socs1基因在控制脓毒症细胞因子风暴强度和消退中的关键作用 稳定性和平移性。我们的数据有力地表明，内毒素或细菌感染激活了NF-κB途径 刺激Socs1基因转录；细菌感染进一步增加Socs1 m6A甲基化 促进FTO(橡皮擦)mRNA降解，然后YTHDF1(阅读器)与Socs1 m6A位点结合 促进SOCS1mRNA的稳定性，增加其翻译。在本提案中，我们将验证SOCS1是 利用体内和体外模型控制脓毒症反应中巨噬细胞激活的Essential METTL14靶点 (目标1)，验证YTHDF1是促进脓毒症Socs1 mRNA稳定性和翻译的关键读者 响应(目标2)，并验证FTO是其mRNA降解促进SOCS1 m6A的关键橡皮擦 甲基化和对巨噬细胞激活的负反馈控制有很大贡献(目标3)。

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).