Roles of m6A mRNA Methylation in Innate Immunity

项目概要/摘要 mRNA的转录后修饰已经成为遗传学中的一种中心调节机制， 信息流N6-甲基腺苷（m6 A）是在细胞中最丰富的转录后修饰。 真核生物mRNA。m6 A mRNA甲基化是可逆的，并受书写者、橡皮擦和 读者Writers是将甲基安装在腺苷残基上的甲基转移酶，erasers是 去甲基酶，去除甲基，和读者是蛋白质，识别和相互作用的m6 A 绝佳的价钱m6 A甲基化影响mRNA代谢的所有基本方面，包括mRNA加工， 稳定性和翻译。尽管取得了巨大的进展，m6 A mRNA甲基化在体内的作用， 巨噬细胞生物学仍不清楚。脓毒症是一个主要的临床问题和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， 细胞因子风暴作为第一道防线的巨噬细胞是促炎性细胞因子的主要生产者。 感染期间的细胞因子。细胞因子应答的适当解决对于宿主的健康是必不可少的。的 细胞因子风暴的强度和持续时间受到负反馈调节环的微妙调节， SOCS蛋白家族是这种反馈调节机制的核心参与者。我们力求 了解m6 A甲基化在巨噬细胞生物学中的作用， m6 A甲基转移酶的亚基（作者）。我们发现，骨髓中携带胃L14缺失的小鼠， 细胞在盲肠结扎穿孔（CLP）和脂多糖（LPS）诱导的脓毒症中都是超敏的 模型这些组织特异性胃L14突变小鼠产生并维持了高得多的血清 促炎细胞因子，并且比对照小鼠遭受高得多的死亡率。L14缺失的胃 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 其根本原因是胃L14缺失损害了SOCS 1在巨噬细胞中的诱导 细菌感染或LPS攻击后。我们的数据支持这样的假设，即m6 A甲基化在细胞凋亡中起作用。 通过增加Socs 1 mRNA在控制脓毒症中细胞因子风暴的强度和消退中的关键作用 稳定性和翻译。我们的数据强烈提示LPS或细菌感染激活NF-κB通路 刺激Socs 1 mRNA转录; LPS/细菌感染进一步增加Socs 1 m6 A甲基化， 促进FTO（擦除器）mRNA降解，然后YTHDF 1（阅读器）与Socs 1 m6 A位点结合， 促进Socs 1 mRNA的稳定性并增加其翻译。在本提案中，我们将验证SOCS 1是一个 使用体内和体外模型控制脓毒症反应中巨噬细胞活化的必需胃L14靶标 (Aim 1），验证YTHDF 1是促进脓毒症中Socs 1 mRNA稳定性和翻译的关键阅读器。 响应（目标2），并验证FTO是一个关键的擦除器，其mRNA降解促进Socs 1 m6 A 甲基化，并且极大地有助于巨噬细胞活化的负反馈控制（目的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).