Roles of m6A mRNA Methylation in Innate Immunity

m6A mRNA 甲基化在先天免疫中的作用

• 批准号: 10676807
• 负责人: Yan Chun LI
• 金额: $ 40.5万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-22 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10676807
• 关键词: Acute; Adenosine; Bacterial Infections; Binding; Biological Process; Biology; Cause of Death; Clinical; Data; Development; Feedback; Genetic Transcription; Goals; Gram-Negative Bacterial Infections; Immune response; Impairment; In Vitro; Infection; Inflammatory; Intensive Care Units; Learning; Lipopolysaccharides; Macrophage; Macrophage Activation; Mediating; Memory; Messenger RNA; Metabolism; Methylation; Methyltransferase; Modeling; Modification; Molecular; Mus; Mutant Strains Mice; Myeloid Cells; NF-kappa B; Natural Immunity; Organ; Pathway interactions; Patients; Personal Satisfaction; Physiology; Play; Production; Proteins; Reader; Regulation; Resolution; Role; Sepsis; Serum; Site; Stress; Suppressor of Cytokine Signaling Family Protein; Tissues; Transcript; Translations; biological adaptation to stress; cecal ligation puncture; chemokine; cytokine; cytokine release syndrome; genetic information; in vitro Model; in vivo; mRNA Stability; mRNA Transcript Degradation; mRNA Translation; methyl group; mortality; posttranscriptional; response; septic

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).

项目概要/摘要 mRNA 的转录后修饰已成为遗传中的核心调节机制。 信息流。 N6-甲基腺苷 (m6A) 是最丰富的转录后修饰 真核 mRNA。 m6A mRNA 甲基化是可逆的，并受写入器、擦除器和动态调节 读者。书写器是甲基转移酶，将甲基安装在腺苷残基上，擦除器是 去甲基酶可去除甲基，而阅读器是识别 m6A 并与之相互作用的蛋白质 地点。 m6A 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。尽管取得了巨大进展，m6A 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，也是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，该感染会引发禁食 细胞因子风暴。巨噬细胞作为第一道防线，是促炎细胞的主要产生者 感染期间的细胞因子。细胞因子反应的正确解决对于宿主的健康至关重要。这 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，并且 SOCS 蛋白家族是这种反馈调节机制的核心参与者。我们一直致力于 通过基因靶向 METTL14（核心）了解 m6A 甲基化在巨噬细胞生物学中的作用 m6A 甲基转移酶（作家）的亚基。我们发现骨髓中携带 METTL14 缺失的小鼠 细胞对盲肠结扎穿刺 (CLP) 和脂多糖 (LPS) 诱导的脓毒症高度敏感 模型。这些组织特异性 METTL14 突变小鼠产生并维持了更高水平的血清 促炎细胞因子和死亡率比对照小鼠高得多。 METTL14 耗尽 巨噬细胞产生并维持比对照高得多的促炎细胞因子水平 巨噬细胞，根本原因是 METTL14 缺失损害了巨噬细胞中 SOCS1 的诱导 细菌感染或 LPS 攻击后。我们的数据支持 m6A 甲基化发挥作用的假设 通过增加 Socs1 mRNA 在控制脓毒症细胞因子风暴的强度和消退中发挥关键作用 稳定性和翻译。我们的数据强烈表明 LPS 或细菌感染激活 NF-κB 通路 刺激 Socs1 mRNA 转录； LPS/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（读取器）与 Socs1 m6A 位点结合， 促进 Socs1 mRNA 稳定性并增加其翻译。在本提案中，我们将验证 SOCS1 是 使用体内和体外模型控制脓毒症反应中巨噬细胞激活的重要 METTL14 靶点 （目标 1），验证 YTHDF1 是促进脓毒症中 Socs1 mRNA 稳定性和翻译的关键读者 响应（目标 2），并验证 FTO 是一个关键的擦除器，其 mRNA 降解会促进 Socs1 m6A 甲基化并极大地促进巨噬细胞激活的负反馈控制（目标 3）。