The role of m6A RNA modification as modulator of dsRNA induced cell-intrinsic innate immune responses in hematopoiesis

m6A RNA 修饰作为 dsRNA 调节剂在造血过程中诱导细胞内在先天免疫反应的作用

• 批准号: 10454110
• 负责人: Stephanie Halene
• 金额: $ 29.48万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10454110
• 关键词: Adult; Affect; Applications Grants; Biological Assay; Catalytic Domain; Cells; Complex; Data; Defect; Double-Stranded RNA; Dysmyelopoietic Syndromes; Engraftment; Failure; Fetal Development; Fetal Liver; Generations; Genetic; Genetic Transcription; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Immune Response Genes; Immune response; Immune signaling; Immunoprecipitation; In Vitro; Innate Immune Response; Interferons; Knock-out; Knockout Mice; Light; Maps; Mediating; Messenger RNA; Methyltransferase; Modeling; Modification; Nucleotides; Pathway interactions; Phenotype; Play; Process; Proteins; RNA; RNA metabolism; Reader; Reporting; Resolution; Ribonucleases; Role; Signal Transduction; Structure; Transcript; Transcription Initiation Site; Translations; Transplantation; Up-Regulation; Viral; Work; epitranscriptome; functional disability; in vivo; innate immune pathways; knock-down; leukemia; mouse model; novel; oligoadenylate; pathogen; preservation; prevent; progenitor; response; sensor; single-cell RNA sequencing; stem cell function

Project Summary N6-methyladenosine (m6A) is the most abundant RNA modification and plays key roles in RNA metabolism. m6A writers and readers are highly expressed in hematopoietic stem and progenitor cells and dysregulated in myelodysplasia and leukemia. We show that loss of Mettl3 in hematopoietic stem and progenitor cells (HSPCs) resulted in upregulation of an anti-viral innate immune response signature as well as marked proliferation and differentiation defects. In particular, we are the first to report that loss of m6A RNA modification resulted in aberrant dsRNA formation, leading to activation of the MDA5-RIG-I, PKR-eIF2A, and OAS-RNAse L pathways. By differential immunoprecipitation of dsRNAs from Mettl3 KO versus WT cells, we identified a specific subset of long, normally highly m6A modified transcripts with low folding energies, signifying propensity to form extensive RNA secondary structures. Disruption of innate immune signaling via deletion of Mavs or knockdown of Rnasel in part rescued colony formation in vitro and engraftment in competitive transplantation assays in vivo. These data suggest that one of the roles of m6A RNA modifications is to maintain single-strandedness of endogenous RNAs whereby they mark RNAs as “self” and distinguish them from exogenous pathogen-derived dsRNAs. In this SHINE II application we propose to dissect the role of m6A RNA modification in HSPCs specifically as it pertains to its role as suppressor of aberrant innate immune signaling. We will determine the mechanism by which METTL3-mediated m6A RNA modification prevents abnormal dsRNA formation and consequent induction of interferon signaling, we will dissect how m6A loss results in a deleterious immune response that disrupts HSPC function, and we will assess the universality this phenomenon across hematopoietic lineages.

项目摘要 N6-甲基腺苷（m6A）是最丰富的RNA修饰，在RNA代谢中起着关键作用。m6A 作者和读者在造血干细胞和祖细胞中高度表达， 骨髓增生异常和白血病 我们发现，造血干细胞和祖细胞（HSPC）中Mettl3的缺失导致造血干细胞和祖细胞（HSPC）中Mettl3的表达上调。 抗病毒先天免疫应答特征以及显著的增殖和分化缺陷。在 特别地，我们是第一个报道m6A RNA修饰的缺失导致异常dsRNA形成的人， 导致MDA5-RIG-I、PKR-eIF2A和OAS-RNAse L途径的激活。通过差示 通过对来自Mettl3 KO与WT细胞的dsRNA的免疫沉淀，我们鉴定了一个特定的长的、正常的 高度m6 A修饰的转录物具有低折叠能，表明倾向于形成广泛的RNA二级结构 结构.通过删除Mavs或敲除Rnasel来破坏先天免疫信号传导，部分得以挽救 体外集落形成和体内竞争性移植测定中的植入。这些数据表明 m6A RNA修饰的作用之一是维持内源RNA的单链性， 它们将RNA标记为"自身"并将其与外源病原体衍生的dsRNA区分开。 在这个SHINE II应用中，我们建议剖析m6A RNA修饰在HSPCs中的作用，因为它 涉及其作为异常先天免疫信号传导抑制剂的作用。我们将通过以下方式确定机制： 该胃L3介导的m6A RNA修饰阻止了异常dsRNA的形成和随后的诱导， 干扰素信号，我们将剖析如何m6A的损失导致有害的免疫反应，破坏 HSPC功能，我们将评估这种现象在造血谱系中的普遍性。