Endogenous RNA cleavage products that activate the RIG-I dependent interferon response

激活 RIG-I 依赖性干扰素反应的内源性 RNA 裂解产物

• 批准号: 10394282
• 负责人: Rachel Ancar
• 金额: $ 5.18万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10394282
• 关键词: Address; Affinity; Antiviral Response; Autoimmune; Autoimmune Diseases; Autoimmunity; Automobile Driving; Binding; Bioinformatics; Biological Response Modifiers; Biology; Cell Line; Cells; Chronic; Clinical; Defect; Detection; Development; Diphosphates; Discrimination; Disease; Double-Stranded RNA; Endoplasmic Reticulum; Endoribonucleases; Gene Expression; Gene Expression Profile; Genes; Genetic Transcription; Goals; Heat shock proteins; Heterogeneity; Hydroxyl Radical; Immune; Immune signaling; Immunity; Immunoprecipitation; Infection; Inflammatory; Innate Immune Response; Innate Immune System; Interferon Activation; Interferon Type I; Interferons; Investigation; Ligands; Link; Mammalian Cell; Measures; Mediating; Mendelian disorder; Messenger RNA; Metabolism; Methods; Molecular; Molecular Genetics; Mutation; Northern Blotting; Nucleic Acids; Pathogenesis; Pathogenicity; Pathology; Periodicity; Persons; Phenotype; Physicians; Predisposition; Production; Proteins; RNA; RNA Binding; RNA metabolism; RNA replication; Resistance; Role; SKIV2L gene; Scientist; Signal Pathway; Signal Transduction; Training; Transfection; Tretinoin; Trichohepatoenteric syndrome; Up-Regulation; Virus Diseases; Virus Replication; Work; autoinflammatory; cofactor; combat; crosslink; cytokine; cytosolic receptor; exosome; experience; genome-wide; immune RNA; immunogenicity; innate immune sensing; inorganic phosphate; insight; knock-down; mRNA Transcript Degradation; mutant; novel; novel therapeutic intervention; nucleic acid metabolism; prevent; programs; response; sensor; single-cell RNA sequencing; tool; transcriptome sequencing; tripolyphosphate; viral RNA

Project summary/abstract Type I interferons (IFN) are potent antiviral cytokines whose expression must be tightly regulated to combat infection without driving the development of inflammatory and autoimmune pathologies. Upregulation of type I IFN is pathogenic in a group of monogenic disorders termed type I interferonopathies and the majority are caused by mutations in genes involved in nucleic acid metabolism. Defects in RNA metabolism can result in aberrant activation of innate immune RNA sensors by cellular RNAs, initiating type I interferon expression. Chronic type I IFN signaling activates a signature of interferon stimulated gene (ISG) expression associated with autoimmune and autoinflammatory disease. Furthermore, ISG expression is highly heterogeneous and this heterogeneity directs immune cell state and function. Retinoic acid-inducible gene I (RIG-I) is a RNA innate immune sensor that recognizes double-stranded RNAs with 5´-triphosphates – a unique feature of viral RNA replication – to initiate type I IFN expression. Mutations in the RNA exosome (3´à5 degradation) cofactor SKIV2L cause the rare multisystem disorder trichohepatoenteric syndrome; and people with this disorder and SKIV2L mutations have a type I IFN signature. In cells that don't express SKIV2L, activation of the unfolded protein response (UPR) also initiates type I IFN expression dependent on a RIG-I mediated signaling. The RNA endoribonuclease Ire1 cleaves ER- localized mRNAs during the UPR as part of Regulated IRE1-Dependent Decay (RIDD), producing RNA fragments with 5´-hydroxyl and 2´,3´-cyclic phosphate ends. We hypothesize that SKIV2L is required during RIDD to target Ire1 cleaved ER-targeted mRNAs for degradation to prevent activation of a RIG-I dependent type I interferon response. Currently, the RNAs that initiate this response and the consequences of the activated ISG program are unknown. The tools needed to comprehensively identify endogenous cleaved RNAs and direct RIG-I-RNA interactions have limited our understanding of endogenous RNA detection by RIG-I. Our aims are to (1) identify Ire1 cleaved RNAs targeted by SKIV2L for degradation to prevent RIG-I-dependent IFN induction using a novel RNA end-sequencing method to directly identify RNAs cleaved by Ire1 in SKIV2L mutant cells during RIDD; and (2) characterize heterogeneous ISG expression induced by direct activation of RIG-I by Ire1 cleaved RNAs using a cross-linking and immunoprecipitation method to capture and sequence RIG-I bound RNAs and single cell RNA sequencing to profile transcriptional and cellular heterogeneity of the ISG response induced by these endogenous RNAs. These studies will facilitate efforts to understand the molecular basis of endogenous RNA detection by RIG-I and contribute to a developing paradigm highlighting the critical role of RNA sensing by innate immune regulators in interferon-mediated autoinflammatory and autoimmune disease.

项目概要/摘要 I 型干扰素 (IFN) 是有效的抗病毒细胞因子，必须严格调节其表达 对抗感染而不促进炎症和自身免疫性疾病的发展。上调 I 型干扰素在一组称为 I 型干扰素病的单基因疾病中具有致病性，并且大多数 是由参与核酸代谢的基因突变引起的。 RNA 代谢缺陷可能导致 细胞 RNA 异常激活先天免疫 RNA 传感器，启动 I 型干扰素表达。 慢性 I 型干扰素信号传导激活相关干扰素刺激基因 (ISG) 表达的特征 患有自身免疫性疾病和自身炎症性疾病。此外，ISG 表达具有高度异质性， 这种异质性指导免疫细胞的状态和功能。 视黄酸诱导基因 I (RIG-I) 是一种 RNA 先天免疫传感器，可识别双链 带有 5´-三磷酸酯的 RNA（病毒 RNA 复制的独特特征）可启动 I 型 IFN 表达。 RNA 外泌体（3´à5 降解）辅因子 SKIV2L 的突变导致罕见的多系统疾病 毛肝肠综合征;患有这种疾病和 SKIV2L 突变的人具有 I 型干扰素 签名。在不表达 SKIV2L 的细胞中，未折叠蛋白反应 (UPR) 的激活也会启动 I 型 IFN 表达依赖于 RIG-I 介导的信号传导。 RNA 内切核糖核酸酶 Ire1 裂解 ER- 作为调节性 IRE1 依赖性衰变 (RIDD) 的一部分，UPR 期间定位 mRNA，产生 RNA 具有 5'-羟基和 2',3'-环状磷酸末端的片段。我们假设 SKIV2L 在 RIDD 靶向 Ire1 切割的 ER 靶向 mRNA 进行降解，以防止 RIG-I 依赖性激活 I型干扰素反应。目前，启动这种反应的 RNA 及其后果 激活的ISG程序未知。 全面鉴定内源切割 RNA 和直接 RIG-I-RNA 所需的工具 相互作用限制了我们对 RIG-I 内源 RNA 检测的理解。我们的目标是 (1) 确定 Ire1 使用一种新型酶切 SKIV2L 靶向的 RNA 进行降解，以防止 RIG-I 依赖性 IFN 诱导 RNA末端测序方法可直接鉴定RIDD期间SKIV2L突变细胞中被Ire1切割的RNA； (2) 表征由 Ire1 切割的 RNA 直接激活 RIG-I 诱导的异质 ISG 表达 使用交联和免疫沉淀方法来捕获和测序 RIG-I 结合的 RNA 和单 细胞 RNA 测序以分析这些物质诱导的 ISG 反应的转录和细胞异质性 内源性RNA。这些研究将有助于理解内源性 RNA 的分子基础 RIG-I 检测，并有助于发展范例，强调 RNA 传感的关键作用 干扰素介导的自身炎症和自身免疫性疾病中的先天免疫调节剂。