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

项目概要/摘要 I型干扰素（IFN）是有效的抗病毒细胞因子，其表达必须被严格调节以 对抗感染，而不驱动炎症和自身免疫性病理的发展。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是致病性的， 是由参与核酸代谢的基因突变引起的。RNA代谢的缺陷会导致 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IFN信号激活干扰素刺激基因（ISG）表达相关的特征 自身免疫性和自身炎症性疾病此外，ISG表达是高度异质性的， 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， 具有5 ′-三磷酸的RNA-病毒RNA复制的独特特征-启动I型IFN表达。 RNA外泌体（3 ′ à5降解）辅因子SKIV 2L的突变导致罕见的多系统疾病 肝肠综合征;患有这种疾病和SKIV 2L突变的人有I型IFN-γ， 签名.在不表达SKIV 2L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Ire 1切割ER- 在UPR过程中，作为调节性IRE 1依赖性衰变（RIDD）的一部分， 具有5 ′-羟基和2 ′，3 ′-环状磷酸末端的片段。我们假设SKIV 2L是在 RIDD靶向Ire 1切割的ER靶向mRNA进行降解，以防止RIG-I依赖性 I型干扰素反应。目前，启动这种反应的RNA和这种反应的后果是由基因组的结构决定的。 ISG的激活程序未知。 全面鉴定内源性切割RNA和指导RIG-I-RNA所需的工具 相互作用限制了我们对RIG-I检测内源RNA的理解。我们的目标是（1）识别 使用一种新的方法，通过SKIV 2L靶向Ire 1切割RNA进行降解以防止RIG-I依赖性IFN诱导 RNA末端测序法直接鉴定RIDD过程中SKIV 2L突变细胞中Ire 1切割的RNA 和（2）表征由Ire 1切割的RNA直接激活RIG-I诱导的异源ISG表达 使用交联和免疫沉淀方法捕获RIG-I结合的RNA并测序， 细胞RNA测序，以分析这些诱导的ISG应答的转录和细胞异质性， 内源RNA。这些研究将有助于理解内源性RNA的分子基础 通过RIG-I的检测，并有助于突出RNA传感的关键作用的发展模式， 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因子。

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.