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

项目摘要/摘要 I型干扰素是一种有效的抗病毒细胞因子，其表达必须受到严格的调控 在不推动炎症和自身免疫病理发展的情况下与感染作斗争。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的，大多数 是由涉及核酸新陈代谢的基因突变引起的。RNA代谢的缺陷可能会导致 通过细胞RNA异常激活天然免疫RNA感受器，启动I型干扰素的表达。 慢性I型干扰素信号激活干扰素刺激基因(ISG)表达 患有自身免疫性和自身炎症性疾病。此外，ISG的表达具有高度的异质性和 这种异质性决定着免疫细胞的状态和功能。 视黄酸诱导基因I(rig-i)是一种识别双链的rna天然免疫传感器。 含有5‘-三磷酸的RNA--病毒RNA复制的一个独特特征--启动I型干扰素的表达。 RNA外切体(3‘á5降解)辅因子SKIV2L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型干扰素的表达依赖于RIG-I介导的信号。RNA内切核酸酶IRE1裂解ER- 作为受调控的IRE1依赖衰变(RIDD)的一部分，UPR过程中的局部mRNAs，产生RNA 末端含有5‘-羟基和2’，3‘-环磷酸盐的碎片。我们假设SKIV2L在 RIDD靶向IRE1裂解的ER靶向mRNAs进行降解，以防止依赖RIG-I的激活 I型干扰素反应。目前，启动这一反应的RNAs以及 激活的ISG程序未知。 全面识别内源切割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 用一种新的方法抑制由SKIV2L靶向降解的IRE1裂解RNA以防止依赖Rig I的干扰素诱导 RNA末端测序法直接鉴定RIDD过程中SKIV2L突变细胞中IRE1裂解的RNA； 以及(2)表征由IRE1裂解的RNA直接激活RIG-I所诱导的异质性ISG表达 用交联法和免疫沉淀法捕获和测序RIG-I结合的RNA和单个 细胞RNA测序以描述由它们诱导的ISG反应的转录和细胞异质性 内源RNA。这些研究将有助于了解内源rna的分子基础。 通过RIG-I进行检测，并有助于通过以下方式强调RNA传感的关键作用 干扰素介导的自体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.