Molecular mechanisms of cell death induction by double-stranded 5’-triphosphate-RNA

双链5α-三磷酸-RNA诱导细胞死亡的分子机制

• 批准号: 442265435
• 负责人: Dr. Lars König
• 金额: --
• 依托单位: Abteilung für Klinische Pharmakologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2020
• 资助国家: 德国
• 起止时间: 2019-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/442265435?language=en
• 关键词: Molecular; mechanisms; cell; death; induction

Cytoplasmic double-stranded 5’-triphosphate RNA (3P-RNA) released by many viruses during infection is sensed by the cytoplasmic helicase RIG-I. Activation of RIG-I signaling ultimately leads to induction of type I interferons (IFN-I), proinflammatory cytokines and cell death. While IFN-I and cytokine induction by RIG-I signaling is well described, there is contradictory data on the mechanisms leading to cell death. In particular, mechanisms causing IFN-I induction have not been convincingly discriminated from cell death pathways. Currently, both processes are attributed to the same RIG-I-dependent signaling pathway.We analyzed the distinct signaling mechanisms downstream of RIG-I activation by 3P-RNA leading to IFN-I and cell death induction, respectively, using CRISPR/Cas9-mediated knockout (KO) cell lines. IFN-I production and cell death were, as shown earlier by others, strongly dependent on intact RIG-I signaling. Surprisingly, co-culturing RIG-I signaling-deficient and wildtype cells or priming cells with IFN-I, rescued the ability of KO cell lines to undergo apoptosis in response to 3P-RNA suggesting that RIG-I signaling is merely required to prime other 3P-RNA sensors that will ultimately execute cell death. Affinity purification followed by mass spectrometry revealed 3P-RNA-specific binding of oligoadenylate synthetase 1 (OAS1). Cells deficient for RNAse L, the downstream effector of OAS1, showed profoundly impaired ability to undergo cell death. Our analysis of 3P-RNA-induced signaling pathways using KO cell lines provides clear evidence that cytokine release and cell death induction are two separable events downstream of cytoplasmic 3P-RNA recognition by RIG-I and OAS1, respectively.This two-step mechanism consisting of priming and effector phase raises many questions on the discrimination of RIG-I-mediated and OAS/RNase L-mediated processes that have been currently ascribed to RIG-I signaling only. The goals of the present project application are: First, to further elucidate and discriminate the molecular mechanisms of RIG-I and OAS1 activation by 3P-RNA and harmonize conflicting hypotheses apparent in literature; second, to extend our findings to physiological RIG-I ligands produced during viral infection in order to understand if different 3P-RNA concentrations determine whether RIG I or OAS1 is activated, which may help to understand the cell’s decision to either cope or perish upon different viral load; and third, the discrimination between cytokine and cell death pathways makes it possible to investigate the respective contribution to the efficacy of 3P-RNA-based tumor immunotherapy. Through the gained mechanistic insights, the goal is to find biomarkers that predict the cell’s sensitivity to 3P-RNA therapy and to investigate if the combination of 3P-RNA with certain pro-apoptotic stimuli enhances cell death induction for effective tumor therapy.

许多病毒在感染过程中释放的胞质双链5 ' -三磷酸核糖核酸（3P-RNA）被胞质解旋酶RIG-I感知。rig - 1信号的激活最终导致I型干扰素（IFN-I）、促炎细胞因子和细胞死亡的诱导。虽然IFN-I和RIG-I信号诱导的细胞因子得到了很好的描述，但关于导致细胞死亡的机制存在相互矛盾的数据。特别是，引起IFN-I诱导的机制尚未令人信服地从细胞死亡途径中区分出来。目前，这两个过程都归因于相同的rig - i依赖性信号通路。我们利用CRISPR/ cas9介导的敲除（KO）细胞系，分别分析了3P-RNA激活RIG-I下游导致IFN-I和细胞死亡诱导的不同信号机制。如先前其他人所示，IFN-I的产生和细胞死亡强烈依赖于完整的RIG-I信号传导。令人惊讶的是，共培养RIG-I信号缺陷细胞和野生型细胞或IFN-I启动细胞，挽救了KO细胞系在响应3P-RNA时经历凋亡的能力，这表明RIG-I信号仅仅需要启动其他最终导致细胞死亡的3P-RNA传感器。亲和纯化和质谱分析显示寡甘酸合成酶1 （OAS1）与3p - rna特异性结合。缺乏RNAse L （OAS1的下游效应物）的细胞表现出严重受损的细胞死亡能力。我们对KO细胞系中3P-RNA诱导的信号通路的分析提供了明确的证据，表明细胞因子释放和细胞死亡诱导分别是RIG-I和OAS1细胞质中3P-RNA识别的两个可分离的下游事件。这个由启动和效应阶段组成的两步机制提出了许多关于RIG-I介导和OAS/RNase l介导的过程的区别的问题，这些过程目前仅归因于RIG-I信号传导。本课题的目标是：首先，进一步阐明和区分RIG-I和OAS1被3P-RNA激活的分子机制，并协调文献中存在的相互矛盾的假设；其次，将我们的发现扩展到病毒感染期间产生的生理RIG-I配体，以了解不同的3d - rna浓度是否决定RIG-I或OAS1被激活，这可能有助于理解细胞在不同病毒载量下是应对还是死亡的决定；第三，细胞因子和细胞死亡途径之间的区别使得研究各自对基于3p - rna的肿瘤免疫治疗疗效的贡献成为可能。通过获得的机制见解，目标是找到预测细胞对3P-RNA治疗敏感性的生物标志物，并研究3P-RNA与某些促凋亡刺激的组合是否能增强细胞死亡诱导，从而有效治疗肿瘤。