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

许多病毒在感染期间释放的胞质双链5 '-三磷酸RNA（3 P-RNA）被胞质解旋酶RIG-I感应。RIG-I信号传导的激活最终导致I型干扰素（IFN-I）、促炎细胞因子和细胞死亡的诱导。虽然RIG-I信号转导对IFN-I和细胞因子的诱导作用已得到充分描述，但在导致细胞死亡的机制上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与细胞死亡途径区分开来。目前，这两个过程都归因于相同的RIG-I依赖性信号通路。我们使用CRISPR/Cas9介导的敲除（KO）细胞系，分析了3 P-RNA激活RIG-I分别导致IFN-I和细胞死亡诱导的下游信号传导机制。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信号传导缺陷型细胞和野生型细胞或用IFN-1引发细胞，挽救了KO细胞系响应于3 P-RNA而经历凋亡的能力，表明RIG-I信号传导仅仅是引发最终将执行细胞死亡的其它3 P-RNA传感器所需的。亲和纯化，然后通过质谱分析显示寡腺苷酸合成酶1（OAS 1）的3 P-RNA特异性结合。RNA酶L（OAS 1的下游效应子）缺陷的细胞显示出严重受损的细胞死亡能力。我们使用KO细胞系对3 P-RNA诱导的信号传导途径的分析提供了明确的证据，即细胞因子释放和细胞死亡诱导是RIG-I和OAS 1识别胞质3 P-RNA下游的两个可分离的事件，这种由引发和效应阶段组成的两步机制引起了许多关于RIG-I介导的和OAS/RNase L介导的过程的区分的问题，这些过程目前被认为是RIG-I介导的。我只发信号。本项目的目的是：首先，进一步阐明和区分3 P-RNA激活RIG-1和OAS 1的分子机制，并协调文献中明显的相互矛盾的假设;第二，将我们的发现扩展到病毒感染过程中产生的生理RIG-I配体，以了解是否不同的3 P-RNA浓度决定了RIG-I或OAS 1是否被激活，这可能有助于理解细胞在不同病毒载量下科普或死亡的决定;第三，细胞因子和细胞死亡途径之间的区分使得研究各自对基于3 P-RNA的肿瘤免疫疗法的功效的贡献成为可能。通过所获得的机制见解，目标是找到预测细胞对3 P-RNA治疗的敏感性的生物标志物，并研究3 P-RNA与某些促凋亡刺激物的组合是否增强细胞死亡诱导以用于有效的肿瘤治疗。

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.