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

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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.