Molecular mechanisms of cell death induction by double-stranded 5’-triphosphate-RNA
Final Report Abstract
During viral infections, many viruses release double-stranded RNA into the cytoplasm of host cells. This RNA is recognized by RIG-I-like receptors (RLRs), which activate signaling pathways that lead to the production of type I interferons (IFNs), proinflammatory cytokines, and, in some cases, programmed cell death (apoptosis). While the induction of IFNs and cytokines by RLR signaling is well established, the exact mechanisms that trigger cell death remain less clear and have been inconsistently described in the literature. In this project, we investigated the specific molecular pathways that lead to IFN production and cell death in response to stimulation with 5’-triphosphate RNA (3P-RNA), a known ligand of RIG-I. Using human and mouse melanoma cell lines engineered via CRISPR/Cas9 to lack key signaling components, we confirmed that both cytokine production and apoptosis require intact RLR signaling. Interestingly, we found that even in the absence of RIG-I or its direct downstream signal partners, cells could still undergo apoptosis if they were co-cultured with wild-type cells or pre-treated with IFN. This indicates that RLR signaling primarily serves to prime other RNA sensors that ultimately mediate cell death. Further analysis, including affinity purification and mass spectrometry, identified oligoadenylate synthetase 1 (OAS1) as a specific binding partner of 3P-RNA. Overexpression of OAS1 restored apoptosis in RIG-I-deficient cells, and cells lacking RNaseL—the downstream effector of OAS1—showed significantly reduced cell death. These findings highlight the critical role of the OAS/RNaseL pathway in mediating apoptosis in response to 3P-RNA. The ligand requirements of OAS1 showed great similarity to RIG-I. Mechanistically, we demonstrated that effective cell death requires both translational shutdown via RNaseL and upregulation of the pro-apoptotic protein NOXA by RIG-I signaling, which together lead to the depletion of the anti-apoptotic protein MCL-1 and trigger intrinsic apoptosis in melanoma cells. Overall, our findings clearly distinguish two separate pathways activated by cytoplasmic 3P-RNA: RLR-dependent priming for interferon production, and a distinct OAS/RNaseL-driven mechanism responsible for apoptosis. This insight may have important implications for antiviral therapies and cancer treatments that utilize synthetic RNA molecules.
Publications
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Blocking inflammation on the way: Rationale for CXCR2 antagonists for the treatment of COVID-19. Journal of Experimental Medicine, 217(9).
Koenig, Lars M.; Boehmer, Daniel F.R.; Metzger, Philipp; Schnurr, Max; Endres, Stefan & Rothenfusser, Simon
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Defective Interfering Genomes and the Full-Length Viral Genome Trigger RIG-I After Infection With Vesicular Stomatitis Virus in a Replication Dependent Manner. Frontiers in Immunology, 12.
Linder, Andreas; Bothe, Viktoria; Linder, Nicolas; Schwarzlmueller, Paul; Dahlström, Frank; Bartenhagen, Christoph; Dugas, Martin; Pandey, Dharmendra; Thorn-Seshold, Julia; Boehmer, Daniel F. R.; Koenig, Lars M.; Kobold, Sebastian; Schnurr, Max; Raedler, Johannes; Spielmann, Giulia; Karimzadeh, Hadi; Schmidt, Andreas; Endres, Stefan & Rothenfusser, Simon
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Novel Concepts in Innate Immunity 2021, Online/Tübingen Short Talk: Double-stranded Triphosphate-RNA triggers tumor cell death through a RIG-I- dependent priming and an OAS1/RNase L-mediated effector phase,
Thomas Wannerer
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OAS1/RNase L executes RIG-I ligand–dependent tumor cell apoptosis. Science Immunology, 6(61).
Boehmer, Daniel F. R.; Formisano, Simone; de Oliveira Mann, Carina C.; Mueller, Stephan A.; Kluge, Michael; Metzger, Philipp; Rohlfs, Meino; Hörth, Christine; Kocheise, Lorenz; Lichtenthaler, Stefan F.; Hopfner, Karl-Peter; Endres, Stefan; Rothenfusser, Simon; Friedel, Caroline C.; Duewell, Peter; Schnurr, Max & Koenig, Lars M.
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OAS1/RNase L executes RIG-I ligand–dependent tumor cell apoptosis. Science Immunology, 6(61).
Boehmer, Daniel F. R.; Formisano, Simone; de Oliveira Mann, Carina C.; Mueller, Stephan A.; Kluge, Michael; Metzger, Philipp; Rohlfs, Meino; Hörth, Christine; Kocheise, Lorenz; Lichtenthaler, Stefan F.; Hopfner, Karl-Peter; Endres, Stefan; Rothenfusser, Simon; Friedel, Caroline C.; Duewell, Peter; Schnurr, Max & Koenig, Lars M.
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NAI meeting, Edinburgh, 2022 Poster: 5’-Triphosphate RNA triggers cell death through an RIG-I-dependent priming and an OAS1/RNase L mediated effector phase,
Thomas Wannerer
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Annual Immunology Retreat, Harvard Medical Schoon, Boston, 2023: Poster: Cell death-optimized 5’-triphosphate RNAs for improved tumor immunotherapy
Thomas Wannerer
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CIMT Annual Meeting, 2023, Mainz: Poster: Cell death-optimized 5’-triphosphate RNAs for improved tumor immunotherapy
Thomas Wannerer
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i-Target Annual Retreat, Fraueninsel, Chiemsee, 2023: Talk: Cell death-optimized 5’-triphosphate RNAs for improved tumor immunotherapy
Thomas Wannerer
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International Conference on Nucleic Acid Immunity TRR237, Dresden, 2023: Poster: Cell death-optimized 5’-triphosphate RNAs for improved tumor immunotherapy
Thomas Wannerer
