14-3-3ϵ UFMylation promotes RIG-I-mediated signaling

Post-translational modifications regulate RIG-I signaling in diverse ways. We previously showed that UFMylation, the covalent attachment of the ubiquitin-fold modifier UFM1 to proteins, enhances RIG-I signaling by promoting its interaction with its membrane-targeting adaptor 14-3-3{varepsilon}. Here, we map UFM1 conjugation to lysines K50 and K215 on 14-3-3{varepsilon} and demonstrate how these UFMylation events control RIG-I signaling. Using in vitro and cellular UFMylation assays, we reveal that K50R/K215R mutations abolish UFMylation and reduce type I and III interferon induction following RIG-I activation. Unexpectedly, these mutations do not disrupt 14-3-3{varepsilon}-RIG-I interaction. Instead, they paradoxically enhance RIG-I interaction with MAVS while simultaneously reducing 14-3-3{varepsilon}-MAVS interaction. These findings establish UFMylation of 14-3-3{varepsilon} as an important control that shapes MAVS complex architecture to ensure optimal RIG-I signaling and highlights the broader regulatory role of UFMylation in antiviral innate immunity. ImportancePost-translational modifications provide regulatory control of antiviral innate immune responses. Our study reveals that UFMylation of 14-3-3{varepsilon} is required for RIG-I-mediated innate immune signaling. We demonstrate that conjugation of UFM1 to specific lysine residues on 14-3-3{varepsilon} enhances downstream signaling events that facilitate interferon induction. It does this by stabilizing 14-3-3{varepsilon} association with the MAVS signaling complex and coordinating productive complex architecture. By identifying the precise sites of UFMylation on 14-3-3{varepsilon} and their functional consequences, we provide insights into the regulatory layers governing antiviral innate immunity. These findings complement emerging evidence that UFMylation serves as a versatile modulator across diverse immune pathways. Furthermore, our work highlights how protein chaperones like 14-3-3{varepsilon} can be dynamically modified to orchestrate complex signaling cascades, suggesting potential therapeutic approaches for targeting dysregulated innate immunity.