UFMylation of 14-3-3ε coordinates MAVS signaling complex assembly to promote antiviral innate immune induction

Abstract 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 14-3-3ε. Here, we map UFM1 conjugation to lysines K50 and K215 on 14-3-3ε 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 14-3-3ε-RIG-I interaction. Instead, they paradoxically enhance RIG-I interaction with MAVS while simultaneously reducing 14-3-3ε-MAVS interaction. These findings establish UFMylation of 14-3-3ε 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. Importance Post-translational modifications provide regulatory control of antiviral innate immune responses. Our study reveals that UFMylation of 14-3-3ε is required for RIG-I-mediated innate immune signaling. We demonstrate that conjugation of UFM1 to specific lysine residues on 14-3-3ε enhances downstream signaling events that facilitate interferon induction. It does this by stabilizing 14-3-3ε association with the MAVS signaling complex and coordinating productive complex architecture. By identifying the precise sites of UFMylation on 14-3-3ε 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 14-3-3ε can be dynamically modified to orchestrate complex signaling cascades, suggesting potential therapeutic approaches for targeting dysregulated innate immunity. Competing Interest Statement The authors have declared no competing interest. Footnotes This revised manuscript contains substantial changes that better emphasize the mechanistic insights into how 14-3-3ε UFMylation regulates RIG-I signaling. Major revisions include: (1) a revised title and abstract that emphasize coordination of MAVS signaling complex assembly rather than simply promoting RIG-I signaling; (2) expanded Introduction with additional context on MAVS signalosome architecture and reorganized discussion of UFMylation in innate immunity; (3) new experimental data validating findings in A549 cells (new Figure 3C panel) and an additional structural panel (Figure 2B) showing UFMylation sites on the 14-3-3ε structure; and (4) substantially revised and expanded Discussion section that distinguishes global UFMylation loss from site-specific 14-3-3ε modification, provides more mechanistic interpretation of MAVS complex architecture, and explicitly addresses study limitations and future directions.