Abstract

The RIG-I-like receptor (RLR) signaling pathway plays a critical role in the host defense against RNA virus infection. Among the RLR family members, retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are key cytosolic sensors that initiate type I interferon (IFN-I) responses. Their controllable expression, activation, and degradation are essential for maintaining immune homeostasis. However, the precise regulatory mechanisms governing RIG-I and MDA5 function during viral infection remain unclear. Here, we uncover that the E3 ubiquitin ligase RNF20 exerts dual regulatory roles in RLR signaling by modulating the expression and promoting the degradation of RIG-I and MDA5 in a nucleocytoplasmic translocation-dependent manner during viral infection. Under resting conditions, RNF20 resides in the nucleus, where it maintains immune readiness by regulating the basal and inducible transcription of RIG-I and MDA5. Upon RNA virus infection, RNF20 translocates to the cytoplasm via the export receptor CRM1. There, it recognizes the degron motifs of RIG-I and MDA5 through its coiled-coil domain and catalyzes their K27-linked ubiquitination and degradation, thereby preventing excessive antiviral signaling. These findings shed light on the significant and dual regulatory roles of RNF20 in maintaining innate immune homeostasis. The innate immune system uses RIG-I/MDA5 to fight RNA viruses, but their activity must be balanced—too weak and viruses escape, too strong and inflammation harms the body. We found E3 ubiquitin ligase RNF20 acts as a “switch”: in the nucleus, it maintains basal RIG-I/MDA5 levels via transcription to prime defense. Upon infection, RNF20 moves to the cytoplasm via CRM1, binds RIG-I/MDA5 through its coiled-coil domain, and promotes their K27-linked ubiquitination and degradation to curb overactivation. This dual role of RNF20 highlights a sophisticated mechanism that fine-tunes the immune response, offering insights i