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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.RIG-I样受体（RIG-I-like receptor, RLR）信号通路在宿主抗RNA病毒感染的免疫防御中发挥关键作用。在RLR家族成员中，视黄酸诱导基因I（retinoic acid-inducible gene I, RIG-I）与黑色素瘤分化相关基因5（melanoma differentiation-associated gene 5, MDA5）是启动I型干扰素（type I interferon, IFN-I）应答的关键胞质传感器。它们的可控表达、激活与降解对维持免疫稳态至关重要。然而，病毒感染期间调控RIG-I与MDA5功能的精确机制仍未阐明。本研究发现，E3泛素连接酶（E3 ubiquitin ligase）RNF20在病毒感染过程中，通过依赖核质转运（nucleocytoplasmic translocation）的方式调控RIG-I与MDA5的表达并促进其降解，从而在RLR信号通路中发挥双重调控作用。静息状态下，RNF20定位于细胞核，通过调控RIG-I与MDA5的基础转录与诱导性转录维持免疫就绪状态。当RNA病毒感染发生时，RNF20通过输出受体CRM1转运至细胞质。在此处，RNF20借助其卷曲螺旋结构域（coiled-coil domain）识别RIG-I与MDA5的降解基序（degron motifs），并催化二者发生K27位泛素化（K27-linked ubiquitination）修饰并促进其降解，从而阻断过度的抗病毒信号转导。上述研究结果阐明了RNF20在维持先天免疫稳态中发挥的重要双重调控功能。