RNF20 dual regulation of MDA5 signaling to maintain immune homeostasis in chickens.

RIG-I and MDA5, known as the RIG-I-like receptors (RLRs), play a pivotal role in inducing antiviral responses to RNA viral infections. While chickens lack RIG-I, they possess a functionally enhanced MDA5 that recognizes pathogens and regulates immunity, underscoring the critical role of MDA5 in maintaining immune homeostasis in chickens. However, the precise mechanisms governing the expression and optimal activation of MDA5 remain unclear. Here, we reveal that the chicken E3 ubiquitin ligase RNF20 is essential for modulating MDA5-mediated innate immune homeostasis. Transcriptome sequencing analysis revealed that RNA viral infection of DF-1 cells significantly upregulated the expression of chicken RNF20. Overexpression of RNF20 markedly suppresses the expression of chicken innate immunity-related genes, while RNF20 knockout leads to immune deficiency both IMPORTANCE: Chicken MDA5 is an important RNA viral sensor for initiating the antiviral innate immune response. The protein level of MDA5 must be tightly regulated to maintain antiviral innate immune homeostasis. In this study, we demonstrate that the E3 ubiquitin ligase RNF20 precisely regulates MDA5 protein stabilization through nucleoplasmic translocation. Specifically, in uninfected and during early infection, RNF20 regulates MDA5 transcription in the nucleus. While in the late stages of infection, RNF20 translocates out of the nucleus and catalyzes the ubiquitinated degradation of MDA5. Thus, RNF20 is important in regulating chicken antiviral innate immune homeostasis.