Ctenopharyngodon idellus Mex3C activates the innate immune by promoting the K63-linked ubiquitination of RIG-I

The cytoplasmic RNA sensor RIG-I-like receptors (RLRs) play a pivotal role in initiating antiviral innate immunity. In this study, we identified the grass carp (Ctenopharyngodon idellus) Mex3C gene, named CiMex3C which encodes a protein with a RING domain and two heterogeneous nuclear ribonucleoprotein K homology (KH) domains. Our study revealed that CiMex3C responded to multiple stimuli including GCRV, poly(I:C), B-DNA, Z-DNA, characterized by the heightened sensitivity to GCRV stimulation. Notably, CiMex3C co-localized with RIG-I in the cytoplasm of C.idellus kidney (CIK) cells following viral challenge. Further investigation revealed that CiMex3C overexpression significantly enhanced K63-linked ubiquitination of RIG-I. CiMex3C exhibited E3 ubiquitin ligase activity through its conserved RING domain. It directly catalyzed the K63-linked ubiquitin chains on RIG-I. Conversely, siRNA-mediated knockdown of CiMex3C in CIK cells markedly attenuated the RIG-I ubiquitination and suppressed IFN I production. These findings provided the empirical evidences that CiMex3C functions as a post-translational modulator of RIG-I activation, establishing its essential role in antiviral innate immune response in teleosts.