Zebrafish RNF128a positively regulates innate immunity by promoting the K63-linked ubiquitination of TBK1

RNF128, also known as GRAIL, is a well-studied E3 ubiquitin ligase involved in the induction and maintenance of T cell anergy. It contains PA_GRAIL_like domain, HRD1 domain and RING-H2_like domain. Recent studies have shown that RNF128 is involved in the regulation of innate immunity in mammals. It should be noted that zebrafish, possess two RNF128 paralogs, RNF128a and RNF128b. We focused on RNF128a, which shares the closest sequence homology with mammalian RNF128; however, its role in fish has not yet been elucidated. In this paper, we demonstrated that RNF128a acts as a positive regulator of innate immunity in Zebrafish. Zebrafish RNF128a responds to multiple stimuli including SVCV (Spring Viremia of Carp Virus), poly(I:C), B-DNA, LPS, and is characterized by the heightened sensitivity to SVCV. Notably, Zebrafish RNF128a up-regulates the expression of IFN1, ISG15, MX, as well as the inflammatory cytokines such as IL-6 and TNFα in response to SVCV. Mechanistically, Zebrafish RNF128a interacts with TBK1 through its protease-associated (PA) domain. However, the subcellular localization of RNF128a is associated with its HRD1 domain. In addition, Zebrafish RNF128a is localized to both early endosome RAB5 and late endosome RAB7 but rarely in Golgi apparatus and endoplasmic reticulum. Structurally, only full-length RNF128a promotes the K63-linked ubiquitination of TBK1, while none of the truncation mutants retains this activity. Our study identified Zebrafish RNF128a as an E3 ligase that catalyzes K63-linked ubiquitination and activates TBK1, and delineated its essential role in the antiviral innate immune response in zebrafish.