Phylogenetic Analysis Of Fish TRIM Genes And Their Functional Roles In Innate Immune System

The tripartite motif-containing(TRIM) proteins are characterized by a tripartite motif that comprises a RING zinc finger domain, one or two B-boxes and a Coiled-coil domain, and followed by different C-terminal domains. They are therefore also known as RBCC proteins. The TRIM family is ancient with members existing throughout the metazoan kingdom and varying amounts among species. The family has expanded rapidly during vertebrate evolution. The highly conserved modular structure reveals their common biochemical roles in cellular activity. Recent studies have shown that TRIM protein as an E3 ubiquitin ligase is involved in many biological processes including cell differentiation, cell cycle regulation, apoptosis, transcriptional regulation and innate immune pathways, etc. By comparison with the exhausting discovery of TRIM genes in human, studies on fish TRIM genes are limited and sporadic. The objectives of this study are to analyze the genetic diversity and phylogenesis of fish TRIM genes, to undermine their evolutionary direction and richness, and to study the role of TRIM genes in innate immune pathways upon Aeromonas hydrophila infection.A total of 198 TRIM genes were identified from the zebrafish genome and subject to systemic analysis of chromose location, splice variants and domain organizations. Four TRIM proteins with different domains at the C-terminus were firstly found. Besides, 57 TRIM genes were found based on the Blast analysis of the genome sequence of elephant shark(Callorhinchus milii). Based on the phylogenetic analysis of TRIM sequences from zebrafish, elephant shark and human, three(ftr, btr and trim35) and one(zinc-binding A33 like protein) gene subfamily were identified in zebrafish and elephant shark, respectively. The members of these subfamilies were numerous and had no homologs with human TRIMs. It indicated that fish TRIM genes had a different evolution direction from those from human and were more rich in diversity. Synteny analysis of TRIM genes indicated that TRIM genes underwent adaptive evolution, rapid expansion, duplication, differentiation, loss events, etc to create such a multigenic family. The results demonstrated that under positive selection, fish TRIM genes evolve faster and play important roles in immunity.Two full-length TRIM genes, Cptrim35 and Cptrim39, were firstly identified in whitespotted bamboo shark(Chiloscyllium plagiosum). The c DNAs of Cptrim35 and Cptrim39, 1,935 bp and 1,829 bp in length, encoded 537 and 488 animo acids, respectively. The Cptrim35 gene had three splice variants. The protein structure analysis indicated that the two TRIM proteins had a B30.2 domain at the C-terminus. A phylogenetic tree was constructed based on the amino acids of TRIM proteins, and Cptrim35 and Cptrim39 formed a clade together with those from teleost, but more distant from bony fish TRIMs. The expression profiles of Cptrim35 and Cptrim39 in different tissues were assessed by real-time quantitative PCR(RT-q PCR), and their subcellular localization and self-ubiquitylation were also analyzed. The results revealed that both Cptrim35 and Cptrim39 had a RING domain-dependent E3 ubiqutin ligase activity and play a role in host defense against infection.Zebrafish was selected as a model to study the differential expression of fish TRIM genes after A. hydrophila infection. Several TRIM genes were upregulated upon bacterial infection, including trim1, trim16, trim19, trim23, btr20, btr24, trim35-21, trim35-28, ftr24 and ftr43. Using RT-q PCR and RNAi techniques, the expression profile of btr20 and its interaction with the inflammatory cytokine gene NF-κB were analyzed. The results suggested that btr20 may involve in the anti-microbial activity in the immune system as an E3 ubiquitin ligase. To further study the role of TRIM proteins in innate immune signaling pathways, the yeast two-hybrid system was used to screen the zebrafish spleen c DNA library for proteins interacting with trim1 and trim23. As a result, interactions between sestrin and trim1, Uba52, Ubc E2 I, lectin, cp-2 and trim23 were identified, and the interaction between trim23 and Ubc E2 I was further verified by the pull-down experiment.In conclusion, the phylogenetic analysis and expression profile analysis after bacterial infection indicated that fish TRIM genes evolve faster upon selection pressure. The encoded proteins act as an E3 ubiquitin ligase and play important roles in innate immunity. A few fish TRIM proteins and related signal pathway proteins were obtained, which provide excellent materials for the mechanism study of immune signal pathways.