A large new subset of TRIM genes highly diversified by duplication and positive selection in teleost fish

Abstract

Background: In mammals, the members of the tripartite motif (TRIM) protein family are involved in various cellular processes including innate immunity against viral infection. Viruses exert strong selective pressures on the defense system. Accordingly, antiviral TRIMs have diversified highly through gene expansion, positive selection and alternative splicing. Characterizing immune TRIMs in other vertebrates may enlighten their complex evolution.

Results: We describe here a large new subfamily of TRIMs in teleosts, called finTRIMs, identified in rainbow trout as virus-induced transcripts. FinTRIMs are formed of nearly identical RING/B-box regions and C-termini of variable length; the long variants include a B30.2 domain. The zebrafish genome harbors a striking diversity of finTRIMs, with 84 genes distributed in clusters on different chromosomes. A phylogenetic analysis revealed different subsets suggesting lineage-specific diversification events. Accordingly, the number of fintrim genes varies greatly among fish species. Conserved syntenies were observed only for the oldest fintrims. The closest mammalian relatives are trim16 and trim25, but they are not true orthologs. The B30.2 domain of zebrafish finTRIMs evolved under strong positive selection. The positions under positive selection are remarkably congruent in finTRIMs and in mammalian antiviral TRIM5alpha, concentrated within a viral recognition motif in mammals. The B30.2 domains most closely related to finTRIM are found among NOD-like receptors (NLR), indicating that the evolution of TRIMs and NLRs was intertwined by exon shuffling.

Conclusion: The diversity, evolution, and features of finTRIMs suggest an important role in fish innate immunity; this would make them the first TRIMs involved in immunity identified outside mammals.

Figure 1

Variants of rainbow trout finTRIM ORFs. (A) Schematic representation of the structure of the rainbow trout finTRIMs from SSH and cDNA library. ORFs were predicted from the sequence of full-length transcripts. The RING, B1 and B2 boxes, coiled-coil and PRY/SPRY domains are represented by boxes with specific colors. (B) Quantitative RT-PCR analysis of finTRIM induction in trout leukocytes (Leuk.) incubated with the virus (VHSV) or LPS for 40 h or in RTG-2 fibroblasts (Fibr.) incubated with poly(I:C) for 24 h, respectively. Normalized values for finTRIM expression were determined by dividing the average finTRIM value by the average β-actin value. Then, normalized finTRIM values were subjected to a calibration relative to the basic expression in control (Ctrl) leukocytes or RTG2 cells. The error bars represent standard deviation. (C) Complex profiles produced by 3'RACE amplification of rainbow trout finTRIMs from leukocytes incubated with (V) or without VHSV (Ctrl). (D) 3'RACE profiling of finTRIM from fibroblasts incubated with poly(I:C) (I:C) or without poly(I:C) (Ctrl). (E) Structure of the rainbow trout finTRIM ORFs from the 3'RACE products. Five classes could be distinguished on the basis of motifs and ORF lengths. Striped boxes indicate regions without similarity to the finTRIM consensus, perhaps corresponding to alternative splicing to additional exons. The number of clones corresponding to the different classes from the fibroblasts incubated with poly(I:C), or from leukocytes incubated with or without VHSV are indicated on the right.

Figure 2

Genomic location of zebrafish fintrims and related genes, based on the zv7 assembly. Genes are depicted in different colors according to their subgroup: blue for group A ftrs, green for group B ftrs, brown for group C ftrs; red for btrs, and black for trim16 and trim25. Top: global distribution on the whole zebrafish genome; bottom: detailed views of multigenic loci, with gene orientations. Gene limits depicted here correspond to the predicted start and stop codons (see details on Additional file 2 – Table S1) in the zebrafish genome zv7 assembly

Figure 3

Variants of zebrafish finTRIM ORFs. Schematic representation of the zebrafish expressed ftr sequences from group A. Protein sequences were predicted from 3'RACE clones that included a polyA tail. Since the consensus RACE primer was designed in the coding region (in the RING region), the N-termini of the proteins was extrapolated from the corresponding genomic sequences in zv7. Colors of protein domains are as in Figure 1 (plus the grey box representing a chromodomain).

Figure 4

finTRIMs represent a teleost-specific multigenic family. (A). Multiple alignment of representative teleost finTRIM protein sequences. Rainbow trout (onmy) [GenBank:AM887799], zebrafish (dare) (ftr14, [GenBank:XM_692536]), channel catfish (icpu) ([GenBank:BM424798], medaka (orla) (Ensembl: ENSORLP00000003320), Stickleback (gaac) (Ensembl, Linkage group III: 14324861–14326501; GENSCAN00000022585), Pufferfish (teni) (Ensembl GSTENT00020235001). (B and C) Phylogenetic trees (NJ, boostrap = 1000) of the finTRIM and their relatives, based on the RBB (B) and B30.2 (C) regions. finTRIM accession numbers are as in (A); other sequences from zebrafish: Bty (bloodthirsty) [GenBank: NP_001018311]; TRIM25, [GenBank: NP_956469]; TRIM16, [GenBank: XR_029737]; from human: TRIM16, [SwissProt: Q99PP9]; TRIM25, [SwissProt: Q14258]; TRIM39, [GenBank: NP_742013]; from chicken: TRIM25, [GenBank: XP_415653]; from salmon: TRIM25 [gene index TC35355 accessible at ]; from xenopus: TRIM16a, [GenBank: AAH74300]; TRIM16b, [GenBank: NP_001086184]; TRIM25, [Ensembl Xenopus genome scaffold255: 821309_819660]; TRIM39, [Ensembl Xenopus genome scaffold709: 241758_272825].

Figure 5

Conserved synteny of finTRIM-like genes in teleosts. The locations of ftr82/83, trim25 and trim16 is schematically represented in the context of gene markers in zebrafish, stickleback, medaka, human, mouse and chicken to show conserved synteny groups. The gene locations are indicated (in Kb) in the table, according to the last assembly available for each species at the ensembl website (medaka: HdrR; stickleback: BROAD S1; zebrafish: zv7; human: NCBI 36; mouse: NCBI m37).

Figure 6

Positive selection in the B30.2 domain. Distribution of hypervariable and positively selected residues in a multiple alignment of B30.2 domains from representative zebrafish and rainbow trout finTRIMs (Dareftr13: [GenBank: XM_695031], Onmyref21: [GenBank: XM_695031], human TRIM21 (HosaTRIM21) and TRIM5α (HosaTRIM5α). Sites that are less than 80% conserved among zebrafish or among trout sequences are indicated in red. Positively selected sites (among zebrafish finTRIMs: this study; among primate TRIM5α : previous work of Sawyer et al [44]) are boxed in blue when detected under models 2a and 8. One site was positive under M8 but not under M2 and is boxed in green. β-strands identified from the TRIM21 structure B30.2 sequence are indicated by dark (PRY) or light (SPRY) blue arrows [16]. The variable loop-connecting strands are named VL1–VL6. The four hypervariable regions of the TRIM5α B30.2 domain are underlined [27]. The detailed PAML results for each position under positive selection are available in Additional file 6, Table S4. The purple 'R' indicates the recombination site identified by GARD.

Figure 7

Distance tree of finTRIM B30.2 domains and related sequences. The domain organization of finTRIM and NLR proteins is represented in (A). A typical alignment of the B30.2 protein sequences from group A ftr (ftr35) and NLR (chr5_32382364) is shown in (B). B30.2 domains of the zebrafish fintrims and btrs as well as those extracted from related trims of other vertebrates (human and xenopus trim16, human trim39, Atlantic salmon, zebrafish, xenopus, chicken and human trim25; see Figure 4 for accession numbers) were aligned with related B30.2 sequences from nlrs, and a distance tree was produced using mega4 (NJ, boostrap = 1000); high bootstrap values of key nodes are indicated (C).

Figure 8

Tentative evolutionary pathway of fintrims and their relatives in vertebrates. The genes identified in the genomes of different vertebrate groups are indicated on the right. Since the current draft of the elephant shark genome is still partial, the absence of fintrim and btr is unsure, which is indicated by '?'.