# Origin and evolution of TRIM proteins: new insights from the complete TRIM repertoire of zebrafish and pufferfish

Pierre Boudinot, Lieke M van Der Aa, Luc Jouneau, Louis Du Pasquier, Pierre Pontarotti, Valérie Briolat, Abdenour Benmansour, Jean-Pierre Levraud

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Pierre Boudinot, Lieke M van Der Aa, Luc Jouneau, Louis Du Pasquier, Pierre Pontarotti, et al. Origin and evolution of TRIM proteins: new insights from the complete TRIM repertoire of zebrafish and pufferfish. PLoS ONE, 2011, 6 (7), pp.e22022. 10.1371/journal.pone.0022022. hal-00831143

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# Origin and Evolution of TRIM Proteins: New Insights from the Complete TRIM Repertoire of Zebrafish and Pufferfish

Pierre Boudinot¹*, Lieke M. van der Aa¹,², Luc Jouneau¹, Louis Du Pasquier³, Pierre Pontarotti⁴, Valérie Briolat⁵,⁶, Abdenour Benmansour¹, Jean-Pierre Levraud⁵,⁶*

¹Virologie et Immunologie Moléculaires, Institut National de la Recherche Agronomique, Jouy-en-Josas, France, ²Cell Biology and Immunology Group, Wageningen University, Wageningen, The Netherlands, ³Institute of Zoology and Evolutionary Biology, University of Basel, Basel, Switzerland, ⁴Equipe Evolution Biologique et Modélisation UMR 6632 Université de Aix Marseille I/CNRS, Centre St Charles, Marseille, France, ⁵Unité Macrophages et Développement de l'Immunité, Institut Pasteur, Paris, France, ⁶URA 2578 du Centre National de la Recherche Scientifique, Paris, France

## Abstract

Tripartite motif proteins (TRIM) constitute a large family of proteins containing a RING-Bbox-Coiled Coil motif followed by different C-terminal domains. Involved in ubiquitination, TRIM proteins participate in many cellular processes including antiviral immunity. The TRIM family is ancient and has been greatly diversified in vertebrates and especially in fish. We analyzed the complete sets of *trim* genes of the large zebrafish genome and of the compact pufferfish genome. Both contain three large multigene subsets - adding the *hs15/trim35*-like genes (*htr*) to the *ftr* and the *btr* that we previously described - all containing a B30.2 domain that evolved under positive selection. These subsets are conserved among teleosts. By contrast, most human *trim* genes of the other classes have only one or two orthologues in fish. Loss or gain of C-terminal exons generated proteins with different domain organizations; either by the deletion of the ancestral domain or, remarkably, by the acquisition of a new C-terminal domain. Our survey of fish *trim* genes in fish identifies subsets with different evolutionary dynamics. *trims* encoding RBCC-B30.2 proteins show the same evolutionary trends in fish and tetrapods: they evolve fast, often under positive selection, and they duplicate to create multigenic families. We could identify new combinations of domains, which epitomize how new *trim* classes appear by domain insertion or exon shuffling. Notably, we found that a cyclophilin-A domain replaces the B30.2 domain of a zebrafish *fintrim* gene, as reported in the macaque and owl monkey antiretroviral TRIM5α. Finally, *trim* genes encoding RBCC-B30.2 proteins are preferentially located in the vicinity of MHC or MHC gene paralogues, which suggests that such *trim* genes may have been part of the ancestral MHC.

**Citation:** Boudinot P, van der Aa LM, Jouneau L, Du Pasquier L, Pontarotti P, et al. (2011) Origin and Evolution of TRIM Proteins: New Insights from the Complete TRIM Repertoire of Zebrafish and Pufferfish. PLoS ONE 6(7): e22022. doi:10.1371/journal.pone.0022022

**Editor:** Nikolas Nikolaidis, California State University Fullerton, United States of America

**Received** March 9, 2011; **Accepted** June 12, 2011; **Published** July 15, 2011

**Copyright:** © 2011 Boudinot et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

**Funding:** This work has been funded by the Institut National de la Recherche Agronomique (INRA) and by the Wageningen University (WUR), and by a grant from Agence Nationale de la Recherche (ANR-07-MIME-029-01). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

**Competing Interests:** The authors have declared that no competing interests exist.

* E-mail: pierre.boudinot@jouy.inra.fr (PB); jlevraud@pasteur.fr (JPL)

## Introduction

The tripartite motif (TRIM) family –also known as the N-terminal RING finger/B-box/coiled coil (RBCC) family– play major roles in development, tumor suppression, disease pathology and viral restriction/sensing [1,2]. This tripartite motif is associated with diverse C-terminal domains, which often determine the specificity of the interactions of TRIMS with other proteins. Hence, TRIM proteins associate a RING-dependent E3 ubiquitin ligase activity with the capacity to build multiprotein complexes though interactions with their CC and C-terminal domains. Human TRIM proteins have been classified into 9 architectural subsets on the basis of their C-terminal domains, subcellular compartmentalization and functionality ([3], Figure 1). The B30.2 domain [4] found in Class-I and Class-IV TRIM proteins is constituted by the juxtaposition of a PRY and a SPRY domain, and is also known as PRY/SPRY domain [5].

In a survey of the TRIM family in various species, Sardiello *et al.* distinguished two groups: the *trim* genes from group 1 contain a

variety of C-terminal domains and are generally well conserved among distant species, while members of group 2 correspond to the structural Class IV subgroup which evolve much faster, display lower levels of amino acid conservation in distant species and are subjected to different selection pressures [6]. Importantly, the Class IV TRIM proteins include multiple members involved in antiviral immunity at various levels of the interferon (IFN) signalling cascade. For instance TRIM25 is required for viral RNA sensing performed by the cytoplasmic helicase RIG-I, leading to IFN production [7]. Other class IV TRIM proteins also control signalling pathways that lead to IFN production: TRIM27 represses NFκB and IRF3/IRF7 [8] while TRIM21 ubiquitylates IRF3/IRF7 and IRF8 [9,10,11]. On the other hand, TRIM5α - which was described as a strong restriction factor for HIV-1 in rhesus macaque - directly targets retroviruses [12]. The TRIM5α B30.2 domain binds the nucleocapsid of incoming viral particles and accelerates the uncoating of the viral core, while the RING/ B-box domains are essential for the localization in specific cytoplasmic ‘bodies’ [13,14,15] and mediate a TRIM5α higher-order

<figure><img src="image_2.png" /></figure>
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>
**Figure 1. TRIM proteins from zebrafish and pufferfish.** Classification of fish TRIM proteins based on their C-terminal domain(s) and the categories proposed by Short & Cox in Ref. 3. Previously unreported TRIM proteins found in fish were tentatively numbered TRIM101-111. Conserved TRIM proteins are represented on the left panel (Classes I–III and V–IX). Other TRIM proteins are shown on the right panel (Class IV). Dotted lines delimit groups of closely related human TRIM (modified from Ref. 6) corresponding to a diversification that occurred during tetrapod evolution. Blue frames indicate multigenic families observed in teleost fish. RBCC: Ring-BBox-Coiled Coil; COS: C-terminal subgroup one signature; FN3: Fibronectin, type III; B30.2: PRY/SPRY domain; PHD: Plant Homeo Domain; NHL: NCL-1, HT2A and Lin-41 repeat; Filamin: named from the protein Filamin; Bromo: acetylated lysine binding domain; ARF/SAR: from ARF and SAR GTP binding proteins; Pyrin: a member of the six-helix bundle death domain-fold superfamily; TM: transmembrane; Math: meprin and TRAF homology domain; Chromo: CHRromatin Organization Modifier domain.
doi:10.1371/journal.pone.0022022.g001

self association that increases avidity for retroviral capsids [16,17]. The structure of the B30.2 domain is a β-sandwich core with ligand-binding loops at the top that are variable and determine the specificity of the interaction. Ligand-binding loops of the TRIM5α B30.2 domain diversified during the evolution of primates, ensuring efficient restriction of specific retroviruses in the different species [18,19]. Thus, while TRIMs constitute an ancient family with members involved in basic cellular processes in practically all bilateria and pre-bilateria phyla [20], it seems that several subsets have been recruited and diversified for antiviral immunity during the evolution of vertebrates. However, the specific modalities of these apparently independent multiplication and diversification events are still poorly understood.

Teleost fishes offer an intriguing model for a comparative study of the TRIM family because of their ancient separation from the tetrapods, their great diversity and the considerable variation in the number of *trim* genes in their genome. In addition, the zebrafish (*Danio rerio*, Hamilton) is an important vertebrate model for developmental biology, and more recently, for host-pathogen interactions. Therefore the identification and classification of its many *trim* genes is important for these fields of biological study— and the further development of zebrafish as a model for vertebrates. Sardiello *et al*. listed 240 zebrafish *trim* genes, but without providing classification [6]. During our investigation of IFN-inducible class IV trim homologues in trout, we identified 84 *fintrim* (*ftr*) and 33 *trim39/bloodthirsty*-like (*btr*) genes [21], implying that the zebrafish genome contains at least 117 class IV genes, and probably many more. *Ftrs* do not have true orthologues in mammals, thus should have a specific function in fish defense. Apart from *ftrs* and *btrs*, the zebrafish gene database at zfin.org currently lists only 21 additional *trim* genes.

We therefore performed an exhaustive description of *trim* genes in two fish species that followed different genomic evolutionary histories – zebrafish (*Danio rerio*) and spotted green pufferfish (*Tetraodon nigroviridis*) – updating and completing the lists provided in [6]. In contrast to other *trim* genes that are generally conserved through vertebrates with conserved expression patterns, the vast majority of fish class IV *trim* genes belongs to three multigenic families of which the B30.2 domain has evolved under positive selection. Our systematic analysis of *trim* genes also led to the identification of genes that have lost, gained or replaced their C terminus domain, providing a good illustration of the mechanisms giving birth to new *trim* classes.

## Results

### TRIM classes reflect two distinct evolutionary pathways in fish

The complete repertoire of *trim* genes was determined in two fish species using combined approaches of genome scanning for protein domains and sequence comparison (see Material and Methods for details). Among fish species with available genomes, we chose the zebrafish and the spotted green pufferfish because they are phylogenetically distinct with an estimated 300 My time of divergence [22] and followed distinct genomic evolutions. They

have different gene contents (15315 genes in the pufferfish compared to 23569 in the zebrafish, in Ensembl release 57) and have been subjected to different events of genome expansion/contraction. Thus, we expected that zebrafish genome would contain an expanded repertoire of *trim* genes while the compact pufferfish genome may have a “minimal” *trim* repertoire.

Zebrafish and pufferfish *trim* repertoires are presented in Figure 1, where they are compared to the human repertoire. Detailed information about the genes is provided in Supplemental Figures S1, S2 and S3. Fish *trim* genes were named after the human orthologues, following the Ensembl annotations confirmed by the analysis of the domain organization of the protein. When a close paralogue of such a fish *trim* was found with a highly similar organization but lacking the terminal domain, it was considered as another orthologue (i.e. a co-orthologue) and was therefore given the same name with a “like” suffixe. When a fish *trim* had no orthologue in human or in the mouse, we named it from available publications or we attributed a temporary “trim101-110” annotation, waiting for a definitive nomenclature.

We found 208 *trim* genes in the zebrafish (Zv9 assembly) and 66 in pufferfish (Figure 1), compared to 75 and 67 genes reported in human and in the mouse respectively [23,24]. Sardiello *et al*. had reported 240 *trim* genes in zebrafish and 58 in pufferfish [6]; the large discrepancy observed in zebrafish was due to the fact the list established by Sardiello *et al*. was derived from a search in ESTs that were not matched to the genome sequence. This procedure resulted in frequent inclusion of the same sequence under two accession numbers, or inclusion of allelic variants. Orthologues of human *trim* genes for all classes excluding C-III (RBCC-COS-FN3) were present in both zebrafish and pufferfish. The main *trim* categories, with the possible exception of C-III, were therefore already defined in the common ancestor of fishes and tetrapods.

As shown below, *trim* genes could be separated into two main subsets reflecting their evolutionary dynamics, in complete agreement with Sardiello *et al*. [6].

One or two counterparts were found in fish for the majority of the human *trim* genes belonging to the classes I, II and VI–IX (Figure 1, left column). The presence of two co-orthologues of a given human gene in both fish species likely reflects the ancestral duplication of teleost genomes [25,26]. The term “co-orthologue” is employed here to describe the evolutionary relationship of two or more paralogous genes with their counterpart in another species. Co-orthologue is synonymous of “inparalogue” [27]. For these *trim* classes, pufferfish and zebrafish *trim* repertoires were overall very similar, with a few exceptions: *trim40* (ClassV), *mf207* (ClassV), *trim102* (ClassVI), as well as a *trim1*-like, a *trim54*-like and a *trim3*-like were found in zebrafish only while *trim20*, *trim66*, *trim18-2* and *trim2*-like were found only in pufferfish. These exceptions are likely due to local events of gene duplication or loss.

TRIM lacking a unique C terminal domain (Class C–V) could also be included into this subset as they never have more than two fish orthologues, even though many class V human genes lack a fish counterpart: only 6 zebrafish and 2 pufferfish counterparts were found for 12 human genes. In fact, genes closely related to members of other classes that have lost their C terminal domain - for example

<figure><img src="image_5.png" /></figure>
zebrafish *trim54like* - could fall into the class V as defined above. Thus, at least in fish, *trim* genes with no specific C-terminal domain do not constitute a homogeneous group.

The conservation of these TRIM proteins between teleosts and mammals strongly suggests that they carry out conserved functions. Such a hypothesis would be reinforced if these proteins had similar tissue-specific expression patterns. To test this hypothesis, we selected a subset of *trim* genes with a clear orthology relationship between human and zebrafish (Supplemental Figure S4) and tissue-specific expression described in mammals, and measured their expression in various organs of adult zebrafish by qRT-PCR (Figure 2). *Trim1* (also known as *FXT2* or *MID2*) has been reported to be expressed “in low abundance in brain and lung, with even lower levels in heart, liver and kidney” by northern blot analysis of mouse tissues [28]. Indeed, in zebrafish, *trim1* was expressed at a higher level in brain than in heart, liver or kidney (Figure 2). In the case of *trim13* (aka *RFP2*) strongest expression was found in the testis (ovary was not tested) for both mouse and human [29]; the situation was different in the zebrafish, where only moderate levels of *trim13* were measured in the testis, although levels were high in the ovary (Figure 2). Apart from gonads, the zebrafish tissue with the highest level of expression was the brain, in agreement with mouse, but not human northern blot data [29] – note however that strong staining with anti-RFP2 antibody is detected in human brain samples (www.proteinatlas.org). Expression of *trim25* (also known as *effp*) –the function of which may suggest a rather uniform expression [7]– has been tested by RNAse protection assay in mouse tissues; abundant levels were observed in uterus, ovary and placenta, medium levels in the mammary gland, and lower levels in liver, spleen, kidney, heart, lung and thymus, and only a faint band in brain [30]. A rather ubiquitous pattern was found in zebrafish (Figure 2); a discrepancy with mouse data was the relatively high expression in brain. By Northern blot, *trim33* (or *TIF1γ*) expression was found to be

highest in mouse testis, then in liver, heart, brain and kidney, weak in spleen and lung and very low in skeletal muscle [31]. Among the corresponding zebrafish organs, *trim33* expression was highest in brain, then in testis; however, it was fairly low in liver, while intermediate in muscle (Figure 2). *Trim54* (also known as *MURF*) constitutes the most clear-cut example or tissue-specific expression, with an almost exclusive expression in heart and skeletal muscle [32]. A similar pattern was observed in zebrafish: expression was strong in skeletal muscle, and extremely low in visceral organs – the low heart expression was, however, a remarkable difference (Figure 2). Finally, *trim59* (or *Mif1*) expression data in mammals are rather conflicting; in mouse, expression was found to be strong in testis, moderate in spleen, weak in brain and heart, and very low in lung, liver, kidney or skeletal muscle [33]; while in humans, highest levels were detected in skeletal muscle, heart, liver and lung [34]. In zebrafish, strong expression was found in ovary, and low levels in gut, level or muscle (Figure 2). In conclusion, although the variety of techniques and organs sampled in the various published studies makes comparisons quite difficult, similar patterns of expression can often be observed between mammalian and fish tissues (if one excludes gonads, where extreme expression levels are frequent), likely reflecting conservation of function for these genes.

Class IV trims (RBCC-B30.2) followed a different evolutionary pathway. No obvious counterpart could be found in fish of the majority of human and mouse *trim* genes belonging to this class (Figure 1, right column). Our analysis identified fish orthologues only for *trim16*, *trim25*, *trim35*, *trim39* (the *btr* family), *trim47* and *trim62*. Reciprocally, several fish class IV genes had no counterparts in mammals, such as the *fintrims* and the *trim103-110*. Strikingly, several Class-IV members constitute multigenic subsets in fish. Two of these multigene subsets possess a unique human counterpart *trim39* and *trim35*, respectively. The third multigene set is composed of sequences that lack counterparts in tetrapods,

<figure><img src="image_6.png" /><figcaption>**Figure 2. Expression profile of selected trim genes.** The expression of 6 zebrafish *trim* genes was measured by quantitative RT-PCR in pools of tissues from 10–12 animals. E1f-α was used as a housekeeping gene, and the relative expression levels of *trim* genes were normalized on the geometric mean of the values measured for “whole males” and “whole females”, to take both sexes into account in the normalization. The data are represented as a heat map, with expression level and standard deviation is indicated for each condition.
doi:10.1371/journal.pone.0022022.g002</figcaption></figure>

<figure><img src="image_7.png" /></figure>
<figure><img src="image_8.png" /><figcaption>ftr06</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>ftr52</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>btr31</figcaption></figure>

**Figure 3.** Three different ways to generate TRIM proteins with new domains. For these three cases observed in the zebrafish genome, the new gene is shown on the bottom of panel, and is compared with a closely related, typical member of the *ftr* or *btr* family illustrated on top. Percentages of identity refer to DNA sequences. Rectangles represent exons, numbers refer to nucleotides of coding sequence, stop included. Intron

<figure><img src="image_11.png" /></figure>
not to scale. A: the *ftr06* gene, contained within a large cluster of tandem *ftr* genes on chromosome 2, encodes a protein with a C-terminal chromodomain instead of a B30.2. This is due to the insertion of a single chromodomain-encoding exon just upstream of the usual exon 6. The previous B30.2 exon, shown in parenthesis, is still present downstream, nonmutated, but is not included in the chromodomain-encoding transcript. B: the *ftr52* gene, isolated on chromosome 9, encodes for a TRIM protein with a C-terminal RanBD/cyclophilin A domain instead of a B30.2. In this case, the new C-terminal domain is encoded by multiple exons; no B30.2-encoding sequence can be detected in this genomic area. C. The *btr31* gene, located on chromosome 19 tandemly to its close relative *btr32*, encodes for a protein with the typical N- and C- ends of bloodthirsty-like proteins, but the B-boxes and the coiled-coil regions in the middle have been replaced by a transmembrane domain.
doi:10.1371/journal.pone.0022022.g003

the *fintrims* (*ftr*) [21]. These different gene expansion events explain the high *trim* numbers observed in fish compared to human.

Interestingly, the repertoire of class IV genes was also more divergent between zebrafish and pufferfish than for the other classes. Indeed, the multigenic class IV *trim* subsets contains much less genes in pufferfish compared to zebrafish: 12 *ftr* for 89, 6 *trim35* instead of 37 and 10 *btr* instead of 33. Additionally, several genes including *trim106-109* and *trim25* were absent from pufferfish while found in zebrafish.

Most remarkably, three instances of gain of domain were also detected (Figure 3). The *ftr06* gene acquired a C-terminal chromodomain via the insertion of a single exon between the original 5th and 6th exons (Figure 3A). Thus, although the sequence encoding for a B30.2 domain is still present in the genome downstream of the chromodomain, it is not included in the transcribed gene, as established previously by RACE analysis ([21] see sequences # AM941366 and AM941342). Along the same line, just downstream of the *ftr52* gene, one can find a Ran binding-domain (RanBD) and a cyclophilin A (CypA) domain, encoded by four exons, while no B30.2 domain can be detected in this genomic region (Figure 3B). Such a configuration could happen in one single step by the insertion of a piece of DNA containing exons 1 to 4 of a *ftr* gene within a pre-existing *RanBP2*-like gene. *Ftr52* was believed to be a pseudogene since it contains a predicted stop codon in the N-terminal *RING-encoding* region (found on the previous and current genomic assemblies). To test whether *ftr52* was effectively transcribed into a *trim* mRNA, we PCR-amplified cDNA from AB fish with primers upstream of the *RING* and downstream of the CypA regions, and cloned and sequenced the product (accession number JF295002). The retrieved sequence does correspond to a properly spliced transcript that would encode a RBCC-RanBD-CypA protein if not for a premature stop codon in the N-terminal exon. This stop codon is identical to the one found in the current genomic sequence, derived from a Tü strain fish. We PCR-amplified genomic DNA of several independent AB and Tü zebrafish (the most used “wild-type” laboratory strains) and found this stop codon in all products. A slightly more complicated sequence of events took place to generate the *btr31* gene that encodes a protein with a predicted TM domain between a N terminal *RING* domain and a C terminal B30.2 domain, while B-Boxes and the Coiled Coil have disappeared. This gene is clearly a product of recent duplication of a *btr* gene, and is most similar to its neighbour *btr32* that possesses the bona fide domains. The genesis of *btr31* would require at least two genomic rearrangement events: the replacement of exons 3 and 4 by a DNA stretch containing a TM-encoding exon, and the deletion of the end of exon 1; however the gene structure is confirmed by several ESTs (i.e., EH489524 and EH515884), excluding an assembly artefact. Similar to the first subset of *trim* genes, the loss of the specific C-terminal domains was also frequently observed in pairs of co-orthologues. Such events of gain of domain were not found in the pufferfish.

**trim39/btrs and trim35-related multigene families derive from ancient duplications**

Fish genomes contain three large multigene subsets of class IV trims: *finTRIMs* (*ftrs*), bloodthirsty-like trims (*bts*) and Hematopoietic

lineage switch-5/trim35-like trims (*hltrs*). In an attempt to understand the selective constraints that give rise to such large families, we analyzed the diversity of the *ftr* family from a prior study [21] and extend this analysis to include *btr* and *trim35/hltrs* genes.

The *btr* family, orthologous to *trim39*, has been previously reported in zebrafish and other teleosts [21]. These genes were named «bloodthirsty related genes» or *btr* from the first described member of the family, involved in erythropoiesis, *bloodthirsty* [35]. The *btr* genes are relatively dispersed in the zebrafish genome but do not colocalize with the *ftr* clusters. *btr* clusters are found on chromosomes 7, 15 and 19 (Figure 4). As previously seen for *ftr*, a minority of *btr* genes are part of synteny groups conserved in zebrafish, pufferfish and in other fish. *btr1* (chr1), *btr2* (chr3) and *btr33* (chr24) belong to gene sets found in conserved synteny (Figure 5). Additionally, *btr* genes located on zebrafish chr5 and 15 are linked to the paralogous markers encoding the alpha-crystallins *cryabb* and *cryaa* respectively, suggesting a common origin for the corresponding regions. Thus, at least the *btr* that are involved in conserved syntenies were produced by regional and global duplications which occurred relatively early during fish evolution. The other *btr* constituting clusters are probably more recent.

Thirty-seven *trim35/hltr* genes were identified in the zebrafish genome (*Zv9* assemby), all containing a B30.2 domain. In contrast, only six *trim35/hltr* were found in the pufferfish genome. When all zebrafish *trim35/hltr* sequences were included in a distance tree with representative genes from class IV, they grouped in a specific cluster, confirming that they constitute a distinct subset in the *trim* family (Figure 6A). Separate phylogenetic analyses were performed for RBB and B30.2 regions using NJ (Figure 6B &6C) and PHYML. These analyses indicate that the fish *TRIM35/HLTR* sequences group with the reptile and mammalian *TRIM35*, while its closest relatives *TRIM21*, 11 and 60 as well as FTR and *TRIM25* determine distinct clusters each supported by high bootstrap values in phylogenetic trees. Both RBB and B30.2 trees are congruent and strongly suggest that fish *trim35/hltr* genes are good co-orthologs of their mammalian unique (i.e. non-duplicated) counterpart. This hypothesis could not be further validated by examining conserved synteny because the markers defining a conserved 2 Mbp-neighbourhood of *trim35* in tetrapods (Figure 7A) are not found in the same synteny group in teleosts. In contrast, more than 25 of the *trim35/hltr* genes found in the teleost stickleback (*Gasterosteus aculeatus*) are part of synteny clusters conserved in medaka (*Oryzias latipes*) and even in pufferfish (Figure 7B), indicating that some duplications predated the split between these lineages. Only the regions involving *trim35-12* and *trim35-28* have counterparts in zebrafish. In zebrafish, the multiple copies of *trim35/hltr* are scattered on 15 different chromosomes (Figure 4) and they are often grouped in clusters as previously observed for *ftr* and *btr* ([21], see above). In striking similarity with the *ftr* genes, the *trim35/hltr* genes involved in conserved syntenies are not found among the large sets of recently duplicated sequences represented by the distal branches in the phylogenetic tree.

**Positive selection of B30.2 domains of btr and trim35 families**

The B30.2 domain consists of two subdomains, PRY and SPRY and forms a distorted β-sandwich of two layers of antiparallel

<figure><img src="image_12.png" /></figure>
<figure><img src="image_13.png" /><figcaption>**Figure 4.** Genomic location of zebrafish *trim* genes. *trim* genes are depicted in different colors according to each *trim* class: class I in red, class II in yellow and boxed, class IV in blue, class V in green, class VI in pink, class VII in black, class VIII in yellow and red, class IX in orange. The different *trim* subsets belonging to class IV are indicated in shades of blue. This representation is based on the Zv8 assembly. RBCC: Ring-Bbox-Coiled Coil; COS: C-terminal subgroup one signature; FN3: Fibronectin, type III; B30.2: PRY/SPRY domain; PHD: Plant Homeo Domain; NHL: NCL-1, HT2A and Lin-41 repeat; Filamin: named from the protein Filamin; Bromo: acetylated lysine binding domain; ARF/SAR: from ARF and SAR GTP binding proteins; Pyrin: a member of the six-helix bundle death domain-fold superfamily; TM: transmembrane; Math: meprin and TRAF homology domain; Chromo: CHRromatin Organization Modifier domain.
doi:10.1371/journal.pone.0022022.g004</figcaption></figure>

β-sheets [36,37,38]. The β-strands are connected by six variable loops (VL) that define hypervariable regions and form the ligand-binding surface in TRIM5α. The loops also contain the Ig-binding regions in TRIM21 [39]. We observed earlier in FTRs hypervariable regions similar to those of TRIM5α [21], and we showed that they evolved under positive selection. To determine whether zebrafish *trim35/hltr* and *btr* share the same evolutionary pattern and show diversified regions in their B30.2 domains, these genes were subjected to a similar analysis calculating the Shannon entropy site by site (see Supplemental Figure S4).

The distribution of variable sites in TRIM35/HLTR and BTR is remarkably consistent with the patterns observed for FTRs and for TRIM5α: 39 among 59 and 26 among 37 hypervariable sites of TRIM35/HLTR and BTR, respectively, are shared with FTR (Figure 8). Variable regions corresponding to the loops joining the B30.2 domain β-strands were retrieved, including those involved in the binding of the virus by TRIM5α. Interestingly, conserved variable sites were concentrated in the β2-β3 loop (VL1), which is responsible of retroviral binding specificity of TRIM5α and was identified as an evolutionary hotspot in TRIM5α and TRIM22 [40].

To test whether *trim35/hltr* and *btr* B30.2 domains evolved under diversifying selection in zebrafish, we used a test based on the estimation of synonymous (*dS*, silent mutations) and non-synonymous (*dN*, amino acid altering) substitution rates of all codons among a set of sequences: the ratio ω = *dN*/(*dS*) is an indication for negative (purifying) selection of deleterious changes (ω<1), neutral evolution (ω = 1), or positive (diversifying) selection when changes offer a selective advantage (ω>1). This approach is often used on paralogues to detect the accumulation of non-synonymous changes that suggests a positive selection driving the evolution of new functions following gene duplications [41,42]. This method requires that the paralogue sequences are not too divergent i.e. that good quality multiple alignments can be easily produced. We verified that this condition was met for each dataset subjected to PAML analysis (Supplemental Figure S5).

B30.2 domain sequences of zebrafish *trim35/hltr* and *btr* were analyzed under different evolutionary models M1a, M2a, M7 and M8 by PAML. Positive selection was detected for ~10% of sites of *trim35/hltr* under both M2a and M8, while for *btr* 4.5% of sites were positively selected under M2a and 8.5% of sites under M8 (Table 1). These results were validated by significant likelihood

<figure><img src="image_14.png" /></figure>
<figure><img src="image_15.png" /><figcaption>**Figure 5.** Group of conserved synteny around *btr* 01, 02 and 33. Synteny groups were determined from Ensembl assemblies using the genomicus database and browser (http://www.dyogen.ens.fr/genomicus-59.01/cgi-bin/search.pl) [85]. The figure is edited from the PhyloView taking *btr* 01 (A), 02 (B) and 33 (C) genes as references. The reference *btr* gene and its orthologues are shown in light green over a thin vertical line, and are indicated in bold.
doi:10.1371/journal.pone.0022022.g005</figcaption></figure>

ratio test (LRT) with *p*<0.001 for both models for *trim35/hltr* and *btr* (see Supplemental Figure S6).

To investigate whether the estimation of positive selection under PAML was not perturbed by recombination between similar *trim* sequences during the evolution of the zebrafish genome, we re-analyzed our dataset with the algorithm PARRIS, which uses a partitioning approach to test whether sequences have been subjected to positive selection even if recombination occurred. Positive selection was still indicated by the LRT with *p*<0.001 for both TRIM35/HLTR and BTR. This indicated that whether or not recombination did occur, the B30.2 domains of TRIM35/HLTR and BTR have most probably evolved under positive selection.

To search for recombination sites, we used the program GARD, which subdivides a sequence alignment in putative non-recombinant fragments, infers phylogenies for each fragment and assesses the quality of the fit for these phylogenies. This comparison therefore determines if the fragments are derived from two different ancestor sequences due to recombination. No evidence for recombination was detected for *trim35/hltr*. In contrast, seven breakpoints were identified between *btr* sequences at the positions 262, 269, 271, 288, 399, 405 and 427 of the *btr* B30.2 multiple alignment (see the multiple alignment in Supplemental Figure S5 and Δc-AIC values in Supplemental Figure S7). These breakpoints suggested the existence of three segments of *btr* B30.2 domain

<figure><img src="image_16.png" /></figure>
A

<figure><img src="image_17.png" /></figure>

B

<figure><img src="image_18.png" /></figure>

C

<figure><img src="image_19.png" /></figure>

**Figure 6.** Fish counterparts of *trim35* constitute multigenic subsets. (A) Distance tree produced by ClustalW (Neighbor joining; bootstrap = 1000) for the zebrafish TRIM35/HLTR sequences and representative TRIM sequences from other species. Relevant bootstrap values are indicated. Separate phylogenetic analyses of the RBB (B) and B30.2 (C) regions of TRIM35 and other representative TRIM using Clustalw (Neighbor joining; bootstrap = 1000). The same analyses were performed with PHYML and led to consistent trees. Sequences integrated into the trees: DareBty: zebrafish bloodthirsty (NP_001018311); DareFtr: zebrafish fintrim (XM_692536); GaacFtr: stickleback fintrim; OrlaFtr: medaka fintrim (ENSORLP00000003320); OnmyFtr: rainbow trout fintrim (AM887799); DareTr25: zebrafish trim25 (NP_956469); SasaTr25: salmon trim25 (gene index TC35355 accessible at http://compbio.dfci.harvard.edu/); GagaTr25: chicken trim25 (XP_415653); XetrTr25: Xenopus tropicalis Trim25 (Ensembl Xenopus genome scaffold255: 821309_819660); HosaTr25: human Trim25 (Q14258); GagaTr35: chicken trim35 (ENSGALP00000026735); AncaTr35:

<figure><img src="image_20.png" /></figure>
lizard trim35 (ENSACAP00000002320); HosaTr35: human trim35 (NP_741983.2); MumuTr35: mouse trim35 (ENSMUSP00000022623); GaacTr35: stickleback TRIM35 (ENSGACP00000004694); OrlaTr35: medaka Trim35; TeniTr35: pufferfish Trim35; dareTr35: zebrafish Trim35-8 (ENS-DARP00000064945); HosaTr21: human Trim 21 (NP_003132); HosaTr11: human Trim 11 (NP_660215); HosaTr60: human Trim 60 (AAI00986). The IDs of the other TRIM35 sequences from zebrafish used in (A) are available in Figure S1.
doi:10.1371/journal.pone.0022022.g006

where no recombination has occurred (see the segments 1, 2 and 3
in Figure 8). We detected positive selection in fragment one and
three, with p<0.001 in the LRT under M1a–M2a and M7–M8
models. No positive selection was detected in fragment two
(Supplemental Figure S8).

Finally, the specific sites where non synonymous changes
accumulated were identified by a Bayesian approach using the
complete gene set for *trim35/hltr* and the fragmented gene set for
*btr*. For *trim35/hltr*, we found 12 sites under M2a and 11 sites
under the more restrictive model M8. The majority of the sites fall
in the predicted variable loops corresponding to those reported for
TRIM21 and TRIM5α. For the *btrs*, we found 7 sites under both
M2a and M8.

Hence, a significant number of sites showing hints of positive selection in *trim35/hltr* and *btr* B30.2 domains were located in the β2-β3 loop, at positions matching well those previously noted for *ltr* and for the same region in TRIM5α (Figure 8).

Do fish *trim* genes colocalize with the MHC paralogs?

The Major histocompatibility complex (MHC) is a genetic
region that plays a key role in self/non-self recognition and T cell
responsiveness. The presence of Class IV *trim* genes in the MHC is
a conserved feature in mammals and birds [43,44,45]. Based upon
this feature we addressed whether the co-localization predates the
split (>450 My) between fish and tetrapods. This gene dense
region has an ancient history as the mouse and human genome
contain four well established MHC paralogous regions of the
MHC, that are believed to be the result of two whole genome
duplications in the early evolution of vertebrates [46]. In teleosts,
a variable number of global genome duplications followed by
genome contraction and rearrangement events have “broken” the
MHC into multiple regions in the genome of fish such as the
MHC class I and II regions are on different chromosomes [47]. To
trace the existence of an early association of ClassIV *trim* genes
with the “primordial” MHC, it was therefore relevant to examine
the different MHC regions and all of their associated paralogues in
fish genomes.

A loose linkage of *ltr*, *btr* and the MHC or its paralogues has
been previously reported in zebrafish [20]. The MHC regions and
their paralogues also contain RBCC-B30.2 genes in another fish
species that possesses fewer Class IV *trim* genes than zebrafish: in
stickleback, *notch1.1*, *notch1.2* and *notch3* are associated to 7 genes
belonging to the *trim* Class IV grouping. Since in humans, *notch4* is
found within the MHC and other *notch* genes in paralogous loci
[46], this was the first indication that the linkage might be older
than tetrapods. We therefore performed a systematic survey of the
distribution of *trim* genes and MHC markers in the zebrafish
genome, looking for a co-localization pattern. We searched the
homologues of a set of classical MHC markers and their para-
logues described in other vertebrates [48,49,50,51,52,53] (See
Supplemental Figure S9). Since it was not always possible to
attribute the zebrafish homologues to one given member of the
tetrad of MHC paralogues in humans [54], we defined “MHC
neighbourhoods” as regions extending 5 Mb (size of the MHC
proper) upstream and downstream from each MHC or paralogue
marker. The MHC neighbourhood represented 376 megabases in
zebrafish containing 7884 genes compared to 1187 megabases and
16072 genes for the rest of the genome. We then compared the

numbers of *trim* genes that were located in these MHC neigh-
bourhoods versus that of the rest of the genome (see Supplemental
Figure S9); counting each cluster of tandemly duplicated *trim* genes
as a single occurrence to avoid skewing of the analysis. In-
terestingly, the Class IV *trim* genes were significantly enriched in
the MHC neighbourhoods (Independence χ²-test, p value =
0.0035), while no bias could be detected for the other *trim* genes
(Table 2).

Discussion

TRIMs are widely distributed in metazoans, and these
intracellular proteins are involved in the regulation of multiple
pathways. In this report, a systematic survey of *trim* genes was
performed in zebrafish and pufferfish to examine the character-
istics of this family in two fish species with different genome
dynamics. The zebrafish genome is large (about 1600 Mb/24000
genes) and contains an abundance of repeated DNA elements [55]
as well as many highly expanded gene families. In contrast, the
pufferfish genome is compact (about 350 Mb/15000 genes) and
the multigenic families are smaller than found in zebrafish, at least
those involved in the immune system. The pufferfish belongs to
*Tetraodontidae* in the vast group of percomorphs, and it is phy-
logenetically distinct from zebrafish, with an estimated 300 My of
divergence [22]. We therefore attempted a comparison of an
extensive versus a minimal repertoire of *trim* genes in teleosts to
better understand their evolutionary histories.

We retrieved a large number of *trim* genes in both species,
representing almost all the classes defined by Short and Cox [3] in
human. Our data indicate that the main TRIM classes were
already defined in the common ancestor of fishes and tetrapods.
However, a few genes show specific features that illustrate the
evolutionary pathways leading to the generation of new *trim*
classes. There is only one class III *trim* gene in humans (*trim42*) with
orthologues in amniotes but not in fish. However, if class III genes
are defined by domain organization alone (RBCC-FN3), fish do
possess a class III *trim* gene, which is one of the two co-orthologues
of *trim46*, a class I gene (RBCC-FN3-B30.2). One can then
hypothesize that the human *trim42* itself derives from a class I *trim*
gene through an ancient event involving the loss of the B30.2
domain. In the same line, the frequent loss of various C-terminal
domains led to the birth of new class V *trims* found in zebrafish
(e.g. *trim25like*, *trim54like*, several *ftrs*). This is also likely to be the
case for some human class V *trim* genes; a relatively recent origin
by such a mechanism would explain why few human members of
this class have fish counterparts.

At the N terminus, loss of the RING domain is also observed
in several instances (*trim24like*, *trim32*, *trim1* and *trim2like*). As
described above, this event results in truncated *trim*-like genes, but
could not result from the deletion of an entire exon, which suggests
that the loss of the RING was positively selected. In fact, such
events can be sometimes dated before the split between the
pufferfish and zebrafish lineages, or some are even much older
such as *trim16* that is retrieved in fish and tetrapods. The loss of B
Boxes and Coiled Coil is also sometimes observed – mainly in
Class IV genes – but does not seem to be fixed as easily.

In contrast, insertions of single- or multi-exon domains down-
stream of a RBCC module were found to generate new *trim*

<figure><img src="image_21.png" /></figure>
A

<figure><img src="image_22.png" /></figure>

B

<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /></figure>
**Figure 7. Group of conserved synteny around trim35 genes and gene clusters.** Synteny groups were determined from Ensembl assemblies using genomicus database and browser (http://www.dyogen.ens.fr/genomicus-59.01/cgi-bin/search.pl) [85]. The figure is edited from the PhyloView taking the human *trim35* (ENSG000000104228) as a reference (A), or taking the stickleback *trim35-01*, *trim35-02*, *trim35-04*, *trim35-10*, *trim35-14*, *trim35-24*, *trim35-26* and *trim35-27* genes as references (B). The reference gene and its orthologues is shown in light green over a thin vertical line and is indicated with its Ensembl ID.
doi:10.1371/journal.pone.0022022.g007

configuration in the zebrafish genome (i.e. *ftr06*, *ftr52*, *btr31*). When this occurred is unknown, but the insertion of the chromodomain in the *ftr06* gene appears to be recent, considering the dynamics of the *ftr* family and the absence of inactivating mutations in the B30.2 exon displaced by the “usurper” exon. Whether this change has a functional consequence for the encoded gene remains to be tested experimentally; a detailed phylogenetic reconstruction in close relatives of the zebrafish would therefore be informative. As chromodomains are involved in chromatin remodelling such a protein would be expected to regulate gene expression; similar functions have been described for TRIM proteins with a C-terminal bromodomain, structurally distinct from chromodomains but with comparable functions. In contrast, the replacement of the B30.2 exon of *ftr52* by exons encoding a

cyclophilin A (CypA) domain could have given rise to a TRIM protein with affinity to different viral proteins, because most remarkably, TRIM5-cypA proteins have also appeared at least twice independently (by retrotransposition of a cypA sequence in the *trim5* locus) in the primate lineage, leading to proteins with demonstrated anti-retroviral activity involving capsid binding by CypA [56,57,58,59,60,61]. In spite of this, no *trim* gene with a CypA domain has been reported in humans or in non primate species with a fully sequenced genome. The early stop codon found in the zebrafish *ftr52* gene leads us to speculate that although such a domain combination may provide a transient benefit against some viral infections, it may have some drawbacks that impairs its definitive fixation in a lineage. For *btr31*, the recombination events led to a unique configuration *RING-TM-B30.2*

<figure><img src="image_25.png" /></figure>

**Figure 8. Positive selection in the B30.2 domain of BTR and TRIM35/HLTR.** Distribution of hypervariable and positively selected residues in a multiple alignment of B30.2 domains from representative zebrafish BTR and TRIM35/HLTR, compared with a typical FTR sequence (Dareftr13: [GenBank: XM_695031]), and with TRIM5α (HosaTRIM5a). Hypervariable sites (shannon entropy >2) are indicated in red. Hypervariable sites previously described [21] are indicated in pink in the FTR13 sequence. The four hypervariable regions of the TRIM5α B30.2 domain are underlined. The variable loop-connecting strands of the domain are named VL1-VL6. β-strands of the B30.2 domain are indicated by dark (PRY region) or light (SPRY region) blue arrows from [36]. Segments 1, 2 and 3 determined by the recombination GARD analysis in the BTR multiple alignment are shown under the BTR01 sequence. Positively selected sites (among zebrafish TRIM35/HLTR & BTRs: this study; among FTRs: [21] and among primate TRIM5α: [40] are boxed in blue when detected under models 2a and 8. Sites positive under M8 but not under M2 are boxed in green. In TRIM35/HLTR, Q (β-strand 1) was detected under M2 not M8. In BTR, R (β-strand 1) has been detected in the BTR analysis only under M8 with complete domain, not in segment 1. In BTR, S (β-strand 12) has been detected only under M2a and M8 of BTR segment 3, not in the analysis using the complete domain. The detailed PAML results for each position under positive selection are available in Additional data file 8.
doi:10.1371/journal.pone.0022022.g008

<figure><img src="image_26.png" /></figure>
<table><caption>**Table 1.** PAML results.</caption><thead><tr><th>Region<sup>1</sup></th><th>n<sup>2</sup></th><th>c<sup>3</sup></th><th>Parameters in ω distribution under M2a<sup>4</sup></th><th>Parameters in ω distribution under M8<sup>5</sup></th></tr></thead><tbody><tr><td>TRIM35</td><td>38</td><td>122</td><td>ω<sub>&gt;1</sub> = 5.68162<br>p<sub>&gt;1</sub> = 0.09952</td><td>ω<sub>&gt;1</sub> = 4.13995<br>p<sub>1</sub> = 0.10057</td></tr><tr><td>complete B30.2</td><td></td><td></td><td>ω<sub>1</sub> = 1,000<br>p<sub>1</sub> = 0.43562</td><td>ω<sub>1</sub> = 0.89943<br>p<sub>0</sub> = 0.89943</td></tr><tr><td></td><td></td><td></td><td>ω<sub>&lt;1</sub> = 0.19870<br>p<sub>&lt;1</sub> = 0.46485</td><td>p = 1.13670<br>q = 1.81193</td></tr><tr><td>TRIM39</td><td>25</td><td>171</td><td>ω<sub>&gt;1</sub> = 3.33719<br>p<sub>&gt;1</sub> = 0.04501</td><td>ω<sub>&gt;1</sub> = 1.99982<br>p<sub>1</sub> = 0.08529</td></tr><tr><td>complete B30.2</td><td></td><td></td><td>ω<sub>1</sub> = 1,000<br>p<sub>1</sub> = 0.31535</td><td>ω<sub>1</sub> = 0.91471<br>p<sub>0</sub> = 0.91471</td></tr><tr><td></td><td></td><td></td><td>ω<sub>&lt;1</sub> = 0.19082<br>p<sub>&lt;1</sub> = 0.63964</td><td>p = 0.88108<br>q = 1.86498</td></tr><tr><td>TRIM39 B30.2</td><td>25</td><td>84</td><td>ω<sub>&gt;1</sub> = 3.96823<br>p<sub>&gt;1</sub> = 0.08146</td><td>ω<sub>&gt;1</sub> = 2.74731<br>p<sub>1</sub> = 0.08977</td></tr><tr><td>Fragment 1–252</td><td></td><td></td><td>ω<sub>1</sub> = 1.000<br>p<sub>1</sub> = 0.39488</td><td>ω<sub>1</sub> = 0.91023<br>p<sub>0</sub> = 0.91023</td></tr><tr><td></td><td></td><td></td><td>ω<sub>&lt;1</sub> = 0.14824<br>p<sub>&lt;1</sub> = 0.52366</td><td>p = 0.89035<br>q = 1.87745</td></tr><tr><td>TRIM39 B30.2</td><td>25</td><td>30</td><td>ω<sub>&gt;1</sub> = 1.000<br>p<sub>&gt;1</sub> = 0.13938</td><td>ω<sub>&gt;1</sub> = 1.00000<br>p<sub>1</sub> = 0.09928</td></tr><tr><td>fragment 291–393</td><td></td><td></td><td>ω<sub>1</sub> = 1.000<br>p<sub>1</sub> = 0.32559</td><td>ω<sub>1</sub> = 0.90072<br>p<sub>0</sub> = 0.90072</td></tr><tr><td></td><td></td><td></td><td>ω<sub>&lt;1</sub> = 0.19144<br>p<sub>&lt;1</sub> = 0.5350</td><td>p = 2.35230<br>q = 2.35230</td></tr><tr><td>TRIM39 B30.2</td><td>25</td><td>39</td><td>ω<sub>&gt;1</sub> = 2.21718<br>p<sub>&gt;1</sub> = 0.08741</td><td>ω<sub>&gt;1</sub> = 1.90649<br>p<sub>1</sub> = 0.12627</td></tr><tr><td>Fragment 430–557</td><td></td><td></td><td>ω<sub>1</sub> = 1.000<br>p<sub>1</sub> = 0.12322</td><td>ω<sub>1</sub> = 0.87373<br>p<sub>0</sub> = 0.87373</td></tr><tr><td></td><td></td><td></td><td>ω<sub>&lt;1</sub> = 0.19452<br>p<sub>&lt;1</sub> = 0.78936</td><td>p = 4.37468<br>q = 4.37468</td></tr></tbody></table>

where a membrane separates a RING and a B30.2 domain, which has completely unknown functional consequences.

Domain organization, sequence similarity and phylogenetic analyses indicate that one or two orthologues of multiple human *trim* genes that belong to classes I, II, V, VI, VII, VIII, IX i.e. to the “group 1” defined by Sardiello *et al.* [6] are present in both zebrafish and pufferfish. Often, when two co-orthologues are found for such genes, one of these has lost the C-terminal domain, while the other has retained the complete domain organisation and thus probably constitutes a true functional counterpart. This notion is also supported by similar expression patterns that were observed for genes selected in this category in vertebrates. The correspondence between the repertoires of “group 1” *trim* genes in zebrafish, pufferfish, and mammals indicates that strong purifying selection pressures were exerted to keep one (or few) copy(ies) of these genes in vertebrate genomes, illustrating their key functions in the basic biology of the cell. This is in sharp contrast with the evolutionary pathway of the Class IV RBCC-B30.2 *trim* genes.

The RBCC-B30.2 *trim* genes from Class IV represent unique sets in the different species of mammals and other tetrapods examined in detail by Sardiello and colleagues [6]. Our survey of zebrafish and pufferfish *trim* genes generally confirms and extends this conclusion. Most human ClassIV *trim* genes have no counterpart in the zebrafish or the pufferfish, and fish possess many ClassIV *trim* genes that do not exist in human nor in the mouse. Another feature of Class IV *trim* genes that was well exemplified in our previous report on *fintrim* [21] is their propensity to expand into multigenic subsets. In this study we demonstrated that fish possess in fact three multigenic subsets of *trim* genes all belonging to the Class IV: *ftr* (i. e. *fintrim*), *btr* (i.e. *bloodthirsty-related trim/trim39*) and *trim35/hltr*. The number of *ftr*, *btr* and *trim35/hltr* is different between fish species belonging to distant families, indicating different degrees of expansion. This is particularly striking from the comparison of zebrafish with expanded subsets and pufferfish with a “minimal” repertoire. This contrast likely reflects the high level of genomic rearrangement of the zebrafish genome – as

<table><caption>**Table 2.** Class IV trims genes are concentrated in the MHC and MHC paralogues.</caption><thead><tr><th></th><th>MHC neighbourhood</th><th>Rest of the genome</th><th>Total</th><th>Chi square test</th></tr></thead><tbody><tr><td>Length (megabase)</td><td>376<sup>1</sup></td><td>1187</td><td>1563</td><td></td></tr><tr><td>Number of genes (total)</td><td>7884</td><td>16263</td><td>24147</td><td></td></tr><tr><td>Number of Class IV trim<sup>2</sup></td><td>31</td><td>31</td><td>62</td><td>p = 0.0035</td></tr><tr><td>Number of other trim</td><td>10</td><td>23</td><td>33</td><td>NS</td></tr></tbody></table>

<figure><img src="image_27.png" /></figure>

<footnote n="1">¹for sequence fragments, the numbers correspond with the position of first and last nucleotides in the alignment with excluded gaps.</footnote>

<footnote n="2">²n, the number of sequences in the alignment and tree.</footnote>

<footnote n="3">³c, the number of codons.</footnote>

<footnote n="4">⁴parameters determined under M2a with ω the ratio of non-synonymous rates (dN) and synonymous rates (dS) and p the corresponding proportion of sites for each ω-class.</footnote>

<footnote n="5">⁵parameters determined under M8 with ω the ratio dN/dS, the corresponding proportion (p₁ = 1 – p₀) of sites and p- and q-estimates in the β(p,q)-distribution.
doi:10.1371/journal.pone.0022022.t001</footnote>

<footnote n="1">¹The results are based on the genome assemblies available at http://www.ensembl.org (release 58). The detailed calculations and a map with MHC and MHC paralogues considered in the analysis are available in SupplMat 8.</footnote>

<footnote n="2">²To avoid skewing the analysis by the numerous *trim* recently duplicated, we counted each clusters of *trim* genes as only one event From the Zv8 assembly of the zebrafish genome.</footnote>

<footnote>doi:10.1371/journal.pone.0022022.t002</footnote>
indicated by short conserved synteny blocks compared to other
fish versus mammals – and the strong compaction of the
tetraodon/fugu genome [26,62]. However, functional data would
provide a better understanding of these sharp differences of class
IV among teleosts. Besides, this underlines the strong constraints
that maintained the conservation of *trim* belonging to the other
trim classes (the “group 1” defined by Sardiello *et al.* ) in different
lineages. Some members of *ftr*, *btr* and *trim35/hltr* are part of
conserved synteny groups conserved among teleosts, showing that
their initial emergence and subsequent diversification is ancient in
the evolution of teleosts and predates the differentiation of the
main fish lineages. Consistently, these genes involved in syntenies
appear to be the most ancient genes in their subset. They branch
close to the basis of their multigenic subset in phylogenetic trees
and generally do not belong to large genomic clusters. This is the
case for *ftr* 82/83, *btr*1 & -33, *trim35-12* & 28. In contrast, the
genes composing large genomic clusters such as zebrafish *ftr* on
chromosome 2, are not included in conserved synteny groups and
probably represent more recent, lineage-specific diversification
events. The mechanisms for the amplification of *trim* are likely
different for *ftr*, *btr* and *trim35/hltr* within a species: for example in
medaka the *trim35/hltr* expansion occurred by duplication, while
*ftr* expansion involved retrotransposition. Interestingly, only three
sets of class IV genes are retrieved as multigenic groups in any fish
species for which a complete genome assembly is available. The
diversification of *ftr*, *btr* and *trim35/hltr* therefore appears to be
rooted in ancient duplication events, followed by parallel diver-
sification processes, which reflects similar functional constraints in
different fish lineages. Multiplication of some class IV *trim* genes
has also occurred in mammals, albeit to a smaller scale; thus,
human *trim5*, *trim6*, *trim22* and *trim34* likely result from such a
duplication event, while in cow, the *trim5* gene has further
expanded into eight tandem copies, five of which encode a
functional protein [40].

To date, functions of the multiple Class IV fish trims are still
largely unknown and do not provide an obvious explanation for
their extensive expansion. A non-redundant role in erythropoiesis
has been reported for Bloodthirsty (bty) [35] which is quite difficult to
understand in the context of the large multigene btr family - it is
also noteworthy that this role is deduced from morpholino-based
transient inactivation in embryos and the original bty gene is not
found in the current zebrafish assembly zv9 (www.ensembl.org;
Tübingen background). The closest zv9 gene is btr18, and it
remains to be established if bty is unique to the original genetic
background used by Yergeau et al. or is an allelic variant of a zv9
gene. At least some finTRIMS are induced by IFN and virus
infection in rainbow trout [21,63], and a btr is upregulated by poly
I:C in Atlantic cod [64]. In fact, these trim genes were not only
duplicated many times, but also diversified after gene expan-
sion with an accumulation of non synonymous changes. Thus,
apparent signatures of diversifying selection were found in the β2–
β3 loop in the B30.2 domain of btr, trim35/hltr (this study, Table 1)
as previously reported for ftr [21] in zebrafish. Interestingly, the
B30.2 domain – especially the β2-β3 loop - was subjected to a
strong diversification in primates and accounts for the species-
dependent retrovirus restriction of TRIM5α in the different
species [18,19,65]. Moreover, several copies of trim5 can be found
in the genome of certain species such as cow [40]. Considering the
importance of trim genes for antiviral immunity [66,67] and the
role of the B30.2 domain, we believe that virus sensing/restriction
may be the driving force in the diversification of the fish trim
multigene subsets under positive selection. However, the approach
we followed to find sites under positive selection may lead to false
positive, and accumulation of non synonymous changes does not

necessarily imply functional changes [68]. Experimental evidence – for example of multiple B30.2/virus binding - would be required for a definitive proof of the functional impact of B30.2 diversification.

Our simple analysis of localization of *trim* genes relatively to genes of the MHC and MHC paralogues would have to be complemented by a detailed phylogenomic analysis of these regions through the whole vertebrate evolution from lamprey and sharks to fish and mammals. This will become possible with the publication of good quality genomes. However, the co-location pattern that we report suggests that *trim* and B30.2 are associated with the MHC and MHC paralogues in fish as well as already reported in mammals and birds [43,44,45]. Could it be for the benefit of immunity? An interesting question then would be to determine if the B30.2 domain was first associated to the ancestral MHC as a part of a pre-existing Class IV TRIM molecule. The existence of *trim*-like genes with canonical B30.2 domains in *Branchiostoma* (Cephalochordates), *Drosophila* (Arthropods), *C. elegans* (Nematods) *Nematostella* (Cnidarians) and *Trichoplax* (Placozoa) ([20] and unpublished observations) indicates that genes resembling Class IV *trims* are probably very ancient and could have been inherited from a common ancestor to vertebrates and these different groups of invertebrates. Considering the role of the B30.2 domains in mammals, we propose that ancestral Class IV TRIMS participated in defence and were part of a gene complex, the proto MHC, equipped in genes selected for processing (and later presenting) viral peptides. Indeed, intense duplication is typical of genes families involved in immunity. Such genes belonging to different families have diverged rapidly and independently within different classes of organisms in function of the pressures exerted by the pathogenic environment [69,70,71,72,73,74]. Besides, *trim* genes may have been kept in a genomic cluster with proteasome components because they were involved in targeting virus particles to the ubiquitin-dependent proteasome system in a manner ana-logous to LMP/TAP genes that form a tight cosegregating unit in practically all vertebrates. This simple antiviral axis might have been very ancient and could have participated in the establishment of a proto-MHC selected for proteasome-mediated destruction of virus proteins and therefore production of peptides to which the antigen presenting machinery would be added later in evolution.

In support of this hypothesis one can remark that several class
IV TRIM such as *trim11* and *trim17*, are located in human MHC
paralogous regions and participate in the ubiquitin-proteasome
system [75,76]. The binding of TRIM5α to the retroviral capsid
induces a rapid degradation of TRIM5α by the proteasome,
providing an additional link between TRIM-dependent virus
restriction and proteasome activity [77]. Another class IV TRIM
involved in the ubiquitin-proteasome axis is TRIM21 that binds Ig
constant region with very high affinity and targets viral particles
coated with antibodies to the proteasome [78].

Given the abundance of viruses in the aquatic environment where early metazoa developed, the necessity for diverse protective measures against viruses certainly played a major role in shaping the immune system. The recruitment and diversification of IgSF TCR-like antigen receptors from proteins used by viruses to enter cells would be a good example of the consequences of such measures [79]. Similarly, the *trim* connection with the MHC could be a remnant of the early steps towards the construction of an adaptive immune system with associative recognition (TCR, MHC-peptide) by recruitment of antiviral primary defence systems. In the genomes of modern species, the *trim* family provides a good model to study the evolution of multigene families and functional diversification. The identification of the ligands and

<figure><img src="image_28.png" /></figure>
functions of such diversified subsets should provide new insights on the molecular pathways developed in the main vertebrate lineages.

Our survey of fish *trim* genes in two fish species identifies subsets with very different evolutionary dynamics. Thus, *trims* encoding RBCC-B30.2 proteins show the same evolutionary trends in fish and tetrapods: they are fast evolving, often under apparent positive selection, and they duplicate to create multigenic families that can be very large such as zebrafish *ftrs*. Among these multigenic subsets, we could identify several new combinations of domains, which epitomize how new *trim* classes appear by domain insertion or exon shuffling. Finally, *trim* encoding RBCC-B30.2 proteins are preferentially located in the MHC and in MHC paralogues, which suggests that such *trim* genes with a B30.2 exon may have been part of the ancestral MHC.

## Materials and Methods

### Identification of a complete array of genes from the *trim* family in zebrafish

Zebrafish *trim* genes – defined as encoding proteins with a RING-B-Box-Coiled Coil (RBCC) motif – were searched in the Zv8 genome assembly available at http://www.ensembl.org/. The survey was later updated from the current assembly (Zv9, made available at the end of 2010). The new assembly Zv9 did no show any major change in number, structure or location of *trim* genes. Both lists are given in Supplemental Figure S1 for an easier comparison with previous reports.

Several strategies were followed in parallel to try to get a complete list of zebrafish *trim*. First, all zebrafish ensembl proteins with a motif RING (ipr IPR001841) or B box (ipr IPR000315) were extracted using the biomart tool, and the intersection of the two lists kept as a first set of trim sequences (Set#1). The ensembl Ids, annotation, locations and status were also extracted. Second, the protein sequences belonging to the TRIM Ensembl families detected in zebrafish (ENSFM00300000079125, ENSFM0040000131833, ENSFM00250000004079, ENSFM00250000005797, ENSFM00390000126422, ENSFM00500000272256, ENSFM00500000271543, ENSFM00500000272036, ENSFM00390000126385, ENSFM00250000006428, ENSFM00250000001082, ENSFM00500000270185, ENSFM00250000001642, ENSFM0040000131788, ENSFM00250000004429, ENSFM00250000008223, ENSFM00500000287404): were collected and combined with the set #1 (set#2). Third, the zebrafish ensembl orthologs of all human trim gene were collected; the human orthologue of each gene was then checked, and this information was used to annotate the genes previously identified.

To compare the sequences to our previous work on two multigenic *trim* subsets performed on the zebrafish Zv7 assembly – the *funtrims* and the bloodthirsty-related (*btr*) *trims* – we used the TBLASTN program at http://www.ensembl.org/ to align the FTR and BTR protein sequences with the current genome assembly. We also compared the sequences of *ftr* and *btr* genes extracted from Zv7 to the current assembly. Using both alignment scores and hit location, the *ftr* and *btr* sequences were identified in the set#2. For the new *ftr* and *btr* present in Zv8 as well as for the *Trim35* multigenic family, sequences were manually edited from gene models available in both Ensembl and Genbank. When the *ftr* or *btr* genes were fully retrieved in Zv8, we kept our previous manual annotation rather than the Ensembl automatic assignment. For the other *trim*, the protein models from Zv8 and Zv9 were considered, and the most recent annotation available. Finally, the protein sequences corresponding to this *trim* list was subjected to a domain analysis using Interproscan. The sequences unassigned yet were then manually annotated one by one. Starting

from each zebrafish *trim*, we searched for the possible orthologues and paralogues in pufferfish. The orthologues of each human and zebrafish *trim* were searched in the Ensembl database. All proteins including a RING and a B30.2 domains were also extracted, which confirmed that the previous list was comprehensive.

### Cloning of *ftr52* sequences

Transcript sequences were amplified from cDNA of pooled 5dpf AB larvae with AccuStar DNA polymerase (Eurogentec) using primers ATGAATTCGTGTAAATACAGCGAAATGGCA and ATGCGGCCGCACCTAGGCTCACAGCTG. A band of ~2 kb was gel-purified, digested with EcoRI and NotI, and cloned in the pBK-CMV plasmid. The genomic region encompassing the RING-encoding domain was PCR-amplified with primers TA-CAGTGGCTCGTCAAGTGA and TGCACTCTTCATCCG TGTGA.

### Detection of positive selection in B30.2 domain

The dataset for positive selection analysis was prepared from *btr* and *trim35/hlr* sequences that were found on the Ensembl zebrafish assembly. Domains were identified by the web-based tool Simple Modular Architecture Research Tool (SMART) at http://smart.embl-heidelberg.de/. A multiple sequence alignment was made for each domain with ClustalW within the *MEGA4* software and gaps were removed from the alignment.

The Codeml program of the Phylogeny Analysis by Maximum Likelihood (PAML) package [80], retrieved from http://abacus.gene.ucl.ac.uk/software/paml.html, was used for the detection of positive selection. The models M0, M1a, M2a, M7 and M8 were employed. The ratio of synonymous (*dS*) to non-synonymous (*dN*) substitution rates, $\omega = dS/dN$, is determined by the program. We used the site-specific model that allows $\omega$ to vary among sites. The null models M0, M1a and M7 do not allow the existence of positively selected sites ($\omega>1$), while the alternate models M2a and M8 allow $\omega>1$. M8 follows a beta(*p*,*q*)-distribution and is less stringent than M2a. Within the models, a Maximum Likelihood algorithm is used, whereby the sites are allocated under classes of different $\omega$ probabilities. Sites allocated under the class with $\omega>1$ are considered as being under positive selection and were identified by a Bayes Empirical Bayes (BEB) analysis. Significance of outcome was confirmed by a likelihood ratio test (LRT). In the LRT we took twice the difference in log likelihood ($2\Delta\ln L$) between the nested models and used the chi-square test with the degrees of freedom (df) being the difference in free parameters between the two models (M1a vs. M2a and M7 vs. M8). Tests were considered positive when $p<0.001$. Sites identified by BEB with a posterior probability higher than 95 percent were considered significant.

### Analysis for recombination

To test for interference of recombination on the PAML results, we implemented a test by the algorithm PARRIS [81]. Under PARRIS, the PAML models M1a-M2a are employed with incorporation of site-to-site variation in synonymous substitutions rates and partitioning of data. We used the codon model for evolution GY94×HKY85 and a discrete distribution of three bins for synonymous and for non-synonymous rates. Significance of results was tested by a LRT.

We detected recombination breakpoints by the algorithm GARD [82]. We used the HKY85 model with general discrete distribution of rates across sites. We performed two screenings, for 2 or 20 breakpoints. The detection was validated by corrected Akaike's information criterium (c-AIC) for best-fitted model

<figure><img src="image_29.png" /></figure>
selection. Both PARRIS and GARD are integrated in the HyPhy
software package that was retrieved from http://www.hyphy.org.

Fish, RNA isolation and real time quantitative PCR

RNA was extracted from either single fish or pooled organs
from five to ten two-year old zebrafish of AB background. All the
animal experiments described in the present study were conducted
at the Institut Pasteur according to the European Union guidelines
for the handling of laboratory animals (http://ec.europa.eu/
environment/chemicals/lab_animals/home_en.htm) and were
approved by the Institut Pasteur animal care and use committee
and by Direction Sanitaire et Vétérinaire de Paris under permit
#A-75-12-22. Dissected organs, or entire fish cut in 3 mm pieces,
were stored for a few days in RNALater (Ambion) before RNA
extraction using TriZol (Invitrogen). DNA contaminations were
removed by DNase I treatment followed by phenol-chloroform
extraction; integrity of the resulting RNA was checked on an 2100
bioanalysis station with a RNA nano chip (Agilent). cDNA was
generated using M-MLV H- reverse-transcriptase (Promega) with
a dT₁₇ primer. Quantitative PCR was then performed on an
ABI7300 thermocycler (Applied Biosystems) using SYBR green
reaction power mix (Applied Biosystems). The following pairs of
primers were used:

EF1α: GCTGATCGTTGGAGTCAACA and ACAGACTT-GACCTCAGTGGT

trim1: CAAAACCAACAGTCAGCCTTT and AAGAGCGTACCATGTAGAGG

trim13: CAGGTAGACAAACTTTGCGC and CAGTCCGACGGAAGAAAGTT

trim25: GAGCGGCGCTTCAAACAAAA and ATCAATTG
CCAGCATGGCCT

trim33: GTTCCTACCTCGGTTCCTAA and GAATCGGC
CTGGACATTACT

trim54: GGAGCATCAAGGACAATGGT and CTTCGTG
CTCTGCAGGAATA

trim59: CTGGTGCAGAAAGATCGAGA and CTCGTAG
GCCTGATTGAGAA

Quantifications were performed on triplicate wells, and taking
into account the previously measured yield of the reaction as
described in [83]. To normalize cDNA amounts, we have used the
housekeeping gene *EF1α*, chosen for its high and stable level of
expression over development and among tissues [84]. After
calculations of *trim/Ef1α* transcript expression ratios, data have
been normalized to the average expression in entire fish (using the
geometric mean of the results obtained on the whole male and the
whole female), to highlight which organs express higher or lower
levels of a given gene compared to the rest of the body. Results are
reported as mean ± standard deviation of the measured ratios.

Supporting Information

**Figure S1** A table listing the *trim* genes of zebrafish, with domain structure, annotations and genomic locations in Zv8 and Zv9 assemblies.
(XLS)

References

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**Figure S2** A table listing the *trim* genes of pufferfish, with annotations and genomic locations.
(XLS)

**Figure S3** A table showing the correspondence between *trim* genes in zebrafish and pufferfish, allowing an easy comparison of their domain structures.
(XLS)

**Figure S4** A phylogenetic tree showing the evolutionary branching of the genes selected for the qRTPCR expression analysis.
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**Figure S5** Figure S5 shows the profiles of Shannon entropy calculated site by site from zebrafish *trim35/hltr* and *btr* alignments to determine the hypervariable regions, and the multiple sequence alignments used for positive selection calculations.
(DOC)

**Figure S6** A table showing the results if of the likelihood ratio test (LRT) of positive selection of zebrafish *trim35/hltr* and *btr* B30.2 domains.
(DOC)

**Figure S7** A table showing the results of the GARD program for recombination between zebrafish *trim35* and *btr* B30.2 domains.
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**Figure S8** A table that summarizes the positive selection analysis for the different regions of zebrafish BTR B30.2 domains.
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**Figure S9** A table listing *trim* genes and the zebrafish homologues of a set of classical MHC markers and their paralogues, with their genomic location.
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Acknowledgments

We are grateful to C. Caron from the CNRS/UPMC ABIMS bioinformatics platform (http://abims.sb-roscoff.fr) for providing help and support for the recombination site computing. We thank John D Hansen for discussions and proofreading of the manuscript, Olivier Chabrol, Anthony Levasseur and Manuela Royer-Carenzi for support and helpful discussions about gene distributions and positive selection analysis, and Philippe Herbomel for discussions and support.

Author Contributions

Conceived and designed the experiments: PB LMvdA LJ LDP AB J-PL.
Performed the experiments: PB LMvdA LJ LDP VB J-PL. Analyzed the
data: PB LMvdA LJ LDP PP J-PL. Contributed reagents/materials/
analysis tools: PB LDP LMvdA LJ PP AB J-PL. Wrote the paper: PB
LMvdA LDP PP J-PL.

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