Identification of a genomic reservoir for new TRIM genes in primate genomes

Abstract

Tripartite Motif (TRIM) ubiquitin ligases act in the innate immune response against viruses. One of the best characterized members of this family, TRIM5α, serves as a potent retroviral restriction factor with activity against HIV. Here, we characterize what are likely to be the youngest TRIM genes in the human genome. For instance, we have identified 11 TRIM genes that are specific to humans and African apes (chimpanzees, bonobos, and gorillas) and another 7 that are human-specific. Many of these young genes have never been described, and their identification brings the total number of known human TRIM genes to approximately 100. These genes were acquired through segmental duplications, most of which originated from a single locus on chromosome 11. Another polymorphic duplication of this locus has resulted in these genes being copy number variable within the human population, with a Han Chinese woman identified as having 12 additional copies of these TRIM genes compared to other individuals screened in this study. Recently, this locus was annotated as one of 34 "hotspot" regions that are also copy number variable in the genomes of chimpanzees and rhesus macaques. Most of the young TRIM genes originating from this locus are expressed, spliced, and contain signatures of positive natural selection in regions known to determine virus recognition in TRIM5α. However, we find that they do not restrict the same retroviruses as TRIM5α, consistent with the high degree of divergence observed in the regions that control target specificity. We propose that this recombinationally volatile locus serves as a reservoir from which new TRIM genes arise through segmental duplication, allowing primates to continually acquire new antiviral genes that can be selected to target new and evolving pathogens.

Figure 1. TRIM73 and TRIM74 arose in our recent primate ancestors.

(A) A generic schematic of a TRIM protein is shown. There may be one or two B-boxes (yellow), and the final domain is variable although commonly a B30.2 domain. (B) An illustration summarizing the results of a previous analysis of human (h) and mouse (m) TRIM genes . Most TRIM genes have strict 1∶1 orthologs between the two species, as illustrated by the pairs in gray boxes. Two clades of human-expanded TRIM genes were also noted (orange boxes). (C) The relationships of the species discussed in this study are shown, along with approximate dates of divergence , . (D) A cladogram illustrates the relationship of TRIM72/TRIM50/TRIM73/TRIM74 homologs present in the genomes of human, chimpanzee, orangutan, rhesus macaque, and mouse. The domain structure of the proteins encoded by these genes is indicated (R-B-CC is the tripartite motif discussed in the text). The asterisk (*) denotes orangutan TRIM73, which is an un-annotated gene located on an unassembled contig in the ponAbe2 genome assembly (7_random; positions 15,637,224–15,648,145). The tree was made from approximately 7,000 aligned bases in the R-B-CC region of these genes (introns and exons). Bootstrap values are shown for both neighbor joining and maximum likelihood methods (NJ/ML). A maximum parsimony tree was also constructed (not shown), and all nodes are supported by 88% or greater of bootstrap replicates regardless of the method used.

Figure 2. A dynamic clade of TRIM genes in the human genome.

(A) 31 human paralogs were identified that group into a single phylogenetic clade. Seven of them have already been annotated with standard TRIM genes names (TRIM48, TRIM51, TRIM77, TRIM49, TRIM53, TRIM64, and TRIM43), including the four genes that were originally being investigated here (bold type). The rest are predicted genes that have been given temporary names reflecting their phylogenetic subclades (i.e. “A1”). Subclades of genes are color-coded for naming purposes. Pink boxes indicate TRIM genes located on chromosome 2, all other genes are on chromosome 11. The neighbor joining tree was based on an alignment of the predicted coding regions. Bootstrap values are shown for both neighbor joining and maximum likelihood methods (NJ/ML). Nodes are collapsed where support by both methods is <75%. The two methods yield different branching orders in only one case, in the B subclade at the node indicated (∧). (B) The genomic positions of these 31 TRIM genes are illustrated, according to the hg19 human genome assembly. Pentagons represent TRIM genes, with strand orientation designated by the direction of the symbol. The color of the gene symbol reflects the phylogenetic subclade to which the gene belongs (according to the tree in panel A). Green and yellow bars indicate two apparent inverted segmental duplication events. The segmental duplication at the chromosome 11 centromere actually spans the centromere , and is therefore likely to be substantially longer than the 310 kilobases indicated.

Figure 3. Primate comparative genomics reveals 11 African ape-specific and 6 human-specific TRIM genes.

The chromosome 11 TRIM genes are diagrammed as they occur in the latest versions of four available primate genome projects. The dashed-empty pentagons represent genes that are presumed to be present, but could not be identified due to large regions of poor sequence quality in several of the genome projects. The dashed lines spanning the centromere in the orangutan and rhesus macaque genomes denote sequence that is not syntenic between genomes, due to a large rearrangement that has been described . On the right-hand end of the diagrams, a 1 megabase block of synteny (grey bar) was identified that sits adjacent to segment 1 or segment 3 in all four genomes.

Figure 4. TRIM genes in segments 1 and 3 are copy number variable.

(A) The schematic illustrates the Multiplex Ligation-dependent Probe Amplification (MLPA) assay. This assay utilizes pairs of probes designed to sit directly adjacent to one another at a particular genomic region. When the two probes anneal to the correct target region on denatured genomic DNA, the addition of a ligase results in their joining into a single, larger probe. Universal primer sites (black bars) at each end allow amplification of fragments from ligated pairs. Each probe pair yields a PCR product of unique length due to a “stuffer sequence” (orange) that is placed internally to one of the universal primer binding sites. The universal PCR primers are labeled with a fluorescent dye, and the quantity of each uniquely-sized fragment produced is measured with a fragment analyzer. (B) In this panel, ligated probe pairs are now illustrated with a single green bar. Eighteen probe pairs were designed to span segment 1, and are also a perfect match to their target sequence in segment 3. Thus, each is expected to anneal four times in a diploid genome. The one exception is the probe “M-uniq” which sits in a unique region between the segmental duplications. (C) The results of the MLPA assay are shown for 12 geographically diverse human samples. For each individual, the fragment intensity produced from each probe pair was normalized to the intensity produced by the Utah reference sample (NA10851). Control probes recognize single copy genes at the chromosomal locations indicated along the X-axis. For this reason, they yield the same quantity of fragments in experimental and reference genomes (all values hover between 0.65 and 1.35, the cut-offs for deletion and duplication). This is also true for the experimental probes. The one exception is in the genome of a Han Chinese sample (NA18573), where 16 adjacent experimental probe pairs (GAP1-1 through M-uniq) all yielded a quantity of fragments averaging 1.6× greater than the reference Utah genome. In all, 72 geographically-diverse human samples were assayed by MLPA, and a table of full results can be found in Table S5. (D) The MLPA results are consistent with a heterozygous duplication of most of the segment 1 – segment 3 locus.

Figure 5. Positive selection has shaped the sequence of the novel TRIM genes on chromosomes 2 and 11.

(A) A schematic where tick marks represent the 10 residue positions found to be evolving under positive selection in the novel TRIMs. Also indicated is a phylogenetic breakpoint which was detected in the sequence alignment, located between the RING and B-box 2 domains (after base position 237 in the coding sequence). (B) The crystal structure (PDB 3KB5) of a TRIM B30.2 domain is shown , with the β2-β3 loop highlighted in red. (C) The phylogenetic trees of the two halves of the alignment differ only in the placement of two branches (in red). Bootstrap support for each node is shown by both neighbor joining and maximum likelihood (NJ/ML) methods. On each branch, the estimated value of dN/dS is given, followed by the number of non-synonymous and synonymous mutations predicted to have occurred along that branch (N∶S). dN/dS is indicated as infinity where dS = 0. Text is highlighted in red where dN/dS >1 (or, arbitrarily, where N∶S≥3∶0 in cases where dS is zero). Genes A1 and A2, and genes C1 and C2, are identical along the length of their coding sequence. Stars on the right indicate genes that were functionally tested in retroviral restriction assays. Finally, each of the two trees has one poorly supported node (in green).