Evolutionary mode and functional divergence of vertebrate NMDA receptor subunit 2 genes

Abstract

Background: Ionotropic glutamate receptors in the central nervous system play a major role in numerous brain functions including learning and memory in many vertebrate species. NR2 subunits have been regarded as rate-limiting molecules in controlling the optimal N-methyl-D-aspartate (NMDA) receptor's coincidence-detection property and subsequent learning and memory function across multi-species. However, its evolutionary mode among vertebrate species remains unclear.

Results: With extensive analysis of phylogeny, exon structure, protein domain, paralogon and synteny, we demonstrated that two-round genome duplication generated quartet GRIN2 genes and the third-round fish-specific genome duplication generated extra copies of fish GRIN2 genes. In addition, in-depth investigation has enabled the identification of three novel genes, GRIN2C_Gg, GRIN2D-1_Ol and GRIN2D-2_Tr in the chicken, medaka and fugu genome, respectively. Furthermore, we showed functional divergence of NR2 genes mostly occurred at the first-round duplication, amino acid residues located at the N-terminal Lig_chan domain were responsible for type I functional divergence between these GRIN2 subfamilies and purifying selection has been the prominent natural pressure operating on these diversified GRIN2 genes.

Conclusion and significance: These findings provide intriguing subjects for testing the 2R and 3R hypothesis and we expect it could provide new insights into the underlying evolution mechanisms of cognition in vertebrate.

Figure 1. The phylogenetic tree (A) of NR2 proteins and their protein domain structure (B).

The bootstrap consensus phylogenetic tree was constructed with the fruitfly Nmdar2 and nematode nmr-2 protein as outgroup using the neighbor-joining method in MEGA4, and the numbers indicate the percentage bootstrap support. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. Red circles indicate the gene duplication event supported by the phylogenetic analysis. Dm, Drosophila melanogaster; Ce, Caenorhabditis elegans; Dr, Danio rerio; Tr, Takifugu rubripes; Tn, Tetraodon nigroviridis; Ol, Oryzias latipes; Ac, Anolis carolinensis; Gg, Gallus gallus; Tg, Taeniopygia guttata; Mm, Mus musculus; Hs, Homo sapiens. Protein domains are shown as boxes based on identification by Pfam.

Figure 2. Exon structures (5′→3′) of vertebrate GRIN2A (A), GRIN2B (B), GRIN2C (C) and GRIN2D (D) genes and two invertebrate NR2 orthologues (E).

The exons that transverse or flank the splice sites are indicated above each exon boundary. Numbers in boxes are nucleotide numbers. Exons coloured green are conserved in length among all NR2 subunit genes from all species. Exons coloured red, blue, pink or brown are conserved in length between each of GRIN2A, GRIN2B, GRIN2C, GRIN2D orthologues from all species. The size of each exon is not drawn to scale.

Figure 3. Paralogons surrounding the human (A), mouse (B), zebra finch (C) GRIN2A, GRIN2B, GRIN2C and GRIN2D genes.

Within each connected block on each chromosome, genes are shown in actual order, although not to scale. Gene families are indicated by lines connecting the chromosomes. Paralogous genes flanking each GRIN2 genes share high degree of conservation among human, mouse and zebra finch.

Figure 4. Comparison of genes surrounding GRIN2A (A), GRIN2B (B), GRIN2C (C) and GRIN2D (D) in chromosomes of different species.

Orthologous genes flanking each GRIN2 genes define a synteny with high degree of conservation among human, mouse, zebra finch and chicken. (A) The tetrapod GRIN2A paralogon has two fish paralogons, the GRIN2A-1 paralogon and the GRIN2A-2 paralogon. AXIN1 on human chromosome 16, zebra finch chromosome 14 and chicken chromosome 14 near GRIN2A have two co-orthologs located in medaka chromosomes 1 and 8. The tetrapod GRIN2B paralogon has two fish paralogons, the GRIN2B-1 paralogon and the GRIN2B-2 paralogon (B). PDE6H on human chromosome 12, mouse chromosome 6, zebra finch chromosome 1A and chicken chromosome 1 near GRIN2B have two co-orthologs located in medaka chromosomes 1 and 8. (C) The tetrapod GRIN2C paralogon has two fish paralogons, the GRIN2C-1 paralogon and the GRIN2C-2 paralogon. Each of the SOX9, AATK and CYTH1 on human chromosome 17 near GRIN2C has two co-orthologs in zebrafish chromosomes 3 and 12 and, also, two co-orthologs in tetraodon chromosomes 2 and 3. Each GRIN2D paralogons (D) define a synteny with high degree of conservation among the studied tetrapod and fish species. The positions of genes on chromosomes are not drawn to scale.

Figure 5. Evolutionary mode and functional divergence of the vertebrate GRIN2s genes.