The evolutionary fate of alternatively spliced homologous exons after gene duplication

Abstract

Alternative splicing and gene duplication are the two main processes responsible for expanding protein functional diversity. Although gene duplication can generate new genes and alternative splicing can introduce variation through alternative gene products, the interplay between the two processes is complex and poorly understood. Here, we have carried out a study of the evolution of alternatively spliced exons after gene duplication to better understand the interaction between the two processes. We created a manually curated set of 97 human genes with mutually exclusively spliced homologous exons and analyzed the evolution of these exons across five distantly related vertebrates (lamprey, spotted gar, zebrafish, fugu, and coelacanth). Most of these exons had an ancient origin (more than 400 Ma). We found examples supporting two extreme evolutionary models for the behaviour of homologous axons after gene duplication. We observed 11 events in which gene duplication was accompanied by splice isoform separation, that is, each paralog specifically conserved just one distinct ancestral homologous exon. At other extreme, we identified genes in which the homologous exons were always conserved within paralogs, suggesting that the alternative splicing event cannot easily be separated from the function in these genes. That many homologous exons fall in between these two extremes highlights the diversity of biological systems and suggests that the subtle balance between alternative splicing and gene duplication is adjusted to the specific cellular context of each gene.

Keywords: alternative splicing; gene duplication; homologous exons; protein diversity; subfunctionalization.

Fig. 1.—

(A) The date of origin and loss of the 97 human MEHE AS patterns shown against the phylogeny of human and five distant vertebrate species. Gain of AS event is shown in green, and the inferred number of AS losses in red. (B) The percentage of conservation of the 97 human AS events in each species.

Fig. 2.—

Splice isoform separation of CALU in teleosts by differential retention of ancestral MEHEs (A) that code for the first EF-hand domain (B) is strongly supported by the position in the ML exon tree of two distinct teleost genes, CALUA and CALUB, each within the group of monophyly defined by each ancestral MEHE (C; with the best-fit evolutionary model LG+I+G). Numbers close to nodes indicate cases with more than 70% of bootstrap support based on 1,000 replicates. The multiple sequence alignment reveals some positions (blue arrows) with specific conservation patterns between MEHEs of human, spotted gar and coelacanth, and between duplicated genes in zebrafish and other teleosts (D).

Fig. 3.—

The ML phylogenetic tree of MARVELD3 exons (LG+I+G+F evolutionary model), which shows the evolutionary relationship between equivalent homologous exons in different species. The exons exist either in the form of alternatively spliced exons or as constitutively spliced exons in separate genes. The numbers at each internal node indicate bootstrap support.

Fig. 4.—

The 3D-structure of human MAPK8 (pdb code 3O17) is shown in (A) emphasizing the region corresponding to the MEHEs (blue), which of the residues coded by the MEHEs differ between alternative MAPK8 isoforms (purple) and the location of the active ATP-binding site (orange). (B) Direct comparison between the two alternative human MAPK8 isoforms (3O17 in blue, 1UKH in red), showing that most differences are found within the loop. (C) Multiple sequence alignment of MEHEs of JNKs (E6a and E6b in MAPK8), highlighting residues that are specifically conserved within each ancestral exon (blue dots) or that are conserved in one but variable in the other (orange dots).

Fig. 5.—

The multiple sequence alignment (A) of a pair of MEHEs from different alpha actinins (corresponding to exons 8a and 8b in human ACTN2) reveals the ancient ancestry of this AS event (it first appeared in the ancestor of bilaterians) and how the original pattern has been conserved in multiple gene lineages despite several GD events. Alternatively spliced MEHEs are highlighted by using same colors. Human ACTN4 has two MEHE events, one conserved in ACTN2 (see above) and another that is found in ACTN1, which are spatially close in the 3D dimeric structure of alpha actinin, within the actin-binding regions shown in (B). The structure corresponds to the cryoEM model of chicken ACTN1 (pdb:1SJJ; Liu et al. 2004).

Fig. 6.—

Multiple sequence alignments of two sets of homologous exons from human genes CACNA1C and CACNA1D, along with the equivalent exons from the CACNA1F and CACNA1S paralogs. After duplication CACNA1F retained one homologous exon from each pair of ancestral MEHEs and CACNA1S the other. CACNA1C also has a third pair of MEHEs at the beginning of the third ion transport domain (blue). Exon numbering is distinct in CACNA1C and CACNA1D.