Tracking the evolution of alternatively spliced exons within the Dscam family

Abstract

Background: The Dscam gene in the fruit fly, Drosophila melanogaster, contains twenty-four exons, four of which are composed of tandem arrays that each undergo mutually exclusive alternative splicing (4, 6, 9 and 17), potentially generating 38,016 protein isoforms. This degree of transcript diversity has not been found in mammalian homologs of Dscam. We examined the molecular evolution of exons within this gene family to locate the point of divergence for this alternative splicing pattern.

Results: Using the fruit fly Dscam exons 4, 6, 9 and 17 as seed sequences, we iteratively searched sixteen genomes for homologs, and then performed phylogenetic analyses of the resulting sequences to examine their evolutionary history. We found homologs in the nematode, arthropod and vertebrate genomes, including homologs in several vertebrates where Dscam had not been previously annotated. Among these, only the arthropods contain homologs arranged in tandem arrays indicative of mutually exclusive splicing. We found no homologs to these exons within the Arabidopsis, yeast, tunicate or sea urchin genomes but homologs to several constitutive exons from fly Dscam were present within tunicate and sea urchin. Comparing the rate of turnover within the tandem arrays of the insect taxa (fruit fly, mosquito and honeybee), we found the variants within exons 4 and 17 are well conserved in number and spatial arrangement despite 248-283 million years of divergence. In contrast, the variants within exons 6 and 9 have undergone considerable turnover since these taxa diverged, as indicated by deeply branching taxon-specific lineages.

Conclusion: Our results suggest that at least one Dscam exon array may be an ancient duplication that predates the divergence of deuterostomes from protostomes but that there is no evidence for the presence of arrays in the common ancestor of vertebrates. The different patterns of conservation and turnover among the Dscam exon arrays provide a striking example of how a gene can evolve in a modular fashion rather than as a single unit.

Figure 1

Phylogenetic relationships among the organisms included in this study, after Hedges [26]. Taxonomic categories mentioned in the text are those used by NCBI [42].

Figure 2

Multiple sequence alignment for homologs to fly Dscam exons 4 (panel A), 9 (panel B), and 17 (panel C). Representative sequences from fly, mosquito and honeybee Dscam exon arrays are aligned with homologous sequences from eight vertebrate genomes. Shaded areas indicate columns whose residues produced gaps within the alignment and were excluded from further phylogenetic analyses as discussed in the Methods section. Boxed residues distinguish mammalian Dscam and Dscam-like sequences.

Figure 3

Bayesian phylogeny of Dscam exon 4 homologs. Only branches with posterior probabilities greater than 0.5 are shown (probabilities are shown beside each branch). Roman numerals (I, II and III) and colored branches denote the three major clades (magenta, orange and cyan, respectively). Subclades (A and B) of Clade III are denoted with colored text labels, blue (Dscam_suffix) and green (DscamL_suffix).

Figure 4

Bayesian phylogeny of Dscam exon 17 homologs. Only branches with posterior probabilities greater than 0.5 are shown (probabilities are shown beside each branch). Roman numerals (I and II) and colored branches denote the two major clades (magenta, and cyan, respectively). Subclades (A and B) of Clade I are denoted with colored text labels, orange (prefix_01) and black (prefix_02). Subclades (A and B) of Clade II are denoted with colored text labels, blue (Dscam_suffix) and green (DscamL_suffix).

Figure 5

Phylogeny of the annotated insect (fly, mosquito and honeybee) Dscam exon 4 homologs and organization of tandem arrays. (A) Only branches with a posterior probability greater than 0.5 are shown (probabilities are shown beside each branch). Roman numerals (I thru V) and colored branches denote the five major subclades of the Clade I (Figure 3) sequences. Each major subclade contains at least one sequence from each fly, mosquito and honeybee. 5B. (B) Comparison between tandem arrays of honeybee (top), fly (middle) and mosquito (bottom) annotated Dscam exon 4 variants. Colored boxes represent the exon variants and the numbers below indicate the position of the variant within the tandem array. The box colors correspond to the colors of the text labels (rather than branch colors) shown in Figure 5A.

Figure 6

Phylogeny of Dscam exon 6 homologs. Only branches with posterior probabilities greater than 0.5 are shown (probabilities are shown beside each branch). The black pie-wedge represents a confluence of lineages whose branches all radiate from a common node and these branches remain unresolved.

Figure 7

Phylogeny of Dscam exon 9 homologs. Only branches with posterior probabilities greater than 0.5 are shown (probabilities are shown beside each branch). Roman numerals (I and II) and colored branches denote the two major clades (magenta, and cyan, respectively). Subclades (A and B) of Clade II are denoted with colored text labels, blue (Dscam_suffix) and green (DscamL_suffix).

Figure 8

Comparative protein domain structure of Human Dscam, Dscam-like and fly Dscam. Based on results from Ensembl, UniProt, and InterProScan, the four exons in fly that undergo mutually-exclusive alternative splicing are marked above the protein subdomains (exons 4 and 6) or domains (exons 9 and 17) that they encode. The homologous exons in human Dscam and Dscam-L are marked above their corresponding domains in the encoded proteins, with the homologous fly exon for each in parentheses. The vertebrate homologs to fly exon 6 were located for the figure by their position in the global alignment, though the overall similarity was low for these compared to the other homologous exon pairs.