The Emergence of Alternative 3′ and 5′ Splice Site Exons from Constitutive Exons
Figure 5
Mutations That Introduced a New Competing 5′ss Shifted Splicing from Constitutive to Alternative during Evolution
Multiple alignment of homolog exons and flanking intronic sequences among seven vertebrate species was constructed according to a known evolutionary tree (see Table S1B for the sequences accessions) using ClustalW ([46]; http://www.ebi.ac.uk/clustalw).
(A,B) The analysis was conducted on “group 1” and “group 2” alternative 5′ss exons, respectively. In the top panel (i) is a schematic representation of the handled exon. Exon sequence is represented by a yellow box and the alternative sequence (extension) by a light blue box. Introns are represented by black lines. The human–mouse KA/KS values and identity percentage are shown above the boxes. Major and minor splice site is indicated below the boxes as well. The organisms that have a potential (according to their splice site content) to have either both alternatively spliced forms or only one of the forms are shown on the left of the schema. In the middle (ii) panel is a multiple alignment, among the seven species, of the exon and flanking 3′ and 5′ splice sites. The major 5′ss (M), minor 5′ss (m), and 3′ss are marked in red and pointed to by an arrow. Intronic regions are highlighted in light grey. On the bottom panel (iii) is an RT–PCR analysis on normal brain cDNA of human, mouse, and rat (H, M, and R, respectively), chicken 5-d embryo (C), adult zebrafish whole body (Z), and xenopus oocytes (X). PCR products were amplified using species-specific primers, and splicing products were separated on a 3.5% agarose gel and sequenced. Asterisks point to the alternative isoform; an illustration of both alternative isoforms is shown on the right by grey, cyan, and white boxes (alternative exon, extension, and flanking exons, respectively).