Figure 5.

Prediction and validation of splicing regulatory hexamers by rate shift analysis. (A) Twelve vertebrate species divided in two sub-trees: mammals (black) vs. nonmammalian vertebrates (light gray). (B) An example of motif identified using rate shift analysis. The position is variable among nonmammalian vertebrates (two species of fish, frog, and chicken) and is highly conserved among mammals. Marked in light gray is one of six possible hexamers that are formed by the rate shifting substitution. (C) The effect of putative enhancing hexamers and their mutants in SXN13 reporter system. Each tested hexamer was inserted into the second exon of a reporting minigene comprised of four exons. Splicing patterns of each hexamer and its mutant are demonstrated in lanes 2, 4, 6, 8, and 10 and 3, 5, 7, 9, and 11, respectively. (Lane 1) SXN13 wild type. The upper bands indicate exon inclusion while the lower bands indicate exon skipping.