Efficient internal exon recognition depends on near equal contributions from the 3′ and 5′ splice sites

Pre-mRNA splicing is carried out by the spliceosome, which identifies exons and removes intervening introns. In vertebrates, most splice sites are initially recognized by the spliceosome across the exon, because most exons are small and surrounded by large introns. This gene architecture predicts that efficient exon recognition depends largely on the strength of the flanking 3′ and 5′ splice sites. However, it is unknown if the 3′ or the 5′ splice site dominates the exon recognition process. Here, we test the 3′ and 5′ splice site contributions towards efficient exon recognition by systematically replacing the splice sites of an internal exon with sequences of different splice site strengths. We show that the presence of an optimal splice site does not guarantee exon inclusion and that the best predictor for exon recognition is the sum of both splice site scores. Using a genome-wide approach, we demonstrate that the combined 3′ and 5′ splice site strengths of internal exons provide a much more significant separator between constitutive and alternative exons than either the 3′ or the 5′ splice site strength alone.