Activation of the intronic cryptic 5' splice site depends on its distance to the upstream cassette exon.

Splice site selection is a key step that determines the mRNA isoforms generated from a single transcript. The large diversity in splice site sequences emphasizes the plasticity of splice site recognition and selection. In this report, a cell-based reporter system using a SMN1/2 cassette exon was applied to study the roles governing the activation of a cryptic 5'SS from the intron 4 of the CT/CGRP gene. We found that the cryptic site was activated when placed within 124nt downstream the cassette exon, and the level of activation was negatively correlated with its distance from the exon. In addition, activation was not affected by PTB but was eliminated by an insertion extending the exon length. Activated cryptic 5'SSs in intron or exon could override the original alternative 5'SS, obeying the U1 base-pairing rule. These results suggest that the exon length itself could represent a factor in determining the splice site selection.