Biased exon/intron distribution of cryptic and de novo 30 splice sites

We compiled sequences of previously published aberrant 30 splice sites (30ss) that were generated by mutations in human disease genes. Cryptic 30ss, defined here as those resulting from a mutation of the 30YAG consensus, were more frequent in exons than in introns. They clustered in 20 nt region adjacent to authentic 30ss, suggesting that their underrepresentation in introns is due to a depletion of AG dinucleotides in the polypyrimidine tract (PPT). In contrast, most aberrant 30ss that were induced by mutations outside the 30YAG consensus (designated ‘de novo’) were in introns. The activation of intronic de novo 30ss was largely due to AG-creating mutations in the PPT. In contrast, exonic de novo 30ss were more often induced by mutations improving the PPT, branchpoint sequence (BPS) or distant auxiliary signals, rather than by direct AG creation. The Shapiro– Senapathy matrix scores had a good prognostic value for cryptic, but not de novo 30ss. Finally, AG-creating mutations in the PPT that produced aberrant 30ss upstream of the predicted BPS in vivo shared a similar ‘BPS-new AG’ distance. Reduction of this distance and/or the strength of the new AG PPT in splicing reporter pre-mRNAs improved utilization of authentic 30ss, suggesting that AG-creating mutations that are located closer to the BPS and are preceded by weaker PPT may result in less severe splicing defects.