The molecular basis of the skipping of constitutive exons in many messenger RNAs is not fully understood. A well-studied example is exon 9 of the human cystic fibrosis transmembrane conductance regulator gene (CFTR), in which an abbreviated polypyrimidine tract between the branch point A and the 3′ splice site is associated with increased exon skipping and disease. However, many exons, both in CFTR and in other genes and have short polypyrimidine tracts in their 3′ splice sites, yet they are not skipped. Inspection of the 5′ splice sites immediately up- and downstream of exon 9 revealed deviations from consensus sequence, so we hypothesized that this exon may be inherently vulnerable to skipping. To test this idea, we constructed a CFTR minigene and replicated exon 9 skipping associated with the length of the polypyrimidine tract upstream of exon 9. We then mutated the flanking 5′ splice sites and determined the effect on exon skipping. Conversion of the upstream 5′ splice site to consensus by replacing a pyrimidine at position +3 with a purine resulted in increased exon skipping. In contrast, conversion of the downstream 5′ splice site to consensus by insertion of an adenine at position +4 resulted in a substantial reduction in exon 9 skipping, regardless of whether the upstream 5′ splice site was consensus or not. These results suggested that the native downstream 5′ splice site plays an important role in CFTR exon 9 skipping, a hypothesis that was supported by data from sheep and mouse genomes. Although CFTR exon 9 in sheep is preceded by a long polypyrimidine tract (Y14), it skips exon 9 in vivo and has a nonconsensus downstream 5′ splice site identical to that in humans. On the other hand, CFTR exon 9 in mice is preceded by a short polypyrimidine tract (Y5) but is not skipped in vivo. Its downstream 5′ splice site differs from that in humans by a 2-nt insertion, which, when introduced into the human CFTR minigene, abolished exon 9 skipping. Taken together, these observations place renewed emphasis on deviations at 5′ splice sites in nucleotides other than the invariant GT, particularly when such changes are found in conjunction with other altered splicing sequences, such as a shortened polypyrimidine tract. Thus, careful inspection of entire 5′ splice sites may identify constitutive exons that are vulnerable to skipping.
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