Conservation of an open-reading frame as an element affecting 5′ splice site selection

Splice site selection is a key element of pre-mRNA splicing and involves specific recognition of consensus sequences at the 5′ and 3′ splice sites. Evidently, the compliance of a given sequence with the consensus 5′ splice site sequence is not sufficient to define it as a functional 5′ splice site, because not all sequences that conform with the consensus are used for splicing. We have previously hypothesized that the necessity to avoid the inclusion of premature termination codons within mature mRNAs may serve as a criterion that differentiates normal 5′ splice sites from unused (latent) ones. We further provided experimental support to this idea, by analyzing the splicing of pre-mRNAs in which in-frame stop codons upstream of a latent 5′ splice site were mutated, and showing that splicing using the latent site is indeed activated by such mutations. Here we evaluate this hypothesis by a computerized survey for latent 5′ splice sites in 446 protein-coding human genes. This data set contains 2311 introns, in which we found 10 490 latent 5′ splice sites. The utilization of 10 045 (95.8%) of these sites for splicing would have led to the inclusion of an in-frame stop codon within the resultant mRNA. The validity of this finding is confirmed here by statistical analyses. This finding, together with our previous experimental results, invokes a nuclear scanning mechanism, as part of the splicing machine, which identifies in-frame stop codons within the pre-mRNA and prevents splicing that could lead to the formation of a prematurely terminated protein.