Genomic splice-site analysis reveals frequent alternative splicing close to the dominant splice site

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Abstract
Alternative pre-mRNA splicing may be the most efficient and widespread mechanism to generate multiple protein isoforms from single genes. Here, we describe the genomic analysis of one of the most frequent types of alternative pre-mRNA splicing, alternative 5′- and 3′-splice-site selection. Using an EST-based alternative splicing database recording >47,000 alternative splicing events, we determined the frequency and location of alternative 5′- and 3′-splice sites within the human genome. The most common alternative splice sites used in the human genome are located within 6 nucleotides (nt) of the dominant splice site. We show that the EST database overrepresents alternative splicing events that maintain the reading frame, thus supporting the concept that RNA quality-control steps ensure that mRNAs that encode for potentially harmful protein products are destroyed and do not serve as templates for translation. The most frequent location for alternative 5′-splice sites is 4 nt upstream or downstream from the dominant splice site. Sequence analysis suggests that this preference is a consequence of the U1 snRNP binding sequence at the 5′-splice site, which frequently contains a GU dinucleotide 4 nt downstream from the dominant splice site. Surprisingly, ∼50% of duplicated 3′-YAG splice junctions are subject to alternative splicing. This high probability of alternative 3′-splice-site activation in close proximity of the dominant 3′-splice site suggests that the second step of the splicing may be prone to violate splicing fidelity.