Abstract
BackgroundThe splicing of pre-mRNAs is conspicuously often variable and produces multiple alternatively spliced (AS) isoforms that encode different messages from one gene locus. Computational studies uncovered a class of highly similar isoforms, which were related to tandem 5'-splice sites (5'ss) and 3'-splice sites (3'ss), yet with very sparse anecdotal evidence in experimental studies. To compare the types and levels of alternative tandem splice site exons occurring in different human organ systems and cell types, and to study known sequence features involved in the recognition and distinction of neighboring splice sites, we performed large-scale, stringent alignments of cDNA sequences and ESTs to the human and mouse genomes, followed by experimental validation.ResultsWe analyzed alternative 5'ss exons (A5Es) and alternative 3'ss exons (A3Es), derived from transcript sequences that were aligned to assembled genome sequences to infer patterns of AS occurring in several thousands of genes. Comparing the levels of overlapping (tandem) and non-overlapping (competitive) A5Es and A3Es, a clear preference of isoforms was seen for tandem acceptors and donors, with four nucleotides and three to six nucleotides long exon extensions, respectively. A subset of inferred A5E tandem exons was selected and experimentally validated. With the focus on A5Es, we investigated their transcript coverage, sequence conservation and base-paring to U1 snRNA, proximal and distal splice site classification, candidate motifs for cis-regulatory activity, and compared A5Es with A3Es, constitutive and pseudo-exons, in H. sapiens and M. musculus. The results reveal a small but authentic enriched set of tandem splice site preference, with specific distances between proximal and distal 5'ss (3'ss), which showed a marked dichotomy between the levels of in- and out-of-frame splicing for A5Es and A3Es, respectively, identified a number of candidate NMD targets, and allowed a rough estimation of a number of undetected tandem donors based on splice site information.ConclusionThis comparative study distinguishes tandem 5'ss and 3'ss, with three to six nucleotides long extensions, as having unusually high proportions of AS, experimentally validates tandem donors in a panel of different human tissues, highlights the dichotomy in the types of AS occurring at tandem splice sites, and elucidates that human alternative exons spliced at overlapping 5'ss posses features of typical splice variants that could well be beneficial for the cell.
Highlights
The splicing of pre-mRNAs is conspicuously often variable and produces multiple alternatively spliced (AS) isoforms that encode different messages from one gene locus
Biased extensions of alternative 5'ss and 3'ss exons Exon-skipping is the most prevalent AS type produced by the human spliceosome, as well as by all other mammals investigated to date, when averaged across different organ systems and cell types that can exhibit tissue-enriched splice forms [21,22]
Out of a collection of ~37,400 transcript-inferred human alternative exons maintained in the HOLLYWOOD database [23], AS events of about 10,300 alternative 5'ss exons (A5Es) and 9,200 alternative 3'ss exons (A3Es) were filtered for exon splice variants of solely one proximal/one distal 5'ss, while being constitutively spliced at the opposite site, and resulted to 5,275 A5Es and 4,497 A3Es; either exon set had no other inferred AS type, respectively
Summary
The splicing of pre-mRNAs is conspicuously often variable and produces multiple alternatively spliced (AS) isoforms that encode different messages from one gene locus. To compare the types and levels of alternative tandem splice site exons occurring in different human organ systems and cell types, and to study known sequence features involved in the recognition and distinction of neighboring splice sites, we performed large-scale, stringent alignments of cDNA sequences and ESTs to the human and mouse genomes, followed by experimental validation. The recognition of authentic pre-mRNA splice sites out of many possible pseudosites, the precise excision of introns, and the ligation of exons to produce a correct message are catalyzed by a large ribonucleoprotein (RNP) complex known as the spliceosome, which is composed of several small RNPs and perhaps over two-hundred proteins [1]. When a pair of closely spaced 3'ss-5'ss signals is recognized, the exon is roughly defined by interactions between U2 snRNP:3'ss, U1 snRNP:5'ss as well as additional splicing factors, including U2AF65:branch site and U2AF35:poly-(Y) site interactions
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