Dataset Information

Violating the splicing rules: TG dinucleotides function as alternative 3' splice sites in U2-dependent introns.

ABSTRACT:

Background

Despite some degeneracy of sequence signals that govern splicing of eukaryotic pre-mRNAs, it is an accepted rule that U2-dependent introns exhibit the 3' terminal dinucleotide AG. Intrigued by anecdotal evidence for functional non-AG 3' splice sites, we carried out a human genome-wide screen.

Results

We identified TG dinucleotides functioning as alternative 3' splice sites in 36 human genes. The TG-derived splice variants were experimentally validated with a success rate of 92%. Interestingly, ratios of alternative splice variants are tissue-specific for several introns. TG splice sites and their flanking intron sequences are substantially conserved between orthologous vertebrate genes, even between human and frog, indicating functional relevance. Remarkably, TG splice sites are exclusively found as alternative 3' splice sites, never as the sole 3' splice site for an intron, and we observed a distance constraint for TG-AG splice site tandems.

Conclusion

Since TGs splice sites are exclusively found as alternative 3' splice sites, the U2 spliceosome apparently accomplishes perfect specificity for 3' AGs at an early splicing step, but may choose 3' TGs during later steps. Given the tiny fraction of TG 3' splice sites compared to the vast amount of non-viable TGs, cis-acting sequence signals must significantly contribute to splice site definition. Thus, we consider TG-AG 3' splice site tandems as promising subjects for studies on the mechanisms of 3' splice site selection.

SUBMITTER: Szafranski K

PROVIDER: S-EPMC2374985 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:BACKGROUND: Alternative splicing (AS) is a key molecular process that endows biological functions with diversity and complexity. Generally, functional redundancy leads to the generation of new functions through relaxation of selective pressure in evolution, as exemplified by duplicated genes. It is also known that alternatively spliced exons (ASEs) are subject to relaxed selective pressure. Within consensus sequences at the splice junctions, the most conserved sites are dinucleotides at both ends of introns (splice dinucleotides). However, a small number of single nucleotide polymorphisms (SNPs) occur at splice dinucleotides. An intriguing question relating to the evolution of AS diversity is whether mutations at splice dinucleotides are maintained as polymorphisms and produce diversity in splice patterns within the human population. We therefore surveyed validated SNPs in the database dbSNP located at splice dinucleotides of all human genes that are defined by the H-Invitational Database. RESULTS: We found 212 validated SNPs at splice dinucleotides (sdSNPs); these were confirmed to be consistent with the GT-AG rule at either allele. Moreover, 53 of them were observed to neighbor ASEs (AE dinucleotides). No significant differences were observed between sdSNPs at AE dinucleotides and those at constitutive exons (CE dinucleotides) in SNP properties including average heterozygosity, SNP density, ratio of predicted alleles consistent with the GT-AG rule, and scores of splice sites formed with the predicted allele. We also found that the proportion of non-conserved exons was higher for exons with sdSNPs than for other exons. CONCLUSIONS: sdSNPs are found at CE dinucleotides in addition to those at AE dinucleotides, suggesting two possibilities. First, sdSNPs at CE dinucleotides may be robust against sdSNPs because of unknown mechanisms. Second, similar to sdSNPs at AE dinucleotides, those at CE dinucleotides cause differences in AS patterns because of the arbitrariness in the classification of exons into alternative and constitutive type that varies according to the dataset. Taking into account the absence of differences in sdSNP properties between those at AE and CE dinucleotides, the increased proportion of non-conserved exons found in exons flanked by sdSNPs suggests the hypothesis that sdSNPs are maintained at the splice dinucleotides of newly generated exons at which negative selection pressure is relaxed.

Project description:BackgroundThe signals that determine the specificity and efficiency of splicing are multiple and complex, and are not fully understood. Among other factors, the relative contributions of different mechanisms appear to depend on intron size inasmuch as long introns might hinder the activity of the spliceosome through interference with the proper positioning of the intron-exon junctions. Indeed, it has been shown that the information content of splice sites positively correlates with intron length in the nematode, Drosophila, and fungi. We explored the connections between the length of vertebrate introns, the strength of splice sites, exonic splicing signals, and evolution of flanking exons.ResultsA compensatory relationship is shown to exist between different types of signals, namely, the splice sites and the exonic splicing enhancers (ESEs). In the range of relatively short introns (approximately, < 1.5 kilobases in length), the enhancement of the splicing signals for longer introns was manifest in the increased concentration of ESEs. In contrast, for longer introns, this effect was not detectable, and instead, an increase in the strength of the donor and acceptor splice sites was observed. Conceivably, accumulation of A-rich ESE motifs beyond a certain limit is incompatible with functional constraints operating at the level of protein sequence evolution, which leads to compensation in the form of evolution of the splice sites themselves toward greater strength. In addition, however, a correlation between sequence conservation in the exon ends and intron length, particularly, in synonymous positions, was observed throughout the entire length range of introns. Thus, splicing signals other than the currently defined ESEs, i.e., potential new classes of ESEs, might exist in exon sequences, particularly, those that flank long introns.ConclusionSeveral weak but statistically significant correlations were observed between vertebrate intron length, splice site strength, and potential exonic splicing signals. Taken together, these findings attest to a compensatory relationship between splice sites and exonic splicing signals, depending on intron length.