The architecture of pre-mRNAs affects mechanisms of splice-site pairing

Abstract

The exon͞intron architecture of genes determines whether components of the spliceosome recognize splice sites across the intron or across the exon. Using in vitro splicing assays, we demonstrate that splice-site recognition across introns ceases when intron size is between 200 and 250 nucleotides. Beyond this threshold, splice sites are recognized across the exon. Splice-site recognition across the intron is significantly more efficient than splice-site recognition across the exon, resulting in enhanced inclusion of exons with weak splice sites. Thus, intron size can profoundly influence the likelihood that an exon is constitutively or alternatively spliced. An EST-based alternative-splicing database was used to determine whether the exon͞intron architecture influences the probability of alternative splicing in the Drosophila and human genomes. Drosophila exons flanked by long introns display an up to 90-foldhigher probability of being alternatively spliced compared with exons flanked by two short introns, demonstrating that the exon͞ intron architecture in Drosophila is a major determinant in governing the frequency of alternative splicing. Exon skipping is also more likely to occur when exons are flanked by long introns in the human genome. Interestingly, experimental and computational analyses show that the length of the upstream intron is more influential in inducing alternative splicing than is the length of the downstream intron. We conclude that the size and location of the flanking introns control the mechanism of splice-site recognition and influence the frequency and the type of alternative splicing that a pre-mRNA transcript undergoes.alternative splicing ͉ bioinformatics ͉ EST database ͉ intron length P re-mRNA splicing is an essential process that accounts for many aspects of regulated gene expression. Of the Ϸ25,000 genes encoded by the human genome (1), Ͼ60% are believed to produce transcripts that are alternatively spliced. Thus, alternative splicing of pre-mRNAs can lead to the production of multiple protein isoforms from a single pre-mRNA, exponentially enriching the proteomic diversity of higher eukaryotic organisms (2, 3). Because regulation of this process can determine when and where a particular protein isoform is produced, changes in alternative-splicing patterns modulate many cellular activities.The spliceosome assembles onto the pre-mRNA in a coordinated manner by binding to sequences located at the 5Ј and 3Ј ends of introns. Spliceosome assembly is initiated by the stable associations of the U1 small nuclear ribonucleoprotein particle with the 5Ј splice site, branch-point-binding protein͞SF1 with the branch point, and U2 snRNP auxiliary factor with the pyrimidine tract (4). ATP hydrolysis then leads to the stable association of U2 snRNP at the branch-point and functional splice-site pairing (5).Intron size has been correlated with rates of evolution (6) and the regulation of genome size (7,8). The exon͞intron architecture has also been shown to influence splice-site recognition (9-11)....

Coevolutionary networks of splicing cis -regulatory elements

“…Drosophila represented an interesting case, with slightly higher levels of exon skipping than intron retention detected, despite the predominant intron definition signature observed in splicing network analysis of constitutive introns. This observation suggests that intron definition may be favored in constitutively spliced Drosophila transcripts, with AS more common in situations involving exon definition, consistent with the long intron lengths associated with many known AS events in this organism (25).…”

“…27). In both the fishes and the flies, splice site strength changed in a correlated fashion with intron length, suggesting that shorter intron lengths increase splicing efficiency in both of these systems (25).…”

“…The 3Јss and 5Јss of pairs of consecutive constitutive exons (i.e., a total of four splice site variables) were considered. The length of the intervening intron was considered as a fifth variable (except in plants, where the sample size was insufficient), because intron length is known to influence splicing in a number of organisms (24,25); exon length did not vary suffi- ciently between orthologs to merit consideration. Throughout this study, we have included the PPT as part of the 3Јss score and have excluded the BPS because for most introns the location of the BPS has not been mapped.…”

Concurrent splicing and transcription are not sufficient to enhance splicing efficiency

“…It has been previously shown that the efficiency of splicing in NE drops sharply when intron length exceeds 200 nt (Fox-Walsh et al 2005). Consequently, we investigated whether splicing of long introns is enhanced by RNAP II transcription.…”

Genome‐wide analysis of alternative splicing events in Hordeum vulgare: Highlighting retention of intron‐based splicing and its possible function through network analysis

“…Intron and exon lengths reflect the constraints imposed by splicing recognition and frequency of AS [18]. Introns flanking alternatively spliced exons are long, whereas constitutively spliced exons are flanked by short introns [60]. Also, enlarged exons lead to exon skipping, but if the flanking introns are short, the enlarged exon is included [61].…”