Dichotomous splicing signals in exon flanks

Abstract

Intronic elements flanking the splice-site consensus sequences are thought to play a role in pre-mRNA splicing. However, the generality of this role, the catalog of effective sequences, and the mechanisms involved are still lacking. Using molecular genetic tests, we first showed that the ∼50-nt intronic flanking sequences of exons beyond the splice-site consensus are generally important for splicing. We then went on to characterize exon flank sequences on a genomic scale. The G+C content of flanks displayed a bimodal distribution reflecting an exaggeration of this base composition in flanks relative to the gene as a whole. We divided all exons into two classes according to their flank G+C content and used computational and statistical methods to define pentamers of high relative abundance and phylogenetic conservation in exon flanks. Upstream pentamers were often common to the two classes, whereas downstream pentamers were totally different. Upstream and downstream pentamers were often identical around low G+C exons, and in contrast, were often complementary around high G+C exons. In agreement with this complementarity, predicted base pairing was more frequent between the flanks of high G+C exons. Pseudo exons did not exhibit this behavior, but rather tended to form base pairs between flanks and exon bodies. We conclude that most exons require signals in their immediate flanks for efficient splicing. G+C content is a sequence feature correlated with many genetic and genomic attributes. We speculate that there may be different mechanisms for splice site recognition depending on G+C content.[Supplemental material is available online at www.genome.org.]Pre-mRNA splicing is a fundamental step in gene expression. During this process, splice sites must be recognized within large introns before subsequent steps occur. How the splicing machinery manages to recognize splice sites remains a central problem in the study of splicing and its regulation. Three conserved sequence motifs, the 5Ј splice site, the 3Ј splice site and the branchpoint (BP), are known to be necessary for the two sequential transesterification reactions by which introns are removed and exons are joined (Adams et al. 1996;Jurica and Moore 2003). However, these sequences are quite degenerate; one can find about two orders of magnitude more intronic sequences that match either splice-site consensus, as well as or better than the sites that are actually used (Sun and Chasin 2000;Zhang et al. 2003). The BP consensus is even more degenerate Senapathy et al. 1990).There is strong genetic and biochemical evidence for the idea that the exon is the unit of initial recognition, and that there is coupling between the recognition of 5Ј and 3Ј splice sites across the exon (Robberson et al. 1990;Carothers et al. 1993;Berget 1995). However, even if pairing of 3' and 5' splice site-like sequences is stipulated and the distance between them constrained to a range typical for real exons, these "pseudo exons" still outnumber the real exons by at least an order of magnitu...

Positive selection acting on splicing motifs reflects compensatory evolution

“…As an estimate of neutral mutation rates, we enumerated changes in intron sequences. We ignored sequences located within 100 nt of exons, as these regions are known to harbor splicing regulatory signals (Louie et al 2003;Zhang et al , 2005c) and tend to be conserved (Sorek and Ast 2003;Sugnet et al 2004;Xing and Lee 2005;Yeo et al 2005). We also excluded highly repeated sequences, as they may be subject to distinctive evolutionary pressures, and we purposely ignored changes at CpG sites to avoid domination of the data by that highly mutable dinucleotide.…”

“…An ESE can act as an ESS and vice versa depending on position (Kanopka et al 1996;Goren et al 2006). Finally, compensatory changes may be acting via changes in intronic motifs flanking these exons (Zhang et al , 2005cYeo et al 2005); we did not search for intronic changes here in the absence of validated global sets of such motifs. The predicted compensatory trends were evident in our experiments despite these limitations.…”

“…For introns, we ignored the highly repeated sequences, which have been premasked in the alignment files. We also ignored intronic sequences within 100 nt of exons, since these sequences are likely to harbor intronic splicing signals (Zhang et al , 2005c and so be subject to selection. We surveyed 16.7 million nt of introns.…”

Splicing of a Critical Exon of Human Survival Motor Neuron Is Regulated by a Unique Silencer Element Located in the Last Intron

“…It is not known if the mouse sequence that corresponds to ISS-N1 contains an enhancer element that is recognized by a splicing factor. Interestingly, this region in the mouse contains a UUUAA motif, which has been found to be a winning pentamer within low GϩC content intronic sequences downstream of the 5Ј ss (62). Winning pentamers supposedly regulate splicing by an as yet unknown mechanism.…”

“…These sequences are highly diverse and could be broadly categorized into GϩC-rich and AϩU-rich regions that constitute distinct pentamer motifs (62). The intron 7 sequence downstream of the 5Ј ss is rich in A and U residues but lacks characteristic pentamer motifs.…”