Differentiated evolutionary rates in alternative exons and the implications for splicing regulation.

Mireya Plass,Eduardo Eyras

doi:10.1186/1471-2148-6-50

Abstract

BackgroundAlternatively spliced exons play an important role in the diversification of gene function in most metazoans and are highly regulated by conserved motifs in exons and introns. Two contradicting properties have been associated to evolutionary conserved alternative exons: higher sequence conservation and higher rate of non-synonymous substitutions, relative to constitutive exons. In order to clarify this issue, we have performed an analysis of the evolution of alternative and constitutive exons, using a large set of protein coding exons conserved between human and mouse and taking into account the conservation of the transcript exonic structure. Further, we have also defined a measure of the variation of the arrangement of exonic splicing enhancers (ESE-conservation score) to study the evolution of splicing regulatory sequences. We have used this measure to correlate the changes in the arrangement of ESEs with the divergence of exon and intron sequences.ResultsWe find evidence for a relation between the lack of conservation of the exonic structure and the weakening of the sequence evolutionary constraints in alternative and constitutive exons. Exons in transcripts with non-conserved exonic structures have higher synonymous (dS) and non-synonymous (dN) substitution rates than exons in conserved structures. Moreover, alternative exons in transcripts with non-conserved exonic structure are the least constrained in sequence evolution, and at high EST-inclusion levels they are found to be very similar to constitutive exons, whereas alternative exons in transcripts with conserved exonic structure have a dS significantly lower than average at all EST-inclusion levels. We also find higher conservation in the arrangement of ESEs in constitutive exons compared to alternative ones. Additionally, the sequence conservation at flanking introns remains constant for constitutive exons at all ESE-conservation values, but increases for alternative exons at high ESE-conservation values.ConclusionWe conclude that most of the differences in dN observed between alternative and constitutive exons can be explained by the conservation of the transcript exonic structure. Low dS values are more characteristic of alternative exons with conserved exonic structure, but not of those with non-conserved exonic structure. Additionally, constitutive exons are characterized by a higher conservation in the arrangement of ESEs, and alternative exons with an ESE-conservation similar to that of constitutive exons are characterized by a conservation of the flanking intron sequences higher than average, indicating the presence of more intronic regulatory signals.

Highlights

Spliced exons play an important role in the diversification of gene function in most metazoans and are highly regulated by conserved motifs in exons and introns
For this set we found lower synonymous substitution rates (Kolmogorov-Smirnov (KS) test p-value < 2.2e-16) and higher non-synonymous substitution rates (KS test p-value < 2.2e-16) for alternative exons compared to constitutive ones
After separating the exons according to the conservation of the exonic structure, we found that most of the differences in evolutionary rates can be explained by the pattern of conservation of the exonic structure

Summary

Introduction

Spliced exons play an important role in the diversification of gene function in most metazoans and are highly regulated by conserved motifs in exons and introns. The higher conservation has been attributed to the fact that alternative exons are in general more regulated than constitutive ones, and contain more conserved sequence motifs, like exonic splicing enhancers and silencers, which function in a coordinated fashion. The conservation of these motifs is important for exon definition [11], and in some cases a single nucleotide mutation can disrupt the splicing and lead to a disease state, like dementia [12] or spinal muscular atrophy [13]. Exonic regions with high density of regulatory motifs have been linked to regions of low SNP density [14], low synonymous SNP density [15], and negative selection against synonymous substitutions [1618]

Objectives

Methods

Results

Conclusion