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

Mireya Plass 1 Eduardo Eyras 0 1 0 Catalan Institution for Research and Advanced Studies (ICREA) , E08010, Barcelona , Spain 1 Research Unit of Biomedical Informatics, IMIM - Pompeu Fabra University , E08003, Barcelona , Spain Background: Alternatively 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 (ESEconservation 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. Results: We 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 nonsynonymous (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. Conclusion: We 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. - Background Alternative splicing (AS) can have a biologically relevant effect on protein structure, as it allows the shuffling of protein domains rather than disrupting them [1]. Consequently, alternative splicing can modulate the function of a gene, affecting, for instance, the signal peptides and the transmembrane segments [2,3]. The importance of AS in many genomes has raised the question of its role in the context of evolution. Modrek and Lee [4] have proposed AS as an evolutionary mechanism that gives an organism the possibility to explore new protein functions by allowing the addition of novel domains while maintaining the rest of a protein intact. This has suggested that alternative exons may have more freedom to change its amino acid sequence. Indeed, recent reports show that conserved alternative exons have higher dN than average [5-8], and can even have higher dS than average [8]. However, the opposite effect has also been observed: alternative exons have been reported to have a DNA sequence conservation higher than average [9,10]. The higher conservation has been attributed to the fact that alternative exons are in general more regulated than constitutive ones, and therefore 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]. In fact, 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]. Orthologous exons with similar splicing regulation show sequence conservation of the cis-acting motifs [18]. On the other hand, it is known that cis-acting regulators of splicing are sometimes not conserved in sequence between orthologous genes [19] or are not located at orthologous positions [20], but still can preserve their function. Furthermore, regulatory elements can function at different distances from the splice sites. This distance has been found to influence the strength of the splicing regulator [21], and there seems to be a distance beyond which a motif becomes inactive [22] or changes its regulating activity [23]. We therefore expect that if two orthologous exons have the same regulation, the cis-acting motifs responsible for this regulation should also show some positional conservation. Conversely, two orthologous exons with different regulation should show low conservation in sequence and/or position of the regulatory motifs involved in splicing. Additionally, as mentioned before, a variation in the sequence and/or the arrangement of the regulatory elements is found to affect the splicing pattern, hence we expect that an arrangement of regulatory elements that is not conserved between orthologous exons must be related to a lack of conservation of the exonic structure of the transcripts including that exon. The mRNA produced from the pre-mRNA is determined by the exonic and intronic signals regulating its splicing, hence the conservation of these signals during evolution implies a conservation of the definition of the exon-intron boundaries. Conversely, transcripts that do not conserve the exonic structure across evolution must be defined by splicing regulatory signals that are not conserved in sequence and/or arrangement. Based on these observations we expect orthologous exons, alternative or constitutive, participating in transcripts with non-conserved exonic structure to have less sequence conservation. A recent report shows that constitutive exons in transcripts with non-conserved exonic structure have greater non-synonymous substitution rate [24]. In this work we generalize these results. We provide further evidence for the differences in sequence evolution between alternative and constitutive exons and link these differences to the pattern of conservat (...truncated)