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

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.

[1]  R. Amann,et al.  Predictive Identification of Exonic Splicing Enhancers in Human Genes , 2022 .

[2]  T. Maniatis,et al.  A splicing enhancer exhibits both constitutive and regulated activities. , 1994, Genes & development.

[3]  H. Akashi,et al.  A test of translational selection at 'silent' sites in the human genome: base composition comparisons in alternatively spliced genes. , 2000, Gene.

[4]  J. Thompson,et al.  CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. , 1994, Nucleic acids research.

[5]  T. Maniatis,et al.  Structural and functional conservation of the Drosophila doublesex splicing enhancer repeat elements. , 1996, RNA.

[6]  C. Pál,et al.  Evidence for purifying selection acting on silent sites in BRCA1. , 2001, Trends in genetics : TIG.

[7]  V. Sievert,et al.  The sex-determining gene doublesex in the fly Megaselia scalaris: conserved structure and sex-specific splicing. , 2000, Genome.

[8]  Michael Q. Zhang,et al.  Distribution of SR protein exonic splicing enhancer motifs in human protein-coding genes , 2005, Nucleic acids research.

[9]  E. Oláh,et al.  Purifying selection on silent sites -- a constraint from splicing regulation? , 2001, Trends in genetics : TIG.

[10]  R. Sorek,et al.  Intronic sequences flanking alternatively spliced exons are conserved between human and mouse. , 2003, Genome research.

[11]  D. Carlini,et al.  Synonymous SNPs Provide Evidence for Selective Constraint on Human Exonic Splicing Enhancers , 2005, Journal of Molecular Evolution.

[12]  M. Gelfand,et al.  Low conservation of alternative splicing patterns in the human and mouse genomes. , 2003, Human molecular genetics.

[13]  Brenton R Graveley,et al.  A computational and experimental approach toward a priori identification of alternatively spliced exons. , 2004, RNA.

[14]  K. H. Wolfe,et al.  Changes in alternative splicing of human and mouse genes are accompanied by faster evolution of constitutive exons. , 2005, Molecular biology and evolution.

[15]  Ewan Birney,et al.  Automated generation of heuristics for biological sequence comparison , 2005, BMC Bioinformatics.

[16]  Christopher J. Lee,et al.  Alternative splicing in the human, mouse and rat genomes is associated with an increased frequency of exon creation and/or loss , 2003, Nature Genetics.

[17]  E. O. Ermakova,et al.  Fast rate of evolution in alternatively spliced coding regions of mammalian genes , 2006, BMC Genomics.

[18]  A. Kornblihtt,et al.  A splicing enhancer in the human fibronectin alternate ED1 exon interacts with SR proteins and stimulates U2 snRNP binding. , 1993, Genes & development.

[19]  C. Lorson,et al.  A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[20]  Yi Xing,et al.  Widespread production of novel soluble protein isoforms by alternative splicing removal of transmembrane anchoring domains , 2003, FEBS letters.

[21]  Simon C. Potter,et al.  An overview of Ensembl. , 2004, Genome research.

[22]  Feng-Chi Chen,et al.  Alternatively and constitutively spliced exons are subject to different evolutionary forces. , 2006, Molecular biology and evolution.

[23]  L. Chasin,et al.  Computational definition of sequence motifs governing constitutive exon splicing. , 2004, Genes & development.

[24]  Martin Vingron,et al.  Increase of functional diversity by alternative splicing. , 2003, Trends in genetics : TIG.

[25]  M. Tomita,et al.  Computational comparative analyses of alternative splicing regulation using full-length cDNA of various eukaryotes. , 2004, RNA.

[26]  T. Maniatis,et al.  A systematic analysis of the factors that determine the strength of pre‐mRNA splicing enhancers , 1998, The EMBO journal.

[27]  Ziheng Yang,et al.  PAML: a program package for phylogenetic analysis by maximum likelihood , 1997, Comput. Appl. Biosci..

[28]  Tomaso Poggio,et al.  Identification and analysis of alternative splicing events conserved in human and mouse. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[29]  Gene W. Yeo,et al.  Variation in sequence and organization of splicing regulatory elements in vertebrate genes. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[30]  Zhi-Ming Zheng,et al.  Regulation of Alternative RNA Splicing by Exon Definition and Exon Sequences in Viral and Mammalian Gene Expression , 2004, Journal of Biomedical Science.

[31]  T. Andrews,et al.  The Ensembl automatic gene annotation system. , 2004, Genome research.

[32]  Ron Shamir,et al.  A non-EST-based method for exon-skipping prediction. , 2004, Genome research.

[33]  Ann E. Loraine,et al.  THE EFFECTS OF ALTERNATIVE SPLICING ON TRANSMEMBRANE PROTEINS IN THE MOUSE GENOME , 2003 .

[34]  G. Schellenberg,et al.  Missense and silent tau gene mutations cause frontotemporal dementia with parkinsonism-chromosome 17 type, by affecting multiple alternative RNA splicing regulatory elements. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[35]  L. Hurst,et al.  Evidence for purifying selection against synonymous mutations in mammalian exonic splicing enhancers. , 2006, Molecular biology and evolution.

[36]  Yi Xing,et al.  Evidence of functional selection pressure for alternative splicingevents that accelerate evolution of protein subsequences , 2005, Genome Biology.

[37]  Stylianos E. Antonarakis,et al.  Comparative gene finding in chicken indicates that we are closing in on the set of multi-exonic widely expressed human genes , 2005, Nucleic acids research.

[38]  Yongqing Zhang,et al.  Distribution of exonic splicing enhancer elements in human genes. , 2005, Genomics.

[39]  Dirk Holste,et al.  Single Nucleotide Polymorphism–Based Validation of Exonic Splicing Enhancers , 2004, PLoS biology.