Differential GC content between exons and introns establishes distinct strategies of splice-site recognition.

During evolution segments of homeothermic genomes underwent a GC content increase. Our analyses reveal that two exon-intron architectures have evolved from an ancestral state of low GC content exons flanked by short introns with a lower GC content. One group underwent a GC content elevation that abolished the differential exon-intron GC content, with introns remaining short. The other group retained the overall low GC content as well as the differential exon-intron GC content, and is associated with longer introns. We show that differential exon-intron GC content regulates exon inclusion level in this group, in which disease-associated mutations often lead to exon skipping. This group's exons also display higher nucleosome occupancy compared to flanking introns and exons of the other group, thus "marking" them for spliceosomal recognition. Collectively, our results reveal that differential exon-intron GC content is a previously unidentified determinant of exon selection and argue that the two GC content architectures reflect the two mechanisms by which splicing signals are recognized: exon definition and intron definition.

[1]  G. Ast,et al.  Human-mouse comparative analysis reveals that branch-site plasticity contributes to splicing regulation. , 2005, Human molecular genetics.

[2]  B. Frey,et al.  Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing , 2008, Nature Genetics.

[3]  Lesley Collins,et al.  Complex spliceosomal organization ancestral to extant eukaryotes. , 2005, Molecular biology and evolution.

[4]  B. Blencowe,et al.  When chromatin meets splicing , 2009, Nature Structural &Molecular Biology.

[5]  D. Niu.  Exon definition as a potential negative force against intron losses in evolution , 2008, Biology Direct.

[6]  Schraga Schwartz,et al.  Chromatin density and splicing destiny: on the cross‐talk between chromatin structure and splicing , 2010, The EMBO journal.

[7]  G Bernardi,et al.  Misunderstandings about isochores. Part 1. , 2001, Gene.

[8]  G. Ast,et al.  Chromatin organization marks exon-intron structure , 2009, Nature Structural &Molecular Biology.

[9]  Martin W. Simmen,et al.  CpG island libraries from human Chromosomes 18 and 22: landmarks for novel genes , 2000, Mammalian Genome.

[10]  Araxi O. Urrutia,et al.  A unification of mosaic structures in the human genome. , 2003, Human molecular genetics.

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

[12]  Noah Spies,et al.  Biased chromatin signatures around polyadenylation sites and exons. , 2009, Molecular cell.

[13]  Gene W. Yeo,et al.  Discovery and Analysis of Evolutionarily Conserved Intronic Splicing Regulatory Elements , 2007, PLoS Genetics.

[14]  Ravi Sachidanandam,et al.  Intrinsic differences between authentic and cryptic 5' splice sites. , 2003, Nucleic acids research.

[15]  Walter Gilbert,et al.  The evolution of spliceosomal introns: patterns, puzzles and progress , 2006, Nature Reviews Genetics.

[16]  G. Ast,et al.  Alternative splicing and evolution: diversification, exon definition and function , 2010, Nature Reviews Genetics.

[17]  Nuno L Barbosa-Morais,et al.  Systematic genome-wide annotation of spliceosomal proteins reveals differential gene family expansion. , 2005, Genome research.

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

[19]  G. Bernardi,et al.  The vertebrate genome: isochores and evolution. , 1993, Molecular biology and evolution.

[20]  Hyunmin Kim,et al.  Pre-mRNA splicing is a determinant of histone H3K36 methylation , 2011, Proceedings of the National Academy of Sciences.

[21]  Chris Smith,et al.  Large-Scale Trends in the Evolution of Gene Structures within 11 Animal Genomes , 2006, PLoS Comput. Biol..

[22]  A. Krainer,et al.  Pre-mRNA splicing in the new millennium. , 2001, Current opinion in cell biology.

[23]  J. V. Moran,et al.  Initial sequencing and analysis of the human genome. , 2001, Nature.

[24]  Maria Carmo-Fonseca,et al.  Splicing enhances recruitment of methyltransferase HYPB/Setd2 and methylation of histone H3 Lys36 , 2011, Nature Structural &Molecular Biology.

[25]  Tyson A. Clark,et al.  HITS-CLIP yields genome-wide insights into brain alternative RNA processing , 2008, Nature.

[26]  David Haussler,et al.  Unusual Intron Conservation near Tissue-Regulated Exons Found by Splicing Microarrays , 2005, PLoS Comput. Biol..

[27]  E. Levanon,et al.  Human housekeeping genes are compact. , 2003, Trends in genetics : TIG.

[28]  D. Burstein,et al.  Changes in exon-intron structure during vertebrate evolution affect the splicing pattern of exons. , 2012, Genome research.

[29]  P. Baldi,et al.  The architecture of pre-mRNAs affects mechanisms of splice-site pairing. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[30]  T. Cooper,et al.  The regulation of splice-site selection, and its role in human disease. , 1997, American journal of human genetics.

[31]  J. Ahringer,et al.  Differential chromatin marking of introns and expressed exons by H3K36me3 , 2008, Nature Genetics.

[32]  Timothy R. Hughes,et al.  G+C content dominates intrinsic nucleosome occupancy , 2009, BMC Bioinformatics.

[33]  Stephen M. Mount,et al.  Species-specific signals for the splicing of a short Drosophila intron in vitro. , 1993, Molecular and cellular biology.

[34]  Jyoti K. Shah,et al.  Differential expression of 24 , 426 human alternative splicing events and predicted cis-regulation in 48 tissues and cell lines , 2011 .

[35]  Jurg Ott,et al.  Distribution and characterization of regulatory elements in the human genome. , 2002, Genome research.

[36]  D. Cooper,et al.  The mutational spectrum of single base-pair substitutions in mRNA splice junctions of human genes: Causes and consequences , 1992, Human Genetics.

[37]  Gil Ast,et al.  How did alternative splicing evolve? , 2004, Nature Reviews Genetics.

[38]  M. Lynch,et al.  The Origins of Genome Complexity , 2003, Science.

[39]  G Bernardi,et al.  The mosaic genome of warm-blooded vertebrates. , 1985, Science.

[40]  Christoforos Nikolaou,et al.  Nucleosome positioning as a determinant of exon recognition , 2009, Nature Structural &Molecular Biology.

[41]  M. Long,et al.  Intron-exon structures of eukaryotic model organisms. , 1999, Nucleic acids research.

[42]  R. Darnell,et al.  Mapping in vivo protein-RNA interactions at single-nucleotide resolution from HITS-CLIP data , 2011, Nature Biotechnology.

[43]  C. Glover,et al.  Gene expression profiling for hematopoietic cell culture , 2006 .

[44]  J. Reyes,et al.  BRG1 helps RNA polymerase II to overcome a nucleosomal barrier during elongation, in vivo , 2010, EMBO reports.

[45]  G. Ast,et al.  Different levels of alternative splicing among eukaryotes , 2006, Nucleic acids research.

[46]  K. Nakai,et al.  Construction of a novel database containing aberrant splicing mutations of mammalian genes. , 1994, Gene.

[47]  D. Burstein,et al.  Large-scale comparative analysis of splicing signals and their corresponding splicing factors in eukaryotes. , 2007, Genome research.

[48]  S. Berget.  Exon Recognition in Vertebrate Splicing (*) , 1995, The Journal of Biological Chemistry.

[49]  G. Ast,et al.  SR proteins: a foot on the exon before the transition from intron to exon definition. , 2007, Trends in genetics : TIG.

[50]  C. Semple,et al.  Widespread signatures of recent selection linked to nucleosome positioning in the human lineage. , 2011, Genome research.

[51]  Jinhua Wang,et al.  ESEfinder: a web resource to identify exonic splicing enhancers , 2003, Nucleic Acids Res..

[52]  C. Béroud,et al.  Human Splicing Finder: an online bioinformatics tool to predict splicing signals , 2009, Nucleic acids research.

[53]  A. Kornblihtt,et al.  Transcriptional Activators Differ in Their Abilities to Control Alternative Splicing* , 2002, The Journal of Biological Chemistry.

[54]  M. Alló,et al.  Neuronal cell depolarization induces intragenic chromatin modifications affecting NCAM alternative splicing , 2009, Proceedings of the National Academy of Sciences.

[55]  Marvin B. Shapiro,et al.  RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression. , 1987, Nucleic acids research.

[56]  J. Hampe,et al.  Single base‐pair substitutions in exon–intron junctions of human genes: nature, distribution, and consequences for mRNA splicing , 2007, Human mutation.

[57]  D. Sankoff.  Minimal Mutation Trees of Sequences , 1975 .

[58]  K. J. Hertel,et al.  Combinatorial Control of Exon Recognition* , 2008, Journal of Biological Chemistry.

[59]  Dustin E. Schones,et al.  Dynamic Regulation of Nucleosome Positioning in the Human Genome , 2008, Cell.

[60]  Lili Wan,et al.  RNA and Disease , 2009, Cell.

[61]  S. Berget,et al.  Architectural limits on split genes. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[62]  Eric T. Wang,et al.  Alternative Isoform Regulation in Human Tissue Transcriptomes , 2008, Nature.

[63]  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.

[64]  B. Blencowe.  Exonic splicing enhancers: mechanism of action, diversity and role in human genetic diseases. , 2000, Trends in biochemical sciences.

[65]  S. Berget,et al.  Intron definition in splicing of small Drosophila introns , 1994, Molecular and cellular biology.

[66]  G Bernardi,et al.  Isochores and the evolutionary genomics of vertebrates. , 2000, Gene.

[67]  Gil Ast,et al.  Comparative analysis detects dependencies among the 5' splice-site positions. , 2004, RNA.