Exon Inclusion Is Dependent on Predictable Exonic Splicing Enhancers

ABSTRACT We have previously formulated a list of approximately 2,000 RNA octamers as putative exonic splicing enhancers (PESEs) based on a statistical comparison of human exonic and nonexonic sequences (X. H. Zhang and L. A. Chasin, Genes Dev. 18 :1241-1250, 2004). When inserted into a poorly spliced test exon, all eight tested octamers stimulated splicing, a result consistent with their identification as exonic splicing enhancers (ESEs). Here we present a much more stringent test of the validity of this list of PESEs. Twenty-two naturally occurring examples of nonoverlapping PESEs or PESE clusters were identified in six mammalian exons; five of the six exons tested are constitutively spliced. Each of the 22 individual PESEs or PESE clusters was disrupted by site-directed mutagenesis, usually by a single-base substitution. Eighteen of the 22 disruptions (82%) resulted in decreased splicing efficiency. In contrast, 24 control mutations had little or no effect on splicing. This high rate of success suggests that most PESEs function as ESEs in their natural context. Like most exons, these exons contain several PESEs. Since knocking out any one of several could produce a severalfold decrease in splicing efficiency, we conclude that there is little redundancy among ESEs in an exon and that they must work in concert to optimize splicing.

[1]  Michael Q. Zhang,et al.  Exonic Splicing Enhancer Motif Recognized by Human SC35 under Splicing Conditions , 2000, Molecular and Cellular Biology.

[2]  S. Berget,et al.  Exon definition may facilitate splice site selection in RNAs with multiple exons. , 1990, Molecular and cellular biology.

[3]  James Stévenin,et al.  Broad specificity of SR (serine/arginine) proteins in the regulation of alternative splicing of pre-messenger RNA. , 2004, Progress in nucleic acid research and molecular biology.

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

[5]  C. R. Valentine,et al.  The association of nonsense codons with exon skipping. , 1998, Mutation research.

[6]  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.

[7]  T. Maniatis,et al.  Multiple Distinct Splicing Enhancers in the Protein-Coding Sequences of a Constitutively Spliced Pre-mRNA , 1999, Molecular and Cellular Biology.

[8]  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.

[9]  Gene W. Yeo,et al.  Systematic Identification and Analysis of Exonic Splicing Silencers , 2004, Cell.

[10]  L. Chasin,et al.  Direct selection for mutations affecting specific splice sites in a hamster dihydrofolate reductase minigene , 1993, Molecular and cellular biology.

[11]  C. Leslie,et al.  Dichotomous splicing signals in exon flanks. , 2005, Genome research.

[12]  M. Green,et al.  Pre-mRNA splicing of IgM exons M1 and M2 is directed by a juxtaposed splicing enhancer and inhibitor. , 1999, Genes & development.

[13]  L. Chasin,et al.  Multiple Splicing Defects in an Intronic False Exon , 2000, Molecular and Cellular Biology.

[14]  J. Manley,et al.  Determinants of SR protein specificity. , 1999, Current opinion in cell biology.

[15]  A. Sureau,et al.  hnRNP A1 and the SR Proteins ASF/SF2 and SC35 Have Antagonistic Functions in Splicing of β-Tropomyosin Exon 6B* , 2004, Journal of Biological Chemistry.

[16]  A. Krainer,et al.  Identification of Functional Exonic Splicing Enhancer Motifs Recognized by Individual Sr Proteins Using an in Vitro Randomization and Functional Selection Procedure, We Have Identified Three Novel Classes of Exonic Splicing Enhancers (eses) Recognized by Human Sf2/asf, Srp40, and Srp55, Respectively , 2022 .

[17]  T. D. Schneider,et al.  Features of spliceosome evolution and function inferred from an analysis of the information at human splice sites. , 1992, Journal of molecular biology.

[18]  T. Cooper,et al.  Identification of a new class of exonic splicing enhancers by in vivo selection , 1997, Molecular and cellular biology.

[19]  M. Hentze,et al.  Nonsense-mediated mRNA decay: from vacuum cleaner to Swiss army knife , 2004, Genome Biology.

[20]  Michael R. Green,et al.  Arginine-serine-rich domains bound at splicing enhancers contact the branchpoint to promote prespliceosome assembly. , 2004, Molecular cell.

[21]  A. Steinkasserer,et al.  PCR-ligation-PCR mutagenesis: a protocol for creating gene fusions and mutations. , 1995, BioTechniques.

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

[23]  D. Black.  Mechanisms of alternative pre-messenger RNA splicing. , 2003, Annual review of biochemistry.

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

[25]  A. Newman.  Pre-mRNA splicing. , 1994, Current Opinion in Genetics and Development.

[26]  D. Housman,et al.  Alternative splicing and genomic structure of the Wilms tumor gene WT1. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[27]  P. Sharp,et al.  Splicing of precursors to mRNAs by the spliceosomes , 1993 .

[28]  J. Nicklas,et al.  Mutations that alter RNA splicing of the human HPRT gene: a review of the spectrum. , 1998, Mutation research.

[29]  O. Kent,et al.  Characterization of a U2AF-Independent Commitment Complex (E′) in the Mammalian Spliceosome Assembly Pathway , 2005, Molecular and Cellular Biology.

[30]  Stephen M. Mount,et al.  A catalogue of splice junction sequences. , 1982, Nucleic acids research.

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

[32]  T. Nilsen.  The spliceosome: the most complex macromolecular machine in the cell? , 2003, BioEssays : news and reviews in molecular, cellular and developmental biology.

[33]  R. Reed,et al.  An Upstream AG Determines Whether a Downstream AG Is Selected during Catalytic Step II of Splicing , 2001, Molecular and Cellular Biology.

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

[35]  L. Chasin,et al.  Splicing mutants and their second-site suppressors at the dihydrofolate reductase locus in Chinese hamster ovary cells , 1993, Molecular and cellular biology.

[36]  T. Cooper,et al.  Pre-mRNA splicing and human disease. , 2003, Genes & development.

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

[38]  N. Harris,et al.  Distribution and consensus of branch point signals in eukaryotic genes: a computerized statistical analysis. , 1990, Nucleic acids research.

[39]  Steven P. Gygi,et al.  Comprehensive proteomic analysis of the human spliceosome , 2002, Nature.

[40]  T. Maniatis,et al.  The function of multisite splicing enhancers. , 1998, Molecular cell.

[41]  Douglas L. Black,et al.  Roles for SR Proteins and hnRNP A1 in the Regulation of c-src Exon N1 , 2003, Molecular and Cellular Biology.

[42]  N L Harris,et al.  Splice junctions, branch point sites, and exons: sequence statistics, identification, and applications to genome project. , 1990, Methods in enzymology.

[43]  J. Manley,et al.  Functions of SR and Tra2 proteins in pre-mRNA splicing regulation. , 1999, Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine.

[44]  Jun Wang,et al.  Nonsense-associated altered splicing: a frame-dependent response distinct from nonsense-mediated decay. , 2002, Molecular cell.

[45]  L. Chasin,et al.  Nonsense mutations in the dihydrofolate reductase gene affect RNA processing , 1989, Molecular and cellular biology.

[46]  A. Krainer,et al.  Listening to silence and understanding nonsense: exonic mutations that affect splicing , 2002, Nature Reviews Genetics.

[47]  Melissa S Jurica,et al.  Pre-mRNA splicing: awash in a sea of proteins. , 2003, Molecular cell.

[48]  K. Heller,et al.  Sequence information for the splicing of human pre-mRNA identified by support vector machine classification. , 2003, Genome research.

[49]  J. Mendell,et al.  Separable Roles for rent1/hUpf1 in Altered Splicing and Decay of Nonsense Transcripts , 2002, Science.

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

[51]  M. Green.  Pre-mRNA splicing. , 1986, Annual review of genetics.

[52]  M. Wilm,et al.  Protein composition of human prespliceosomes isolated by a tobramycin affinity-selection method , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[53]  Michael R Green,et al.  A pathway of sequential arginine-serine-rich domain-splicing signal interactions during mammalian spliceosome assembly. , 2004, Molecular cell.

[54]  L. Maquat.  Nonsense-mediated mRNA decay: splicing, translation and mRNP dynamics , 2004, Nature Reviews Molecular Cell Biology.

[55]  Tom Maniatis,et al.  Selection and Characterization of Pre-mRNA Splicing Enhancers: Identification of Novel SR Protein-Specific Enhancer Sequences , 1999, Molecular and Cellular Biology.

[56]  J. Stévenin,et al.  The splicing factors 9G8 and SRp20 transactivate splicing through different and specific enhancers. , 1999, RNA.