Discovery and Analysis of Evolutionarily Conserved Intronic Splicing Regulatory Elements

Knowledge of the functional cis-regulatory elements that regulate constitutive and alternative pre-mRNA splicing is fundamental for biology and medicine. Here we undertook a genome-wide comparative genomics approach using available mammalian genomes to identify conserved intronic splicing regulatory elements (ISREs). Our approach yielded 314 ISREs, and insertions of ~70 ISREs between competing splice sites demonstrated that 84% of ISREs altered 5′ and 94% altered 3′ splice site choice in human cells. Consistent with our experiments, comparisons of ISREs to known splicing regulatory elements revealed that 40%–45% of ISREs might have dual roles as exonic splicing silencers. Supporting a role for ISREs in alternative splicing, we found that 30%–50% of ISREs were enriched near alternatively spliced (AS) exons, and included almost all known binding sites of tissue-specific alternative splicing factors. Further, we observed that genes harboring ISRE-proximal exons have biases for tissue expression and molecular functions that are ISRE-specific. Finally, we discovered that for Nova1, neuronal PTB, hnRNP C, and FOX1, the most frequently occurring ISRE proximal to an alternative conserved exon in the splicing factor strongly resembled its own known RNA binding site, suggesting a novel application of ISRE density and the propensity for splicing factors to auto-regulate to associate RNA binding sites to splicing factors. Our results demonstrate that ISREs are crucial building blocks in understanding general and tissue-specific AS regulation and the biological pathways and functions regulated by these AS events.

[1]  D. Rio,et al.  The mechanism of somatic inhibition of Drosophila P-element pre-mRNA splicing: multiprotein complexes at an exon pseudo-5' splice site control U1 snRNP binding. , 1992, Genes & development.

[2]  R. Darnell,et al.  Nova, the paraneoplastic Ri antigen, is homologous to an RNA-binding protein and is specifically expressed in the developing motor system , 1993, Neuron.

[3]  R. Hynes,et al.  Regulation of alternative pre-mRNA splicing by a novel repeated hexanucleotide element. , 1994, Genes & development.

[4]  C. Burd,et al.  The determinants of RNA-binding specificity of the heterogeneous nuclear ribonucleoprotein C proteins. , 1994, The Journal of biological chemistry.

[5]  D L Black,et al.  Conserved intron elements repress splicing of a neuron-specific c-src exon in vitro , 1995, Molecular and cellular biology.

[6]  M. Ashiya,et al.  A neuron-specific splicing switch mediated by an array of pre-mRNA repressor sites: evidence of a regulatory role for the polypyrimidine tract binding protein and a brain-specific PTB counterpart. , 1997, RNA.

[7]  M. Rosenfeld,et al.  Control of alternative pre-mRNA splicing by distributed pentameric repeats. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[8]  S. Berget,et al.  G triplets located throughout a class of small vertebrate introns enforce intron borders and regulate splice site selection , 1997, Molecular and cellular biology.

[9]  H. Jumaa,et al.  The splicing factor SRp20 modifies splicing of its own mRNA and ASF/SF2 antagonizes this regulation , 1997, The EMBO journal.

[10]  P. Sharp,et al.  Alternative Splicing of the Fibronectin EIIIB Exon Depends on Specific TGCATG Repeats , 1998, Molecular and Cellular Biology.

[11]  M. Blanchette,et al.  Modulation of exon skipping by high‐affinity hnRNP A1‐binding sites and by intron elements that repress splice site utilization , 1999, The EMBO journal.

[12]  T. Dawson,et al.  Regulation of alternative splicing by RNA editing , 1999, Nature.

[13]  B. Byrne,et al.  Recruitment of human muscleblind proteins to (CUG)n expansions associated with myotonic dystrophy , 2000, The EMBO journal.

[14]  L. Chasin,et al.  Human Genomic Sequences That Inhibit Splicing , 2000, Molecular and Cellular Biology.

[15]  D. Black,et al.  Cooperative Assembly of an hnRNP Complex Induced by a Tissue-Specific Homolog of Polypyrimidine Tract Binding Protein , 2000, Molecular and Cellular Biology.

[16]  C. Burge,et al.  A computational analysis of sequence features involved in recognition of short introns , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[17]  A Sureau,et al.  SC35 autoregulates its expression by promoting splicing events that destabilize its mRNAs , 2001, The EMBO journal.

[18]  J. Stévenin,et al.  TIA-1 and TIAR Activate Splicing of Alternative Exons with Weak 5′ Splice Sites followed by a U-rich Stretch on Their Own Pre-mRNAs* , 2001, The Journal of Biological Chemistry.

[19]  Alan D. Frankel,et al.  Recognition of RNA Branch Point Sequences by the KH Domain of Splicing Factor 1 (Mammalian Branch Point Binding Protein) in a Splicing Factor Complex , 2001, Molecular and Cellular Biology.

[20]  Christopher J. Lee,et al.  Genome-wide detection of alternative splicing in expressed sequences of human genes , 2001, Nucleic Acids Res..

[21]  T. Cooper,et al.  Finding signals that regulate alternative splicing in the post-genomic era , 2002, Genome Biology.

[22]  W. Reinhold,et al.  Alternative splicing of brain-specific PTB defines a tissue-specific isoform pattern that predicts distinct functional roles. , 2002, Genomics.

[23]  Christopher J. Lee,et al.  Genome-wide detection of tissue-specific alternative splicing in the human transcriptome. , 2002, Nucleic acids research.

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

[25]  P. Harper,et al.  Three proteins, MBNL, MBLL and MBXL, co-localize in vivo with nuclear foci of expanded-repeat transcripts in DM1 and DM2 cells. , 2002, Human molecular genetics.

[26]  Jernej Ule,et al.  CLIP Identifies Nova-Regulated RNA Networks in the Brain , 2003, Science.

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

[28]  J. Castle,et al.  Genome-Wide Survey of Human Alternative Pre-mRNA Splicing with Exon Junction Microarrays , 2003, Science.

[29]  Jurg Ott,et al.  Nucleotide frequency variation across human genes. , 2003, Genome research.

[30]  S. Sugano,et al.  A vertebrate RNA‐binding protein Fox‐1 regulates tissue‐specific splicing via the pentanucleotide GCAUG , 2003, The EMBO journal.

[31]  B. Birren,et al.  Sequencing and comparison of yeast species to identify genes and regulatory elements , 2003, Nature.

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

[33]  Terrence S. Furey,et al.  The UCSC Genome Browser Database , 2003, Nucleic Acids Res..

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

[35]  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.

[36]  D. Haussler,et al.  Ultraconserved Elements in the Human Genome , 2004, Science.

[37]  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.

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

[39]  T. Cooper,et al.  Muscleblind proteins regulate alternative splicing , 2004, The EMBO journal.

[40]  Gene W. Yeo,et al.  Variation in alternative splicing across human tissues , 2004, Genome Biology.

[41]  M. Garcia-Blanco,et al.  Alternative splicing in disease and therapy , 2004, Nature Biotechnology.

[42]  S. Batalov,et al.  A gene atlas of the mouse and human protein-encoding transcriptomes. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[43]  David Haussler,et al.  Transcriptome and Genome Conservation of Alternative Splicing Events in Humans and Mice , 2003, Pacific Symposium on Biocomputing.

[44]  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.

[45]  C. Gooding,et al.  Autoregulation of polypyrimidine tract binding protein by alternative splicing leading to nonsense-mediated decay. , 2004, Molecular cell.

[46]  D. Haussler,et al.  Aligning multiple genomic sequences with the threaded blockset aligner. , 2004, Genome research.

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

[48]  B. Frey,et al.  Revealing global regulatory features of mammalian alternative splicing using a quantitative microarray platform. , 2004, Molecular cell.

[49]  K. Lindblad-Toh,et al.  Systematic discovery of regulatory motifs in human promoters and 3′ UTRs by comparison of several mammals , 2005, Nature.

[50]  Robert B Darnell,et al.  Nova autoregulation reveals dual functions in neuronal splicing , 2005, The EMBO journal.

[51]  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.

[52]  F. Clark,et al.  Understanding alternative splicing: towards a cellular code , 2005, Nature Reviews Molecular Cell Biology.

[53]  Peter Stoilov,et al.  Homologues of the Caenorhabditis elegans Fox-1 Protein Are Neuronal Splicing Regulators in Mammals , 2005, Molecular and Cellular Biology.

[54]  S. Kawamoto,et al.  Tissue-dependent isoforms of mammalian Fox-1 homologs are associated with tissue-specific splicing activities , 2005, Nucleic acids research.

[55]  N. Bresolin,et al.  Analysis of intronic conserved elements indicates that functional complexity might represent a major source of negative selection on non-coding sequences. , 2005, Human molecular genetics.

[56]  Tyson A. Clark,et al.  Nova regulates brain-specific splicing to shape the synapse , 2005, Nature Genetics.

[57]  T. Cooper,et al.  Identification of Putative New Splicing Targets for ETR-3 Using Sequences Identified by Systematic Evolution of Ligands by Exponential Enrichment , 2005, Molecular and Cellular Biology.

[58]  B. Blencowe.  Alternative Splicing: New Insights from Global Analyses , 2006, Cell.

[59]  Gene W. Yeo,et al.  Inference of Splicing Regulatory Activities by Sequence Neighborhood Analysis , 2006, PLoS genetics.

[60]  G. Ast,et al.  Comparative analysis identifies exonic splicing regulatory sequences--The complex definition of enhancers and silencers. , 2006, Molecular cell.

[61]  M. Garcia-Blanco,et al.  Fox-2 Mediates Epithelial Cell-Specific Fibroblast Growth Factor Receptor 2 Exon Choice , 2006, Molecular and Cellular Biology.

[62]  B. Blencowe,et al.  An RNA map predicting Nova-dependent splicing regulation , 2006, Nature.

[63]  Hiram Clawson,et al.  Intronic Alternative Splicing Regulators Identified by Comparative Genomics in Nematodes , 2006, PLoS Comput. Biol..

[64]  Zefeng Wang,et al.  General and specific functions of exonic splicing silencers in splicing control. , 2006, Molecular cell.

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

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