Genome-wide Analysis of Alternative Pre-mRNA Splicing
2007; Elsevier BV; Volume: 283; Issue: 3 Linguagem: Inglês
10.1074/jbc.r700033200
ISSN1083-351X
AutoresClaudia Ben-Dov, Britta Hartmann, Josefin Lundgren, Juan Valcárcel,
Tópico(s)RNA and protein synthesis mechanisms
ResumoAlternative splicing of mRNA precursors allows the synthesis of multiple mRNAs from a single primary transcript, significantly expanding the information content and regulatory possibilities of higher eukaryotic genomes. High-throughput enabling technologies, particularly large-scale sequencing and splicing-sensitive microarrays, are providing unprecedented opportunities to address key questions in this field. The picture emerging from these pioneering studies is that alternative splicing affects most human genes and a significant fraction of the genes in other multicellular organisms, with the potential to greatly influence the evolution of complex genomes. A combinatorial code of regulatory signals and factors can deploy physiologically coherent programs of alternative splicing that are distinct from those regulated at other steps of gene expression. Pre-mRNA splicing and its regulation play important roles in human pathologies, and genome-wide analyses in this area are paving the way for improved diagnostic tools and for the identification of novel and more specific pharmaceutical targets. Alternative splicing of mRNA precursors allows the synthesis of multiple mRNAs from a single primary transcript, significantly expanding the information content and regulatory possibilities of higher eukaryotic genomes. High-throughput enabling technologies, particularly large-scale sequencing and splicing-sensitive microarrays, are providing unprecedented opportunities to address key questions in this field. The picture emerging from these pioneering studies is that alternative splicing affects most human genes and a significant fraction of the genes in other multicellular organisms, with the potential to greatly influence the evolution of complex genomes. A combinatorial code of regulatory signals and factors can deploy physiologically coherent programs of alternative splicing that are distinct from those regulated at other steps of gene expression. Pre-mRNA splicing and its regulation play important roles in human pathologies, and genome-wide analyses in this area are paving the way for improved diagnostic tools and for the identification of novel and more specific pharmaceutical targets. Removal of introns from pre-mRNAs is an essential step in eukaryotic gene expression (see Fig. 1A). Alternative patterns of intron removal allow the synthesis of multiple mRNAs from a single gene encoding different proteins (see Fig. 1B). This mini-review focuses on how the recent application of high-through-put technologies is providing a novel and empowered perspective to address the following four outstanding questions in the field of alternative pre-mRNA splicing (AS). 3The abbreviations used are: ASalternative pre-mRNA splicingSR proteinserine/arginine-rich proteinhnRNPheterogeneous nuclear ribonucleoprotein. 3The abbreviations used are: ASalternative pre-mRNA splicingSR proteinserine/arginine-rich proteinhnRNPheterogeneous nuclear ribonucleoprotein. alternative pre-mRNA splicing serine/arginine-rich protein heterogeneous nuclear ribonucleoprotein. alternative pre-mRNA splicing serine/arginine-rich protein heterogeneous nuclear ribonucleoprotein. Although early estimates suggested that AS affects only a small fraction of human genes, large-scale genome and transcriptome sequencing projects allowing extensive alignments of mRNA with genomic sequences (see Fig. 2A) indicate that the majority of human genes are alternatively spliced (1Zavolan M. van Nimwegen E. Curr. Opin. Struct. Biol. 2006; 16: 362-367Crossref PubMed Scopus (27) Google Scholar). Splicing-sensitive microarrays (see Fig. 2B) provide independent confirmation of this high incidence. Using a variety of experimental designs and biological samples, several of these studies have produced consistent estimates of 70–80% of alternatively spliced genes in the human genome (2Johnson J.M. Castle J. Garrett-Engele P. Kan Z. Loerch P.M. Armour C.D. Santos R. Schadt E.E. Stoughton R. Shoemaker D.D. Science. 2003; 302: 2141-2144Crossref PubMed Scopus (1192) Google Scholar, 3Kampa D. Cheng J. Kapranov P. Yamanaka M. Brubaker S. Cawley S. Drenkow J. Piccolboni A. Bekiranov S. 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Tongprasit W. Barbano P.E. Bussemaker H.J. White K.P. Science. 2004; 306: 655-660Crossref PubMed Scopus (250) Google Scholar), and genes with extraordinarily complex patterns of regulation exist in invertebrates. (A now classical example is the Drosophila Dscam gene, which can generate over 38,000 isoforms important for neural wiring and immune defense (9Chen B.E. Kondo M. Garnier A. Watson F.L. Puettmann-Holgado R. Lamar D.R. Schmucker D. Cell. 2006; 125: 607-620Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar).) It is unquestionable that AS can generate mRNAs with important and distinct biological functions (10Black D.L. Annu. Rev. Biochem. 2003; 72: 291-336Crossref PubMed Scopus (1970) Google Scholar, 11Matlin A.J. Clark F. Smith C.W. Nat. Rev. Mol. Cell Biol. 2005; 6: 386-398Crossref PubMed Scopus (968) Google Scholar, 12Blencowe B.J. Cell. 2006; 126: 37-47Abstract Full Text Full Text PDF PubMed Scopus (832) Google Scholar, 13Stamm S. Ben-Ari S. Rafalska I. Tang Y. Zhang Z. Toiber D. Thanaraj T.A. Soreq H. Gene (Amst.). 2005; 344: 1-20Crossref PubMed Scopus (685) Google Scholar). The more difficult question is what fraction of the extensive transcript variation generated by AS is truly biologically relevant and what fraction may be due to stochastic noise in the splicing process. Biological Incidence—Consistent with the idea that AS plays important roles in cellular function, bioinformatic and array data indicate that the process is more prominent in tissues with diverse cell types and among genes playing regulatory functions (4Clark T.A. Schweitzer A.C. Chen T.X. Staples M.K. Lu G. Wang H. Williams A. Blume J.E. Genome Biol. 2007; 8: R64Crossref PubMed Scopus (231) Google Scholar, 12Blencowe B.J. Cell. 2006; 126: 37-47Abstract Full Text Full Text PDF PubMed Scopus (832) Google Scholar, 14Pan Q. Shai O. Misquitta C. Zhang W. Saltzman A.L. Mohammad N. Babak T. Siu H. Hughes T.R. Morris Q.D. Frey B.J. Blencowe B.J. Mol. Cell. 2004; 16: 929-941Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). An important insight from global studies in a variety of biological situations is that the overlap between genes that show changes in AS and those regulated through changes in transcript levels is relatively limited (but also see Ref. 18Li H.R. Wang-Rodriguez J. Nair T.M. Yeakley J.M. Kwon Y.S. Bibikova M. Zheng C. Zhou L. Zhang K. Downs T. Fu X.D. Fan J.B. Cancer Res. 2006; 66: 4079-4088Crossref PubMed Scopus (69) Google Scholar). This observation suggests the existence of dedicated regulatory programs that coordinately control multiple AS events. Such programs are evident, for example, in Drosophila sex determination (19McIntyre L.M. Bono L.M. Genissel A. Westerman R. Junk D. Telonis-Scott M. Harshman L. Wayne M.L. Kopp A. Nuzhdin S.V. Genome Biol. 2006; 7: R79Crossref PubMed Scopus (79) Google Scholar) and mammalian synaptic transmission (20Ule J. Ule A. Spencer J. Williams A. Hu J.S. Cline M. Wang H. Clark T. Fraser C. Ruggiu M. Zeeberg B.R. Kane D. Weinstein J.N. Blume J. Darnell R.B. Nat. Genet. 2005; 37: 844-852Crossref PubMed Scopus (396) Google Scholar, 21Ule J. Stefani G. Mele A. Ruggiu M. Wang X. Taneri B. Gaasterland T. Blencowe B.J. Darnell R.B. Nature. 2006; 444: 580-586Crossref PubMed Scopus (404) Google Scholar) and strongly argue for the biological relevance of AS. Predicted Functional Effects—Bioinformatic analyses indicate that 75% of AS events affect coding regions, with predicted effects ranging from subtle amino acid substitutions to removal of protein motifs or protein truncations (10Black D.L. Annu. Rev. Biochem. 2003; 72: 291-336Crossref PubMed Scopus (1970) Google Scholar, 11Matlin A.J. Clark F. Smith C.W. Nat. Rev. Mol. Cell Biol. 2005; 6: 386-398Crossref PubMed Scopus (968) Google Scholar, 12Blencowe B.J. Cell. 2006; 126: 37-47Abstract Full Text Full Text PDF PubMed Scopus (832) Google Scholar). Although compelling instances of biologically relevant changes due to a single amino acid difference exist (13Stamm S. Ben-Ari S. Rafalska I. Tang Y. Zhang Z. Toiber D. Thanaraj T.A. Soreq H. Gene (Amst.). 2005; 344: 1-20Crossref PubMed Scopus (685) Google Scholar, 22Raingo J. Castiglioni A.J. Lipscombe D. Nat. Neurosci. 2007; 10: 285-292Crossref PubMed Scopus (134) Google Scholar), predicting the functional impact of the frequent small variations in protein structure generated by AS is often difficult. For instance, single amino acid insertions due to the use of alternative NAGNAG 3′-splice sites (which occur in up to 30% of human genes) can affect function or be the result of stochastic choice (23Hiller M. Szafranski K. Backofen R. Platzer M. PLoS Genet. 2006; 2 (Author Reply e208): e207Crossref PubMed Scopus (21) Google Scholar). Stochasticity could nevertheless offer an evolutionary testing ground to explore novel functions, a concept also applicable to other classes of AS events. Mapping of AS regions onto solved polypeptide structures indicates that most AS events affect coiled or loop regions often located on the protein surface (24Wang P. Yan B. Guo J.T. Hicks C. Xu Y. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 18920-18925Crossref PubMed Scopus (50) Google Scholar, 25Romero P.R. Zaidi S. Fang Y.Y. Uversky V.N. Radivojac P. Oldfield C.J. Cortese M.S. Sickmeier M. LeGall T. Obradovic Z. Dunker A.K. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8390-8395Crossref PubMed Scopus (339) Google Scholar). Such location could either reflect an impact on functional interactions with other factors or simply reflect which regions of the protein are more likely to tolerate amino acid changes. Although compelling examples of domain swapping by AS exist (9Chen B.E. Kondo M. Garnier A. Watson F.L. Puettmann-Holgado R. Lamar D.R. Schmucker D. Cell. 2006; 125: 607-620Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 22Raingo J. Castiglioni A.J. Lipscombe D. 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However, a genome-wide study found that most premature termination codon-containing splice variants are produced at low levels independently of the function of the nonsense-mediated mRNA decay pathway and are rarely tissue-specific or phylogenetically conserved (16Pan Q. Saltzman A.L. Kim Y.K. Misquitta C. Shai O. Maquat L.E. Frey B.J. Blencowe B.J. Genes Dev. 2006; 20: 153-158Crossref PubMed Scopus (181) Google Scholar). Nonetheless, recent reports highlight the remarkable evolutionary conservation and importance of this mechanism in the control of expression of splicing regulators (28Lareau L.F. Inada M. Green R.E. Wengrod J.C. Brenner S.E. Nature. 2007; 446: 926-929Crossref PubMed Scopus (441) Google Scholar, 29Ni J.Z. Grate L. Donohue J.P. Preston C. Nobida N. O'Brien G. Shiue L. Clark T.A. Blume J.E. Ares Jr., M. Genes Dev. 2007; 21: 708-718Crossref PubMed Scopus (379) Google Scholar, 30Boutz P.L. Chawla G. Stoilov P. Black D.L. 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Evolutionary Conservation—Conserved AS events are more likely to be functionally important because they show a higher tendency to preserve the reading frame, to modify protein-coding sequences, and to conserve regulatory sequences and be tissue-specific (12Blencowe B.J. Cell. 2006; 126: 37-47Abstract Full Text Full Text PDF PubMed Scopus (832) Google Scholar). However, computational predictions indicate that only 10–20% of AS events appear to be conserved between human and mouse, a figure substantially lower than the overall gene conservation between these organisms (12Blencowe B.J. Cell. 2006; 126: 37-47Abstract Full Text Full Text PDF PubMed Scopus (832) Google Scholar, 34Pan Q. Bakowski M.A. Morris Q. Zhang W. Frey B.J. Hughes T.R. Blencowe B.J. Trends Genet. 2005; 21: 73-77Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Although this figure may increase as more extensive data sets become available, does it argue against the functional relevance of the majority of AS events? An alternative view is that AS can play important roles in the evolution of individual genes. A compelling example is the exonization of transposable elements present in intronic regions of some vertebrate genes, which evolved mechanisms of limited exon inclusion that facilitate exploration of novel functions without compromising expression of the host gene (35Sorek R. RNA. 2007; 13: 1603-1608Crossref PubMed Scopus (140) Google Scholar). There is substantial evidence for the creation or loss of tens of thousands of exons across vertebrate genomes, which can function as evolutionary "hot spots" (36Alekseyenko A.V. Kim N. Lee C.J. RNA. 2007; 13: 661-670Crossref PubMed Scopus (99) Google Scholar). In summary, neither simple structure/function predictions nor extensive phylogenetic comparisons provide straightforward evidence of biological relevance for the majority of AS events. However, evidence accumulated through classical gene-by-gene studies and persuasive examples of coordinated regulation of biological processes through AS readily justify future systematic functional analyses of AS isoforms. As elaborated in other minireviews in this series (37House A.E. Lynch K.W. J. Biol. Chem. 2008; 283: 1217-1221Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 38Hertel K.J. J. Biol. Chem. 2008; 283: 1211-1215Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 39Stamm S. J. Biol. 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Bioinformatics—Splicing regulatory sequences have been identified through a variety of approaches (reviewed in Ref.40Chasin, L. A. (2007) in Alternative Splicing in the Postgenomic Era (Blencowe, B. J., and Graveley, B. R., eds) pp. 85–106, Landes Bioscience, Austin, TX, in pressGoogle Scholar), including (i) comparisons between exons in genes and in pseudogenes or intronless genes, (ii) phylogenetic nucleotide conservation not explained by conservation of amino acid sequence, and (iii) correlations between sequence motifs and the strength of the neighboring splice sites. Additional regulatory sequences were identified as characteristic signatures of tissue-specific splicing or of activation of specific signaling pathways (10Black D.L. Annu. Rev. Biochem. 2003; 72: 291-336Crossref PubMed Scopus (1970) Google Scholar, 12Blencowe B.J. Cell. 2006; 126: 37-47Abstract Full Text Full Text PDF PubMed Scopus (832) Google Scholar, 40Chasin, L. A. (2007) in Alternative Splicing in the Postgenomic Era (Blencowe, B. J., and Graveley, B. R., eds) pp. 85–106, Landes Bioscience, Austin, TX, in pressGoogle Scholar). When added to motifs identified as preferred binding sites for known regulatory factors, the picture coming from these studies is that a substantial fraction of exonic and flanking intronic sequences, both constitutively and alternatively spliced, play roles in modulating the splicing process. Furthermore, the extent and nature of these effects sometimes depend on their position relative to splicing signals (41Goren A. Ram O. Amit M. Keren H. Lev-Maor G. Vig I. Pupko T. Ast G. Mol. Cell. 2006; 22: 769-781Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar) or to other regulatory motifs (42Han K. Yeo G. An P. Burge C.B. Grabowski P.J. PLoS Biol. 2005; 3: e158Crossref PubMed Scopus (129) Google Scholar). Combinatorial assemblies of regulatory motifs and factors act on individual transcripts to facilitate or preclude splice site recognition by the spliceosome and thus establish patterns of AS. Cell type-specific splicing can be achieved either by expression of cell type-specific regulators or through cell type-specific variations in the levels or activity of more ubiquitous factors (10Black D.L. Annu. Rev. Biochem. 2003; 72: 291-336Crossref PubMed Scopus (1970) Google Scholar, 11Matlin A.J. Clark F. Smith C.W. Nat. Rev. Mol. Cell Biol. 2005; 6: 386-398Crossref PubMed Scopus (968) Google Scholar, 12Blencowe B.J. Cell. 2006; 126: 37-47Abstract Full Text Full Text PDF PubMed Scopus (832) Google Scholar). Splicing-sensitive Microarrays—Various platforms for high-throughput detection of splice variants and changes in AS are contributing to decipher these cellular codes (Fig. 2B and supplemental Table 1). First, results from these approaches have increased substantially the number of known tissue-specific AS events (4Clark T.A. Schweitzer A.C. Chen T.X. Staples M.K. Lu G. Wang H. Williams A. Blume J.E. Genome Biol. 2007; 8: R64Crossref PubMed Scopus (231) Google Scholar, 51Sugnet C.W. Srinivasan K. Clark T.A. O'Brien G. Cline M.S. Wang H. Williams A. Kulp D. Blume J.E. Haussler D. Ares Jr., M. PLoS Comput. Biol. 2006; 2: e4Crossref PubMed Scopus (166) Google Scholar, 52Das D. Clark T.A. Schweitzer A. Yamamoto M. Marr H. Arribere J. Minovitsky S. Poliakov A. Dubchak I. Blume J.E. Conboy J.G. Nucleic Acids Res. 2007; 35: 4845-4857Crossref PubMed Scopus (65) Google Scholar, 53Fagnani M. Barash Y. Ip J. Misquitta C. Pan Q. Saltzman A.L. Shai O. Lee L. Rozenhek A. Mohammad N. Willaime-Morawek S. Babak T. Zhang W. Hughes T.R. van der Kooy D. Frey B.J. Blencowe B.J. 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Genes Dev. 2005; 19: 1306-1314Crossref PubMed Scopus (100) Google Scholar) or ubiquitous (45Blanchette M. Green R.E. Brenner S.E. Rio D.C. Genes Dev. 2005; 19: 1306-1314Crossref PubMed Scopus (100) Google Scholar, 46Boutz P.L. Stoilov P. Li Q. Lin C.H. Chawla G. Ostrow K. Shiue L. Ares Jr., M. Black D.L. Genes Dev. 2007; 21: 1636-1652Crossref PubMed Scopus (393) Google Scholar, 47Karni R. de Stanchina E. Lowe S.W. Sinha R. Mu D. Krainer A.R. Nat. Struct. Mol. Biol. 2007; 14: 185-193Crossref PubMed Scopus (687) Google Scholar) regulators. In addition, results from yeast arrays and systematic RNA interference screens in Drosophila revealed substrate-specific splicing defects and changes in AS caused by depletion of core components of the spliceosome, uncovering the unexpected regulatory potential of these factors (44Clark T.A. Sugnet C.W. Ares Jr., M. Science. 2002; 296: 907-910Crossref PubMed Scopus (314) Google Scholar, 48Pleiss J.A. Whitworth G.B. Bergkessel M. Guthrie C. PLoS. 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Genes Dev. 2005; 19: 1306-1314Crossref PubMed Scopus (100) Google Scholar). For example, the targets of a soma-specific splicing regulator (PSI) were included within a subset of the targets of a ubiquitous hnRNP protein (Hrp48), suggesting that PSI requires Hrp48 as a cofactor, whereas Hrp48 has additional, PSI-independent functions. Finally, important cellular regulatory circuits have been uncovered using splicing microarrays. These include, for instance, changes in AS relevant for cell transformation induced by the SR protein SF2/ASF (47Karni R. de Stanchina E. Lowe S.W. Sinha R. Mu D. Krainer A.R. Nat. Struct. Mol. Biol. 2007; 14: 185-193Crossref PubMed Scopus (687) Google Scholar) (see below) and regulation of a neuron-specific form of PTB (nPTB) by the more ubiquitous isoform of this splicing regulator, a feedback mechanism that controls an extensive alternative splicing program during neural development (46Boutz P.L. Stoilov P. Li Q. Lin C.H. Chawla G. Ostrow K. Shiue L. Ares Jr., M. Black D.L. Genes Dev. 2007; 21: 1636-1652Crossref PubMed Scopus (393) Google Scholar). Combining Technologies—The combined use of experimental and computational approaches has been very fruitful for identifying regulatory sequences (50Wang Z. Rolish M.E. Yeo G. Tung V. Mawson M. Burge C.B. Cell. 2004; 119: 831-845Abstract Full Text Full Text PDF PubMed Scopus (521) Google Scholar, 51Sugnet C.W. Srinivasan K. Clark T.A. O'Brien G. Cline M.S. Wang H. Williams A. Kulp D. Blume J.E. Haussler D. Ares Jr., M. PLoS Comput. Biol. 2006; 2: e4Crossref PubMed Scopus (166) Google Scholar), including motifs involved in tissue-specific AS (52Das D. Clark T.A. Schweitzer A. Yamamoto M. Marr H. Arribere J. Minovitsky S. Poliakov A. Dubchak I. Blume J.E. Conboy J.G. Nucleic Acids Res. 2007; 35: 4845-4857Crossref PubMed Scopus (65) Google Scholar, 53Fagnani M. Barash Y. Ip J. Misquitta C. Pan Q. Saltzman A.L. Shai O. Lee L. Rozenhek A. Mohammad N. Willaime-Morawek S. Babak T. Zhang W. Hughes T.R. van der Kooy D. Frey B.J. Blencowe B.J. Genome Biol. 2007; 8: R108Crossref PubMed Scopus (82) Google Scholar). Interestingly, although muscle-specific sequence motifs were largely overlapping with known binding sites for muscle-specific regulators (52Das D. Clark T.A. Schweitzer A. Yamamoto M. Marr H. Arribere J. Minovitsky S. Poliakov A. Dubchak I. Blume J.E. Conboy J.G. Nucleic Acids Res. 2007; 35: 4845-4857Crossref PubMed Scopus (65) Google Scholar), numerous potential novel regulatory sequences for nervous system-specific splicing were found (53Fagnani M. Barash Y. Ip J. Misquitta C. Pan Q. Saltzman A.L. Shai O. Lee L. Rozenhek A. Mohammad N. Willaime-Morawek S. Babak T. Zhang W. Hughes T.R. van der Kooy D. Frey B.J. Blencowe B.J. Genome Biol. 2007; 8: R108Crossref PubMed Scopus (82) Google Scholar), consistent with the high prevalence of AS in this tissue (4Clark T.A. Schweitzer A.C. Chen T.X. Staples M.K. Lu G. Wang H. Williams A. Blume J.E. 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Interestingly, a significant fraction of the protein products of these genes were known to form complexes, suggesting that AS modulates the function of these complexes to shape the synapse. As illustrated by the examples below, a mounting body of evidence implicates splicing defects and altered splicing regulation as causes or modifiers of numerous pathologies. Systematic screening for mRNA defects indicates that 50% of the mutations identified in patients with neurofibromatosis or ataxia-telangiectasia affect splicing of the NF1 or ATM genes, respectively (55Pagani F. Baralle F.E. Nat. Rev. Genet. 2004; 5: 389-396Crossref PubMed Scopus (463) Google Scholar). Noncoding nucleotide expansion disorders affect the function of splicing factors: CTG expansions characteristic of myotonic dystrophy patients, transcribed into RNA, alter the activity of CUG-binding proteins, resulting in changes in AS of other genes that can explain some associated symptoms (56Ranum L.P. Cooper T.A. Annu. Rev. Neurosci. 2006; 291: 257-277Google Scholar). Overexpression of the SR protein SF2/ASF leads to efficient cell transformation and tumor formation by altering the ratios between isoforms of key regulators of cell growth (47Karni R. de Stanchina E. Lowe S.W. Sinha R. Mu D. Krainer A.R. Nat. Struct. Mol. Biol. 2007; 14: 185-193Crossref PubMed Scopus (687) Google Scholar). High-throughput technologies for detecting AS hold the promise of improved diagnostic and prognostic tools (57Brinkman B.M. Clin. Biochem. 2004; 37: 584-594Crossref PubMed Scopus (214) Google Scholar). Both computational predictions and microarray experiments have identified hundreds of AS and aberrant splicing events associated with disease states, particularly various cancers (18Li H.R. Wang-Rodriguez J. Nair T.M. Yeakley J.M. Kwon Y.S. Bibikova M. Zheng C. Zhou L. Zhang K. Downs T. Fu X.D. Fan J.B. Cancer Res. 2006; 66: 4079-4088Crossref PubMed Scopus (69) Google Scholar, 58Wang Z. Lo H.S. Yang H. Gere S. Hu Y. 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Consistent with the notion that changes in AS can be relevant for cellular phenotypes, correlations between transcript features and lymphoma grade, proper classification of histologically distinct glial brain tumors, and improved diagnosis of prostate cancer based upon differential expression of exons have been reported using splicing microarrays (63Relogio A. Ben-Dov C. Baum M. Ruggiu M. Gemund C. Benes V. Darnell R.B. Valcárcel J. J. Biol. Chem. 2005; 280: 4779-4784Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 66Zhang C. Li H.R. Fan J.B. Wang-Rodriguez J. Downs T. Fu X.D. Zhang M.Q. BMC Bioinformatics. 2006; 7: 202Crossref PubMed Scopus (84) Google Scholar, 67French P.J. Peeters J. Horsman S. Duijm E. Siccama I. van den Bent M.J. Luider T.M. Kros J.M. van der Spek P. Sillevis Smitt P.A. Cancer Res. 2007; 67: 5635-5642Crossref PubMed Scopus (74) Google Scholar). Other medically relevant insights provided by these studies include a potential autocrine mechanism for the development of choriocarcinomas, ectopic functional expression of neuron-specific splicing regulators in lymphomas, and the influence of growth substrate on the expression profile of breast cancer cell lines (61Yeakley J.M. Fan J.B. Doucet D. Luo L. Wickham E. Ye Z. Chee M.S. Fu X.D. Nat. Biotechnol. 2002; 20: 353-358Crossref PubMed Scopus (168) Google Scholar, 63Relogio A. Ben-Dov C. Baum M. Ruggiu M. Gemund C. Benes V. Darnell R.B. Valcárcel J. J. Biol. Chem. 2005; 280: 4779-4784Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 69Li C. Kato M. Shiue L. Shively J.E. Ares Jr., M. Lin R.J. Cancer Res. 2006; 66: 1990-1999Crossref PubMed Scopus (67) Google Scholar). Detailed knowledge about isoform expression provides the possibility to identify novel, more specific, and safer targets for drug design (57Brinkman B.M. Clin. Biochem. 2004; 37: 584-594Crossref PubMed Scopus (214) Google Scholar). For example, voltage-independent inhibition of N-type calcium channels in the pain pathway depends critically on an alternatively spliced exon that makes these channels more sensitive to neurotransmitters and drugs (22Raingo J. Castiglioni A.J. Lipscombe D. Nat. Neurosci. 2007; 10: 285-292Crossref PubMed Scopus (134) Google Scholar). In this regard, individual variation in splicing patterns related to population haplotypes (5Kwan T. Benovoy D. Dias C. Gurd S. Serre D. Zuzan H. Clark T.A. Schweitzer A. Staples M.K. Wang H. Blume J.E. Hudson T.J. Sladek R. Majewski J. Genome Res. 2007; 17: 1210-1218Crossref PubMed Scopus (89) Google Scholar, 6Hull J. Campino S. Rowlands K. Chan M.S. Copley R.R. Taylor M.S. Rockett K. Elvidge G. Keating B. Knight J. Kwiatkowski D. PLoS Genet. 2007; 3: e99Crossref PubMed Scopus (126) Google Scholar) may add yet another dimension to personalized medicine. Likely additions to the arsenal of high-throughput technologies to study AS include proteomic technologies able to distinguish and quantify protein isoforms. The potential of two-dimensional difference gel electrophoresis, which compares protein samples labeled with different fluorescent dyes, has recently been illustrated by the discovery of cross-regulation and functional redundancy between paralogs of the splicing regulator PTB (31Spellman R. Llorian M. Smith C.W. Mol. Cell. 2007; 27: 420-434Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). High-throughput comparative mass spectrometry, e.g. through differential isotope labeling methods, holds the promise to quantitatively measure protein variants. Spectacular advances in sequencing technology output (ultrasequencing) are likely to represent a major breakthrough for biological research. The possibility to directly and unambiguously measure the abundance of individual sequences is bound to have a deep impact on global analyses of AS. Additional pending issues in demand of technical developments include the simultaneous detection of combinations of different splicing events occurring in individual transcripts, which requires single molecule detection techniques (70Zhu J. Shendure J. Mitra R.D. Church G.M. Science. 2003; 301: 836-838Crossref PubMed Scopus (85) Google Scholar, 71Calarco, J. A., Saltzman, A. L., Ip, J. Y., and Blencowe, B. J. (2007) in Alternative Splicing in the Postgenomic Era (Blencowe, B. J., and Graveley, B. R., eds) pp. 65–85, Landes Bioscience, Austin, TX, in pressGoogle Scholar), and the identification of novel AS events, which can be helped by the construction of cDNA libraries enriched in alternatively spliced transcripts (72Watahiki A. Waki K. Hayatsu N. Shiraki T. Kondo S. Nakamura M. Sasaki D. Arakawa T. Kawai J. Harbers M. Hayashizaki Y. Carninci P. Nat. Methods. 2004; 1: 233-239Crossref PubMed Scopus (40) Google Scholar). Technologies enabling genome-wide analyses are likely to become standard tools for addressing virtually every functional, mechanistic, medical, or evolutionary question in gene function and AS. The possibility to combine various technologies (e.g. cross-linking and immunoprecipitation with microarray analyses), particularly merging large-scale data gathering with bioinformatic methods, will be of great value for modeling biological impact and regulatory mechanisms and networks (21Ule J. Stefani G. Mele A. Ruggiu M. Wang X. Taneri B. Gaasterland T. Blencowe B.J. Darnell R.B. Nature. 2006; 444: 580-586Crossref PubMed Scopus (404) Google Scholar). We thank Ben Blencowe and Chris Smith for comments on the manuscript. Download .pdf (.04 MB) Help with pdf files
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