Title: Alternative Splicing: New Insights from Global Analyses
Abstract: Recent analyses of sequence and microarray data have suggested that alternative splicing plays a major role in the generation of proteomic and functional diversity in metazoan organisms. Efforts are now being directed at establishing the full repertoire of functionally relevant transcript variants generated by alternative splicing, the specific roles of such variants in normal and disease physiology, and how alternative splicing is coordinated on a global level to achieve cell- and tissue-specific functions. Recent progress in these areas is summarized in this review. Recent analyses of sequence and microarray data have suggested that alternative splicing plays a major role in the generation of proteomic and functional diversity in metazoan organisms. Efforts are now being directed at establishing the full repertoire of functionally relevant transcript variants generated by alternative splicing, the specific roles of such variants in normal and disease physiology, and how alternative splicing is coordinated on a global level to achieve cell- and tissue-specific functions. Recent progress in these areas is summarized in this review. One of the most remarkable observations stemming from the sequencing of genomes of diverse species is that the number of protein-coding genes in an organism does not correlate with its overall cellular complexity. For example, Drosophila melanogaster has fewer protein-coding genes than the nematode Caenorhabditis elegans (∼14,000 versus ∼19,000). Meanwhile, mammalian species have similar numbers of protein-coding genes as Arabidopsis thaliana (∼20,000–25,000) and only four times the number found in the budding yeast Saccharomyces cerevisiae (∼6,000). These observations indicate that mechanisms acting to regulate and diversify gene functions must have played a major role in the evolution of specialized cell types and activities that are typically associated with complex metazoans. Alternative splicing (AS), the process by which the exons of primary transcripts (pre-mRNAs) from genes can be spliced in different arrangements to produce structurally and functionally distinct mRNA and protein variants, may be one of the most extensively used mechanisms that accounts for the greater macromolecular and cellular complexity of higher eukaryotic organisms. AS has numerous critical roles in metazoan organisms (Black, 2003Black D.L. Mechanisms of alternative pre-messenger RNA splicing.Annu. Rev. Biochem. 2003; 72: 291-336Crossref PubMed Scopus (1866) Google Scholar, Matlin et al., 2005Matlin A.J. Clark F. Smith C.W. Understanding alternative splicing: towards a cellular code.Nat. Rev. Mol. Cell Biol. 2005; 6: 386-398Crossref PubMed Scopus (915) Google Scholar). Despite many focused studies on the functions and mechanisms of AS that are associated with specific transcripts, high-throughput experimental approaches for systematically elucidating the roles of AS events are only now beginning to be used. Considerable effort has been directed at the genome-wide identification of AS events in different cell and tissue types and under different conditions in order to establish the extent of functionally relevant AS events. Initial analyses of the resulting data sets are revealing important global regulatory features of AS. The availability of sequenced genomes and large databases of sequenced transcripts, primarily comprising expressed sequence tags (ESTs) and smaller numbers of cDNA sequences, has provided a rich source of information for the identification and analysis of AS events. EST and cDNA sequences can be aligned to genomic sequences using programs that search for conserved splice-site consensus sequences adjacent to the gaps created by intron sequences between the aligned exons. Contigs of genomic exons extracted in this manner are realigned to the corresponding ESTs and cDNAs such that clusters of aligned transcripts with or without middle exon alignments (indicative of an AS event) can be systematically identified. Large databases of AS events mined in this manner have been established for several species, including human, mouse, and rat (Modrek and Lee, 2002Modrek B. Lee C. A genomic view of alternative splicing.Nat. Genet. 2002; 30: 13-19Crossref PubMed Scopus (1020) Google Scholar, Lee et al., 2003Lee C. Atanelov L. Modrek B. Xing Y. ASAP: the Alternative Splicing Annotation Project.Nucleic Acids Res. 2003; 31: 101-105Crossref PubMed Scopus (135) Google Scholar, Thanaraj et al., 2004Thanaraj T.A. Stamm S. Clark F. Riethoven J.J. Le Texier V. Muilu J. ASD: the Alternative Splicing Database.Nucleic Acids Res. 2004; 32: D64-D69Crossref PubMed Google Scholar, Zheng et al., 2005Zheng C.L. Kwon Y.S. Li H.R. Zhang K. Coutinho-Mansfield G. Yang C. Nair T.M. Gribskov M. Fu X.D. MAASE: an alternative splicing database designed for supporting splicing microarray applications.RNA. 2005; 11: 1767-1776Crossref PubMed Scopus (24) Google Scholar). However, a major limitation of AS analyses employing transcript sequence data is that EST coverage is typically biased toward the 3′ and 5′ ends of transcripts, and in general there are insufficient numbers of sequenced transcripts to infer the frequency with which specific alternative exons are included or skipped in a given cell or tissue source or under particular experimental conditions (Johnson et al., 2003Johnson 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. Genome-wide survey of human alternative pre-mRNA splicing with exon junction microarrays.Science. 2003; 302: 2141-2144Crossref PubMed Scopus (1152) Google Scholar, Pan et al., 2004Pan Q. Shai O. Misquitta C. Zhang W. Saltzman A.L. Mohammad N. Babak T. Siu H. Hughes T.R. Morris Q.D. et al.Revealing global regulatory features of mammalian alternative splicing using a quantitative microarray platform.Mol. Cell. 2004; 16: 929-941Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). Some of the limitations inherent in the analysis of EST/cDNA have been overcome by the development of custom microarrays and computational tools, as well as differential hybridization techniques, that permit the large-scale profiling of AS (Yeakley et al., 2002Yeakley J.M. Fan J.B. Doucet D. Luo L. Wickham E. Ye Z. Chee M.S. Fu X.D. Profiling alternative splicing on fiber-optic arrays.Nat. Biotechnol. 2002; 20: 353-358Crossref PubMed Scopus (168) Google Scholar, Johnson et al., 2003Johnson J.M. 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Several of the AS microarrays that have been described contain thousands of sets of anchored oligonucleotide probes on glass slides, with each set typically combining probes that are specific to individual exons and/or splice-junction sequences formed by inclusion or skipping of exons (Figure 1). This type of format has permitted the discovery of new AS events not previously detected in cDNA or EST sequences (Johnson et al., 2003Johnson 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. Genome-wide survey of human alternative pre-mRNA splicing with exon junction microarrays.Science. 2003; 302: 2141-2144Crossref PubMed Scopus (1152) Google Scholar) and the large-scale detection of cell- and tissue-specific AS events involving exons that were initially identified using EST/cDNA sequence data (Pan et al., 2004Pan Q. Shai O. Misquitta C. Zhang W. Saltzman A.L. Mohammad N. Babak T. Siu H. Hughes T.R. Morris Q.D. et al.Revealing global regulatory features of mammalian alternative splicing using a quantitative microarray platform.Mol. Cell. 2004; 16: 929-941Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). More recently, this microarray format has facilitated the global analysis of alternative exons regulated by specific splicing factors (Blanchette et al., 2005Blanchette M. Green R.E. Brenner S.E. Rio D.C. Global analysis of positive and negative pre-mRNA splicing regulators in Drosophila.Genes Dev. 2005; 19: 1306-1314Crossref PubMed Scopus (99) Google Scholar, Ule et al., 2005Ule J. Ule A. Spencer J. Williams A. Hu J.S. Cline M. Wang H. Clark T. Fraser C. Ruggiu M. et al.Nova regulates brain-specific splicing to shape the synapse.Nat. Genet. 2005; 37: 844-852Crossref PubMed Scopus (373) Google Scholar) and has led to the discovery of sequence motifs that correlate with tissue-specific AS (Sugnet et al., 2006Sugnet 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 M. Unusual intron conservation near tissue-regulated exons found by splicing microarrays.PLoS Comput. Biol. 2006; 2: e4Crossref PubMed Scopus (157) Google Scholar). Another microarray format employing a fiber-optic-based system for the detection of specific splice variants has been described, and this approach has been used to monitor splice variants in different transformed cell lines and tumors (Yeakley et al., 2002Yeakley J.M. Fan J.B. Doucet D. Luo L. Wickham E. Ye Z. Chee M.S. Fu X.D. Profiling alternative splicing on fiber-optic arrays.Nat. Biotechnol. 2002; 20: 353-358Crossref PubMed Scopus (168) Google Scholar, Li et al., 2006Li H.R. Wang-Rodriguez J. Nair T.M. Yeakley J.M. Kwon Y.S. Bibikova M. Zheng C. Zhou L. Zhang K. 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Alternative selection of 5′ or 3′ splice sites within exon sequences are also frequent, together accounting for at least one-quarter of the known AS events (Figure 2). This type of AS is capable of introducing subtle changes into coding sequences, differing by as little as a single codon. For example, approximately 30% of human genes contain NAGNAG sequences at the 3′ ends of introns, which have the potential to act as tandem splice-site acceptors (Hiller et al., 2004Hiller M. Huse K. Szafranski K. Jahn N. Hampe J. Schreiber S. Backofen R. Platzer M. Widespread occurrence of alternative splicing at NAGNAG acceptors contributes to proteome plasticity.Nat. Genet. 2004; 36: 1255-1257Crossref PubMed Scopus (144) Google Scholar). However, it is not clear to what extent such subtle variants are functionally significant, and a recent analysis suggests that a large fraction may arise as a consequence of stochastic binding of the spliceosome at neighboring splice sites (Chern et al., 2006Chern T.M. van Nimwegen E. Kai C. Kawai J. Carninci P. Hayashizaki Y. Zavolan M. A simple physical model predicts small exon length variations.PLoS Genet. 2006; 2: e45Crossref PubMed Scopus (62) Google Scholar) (see below). Other types of AS events include retained introns (Ohler et al., 2005Ohler U. Shomron N. Burge C.B. Recognition of unknown conserved alternatively spliced exons.PLoS Comput. Biol. 2005; 1: 113-122Crossref PubMed Scopus (35) Google Scholar) and exons that are spliced in a mutually exclusive fashion (Figure 2). In addition to the AS mechanisms mentioned above, the exon composition of transcripts is often altered by differential selection of transcription initiation and 3′ end processing/termination sites (Figure 2), and these events can impact on adjacent or distal AS events in the same transcript (Zavolan et al., 2003Zavolan M. Kondo S. Schonbach C. Adachi J. Hume D.A. Hayashizaki Y. Gaasterland T. Impact of alternative initiation, splicing, and termination on the diversity of the mRNA transcripts encoded by the mouse transcriptome.Genome Res. 2003; 13: 1290-1300Crossref PubMed Scopus (157) Google Scholar, Kornblihtt, 2005Kornblihtt A.R. Promoter usage and alternative splicing.Curr. Opin. Cell Biol. 2005; 17: 262-268Crossref PubMed Scopus (181) Google Scholar). In several studies, it has been shown that transcription factors acting at the level of initiation and elongation can impact splice-site selection (Kornblihtt, 2006Kornblihtt A.R. Chromatin, transcript elongation and alternative splicing.Nat. Struct. Mol. Biol. 2006; 13: 5-7Crossref PubMed Scopus (120) Google Scholar). In particular, factors resulting in reduced rates of RNA polymerase II (Pol II) elongation can increase the inclusion of alternative exons. One model that has been proposed to explain this effect is that reducing the rate of Pol II elongation kinetically favors the recognition of relatively weak splicing signals surrounding an alternative exon over the inherently stronger splicing signals that otherwise favor splicing of the neighboring upstream and downstream constitutive exons, resulting in skipping of the alternative exon (Kornblihtt, 2006Kornblihtt A.R. Chromatin, transcript elongation and alternative splicing.Nat. Struct. Mol. Biol. 2006; 13: 5-7Crossref PubMed Scopus (120) Google Scholar). It is worth noting that for each type of AS event mentioned above, a distinction can be made between mechanisms controlling splice-site selection involving the binding of regulatory factors and mechanisms that may operate in a stochastic manner. For example, the selection of mutually exclusive exons 2 and 3 in the α-tropomyosin pre-mRNA involves the repression of exon 3 by trans-acting regulatory factors in smooth muscle cells (Gromak et al., 2003Gromak N. Rideau A. Southby J. Scadden A.D. Gooding C. Huttelmaier S. Singer R.H. Smith C.W. The PTB interacting protein raver1 regulates alpha-tropomyosin alternative splicing.EMBO J. 2003; 22: 6356-6364Crossref PubMed Scopus (85) Google Scholar), whereas a stochastic process involving competing base-pairing interactions between intron sequences appears to play an important role in selection of mutually exclusive alternative exons in the Dscam gene of Drosophila (Graveley, 2005Graveley B.R. Mutually exclusive splicing of the insect Dscam pre-mRNA directed by competing intronic RNA secondary structures.Cell. 2005; 123: 65-73Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Remarkably, this gene, which functions in both axon guidance and immune defense (Schmucker et al., 2000Schmucker D. Clemens J.C. Shu H. Worby C.A. Xiao J. Muda M. Dixon J.E. Zipursky S.L. Drosophila Dscam is an axon guidance receptor exhibiting extraordinary molecular diversity.Cell. 2000; 101: 671-684Abstract Full Text Full Text PDF PubMed Scopus (774) Google Scholar, Watson et al., 2005Watson F.L. Puttmann-Holgado R. Thomas F. Lamar D.L. Hughes M. Kondo M. Rebel V.I. Schmucker D. Extensive diversity of Ig-superfamily proteins in the immune system of insects.Science. 2005; 309: 1874-1878Crossref PubMed Scopus (511) Google Scholar, Chen et al., 2006Chen B.E. Kondo M. Garnier A. Watson F.L. Puettmann-Holgado R. Lamar D.R. Schmucker D. The molecular diversity of Dscam is functionally required for neuronal wiring specificity in Drosophila.Cell. 2006; 125: 607-620Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar), has the potential to encode over 38,000 different splice variants, primarily via tandem arrays of mutually exclusive alternative exons, many of which overlap the coding regions for immunoglobulin-like domains. Finally, it should also be kept in mind that each of the types of AS summarized above and shown in Figure 2 can occur within both translated and untranslated regions (UTRs) of transcripts. Consistent with the evidence that RNA Pol II can influence splice-site selection, experimental work indicates that most splicing events, as well as other steps in pre-mRNA processing, occur on pre-mRNA as it emerges from Pol II (Bentley, 2005Bentley D.L. Rules of engagement: co-transcriptional recruitment of pre-mRNA processing factors.Curr. Opin. Cell Biol. 2005; 17: 251-256Crossref PubMed Scopus (378) Google Scholar). Recent evidence further indicates that the exons of nascent transcripts can be “tethered” to Pol II during pre-mRNA processing (Dye et al., 2006Dye M.J. Gromak N. Proudfoot N.J. Exon tethering in transcription by RNA polymerase II.Mol. Cell. 2006; 21: 849-859Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). In addition to potentially participating in AS, tethering of nascent exons to Pol II, together with the formation of networks of interactions across introns and exons (see below), may help to ensure order in the splicing process such that correct pairs of splice sites are united, even when separated by introns that are tens of thousands of bases in length (Ibrahim el et al., 2005Ibrahim el C. Schaal T.D. Hertel K.J. Reed R. Maniatis T. Serine/arginine-rich protein-dependent suppression of exon skipping by exonic splicing enhancers.Proc. Natl. Acad. Sci. USA. 2005; 102: 5002-5007Crossref PubMed Scopus (96) Google Scholar, Dye et al., 2006Dye M.J. Gromak N. Proudfoot N.J. Exon tethering in transcription by RNA polymerase II.Mol. Cell. 2006; 21: 849-859Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Downstream exons are rarely, if ever, inserted between upstream exons, and trans-splicing, although common in nematodes and trypanosomes, also rarely occurs in most metazoan species (Horiuchi and Aigaki, 2006Horiuchi T. Aigaki T. Alternative trans-splicing: a novel mode of pre-mRNA processing.Biol. Cell. 2006; 98: 135-140Crossref PubMed Scopus (84) Google Scholar). However, given the constraints imposed by the transcription and splicing machineries, AS can result in anywhere from a single skipping event per gene to up to tens of thousands of potential splice variants per gene. It has been inferred from the analyses of EST and AS microarray data that over two-thirds of human genes and over 40% of Drosophila genes contain one or more alternative exons (Johnson et al., 2003Johnson 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. Genome-wide survey of human alternative pre-mRNA splicing with exon junction microarrays.Science. 2003; 302: 2141-2144Crossref PubMed Scopus (1152) Google Scholar, Stolc et al., 2004Stolc V. Gauhar Z. Mason C. Halasz G. van Batenburg M.F. Rifkin S.A. Hua S. Herreman T. Tongprasit W. Barbano P.E. et al.A gene expression map for the euchromatic genome of Drosophila melanogaster.Science. 2004; 306: 655-660Crossref PubMed Scopus (243) Google Scholar). In sharp contrast to these estimates, S. cerevisiae has only several known regulated splicing events and lacks orthologs for many factors associated with regulated splicing in metazoans, such as members of the serine/arginine-repeat (SR) and heterogeneous nuclear ribonucleoprotein (hnRNP) families of proteins (Davis et al., 2000Davis C.A. Grate L. Spingola M. Ares Jr., M. Test of intron predictions reveals novel splice sites, alternatively spliced mRNAs and new introns in meiotically regulated genes of yeast.Nucleic Acids Res. 2000; 28: 1700-1706Crossref PubMed Scopus (129) Google Scholar, Boucher et al., 2001Boucher L. Ouzounis C.A. Enright A.J. Blencowe B.J. A genome-wide survey of RS domain proteins.RNA. 2001; 7: 1693-1701PubMed Google Scholar). The lack of sufficiently large data sets of AS microarray data and sequenced ESTs and cDNAs has prevented reliable estimates of the proportions of genes that undergo AS in other organisms (Brett et al., 2002Brett D. Pospisil H. Valcarcel J. Reich J. Bork P. Alternative splicing and genome complexity.Nat. Genet. 2002; 30: 29-30Crossref PubMed Scopus (381) Google Scholar, Kim et al., 2004Kim H. Klein R. Majewski J. Ott J. Estimating rates of alternative splicing in mammals and invertebrates.Nat. Genet. 2004; 36 (author reply 916–917): 915-916Crossref PubMed Scopus (66) Google Scholar). There are also insufficient data currently available to accurately assess the overall number of AS events in any one organism. In the case of human genes known to undergo AS, based on available transcript sequence and microarray profiling data, it is estimated that there are between one and two AS events per multi-intron gene (Lander et al., 2001Lander E.S. Linton L.M. Birren B. Nusbaum C. Zody M.C. Baldwin J. Devon K. Dewar K. Doyle M. FitzHugh W. et al.Initial sequencing and analysis of the human genome.Nature. 2001; 409: 860-921Crossref PubMed Scopus (16517) Google Scholar, Johnson et al., 2003Johnson 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. Genome-wide survey of human alternative pre-mRNA splicing with exon junction microarrays.Science. 2003; 302: 2141-2144Crossref PubMed Scopus (1152) Google Scholar). However, this number may rise considerably as large-scale analyses of AS are performed in more specialized cell and tissue types. In addition, certain genes that undergo “extreme AS,” such as the Dscam gene in Drosophila, are clearly outliers that confound the derivation of accurate estimates for the overall frequency and complexity of AS. Nevertheless, the results of recent bioinformatic and microarray-based analyses have provided several interesting insights into the nature of the evolutionary forces that can impact overall AS frequencies, as well as on additional global features of AS that will be discussed below. Several recent studies have provided evidence that the number of AS events per gene is inversely correlated with gene or paralog copy number, indicating that gene duplication, followed by divergence of paralog functions, may have reduced selection pressure to diversify gene functions by AS (Kopelman et al., 2005Kopelman N.M. Lancet D. Yanai I. Alternative splicing and gene duplication are inversely correlated evolutionary mechanisms.Nat. Genet. 2005; 37: 588-589Crossref PubMed Scopus (119) Google Scholar, Su et al., 2006Su Z. Wang J. Yu J. Huang X. Gu X. Evolution of alternative splicing after gene duplication.Genome Res. 2006; 16: 182-189Crossref PubMed Scopus (115) Google Scholar). Moreover, sequence- and microarray-based analyses have indicated that AS events occur more often in transcripts from genes expressed in functionally complex tissues with diverse cell types, such as the brain and testis, or from genes expressed within individual cell types that have undergone selection to provide diverse functions, such as in the immune system (Modrek et al., 2001Modrek B. Resch A. Grasso C. Lee C. Genome-wide detection of alternative splicing in expressed sequences of human genes.Nucleic Acids Res. 2001; 29: 2850-2859Crossref PubMed Scopus (517) Google Scholar, Johnson et al., 2003Johnson 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. Genome-wide survey of human alternative pre-mRNA splicing with exon junction microarrays.Science. 2003; 302: 2141-2144Crossref PubMed Scopus (1152) Google Scholar, Yeo et al., 2004Yeo G. Holste D. Kreiman G. Burge C.B. Variation in alternative splicing across human tissues.Genome Biol. 2004; 5: R74Crossref PubMed Google Scholar, Watson et al., 2005Watson F.L. Puttmann-Holgado R. Thomas F. Lamar D.L. Hughes M. Kondo M. Rebel V.I. Schmucker D. Extensive diversity of Ig-superfamily proteins in the immune system of insects.Science. 2005; 309: 1874-1878Crossref PubMed Scopus (511) Google Scholar). Indeed, the number of documented examples of regulated AS in the brain is vast, and many of these events are implicated in complex processes such as the control of synaptic plasticity associated with cognition and other neural processes (Lipscombe, 2005Lipscombe D. Neuronal proteins custom designed by alternative splicing.Curr. Opin. Neurobiol. 2005; 15: 358-363Crossref PubMed Scopus (114) Google Scholar, Ule and Darnell, 2006Ule J. Darnell R.B. RNA binding proteins and the regulation of neuronal synaptic plasticity.Curr. Opin. Neurobiol. 2006; 16: 102-110Crossref PubMed Scopus (133) Google Scholar). One example of particular interest involves AS of the apolipoprotein E receptor (Apoer2) gene, which is important for neuronal cell migration during brain development, as well as for long-term potentiation (LTP) in adult mice. Deletion of a single, activity-dependent alternative exon in the Apoer2 gene prevents tyrosine phosphorylation of NMDA receptor subunits by Reelin, a ligand of Apoer2 (Beffert et al., 2005Beffert U. Weeber E.J. Durudas A. Qiu S. Masiulis I. Sweatt J.D. Li W.P. Adelmann G. Frotscher M. Hammer R.E. Herz J. Modulation of synaptic plasticity and memory by Reelin involves differential splicing of the lipoprotein receptor Apoer2.Neuron. 2005; 47: 567-579Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar). Mice missing this Apoer2 exon perform poorly in memory and learning tasks. Besides demonstrating a role for AS of Apoer2 transcripts in LTP, this study also serves to illustrate the importance of targeting individual alternative exons when attempting to understand the regulation of complex biological processes. Independent sources of experimental data from AS microarray profiling, RT-PCR assays, and analyses of EST and cDNA sequences have provided consistent evidence that many alternatively spliced transcripts are low in abundance, especially those that are not conserved over the ∼80 million year time interval separating human and mouse. In fact, comparisons of sequenced transcripts corresponding to large numbers of ortholog gene pairs in human and mouse indicate that only 10%–20% of cassette-type AS events are conserved between these species (Modrek and Lee, 2003Modrek B. Lee C.J. Alternative splicing in the human, mouse and rat genomes is associated with an increased frequency of exon creation and/or loss.Nat. 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The remaining 80%–90% of human- and mouse-specific cassette-type AS events can be separated into two categories: (1) those involving relatively recently gained “genome-specific” exons, which tend to be included at low levels in spliced mRNA (also referred to as “minor-form” variants) (Lev-Maor et al