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Downloaded from genesdev.cshlp.org on June 10, 2013 - Published by Cold Spring Harbor Laboratory PressCalarco et al.Figure 2. Quantitative alternative splicing microarray profilingreveals the extent of alternative splicing differences betweenhuman and chimpanzee orthologous exons, as well as the degreeof divergence between splicing patterns over different evolutionarytime periods. (A) Color spectrum plots indicating thenumber and magnitude of alternative splicing differences betweenhuman and chimpanzee frontal cortex and heart tissues.The Y-axes indicate the number of alternative splicing eventsprofiled; these are sorted according to the magnitude of the absolutevalue of the percent exon inclusion level (%in) differencebetween the human and chimpanzee tissue being compared.The magnitude of the percentage inclusion difference is indicatedby the color scale on the right. (B) Cumulative distributionplot displaying the distribution of percentage inclusion differenceswhen comparing microarray data for 217 conservedalternative splicing events between the following pairs of tissues:human and chimpanzee frontal cortex (blue line), humanand chimpanzee heart (red line), human frontal cortex andmouse cortex (green line), and human and mouse heart (purpleline). (C) Spearman correlation coefficients are shown for pairwisecomparisons between alternative splicing levels (blacknumbers) and transcript levels (purple numbers) for the set of217 orthologous genes analyzed in human, chimpanzee, andmouse tissues in B. Double arrows indicate the pairs of speciescompared. (Hs) Homo sapiens; (Pt) Pan troglodytes; (Mm) Musmusculus.despite the remarkable degree of conservation betweenthe coding regions of the human and chimpanzee genomes,a substantial number of alternative exons thatare not associated with high substitution rates in thealternative exons and flanking intron regions displaypronounced splicing level differences between the twospecies.Evidence for stabilizing selection pressure acting topreserve the majority of splicing levels of orthologoushuman, chimpanzee, and mouse alternative exonsThe similarity between the splicing levels for the majorityof the profiled orthologous exons could reflect stabilizingselection pressure acting to conserve the inclusionlevels of most human and chimpanzee orthologous exons.However, it is also possible that insufficient evolutionarytime has accumulated to result in a higher incidenceof pronounced inclusion level differences than observedabove. To investigate these possibilities, wecompared the extent of divergence between alternativesplicing profiles representing a longer evolutionary timespan, namely the 80- to 90-million-year period separatingthe common ancestor of human or chimpanzee andmouse. Percent inclusion values for 217 alternativesplicing events conserved between humans and themouse were obtained from RT–PCR-validated, quantitativealternative splicing microarray profiling data frommouse heart and brain cortex tissues (Fagnani et al. 2007)and compared with percent inclusion values from thehuman and chimpanzee datasets described above (Fig.2B,C; see the Supplemental Material).Notably, although the splicing levels of the mouse exonsare also very similar to the orthologous exons in thecorresponding human and chimpanzee tissues, they havediverged to a significantly greater extent in both tissues(P
Downloaded from genesdev.cshlp.org on June 10, 2013 - Published by Cold Spring Harbor Laboratory PressSurveying primate alternative splicingTable 1. Genes associated with different cellular functions with experimentally validated splicing level differences betweenhumans and chimpanzeesFunctional category Gene name GO processes/known featuresAlternativesplicing-alteredprotein domain(s)SignalingInositol hexaphosphate kinaseInositol trisphosphate-3 kinaseIHPK2 (IHPK2)activityHypothetical protein LOC220686 Phoshpatidylinositol-4 kinase activity PI4KADP-ribosylation factor-like 3 (ARL3) G-protein-coupled receptor protein GTPasesignaling pathwayGene regulation TBP-associated factor 6 (TAF6) TFIID complex, regulator ofNotranscriptionSRp40 (SFRS5) Splicing regulation RRM, RSPoly(A)-binding protein cytoplasmic 4 RNA binding, poly(A) bindingND(PABPC4)Immune response Toll/Interleukin-1 receptor 8 (TIR8) Immune response NDHerpesvirus entry mediator (HVEM/ Immune responseTM, cytoplasmicTNFRSF14)OtherGlutathione S-transferase omega Glutathione transferase activity, ND2 (GSTO2)metabolism of xenobioticsRoundabout 1 homolog (ROBO1) Axon guidance, dyslexia candidate NDgeneFSHD region gene 1 (FRG1)Facioscapulohumoral diseaseActin cross-linkingcandidate-Adducin (ADD3)Calmodulin binding, structuralSpectrin bindingconstituent of cytoskeletonZonula Occludens protein (ZO-1) Tight junction protein NDBridging Integrator 1 (BIN1) Regulation of endocytosis BARCalbindin 2 (CALB2) Calcium ion binding EF handChr. 14 ORF 153 Uncharacterized NDUnc-84 homolog (UNC84B)Microtubule binding, mitotic spindleorganization, and biogenesisNDFunctional annotations from the Gene Ontology database or from literature searches are listed. Protein domains predicted to be alteredby differential alternative splicing are also listed. In most cases, differential alternative splicing was detected in both frontal cortex andheart; exceptions are in IHPK2, frontal cortex only; BIN1, frontal cortex only; CALB2, heart only; and UNC84B, heart only. (IHPK)Inositol hexaphosphate kinase domain; (PI4K) phosphatidylinositol-4 kinase domain; (GTPase) Ras-like GTPase domain; (RRM) RNArecognition motif; (RS), arginine/serine-rich domain; (TM) transmembrane domain; (Cytoplasmic) cytoplasmic signaling domain;(Actin cross-linking) actin cross-linking protein fold; (Spectrin binding) C-terminal region of DD3 proximal to a defined spectrininteractingdomain; (BAR) BIN/amphiphysin/Rvs domain involved in endocytosis; (EF hand) calcium-binding domain; (ND) no knowndomain is affected by alternative splicing.the microarray profiled genes. A comparable proportionof orthologous genes has been found to display at leasttwofold transcript level differences between correspondinghuman and chimpanzee tissues (Preuss et al. 2004;Khaitovich et al. 2005). Remarkably, however, despite aparallel divergence in alternative splicing and transcriptionalpatterns during the evolution of humans andchimpanzees, primarily nonoverlapping subsets of genesare associated with these changes (Fig. 3). By sortinggenes according to the magnitude of the microarray-detectedpercent inclusion alternative splicing level differences(Fig. 3, columns 1 and 3), it is evident that relativelyfew genes display coincident changes in transcriptionallevels (Fig. 3 in cf. columns 1 and 2, and columns3 and 4). Among the 160–240 splicing events with predictedpercent inclusion differences >20% in frontal cortexand/or heart tissues, only 25% are predicted to alsohave twofold or greater steady-state transcript levelchanges. This observation extends previous results indicatingthat predominantly different subsets of genes areregulated in a cell-specific/tissue-specific or activity-dependentmanner at the transcriptional and alternativesplicing levels (Le et al. 2004; Pan et al. 2004; Li et al.2006; Fagnani et al. 2007; Ip et al. 2007). In particular, thepresent results show that these two layers of regulationhave evolved rapidly to affect different subsets of genes,and indicate that alternative splicing has served as anadditional mechanism for diversifying gene regulationduring the 5–7 million years of evolution separating humansand chimpanzees.Alternative splicing differences between humans andchimpanzees affect transcripts from genes associatedwith diverse cellular functionsGenes with alternative splicing differences between humansand chimpanzees detected using the methods describedabove, and confirmed by the RT–PCR assays, areassociated with diverse biological processes includinggene expression, signaling pathways, immune defense,GENES & DEVELOPMENT 2967
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