Calarco et al 2007 - University of Toronto

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Downloaded from genesdev.cshlp.org on June 10, 2013 - Published by Cold Spring Harbor Laboratory PressCalarco et al.Figure 3. Transcript and AS level differences between humansand chimpanzees involve largely distinct subsets of genes. Thecolor spectrum plots compare splicing level differences andtranscript level differences for the same set of genes expressed inthe frontal cortex and heart. For each tissue comparison, the leftcolumn shows splicing differences (measured as the magnitudeof percent inclusion difference, columns 1 and 3) and the rightcolumn measures differences in gene transcript level (measuredas the magnitude of the hyperbolic arcsine [arcsinh] difference,columns 2 and 4). An hyperbolic arcsine difference of ∼0.4 correspondsto a 1.5-fold change in expression level, and a differenceof ∼0.7 corresponds to a twofold difference in expressionlevel.Alternative splicing differences are consistent betweenhuman and chimpanzee individualsSince the analyses described above employed samples ofpoly(A) + mRNA pooled, in each case, from several humanindividuals and from several chimpanzee individuals(Supplementary Table 2), it was important to assessthe extent to which the alternative splicing differencesmight be explained by variations between individualsfrom each species. Individual samples comprising thepools of tissue mRNAs were therefore analyzed separatelyfor splicing level differences. In each case examined,similar alternative splicing differences were observedbetween the several human and chimpanzee individuals(Fig. 5B; data not shown). These data indicatethat it is highly unlikely that the overall differences insplicing levels measured using the pooled samples areattributed to differences associated with individualvariation within each species. This conclusion was furthersupported by an analysis of alternative splicing leveldifferences in primary fibroblasts and lymphoblastoidcells, also from multiple additional individuals fromeach species (Supplementary Fig. 1). Again, in all casesexamined, similar species-associated splicing level differenceswere detected between the different individualsas detected in the tissue samples (Supplementary Fig. 1;see below). The results from this last experiment furtherand disease (Table 1). In most cases the alternative splicingdifferences were observed in both frontal cortex andheart tissues (Figs. 4, 5) as well as in cell lines fromsimilar origins from both species (Supplementary Fig. 1;see below). An example of an alternative splicing differencedetected using the comparative genomics approachinvolves transcripts encoding the TATA-box-bindingprotein-associated factor 6 (TAF6) (Fig. 4; SupplementaryFig. 1; see Table 1 and below for additional examples andinformation). Examples of alternative splicing differencesdetected in the microarray data affect transcriptsencoding the herpesvirus entry mediator receptor(HVEM/TNFRSF14), the spectrin-binding protein -Adducin(ADD3), ADP-ribosylation factor-like 3 (ARL3),serine/arginine (SR)-repeat family protein splicing factorof 40 kDa (SRp40/SFRS5), and GSTO2 (Figs. 4, 5; seeTable 1 and below for additional examples and information).To assess whether these observed differences arespecifically associated with the human or chimpanzeelineage, RT–PCR assays were also performed using RNAextracted from frontal cortex and heart tissue from rhesusmacaques, an Old World monkey, as an outgroupcomparison. The results of this analysis are summarizedbelow and in Supplementary Table 3, and representativeexamples are shown in Figure 5A.Figure 4. Examples of alternative splicing differences betweenhumans and chimpanzees confirmed by semiquantitative RT–PCR and sequencing. RT–PCR assays were performed usingprimers specific for sequences in constitutive exons flankingthe alternative exons predicted to be differentially spliced by thecomparative genomic or alternative splicing microarray profilinganalyses. Corresponding tissues from human (Hs) and chimpanzee(Pt) are indicated. Major splice isoforms that include andskip alternative exons (black boxes) are indicated on the right ofeach panel. Diagrams below each gel illustrate the predictedconsequence of alternative splicing changes at the protein and/or transcript levels for TAF6, ADD3, the SR-repeat family proteinsplicing factor of 40 kDa (SFRS5/SRp40) and GSTO2 (referto Table 1 and main text for details). Protein domains are labeledas follows: (H) head domain; (N) neck domain; (C) C-terminal tail region known to interact with spectrin; (RRM1)RNA recognition motif 1; (RRM2) RNA recognition motif 2;(RS) arginine/serine-rich domain; (GST) glutathione S-transferasedomain. The stop sign indicates the insertion of a PTC. Theasterisk indicates an additional TAF6 splice isoform detected inhuman but not chimpanzee tissue, as confirmed by sequencing.2968 GENES & DEVELOPMENT
Downloaded from genesdev.cshlp.org on June 10, 2013 - Published by Cold Spring Harbor Laboratory PressSurveying primate alternative splicingFigure 5. Comparisons of AS levels in RNA samplesfrom macaque, human, and chimpanzee individuals. (A)RT–PCR assays were performed using rhesus macaquetotal RNA from frontal cortex and heart samples from anumber of different individuals. RT–PCR primers weredesigned to anneal to the exons neighboring each alternativeexon, resulting in the amplification of two products(the isoforms including and skipping the alternativeexon, as indicated in each of the panels). Asterisksdenote novel isoforms observed primarily in one speciesbut not the other. The transcripts shown correspond tothe ADD3 (top panel), GSTO2 (second panel), HVEM/TNFRS14 (third panel), and ARL3 (bottom panel) genes.The macaque is used as an outgroup to define the ancestralsplicing pattern for each gene. Examples ofchimpanzee lineage-specific splicing differences (ADD3and ARL3 transcripts) and human lineage-specific splicingdifferences (GSTO2 and TNFRSF14) are shown (seealso Supplementary Table 3 for additional examples).(B) RT–PCR experiments were performed using totalRNA from each of the individual samples that werepooled for analysis in the comparative genomic and microarrayexperiments. The same alternative splicingevents displayed for macaque experiments in A are alsoshown in B for comparison. The labels for each gel lanerepresent the macaque, human, and chimpanzee individualslisted in Supplementary Table 2.indicate that the majority of the splicing level differenceswe observed are unlikely related to possible contributionsfrom environmental or physiological differences,such as diet or stress, since the corresponding celllines from both species were cultured in parallel for severalweeks under identical growth conditions, prior toharvesting.Examples of validated human and chimpanzeealternative splicing differencesThe alternative splicing difference detected in TAF6transcripts is human lineage specific and is pronouncedin both the frontal cortex and heart (Fig. 4). TAF6 is asubunit of the general transcription factor TFIID, whichis involved in gene activation (Sauer et al. 1995). It hasbeen reported that TAF6 isoforms are associated with thecontrol of apoptosis and cell cycle arrest (Wang et al.1997, 2004; Bell et al. 2001). The alternative splicing differencefound between humans and chimpanzees lies inthe 5 untranslated region (UTR), and could thereforeaffect TAF6 regulation at the post-transcriptional ortranslational levels. Consistent with this proposal areprevious observations of alternative splicing events in 5UTRs that affect translational efficiency (Wang et al.1999; Singh et al. 2005). Such a difference impactingTAF6 expression could, in turn, account for some of thedifferences in transcriptional profiles that have been observedbetween humans and chimpanzees (Preuss et al.2004; Khaitovich et al. 2006).The variation in alternative splicing of SRp40, whichis also human lineage specific, is intriguing in light ofthe known roles of SR family members in both constitutiveand regulated splicing (Graveley 2000; Sanford etal. 2005; Lin and Fu 2007). These proteins generally functionin splicing by binding to exonic enhancer sequencesvia their RNA recognition motifs (RRMs) (Fig. 4). Thealternative splicing difference in SRp40 transcripts resultsin the increased inclusion of a highly conservedpremature termination codon (PTC)-containing exon (locatedbetween exons 4 and 5) in human transcripts, inboth frontal cortex and heart tissue, and correspondinglyreduced levels of the shorter transcript encoding the fulllengthprotein, particularly in the heart. Several RNAbindingproteins including SR family members havebeen shown previously to regulate their own expressionlevels by activating the splicing of PTC-containing isoformsthat are subsequently targeted by the process ofnonsense-mediated mRNA decay (Lareau et al. 2007; Niet al. 2007 and references within). Individual SR familymembers are essential and tightly regulated proteins(Graveley 2000; Sanford et al. 2005; Lin and Fu 2007). Itis therefore possible that the differential expression ofthe two SRp40 isoforms described above could be relatedto some of the splicing differences we observed betweenhumans and chimpanzees, including those that are notassociated with nucleotide substitutions.The alternative splicing difference affecting GSTO2transcripts results in pronounced skipping of exon 4 ofGENES & DEVELOPMENT 2969
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