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. 2006 Jan 15;20(2):153-8.
doi: 10.1101/gad.1382806.

Quantitative microarray profiling provides evidence against widespread coupling of alternative splicing with nonsense-mediated mRNA decay to control gene expression

Qun Pan  1 , Arneet L Saltzman, Yoon Ki Kim, Christine Misquitta, Ofer Shai, Lynne E Maquat, Brendan J Frey, Benjamin J Blencowe
Affiliations

Quantitative microarray profiling provides evidence against widespread coupling of alternative splicing with nonsense-mediated mRNA decay to control gene expression

Qun Pan et al. Genes Dev. .

Abstract

Sequence-based analyses have predicted that approximately 35% of mammalian alternative splicing (AS) events produce premature termination codon (PTC)-containing splice variants that are targeted by the process of nonsense-mediated mRNA decay (NMD). This led to speculation that AS may often regulate gene expression by activating NMD. Using AS microarrays, we show that PTC-containing splice variants are generally produced at uniformly low levels across diverse mammalian cells and tissues, independently of the action of NMD. Our results suggest that most PTC-introducing AS events are not under positive selection pressure and therefore may not contribute important functional roles.

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Figures

Figure 1.
Figure 1.
Microarray comparison of AS and transcript levels of genes containing AS events predicted, based on sequence analysis, to introduce a PTC upon exon inclusion (“PTC-upon inclusion”), introduce a premature termination codon upon exon exclusion (“PTC-upon exclusion”), or not to introduce a PTC (“No PTC”) (refer to Supplementary Tables S1, S2 for more information). Alternative splicing and corresponding transcript levels of genes were profiled in 10 diverse mouse tissues, arranged from left to right in each panel (lanes 1-10: brain, heart, intestine, kidney, liver, lung, muscle, salivary, spleen, and testis). (A) Alternative splicing levels are represented by percent exon exclusion (i.e., the percent of the total transcripts from a gene with a skipped exon, indicated by the color scale). The AS events are ordered from low to high percent exon exclusion (based on the average value for the 10 tissues) down the Y-axis. (B) Transcript levels are represented by the averages of arcsinh-transformed intensity measurements of the probes specific for the flanking constant exons (indicated by the color scale). The order of AS events down the Y-axis in B is the same as in A, allowing a direct comparison. Histograms below the microarray clustergrams in A represent the distribution of AS events with the percent exon exclusion level range indicated. Histograms in B represent the distribution of AS events with the transcript levels indicated. The percent exon exclusion and transcript levels measured in the 10 tissues in each of the PTC-upon inclusion, PTC-upon exclusion, and No PTC categories were resampled 10,000 times to normalize for the different numbers of AS events in each category. The line curves (bottom row) show cumulative distributions of percent exon exclusion or transcript levels of the AS events, using the same data as used to plot the histograms. The distributions of percent exon exclusion levels between the PTC-upon inclusion (blue text)/exclusion (red text) and No PTC categories are significantly different (Wilcoxon rank sum test). In contrast, differences in the distributions of transcript levels between the PTC-upon inclusion/exclusion and No PTC categories are not statistically significant.
Figure 2.
Figure 2.
Microarray analysis of the role of the essential NMD factor UPF1 in controlling AS and transcript levels. (A) Western blot of total cellular protein isolated from HeLa cells 3 d after transfection with either an siRNA that reduces expression of UPF1 (UPF1 kd), or a nonspecific control siRNA (Control) (Kim et al. 2005). The protein lysates were probed with an antibody specific for UPF1 (Lykke-Andersen et al. 2000), and with an anti-Calnexin antibody as a loading control. Serial threefold dilutions of protein in the left three lanes indicate that the Western blot is semiquantitative and permitted an estimation of the UPF1 depletion efficiency at ∼95%. (B) Microarray comparison of percent exon exclusion levels profiled in Control and UPF1-depleted HeLa cells. Genes are ordered down the Y-axis from low to high percent exon exclusion in the Control columns, and the same gene orders are preserved in the UPF1 kd columns. Data for percent exon exclusion levels are shown for the three AS event categories (PTC-upon inclusion/exclusion and No PTC). The color scale is as shown in Figure 1A. (C) The bar graphs indicate the percent of AS events (Y-axis) in the three AS event categories that show an increase, decrease, or no change in percent exon exclusion level, measured in each case as the difference in the percent exon exclusion between the UPF1 kd and Control kd. Different cutoffs (ranging from at least 5% to at least 15%) for the difference in percent exon exclusion levels were used, as indicated on the X-axis. The enrichment for percent exon exclusion changes in the PTC-upon inclusion/exclusion categories that result in increased levels of the PTC-containing variant upon UPF1 knockdown is statistically significant (p < 10-4 for 5%-10% difference and p < 0.05 for 15% difference, Fisher's exact test). (D) The difference in percent exon exclusion values (upper panel) and ratios of transcript levels (lower panel) between the UPF1-depleted and Control samples for the three AS event categories are displayed in cumulative distribution plots. These plots were generated using data for the same AS genes as shown in B, which represent all AS events with GenASAP percent exon exclusion values ranking in the top half of the data (refer to the text). The microarray measurements in the PTC-upon inclusion/exclusion and No PTC were resampled 10,000 times to normalize for different numbers of AS events in each category. The changes in percent exon exclusion levels between the PTC-upon inclusion/exclusion and No PTC categories, upon UPF1 knockdown, are significantly different (Wilcoxon rank sum test). The differences in ratios of transcript levels between the PTC-upon inclusion/exclusion and No PTC categories, upon UPF1 knockdown, are not statistically significant.
Figure 3.
Figure 3.
AS events with the potential to trigger NMD are more often represented by species-specific alternative exons than conserved alternative exons. Percentages of total AS events with or without the potential to activate NMD that are conserved or species-specific in human and mouse are shown. Alternative exons were identified in human and mouse ortholog gene pairs using EST and cDNA sequence data, as described previously (Pan et al. 2005) (see also Materials and Methods), and also scored for their potential to introduce a PTC and elicit NMD. “Conserved AS” indicates detection of sequence-conserved alternative and flanking exons in a human and mouse ortholog pair; “Species-specific AS of conserved exons” indicates a conserved exon that is alternatively spliced in one species and constitutively spliced in the other; and “Genome-specific AS” indicates detection of an AS event in one species and sequence evidence for only the spliced constitutive exons in the other species.
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References

    1. Alonso, C.R. 2005. Nonsense-mediated RNA decay: A molecular system micromanaging individual gene activities and suppressing genomic noise. Bioessays 27: 463-466. - PubMed
    1. Baek, D. and Green, P. 2005. Sequence conservation, relative isoform frequencies, and nonsense-mediated decay in evolutionarily conserved alternative splicing. Proc. Natl. Acad. Sci. 102: 12813-12818. - PMC - PubMed
    1. Baker, K.E. and Parker, R. 2004. Nonsense-mediated mRNA decay: Terminating erroneous gene expression. Curr. Opin. Cell Biol. 16: 293-299. - PubMed
    1. Black, D.L. 2003. Mechanisms of alternative pre-messenger RNA splicing. Annu. Rev. Biochem. 72: 291-336. - PubMed
    1. Caceres, J.F. and Kornblihtt, A.R. 2002. Alternative splicing: Multiple control mechanisms and involvement in human disease. Trends Genet. 18: 186-193. - PubMed

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