Expression of 24,426 human alternative splicing events and predicted cis regulation in 48 tissues and cell lines - PubMed (original) (raw)

. 2008 Dec;40(12):1416-25.

doi: 10.1038/ng.264. Epub 2008 Nov 2.

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Expression of 24,426 human alternative splicing events and predicted cis regulation in 48 tissues and cell lines

John C Castle et al. Nat Genet. 2008 Dec.

Abstract

Alternative pre-messenger RNA splicing influences development, physiology and disease, but its regulation in humans is not well understood, partially because of the limited scale at which the expression of specific splicing events has been measured. We generated the first genome-scale expression compendium of human alternative splicing events using custom whole-transcript microarrays monitoring expression of 24,426 alternative splicing events in 48 diverse human samples. Over 11,700 genes and 9,500 splicing events were differentially expressed, providing a rich resource for studying splicing regulation. An unbiased, systematic screen of 21,760 4-mer to 7-mer words for cis-regulatory motifs identified 143 RNA 'words' enriched near regulated cassette exons, including six clusters of motifs represented by UCUCU, UGCAUG, UGCU, UGUGU, UUUU and AGGG, which map to trans-acting regulators PTB, Fox, Muscleblind, CELF/CUG-BP, TIA-1 and hnRNP F/H, respectively. Each cluster showed a distinct pattern of genomic location and tissue specificity. For example, UCUCU occurs 110 to 35 nucleotides preceding cassette exons upregulated in brain and striated muscle but depleted in other tissues. UCUCU and UGCAUG seem to have similar function but independent action, occurring 5' and 3', respectively, of 33% of the cassette exons upregulated in skeletal muscle but co-occurring for only 2%.

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Figures

Figure 1

Figure 1

Interrogation of A2BP1 (Fox-1) isoforms on the alternative splicing microarrays. A) The nineteen exons in the locus, including alternative 5' exons (green), mutually exclusive exons (blue), a cassette exon (gray), and an alternative 3' splice site (red). Exon (red) and exon-exon junction (green) probes are displayed for several splice-related microarrays. B) Expression (log10 fold-change to reference pool) of exons (red dots) and junctions (green crosses) in transcripts NM_018723 and NM_145893 in skeletal muscle (cyan line) and brain (red line). C) Expression of the gene, exon 15, exon 14, and the combined splice event expression level. Red shading represents microarray intensity values; dark pink shading (right) represents more significant p-values.

Figure 2

Figure 2

A) The number of events monitored on the arrays and the percentage of each type differentially expressed in at least one tissue. B) The number of events differentially expressed in each tissue. C) The log10 ratio of the number of cassette exons differentially included to those differentially excluded.

Figure 3

Figure 3

CLK1 and CLK2 gene (left) and splice event expression (right).

Figure 4

Figure 4

A) Identification of alternative splicing motifs enriched in eight regions adjacent cassette exons upregulated in skeletal muscle. Abbreviations: exon5 (exon3), 5' (3') portion of cassette exons; uif (dif), intronic fraction upstream (downstream); uexon3 (dexon5) and udif (duif) are the corresponding regions adjacent the upstream (downstream) exons. Sorted pentamer and hexamer enrichment in three exemplary regions are shown. Green points are −log10 p-values; magenta points are those with a Bonferroni-corrected p-value less than 0.01. Positive (negative) p-values indicate motif enrichment (depletion). The black line represents the average of 200 randomized runs. B) Enrichment of UCUCU in human tissues and cell lines. Left axis: enrichment of UCUCU in the 200 nt upstream of upregulated cassette exons. Right axis: gene expression of PTBP1. C) Enrichment of UGCAUG. Left axis: enrichment of UGCAUG in the 200 nt downstream of upregulated cassette exons. Right axis: gene expressions of Fox proteins A2BP1 and RBM9. Displayed p-values are not Bonferroni corrected.

Figure 5

Figure 5

Motif enrichment upstream of cassette exons upregulated in human tissues and cell lines. Values (−log10 e-value) are positive for enrichment and negative for depletion. Motifs and tissues are clustered using agglomerative clustering of the enrichment values.

Figure 6

Figure 6

Motif enrichment downstream of cassette exons upregulated in human tissues and cell lines. Values (−log10 e-value) are positive for enrichment and negative for depletion. Motifs and tissues are clustered using agglomerative clustering of the enrichment values.

Figure 7

Figure 7

Higher resolution enrichment of UGCAUG adjacent heart regulated cassette exons. Middle: smoothed fraction of the cassette exons with UGCAUG. Red, heart upregulated cassette exons; blue, heart downregulated; green, all monitored exons; filled gray regions, RefSeq exons. Bottom: hypergeometric probability (−log10 p-value) for up- and downregulated exons relative to all monitored exons.

Figure 8

Figure 8

Higher resolution enrichment of UCUCU adjacent regulated cassette exons. A) Smoothed fraction of the cassette exons with UGCAUG in cerebellum (upper) and fetal kidney (lower). Red, upregulated cassette exons; blue, downregulated; green, all monitored exons; filled gray regions, RefSeq exons. Maximum significance in cerebellum/uif is p-value < 1e-22; maximum proportion is 52. B) Hypergeometric probability (−log10 p-value) for up- and down-regulated exons relative to all monitored exons. At each point, the most significant p-value associated with either up or down regulated exons is shown. Samples are ordered alphabetically as per Figure 5.

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