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Fig. 1 | Genome Biology

Fig. 1

From: Dynamics of alternative splicing during somatic cell reprogramming reveals functions for RNA-binding proteins CPSF3, hnRNP UL1, and TIA1

Fig. 1

Dynamics of alternative splicing and gene expression changes during B cell reprogramming. A Schematic representation of C/EBPα-mediated B cell reprogramming time points and related controls analyzed by RNA-seq. Bα cells: B cells treated for 18 h with β-estradiol to activate C/EBPα. B Stacked bar plot representing cumulative number of events differentially spliced between B cells and subsequent reprogramming stages (x-axis), as well as controls (iPS and ES cells). The y-axis represents the number of differentially spliced events compared to B cells. The upper part corresponds to events with positive ∆PSI values compared to B cells (> 10%), the lower part to events with negative ∆PSI values compared to B cells (< − 10%). Red/orange areas: alternative 3′/5′ splice sites (Alt3/Alt5) respectively; grey areas: retained introns; blue-green areas: cassette exons of increasing complexity (see “Methods”). See also Additional file 1: Figure S1A. C Clusters of cassette exons displaying related profiles of inclusion level changes during B cell reprogramming. Six clusters (out of a total of 12 identified, see Additional file 1: Figure S1B-C) are shown and classified into 4 categories, corresponding to the timing of the main shift observed (left). The y-axis represents scaled percent spliced in (PSI) values. The color of each line corresponds to the membership score of each exon relative to the general trend of the cluster. The size (number of events) of each cluster is indicated (n). D RT-PCR validation of selected cassette exon changes inferred from RNA-seq analyses. AS events assigned to different clusters were analyzed by semi-quantitative RT-PCR and quantified by capillary electrophoresis. Each panel includes a gel representation of the inclusion (top) and skipping (bottom) amplification products of one of the replicates and the corresponding quantification of the duplicates (PSI = molarity of inclusion product / molarity of inclusion + skipping products). Light grey columns: PSI values quantified by RT-PCR; dark grey columns: PSI values quantified by RNA-seq analysis using vast-tools software (n = 2). E Validation of Grhl1 exon 6 and Dnmt3b exon 10 inclusion level changes, previously associated with reprogramming and pluripotency, performed as in panel D. Crosses indicate time points for which PSI values were calculated with low coverage (less than 10 actual reads). F Heatmap displaying correlations between B cell reprogramming stages according to gene expression (blue, top heatmap) and AS (red, bottom heatmap). Color scales represent Pearson correlation coefficient values calculated on the cpm values of the 25% most variably expressed genes or upon the PSI values of the 25% most variable cassette exons. G Gene expression patterns of genes containing the exons belonging to each of the AS clusters in panel C. Genes with expression changes correlating with the cluster centroid or its negative (membership > 0.3) are highlighted in blue and green, respectively, while the grey portion represents the (majority of) genes which follow gene expression profiles that do not match the changes in inclusion patterns of their exon(s). Percentage of concordant/contrasting patterns are displayed for each cluster. See also Additional file 1: Figure S1D. H Stacked bar plot representing the percentage of cassette exons in each of the AS clusters in panel C classified according to the following categories: disrupting the open reading frame (ORF) upon exclusion or inclusion, preserving the transcript ORF, mapping in non-coding RNAs, 3′ UTRs or 5′ UTRs or uncertain. The first column represents all exons differentially spliced between at least one pair of conditions, while the following ones represent the exons belonging to the each AS cluster (indicated below the bar). Fractions < 5% are not indicated. Where indicated, statistical significance was calculated by Fisher’s exact test on the proportion of exons in the cluster compared to the general distribution of all AS exons (*, **, *** = p value < 0.05, 0.01, 0.001 respectively)

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