Fig. 3

MSI-driven mutational events in non-coding regions lead to the skipping of mainly alternatively regulated exons in MSI CRC. A Left panel, Venn diagram showing the overlap between exon skipping events in MSI CRC tissue compared to normal colonic tissue and to MSS CRC tissue. B Bar plot displaying the number of mutations in coding MS (expected frameshift mutations) and the number of skipped exons across the whole exome for 46 non-metastatic MSI tumors (TNM stage 2 or 3). C Left panel, pie chart representing the portion of alternative and constitutive exons in normal colorectal tissue (n = 57,030 exons, n = 133 normal tissue samples). Right panel, pie chart presenting the portion of the same alternative and constitutive exons whose expression was deregulated in MSI tumors. The results of the chi-square test between the two pie charts are indicated. D Distribution of exon skipping events according to their frequency in MSI tumors. Left and right panels, exons classified as constitutive (n = 293) or alternative (n = 692), respectively, in normal colonic tissue. Bottom panel, example of IGV-Sashimi plots showing the read coverage for 2 examples of significantly deregulated exons between tumors and normal tissue (right, constitutive and left, alternative). E Percentage overlap of MSI exon skipping between our study cohort and three independent TCGA cohorts. The number of overlapping events and total number of events analyzed are indicated in brackets. The P-values of the enrichments are indicated. F Cell distribution in MSS (Top panel) or in MSI (Bottom panel) according to sample status (tumor or normal tissue) (Left) and cell subset (Right). The total number of cells used was the same that in the Pelka et al. [26] study (n = 371,223) (more details in Fig. S5, also showing the distribution of the different cell types according to the MSI/MSS status of tumors). However, when we performed the splicing analysis, the total number of cells available was reduced to n = 128,370, but with the same distribution of cell types, i.e., no statistically significant difference was observed in the distribution of cell types in MSI/MSS tumor samples in the population and in the dataset with PSI data. The different number of cells were subsampled, and we performed a bootstrapping analysis (10,000 permutations), considering the same distribution of PSI values according to TA or colonocytes in MSS/MSI. In both situations, more than 100 cells in each condition, allowed to identify statistically significant differences. G Boxplot of quantitative index of alternative splicing events in transit amplifying cells (TA) according to MSI tumor status