Fig. 4

Splicing anomalies found in MSI CRCs are frequently caused by MSI-related mutations that occur very early in cancer development, as early as the untransformed dMMR crypt state. A Four examples of genes in which exon skipping is closely related to the status of the DNA MS in the PY at the intronic boundary. B Left panel, boxplots representing the percent spliced included (PSI) values according to deletion size for the 96 MS (ES96). The results of two-sided ANOVA between exons classified as alternative and those classified as constitutive were as follows: ***P-value < .001. In the background, each MS displaying a significant correlation is indicated by a line and dots. Right panel, distribution of 96 MS (ES96) at the intronic boundary (50 to 0 of intron/exon junction), showing an enriched localization very close to the U2AF1/2 binding region in the PY (AG site). C In vitro analysis of HSP110 expression. Upper panel, the RT-PCR products of HSP110 show cDNA fragments with a completed or partial skipped exon (HSP110DE9), in mutated MSI cell lines with large deletion (CO115) and small deletion (HCT116) respectively. Bottom panel: western blotting analysis of HSP110wt and HSP110DE9 mutant proteins in the 3 cell lines (CO115, HCT116 and HCT8). NSB*, non-specific band; MW, molecular weight. Uncropped images are available in additional file 3. D In vitro analysis of TRAF3PIP1 gene. Upper panel, RT-PCR products of TRAF3IP1 showing cDNA fragments with a skipped exon (TRAF3IP1 DE6), in MSI cell lines (CO115 and HCT116). Middle panel, aberrant splicing event is also demonstrated by RT-PCR in endogenous and mutant (intronic MS mutation in the PY) TRAF3IP1 transcripts in both MSI and MSS transfected cells with minigene construction. EV, empty vector; WT, wild type; Splice Ratio = ratio of the intensity of the exon-lacking cDNA fragment to the intensity of the sum of exon-containing + exon lacking cDNA fragments. Lower panel, gel mobility shift assay. Nuclear protein extracts from MSS (left panel) and/or MSS (right panel) cells were incubated with TRAF3IP1 wild type or mutant labeled RNA probe. Shortened RNA probes do no longer allow the formation of large RNA/protein complexes. Uncropped images are available in Additional file 3. E Left panel, bar plots of microsatellites in the ES96 and C129 sub-signatures (consisting of coding MS covered in at least 50% of the samples and mutated in 30% of all lesions, n = 129) according to their mutational frequency in the early stage, i.e., dMMR crypts. The top 25% most frequently mutated MS (ES96/Coding) corresponded to MS in the first quartile when they were ranked by mutational frequency. Coding MS known to be highly mutated in MSI colorectal cancer are write in black. ES96 signature is significantly enriched in the top genes; *P-value < 1.10⁻². Right panel, non-exhaustive list of the most frequently mutated MS (ES96/Coding) in the early stage of MSI CRC tumorigenesis (more details in Additional file 7: Table S6). MS are order by mutational frequency in dMMR crypts