Extended Data Fig. 4: Effect of UGC mutation and insertion on splicing regulation of LS and HS events.
(a) Effect on the response to SRRM4 at MID and HIGH conditions of mutating all UGCs in the orthologous variants. The barplots represent the median ΔPSI for all the orthologous WT constructs in either MID or HIGH SRRM4 expression conditions with respect to GFP. Boxplots show the distribution of the ΔPSI of the UGC mutant variants, with each dot representing a UGC variant from a given species. The total number of UGCs considered for the mutagenesis for each event are indicated by the color of the dots. (b) MaPSy-derived PSI values under four experimental conditions (GFP or LOW, MID, HIGH expression of SRRM4) for selected variants of the KIF1C and PUS7 minigenes tested in Fig. 4b. (c) RT-PCR assays showing the splicing pattern of different variants (WT or mutation from the “UGC-addition walk” combined or not with exon length shortening) of three events under control condition (-) or expression of human SRRM4 (Hsa). PSI and standard deviations (std) from at least three biological replicates are provided. (d) Top: Schematics of the location at which 1, 2 or 3 UGCs have been added in the context of the WT sequence. Bottom left: Variation of the ppt_scr of each variant with respect to its corresponding WT upon addition of 1, 2 or 3 UGCs downstream to the best predicted BP. Outliers were removed for visibility. Statistics: Mann-Whitney-Wilcoxon test. Bottom right: Correlation between the changes in ppt_scr and the variation in PSI in the HIGH condition from variant to WT. Statistics: Pearson correlation and p-value are provided. (e) Left: Location at which 1, 2 or 3 UGCs were added in the context of a modified WT sequence in which the BP and Py tract were displaced together either 10 or 20 nts upstream to their initial position. Right: Variation of ss_dist, ppt_scr and PSI in the HIGH condition of each variant with respect to its corresponding WT upon addition of 1, 2 or 3 UGCs in the 10 or 20 nts downstream to the BP and its associated Py Statistics: Mann-Whitney-Wilcoxon test two-sided with Bonferroni correction (ns: non significant).
