Extended Data Fig. 3: Exonic elements contribute to splicing and regulation by SRRM4.
(a) Approach used to perform a semi-deep mutagenesis scan of the exon. Three successive nucleotides (indicated in red) were mutated at the time using 1-nt sliding windows from the 5’ end to the 3’ end of each microexon and following a systematic pattern of nucleotide substitution: A > C, G > T, C > A, T > G. (b–d) For each event and experimental condition (y-axis), the ΔPSI (VAR-WT) upon mutation at different exonic positions (x-axis) is shown as a heatmap (blue/red represents higher/lower inclusion than the WT) (LS in (b) and HS in (c)). For CS events (d), the positions of the mutations in the short exonic version have been aligned to their corresponding WT version of 42 nts (note that because of the removal, the central nucleotides are different). On the right side of panel (d), the line plots represent the PSI in the same experimental conditions for each of the CS events and their corresponding shortened variants. The boxes show the quantification of the perturbations observed on the heatmap. Statistics: Mann-Whitney-Wilcoxon test two-sided with Bonferroni correction (ns: non significant). (e) Stacked bar plots for LS, HS, CS and CSrem events in the four experimental conditions counting the number of mutations leading to ΔPSI (VAR-WT) > 10 (POS), ΔPSI (VAR-WT) < -10 (NEG) or without effect (ELSE). (f) Examples of changes in PSI and SRRM4 response for variants of a LS (CLASP1-HsaEX0015478), a HS (CPEB4-HsaEX0016976), a CS (SNHG14-HsaEX1038166) and a shortened CS (ARHGAP23-HsaEX7108093-rem-15) event. (g) Top: Representation of the approach used to introduce ESR hexamers at two positions within the exon. Two ESEs and two ESSs were inserted either at the 5′ end (from position 4 to 9) or at the 3′ end (from position n-9 to n-4, n being the exon length) of the exon as indicated. The sequences of each hexamer are shown (Supplementary Table 6). Bottom: Distributions of ΔPSI (VAR-WT) in GFP vs LOW, MID, HIGH expression of SRRM4 for LS, HS, CS and their corresponding CSrem events. The colors indicate both the hexamer and the position where it was inserted.
