Fig. 5

Synergistic interactions highlight the functionally most relevant HNRNPH binding sites. a Minigene variants are differentially spliced upon HNRNPH knockdown (KD). Scatterplot compares AE inclusion under control (ctrl) and HNRNPH KD conditions for all minigene variants. Average behaviour illustrated by running mean and standard deviation (red). Shadings schematically highlight stronger/weaker-than-average KD response. Minigenes with mutations C307G, G310A and G305A in HNRNPH splice-regulatory binding sites (SRBS) cluster 3 are highlighted. b Quantification of synergistic interactions by linear regression modelling. Single mutation effects are determined separately for ctrl and HNRNPH KD using linear regression and subtracted to estimate KD responses compared to wt (z-score based on standard deviation of wt minigenes, colour-coded; see Supplementary Note 3). Right graph shows model-inferred AE skipping-to-inclusion isoform ratios of single mutations in ctrl vs. KD. Regression line indicates average KD effect. Consideration of isoform ratios, as compared to isoform frequencies (a), leads to linearisation of KD response in line with predictions of kinetic splicing model (Supplementary Fig. 3a). c Model-inferred synergistic interactions accumulate in RON exon 11. Bar diagrams quantify significant synergistic interactions affecting AE skipping-to-inclusion isoform ratio using different z-score cutoffs in adjacent 5-nt windows. Line indicates density in 5-nt sliding window. Splice sites ± 2 nt were excluded. Predicted HNRNPH SRBS (brown) are given above. d Mutations in HNRNPH SRBS cluster 3 lead to increased AE inclusion and reduced HNRNPH KD response. Dot plots (top) display single mutation effects (inserted nucleobase, see legend) on AE inclusion (mean, n = 3). Red lines indicate median isoform frequency of wt minigenes ± 2 standard deviations (SD). HNRNPH SRBS (brown) are given above. Heat maps (bottom) show z-scores as measure of synergy (mean, n = 3) per inserted nucleobase. White or grey fields indicate mutations that were not present or filtered out, respectively (see Methods). Purple boxes highlight significant synergistic interactions (0.1% FDR). e Consistent with strong synergistic interactions (colour-coded), mutations in cluster 3 almost completely abolish the HNRNPH KD response in MCF7 cells. Scatterplot compares model-inferred estimates with semiquantitative RT-PCR measurements of AE inclusion (circles) and AE skipping (triangles) upon HNRNPH KD (mean, n = 3) for ten targeted mutations in HNRNPH SRBS (Supplementary Data 8)