Fig. 6
Cooperative HNRNPH binding establishes a splicing switch of RON exon 11. a A single point mutation in an HNRNPH splice-regulatory binding site (SRBS) results in reduced HNRNPH binding in HEK293T cells also at neighbouring SRBS in RON exon 11. Bar diagrams show the number of HNRNPH iCLIP crosslink events on the wt RON minigene (top) and the difference in normalised crosslink events on wt and mutated RON minigenes (mutations G305A, G331C and G348C in different SRBS within cluster 3, marked by dashed lines; bottom) in a sliding 5-nt window along the wt RON minigene. HNRNPH SRBS (brown boxes) indicated below. b Splicing response to gradual HNRNPH KD and overexpression suggests cooperative regulation of RON exon 11 by HNRNPH. Scatterplot shows semiquantitative RT-PCR quantifications of RON exon 11 inclusion (in percent spliced-in/PSI, Supplementary Fig. 15a, b) from endogenous RON gene (orange) and wt RON minigene (blue) against corresponding HNRNPH protein levels (Supplementary Fig. 15c, d). Degree of cooperativity is quantified by fitting Hill equation (solid lines) and compared to theoretical fit for non-cooperativity (dashed line). Error bars denote standard deviation of three biological replicates. c Steep regression slope for HNRNPH2 supports cooperative HNRNPH regulation and switch-like splicing of RON exon 11. Boxplot shows distribution of regression slopes for expression correlations of 190 RBPs with RON exon 11 inclusion in TCGA samples (Supplementary Data 7). Box represents quartiles, centre line denotes 50th percentile and whiskers extend to most extreme data points within 1.5× interquartile range. HNRNPH2 is highlighted. d HNRNPH acts as key regulator of RON splicing by recognising multiple cis-regulatory elements in a cooperative fashion. Schematic model summarises position-dependent effects of HNRNPH on RON exon 11, indicating most strongly effected isoform for each site (left panel). Multiple interdependent HNRNPH binding sites within RON exon 11 exert strong cooperative control on the alternative exon, resulting in a splicing switch upon small changes in HNRNPH abundance (right panel)
