Fig. 1

High-throughput mutagenesis screen provides quantitative splicing information across the RON minigene. a High-throughput detection of splicing-effective mutations. Mutagenic PCR creates mutated minigene library (left) that gives rise to alternatively spliced transcripts (middle). Mutations and corresponding splicing products are characterised by DNA and RNA sequencing, respectively, and linked by unique 15-nt barcode sequence in each minigene (coloured boxes). Black and grey boxes depict constitutive and alternative exons, respectively. b Nine most frequent isoforms found in HEK293T cells. Bar diagram shows total frequency in RNA-seq library (black) and maximal frequency for any individual minigene variant (grey). Asterisks mark non-canonical isoforms from cryptic 3′ splice site usage upon mutations at positions marked in g. AE, alternative exon, IR, intron retention, other, non-canonical isoforms. c Occurrence of distinct splice junctions in HEK293T cells. Line thickness and colour represent number of minigene variants producing a given junction (only junctions accounting for ≥1% of all junctions for a given minigene). d Boxplot showing distribution of AE inclusion frequencies (as % of all isoforms) for all wild-type (wt) and mutated minigenes and a subset with mutations in splice sites (ss) of RON exon 11. Boxes represent quartiles, centre lines denote 50th percentile, and whiskers extend to most extreme values within 1.5× interquartile range (IQR). e Validation of AE inclusion frequencies for 59 randomly selected minigene variants. Scatterplot compares the RNA-seq quantification to semiquantitative RT-PCR for individual minigene variants in HEK293T cells. r, Pearson correlation coefficient and associated P value. f Mutational landscape around the 3′ splice site of RON exon 11. Boxplot of AE inclusion frequencies in HEK293T cells for all minigenes with mutation at indicated positions (x-axis). Box representation as in d. Colours illustrate inserted nucleobase (see legend). Blue and purple lines indicate IQR of AE inclusion frequencies for all mutated and wt minigenes, respectively. Sequence of wt RON minigene given below. g Isoform frequencies arising from mutations along RON minigene. Stacked bar chart shows median frequency of six isoform categories for all minigenes with mutation at a given position. Average of three biological replicates in HEK293T cells. Asterisks highlight positions where mutations lead to non-canonical isoforms depicted in b