Exon definition facilitates reliable control of alternative splicing in the RON proto-oncogene

Alternative splicing is a key step in eukaryotic gene expression that allows the production of multiple protein isoforms from the same gene. Even though splicing is perturbed in many diseases, we currently lack insights into regulatory mechanisms promoting its precision and efficiency. We analyse high-throughput mutagenesis data obtained for an alternatively spliced exon in the proto-oncogene RON and determine the functional units that control this splicing event. Using mathematical modeling of distinct splicing mechanisms, we show that alternative splicing is based in RON on a so-called exon definition mechanism. Here, the recognition of the adjacent exons by the spliceosome is required for removal of an intron. We use our model to analyze the differences between the exon and intron definition scenarios and find that exon definition is crucial to prevent the accumulation of deleterious, partially spliced retention products during alternative splicing regulation. Furthermore, it modularizes splicing control, as multiple regulatory inputs are integrated into a common net input, irrespective of the location and nature of the corresponding cis-regulatory elements in the pre-mRNA. Our analysis suggests that exon definition promotes robust and reliable splicing outcomes in RON splicing. SIGNIFICANCEDuring mRNA maturation, pieces of the pre-mRNA (introns) are removed during splicing, and remaining parts (exons) are joined together. In alternative splicing, certain exons are either included or excluded, resulting in different splice products. Inclusion of RON alternative exon 11 leads to a functional receptor tyrosine kinase, while skipping results in a constitutively active receptor that promotes epithelial-to-mesenchymal transition and contributes to tumour invasiveness. Intron retention results in to deleterious isoforms that cannot be translated properly. Using kinetic modeling, we investigate the combinatorial regulation of this important splicing decision, and find that the experimental data supports a so-called exon definition mechanism. We show that this mechanism enhances the precision of alternative splicing regulation and prevents the retention of introns in the mature mRNA.