Coordinated alternative splicing decisions via stepwise exon definition.

Alternative splicing of pre-mRNA is a fundamental step in human gene regulation, and aberrant splicing is frequently linked to complex diseases, including cancer. All splicing events, such as skipping or inclusion of an alternative exon (AE) and intron retention (IR), are regulated by a common molecular machinery, the spliceosome. However, it remains elusive how the spliceosome coordinates different splicing decisions. Here, we analyzed a large-scale mutagenesis screen and transcriptome-wide RNA sequencing data to show that intron retention adjacent to alternative exons most commonly occurs at intermediate inclusion levels. Using data-driven mathematical modeling, we revealed that multistep exon recognition mediated by spliceosome assembly and maturation explains the observed AE-IR dependency for cis-acting sequence mutations and upon knockdown of trans-acting RNA-binding proteins. Furthermore, we found that multistep exon recognition is commonly perturbed in a transcriptome-wide manner in cancer cells, which leads to the coordinated deregulation of IR and AE decisions. In total, our work suggests that stepwise alternative exon recognition by the spliceosome coordinates AE inclusion and the retention of flanking introns, which may facilitate the search for common molecular mechanisms for mis-splicing in cancer.