Position-dependent effects of RNA-binding proteins in the context of co-transcriptional splicing

Alternative splicing is an important step in eukaryotic mRNA pre-processing which increases the complexity of gene expression programs, but is frequently altered in disease. Previous work on the regulation of alternative splicing has demonstrated that splicing is controlled by RNA-binding proteins (RBPs) and by epigenetic DNA/histone modifications which affect splicing by changing the speed of polymerase-mediated pre-mRNA transcription. The interplay of these different layers of splicing regulation is poorly understood. In this paper, we derived mathematical models describing how splicing decisions in a three-exon gene are made by combinatorial spliceosome binding to splice sites during ongoing transcription. We additionally take into account the effect of a regulatory RBP and find that the RBP binding position within the sequence is a key determinant of how RNA polymerase velocity affects splicing. Based on these results, we explain paradoxical observations in the experimental literature and further derive rules explaining why the same RBP can act as inhibitor or activator of cassette exon inclusion depending on its binding position. Finally, we derive a stochastic description of co-transcriptional splicing regulation at the single-cell level and show that splicing outcomes show little noise and follow a binomial distribution despite complex regulation by a multitude of factors. Taken together, our simulations demonstrate the robustness of splicing outcomes and reveal that quantitative insights into kinetic competition of co-transcriptional events are required to fully understand this important mechanism of gene expression diversity.