Pervasive co-regulation of alternative splicing with transcription initiation and termination.

Nascent RNA processing requires coordination of transcription and splicing to yield accurate gene products. However, the basis of this co-regulation remains enigmatic. Here, we develop longPASS, a system that accurately identifies and quantitates transcription start site (TSS) and polyadenylation site (PAS) usage in long-read RNA sequencing data. longPASS systematically reveals thousands of TSS/PAS-associated alternative splicing events (TASE) in human cells, uncovering pervasive, consistent TASEs across genetic perturbations, human tissues, and individuals. Co-occurring pairs of TASE-exons and TASE-TSS/PASs are proximal and show increased RNA-RNA spatial interactions. Large-scale screening reveals that the RBMX protein specifically binds PAS-TASE pairs. Indeed, RBMX knockdown, but not canonical splicing factors, disrupts the co-regulation of splicing and PAS usage in TASEs. Manipulating RNA polymerase II elongation also affects PAS-TASE co-regulation, with slower elongation rates leading to increased co-occurrence. Overall, our analysis reveals pervasive co-regulation between alternative splicing and TSS/PAS usage is mediated by RBMX interaction, Pol II elongation rates, and linked to ageing.