UC Irvine

Eukaryotic gene expression is coordinated through a series of processes from which RNA is transcribed, processed, and translated into the proteins that serve as the functional building blocks of complex cellular organisms. These steps are highly integrated, often occurring in the same spatial and temporal space. Although this co-transcriptional connection is well described, it remains unclear how the concerted rates of global RNA processing steps affect the final mRNA isoform. Here we disrupt steady-state RNA levels using 4-thiouridine (4sU) metabolic labeling and utilize high-throughput sequencing to determine the global rates of pre-mRNA processing. We find that introns that display higher retention are subject to slower splicing kinetics, with longer introns being removed quicker. Exon skipping is subject to competing splice site pairing kinetics and size constraints that highlight optimal exon recognition features by the spliceosome. Integration of this information permits the determination of the order of intron removal across entire genes, thus producing detailed gene RNA processing maps.

Intron retention and exon skipping (cassette exons) are two types of alternative splicing that can be regulated by SR proteins by strengthening the recognition of introns and exons that are otherwise prone to alternative splicing. To test the hypothesis that SR proteins modulate alternative splicing through changes in splicing kinetics, we depleted SRSF1 in human hepatocellular carcinoma cells and derived RNA processing rates. Loss of SRSF1 leads to higher intron retention and more exon skipping. This is primarily achieved through changes in splicing rates and Pol II density, with a strong dependence on optimal feature length constraints. eCLIP data further demonstrates that SRSF1 binds preferentially to weaker exons that are prone to being skipped.

Together these data suggest that alternatively spliced introns and exons have distinct kinetic profiles, constrained by lengths that favor exon definition. The splicing factor SRSF1 acts primarily as an activator, promoting the constitutive splicing of exons and introns through the modulation of Pol II density and subsequent splicing kinetics.