The expression of eukaryotic protein-coding genes is the product of transcriptional (e.g. initiation, elongation and termination) and co-transcriptional processes (e.g. splicing, modification and export). How diverse input signals are relayed to, and interpreted by transcription factors and associated co-factors to govern RNA Pol II activity remains unclear. Here we developed a gene expression reporter system traceable in real-time by flow cytometry and combined this with CRISPR/Cas9 screens to identify epistatic relationships underpinning transcriptional co-activator inhibition in human cells. We find that loss of the U2 spliceosome factor SF3B3 broadly desensitises cells to transcriptional co-activator inhibition, with short, exon-dense genes displaying the most prominent desensitisation. We show that splicing remains functional despite SF3B3 loss and that the desensitisation effect is mediated by the physical presence, but not the catalytic activity of the acetyltransferase and transcriptional co-activator P300. Mechanistically, SF3B3 depletion drives the redistribution of chromatinised P300 to newly licensed cis regulatory elements enriched for JUN/AP1; and perturbs the pause release and elongation of RNA Pol II. Moreover, the association of key transcriptional complexes, elongation factors and cyclin dependent kinases with RNA Pol II is reduced following SF3B3 depletion. Together these data suggest a splicing-independent functional link between SF3B3, P300 and RNA Pol II required for the coordination and licensing of transcriptional elongation.