CTDP1 governs a unique RNAPII phospho-form and coordinates Mediator recruitment, transcription efficiency, and splicing

We previously reported that the RNA polymerase II (Pol II) subunit RPB7 interacts with the phosphatase CTDP1 to regulate transcription termination-reinitiation. Here, we demonstrate that CTDP1 specifically dephosphorylates a unique phospho-isoform of Pol II, which is modified at tyrosine 1, serine 5, and serine 7 (Tyr1, Ser5, Ser7) of its C-terminal domain. Functionally, this regulation is critical for the recruitment of the Mediator complex to chromatin and for modulating Pol II transcriptional efficiency. Furthermore, by integrating phosphoproteomic and immunoprecipitation-mass spectrometry data, we show that CTDP1 also regulates the phosphorylation of numerous RNA splicing factors. Consequently, depletion of CTDP1 disrupts gene expression oscillations, leading to increased intron retention and aberrant splicing of cassette exons. These findings suggest that CTDP1 serves as a key integrator of transcription and splicing, potentially contributing to the regulation of circadian rhythms and the cell cycle.