CTDP1 governs a Pol II phosphorylation intermediate and coordinates Mediator recruitment, transcription efficiency, and splicing.

RNA Pol II exists in hypo- (II-A) and hyper-phosphorylated (II-O) states on chromatin, which are linked to transcription initiation and elongation, respectively. Here, we identify a phosphorylation intermediate of Pol II (designated II-I) that is predominantly localized in the nucleoplasm and is enriched for phosphorylated Tyr1, Ser5, and Ser7 within the C-terminal domain (CTD). We demonstrate that the abundance of this phosphorylation intermediate is rapidly modulated by the phosphatase activity of CTDP1. Mechanistically, CTDP1-mediated regulation is critical for the recruitment of the Mediator complex to chromatin and for modulating Pol II transcriptional efficiency. Moreover, CTDP1's regulatory function extends to the dephosphorylation of splicing factors. Consequently, its depletion causes widespread intron retention and aberrant splicing of cassette exons. These findings position CTDP1 as a key regulator of Pol II phosphorylation intermediate and splicing regulation, with potential implications for cell cycle control.