The C-terminal domain (CTD) of human RNA polymerase II (RNAPII) acts as a regulatory hub for RNA processing and progression of eukaryotic transcription. Transcriptional control is fine-tuned through dynamic phosphorylation of the 52 repetitive heptads on the CTD to generate post-translational modification patterns that precisely recruit cellular proteins according to transcriptional stages. However, it remains unclear what transcription regulators are differentially recruited by specific phospho-marks. We assembled an enzymatic toolbox to recapitulate the dynamic phosphorylation patterns of CTD during transcription. We mapped the individual modification sites in high precision with advanced mass spectrometry strategies such as ultraviolet photodissociation. Utilizing a label-free quantitative proteomics approach, we identified proteins that interact with different phosphoisoforms of Pol II CTD. Specifically, we identified RNA-binding protein SCAF6 that are recruited for transcription by Ser2 and Thr4 phosphorylation. We characterized the interaction of SCAF6 with differentially phosphorylated CTD. The timely recruitment of SCAF6 is then investigated in cells using genome-wide sequencing. In conclusion, we have expanded the repertoire of RNA-binding proteins that directly interact with the CTD of RNA Pol II and illuminate the concerted interplay between splicing and transcription.
