RPRD1B’s direct interaction with phosphorylated RNA Polymerase II regulates polyadenylation of cell cycle genes and drives cancer progression

RNA Polymerase II (Pol II) regulates eukaryotic gene expression through dynamic phosphorylation of its C-terminal domain (CTD). Phosphorylation at Ser2 and Thr4 on the CTD is crucial for RNA 3’ end processing and facilitating the recruitment of cleavage and termination factors. However, the transcriptional roles of most CTD-binding proteins remain poorly understood. In this study, we focus on RPRD1B, a transcriptional regulator that interacts with the phosphorylated CTD and has been implicated in various cancers. We investigated its molecular mechanism during transcription and found that RPRD1B modulates alternative polyadenylation of cell growth transcripts by directly interacting with the CTD. RPRD1B is recruited to transcribing Pol II near the 3’ end of the transcript, specifically in response to Ser2 and Thr4 phosphorylation, but only after flanking Ser5 phosphorylation is removed. Transcriptomic analysis of RPRD1B knockdown cells revealed its role in cell proliferation via termination of the key cell growth genes at upstream polyadenylation sites, leading to the production of tumor suppressor transcripts that lack AU-rich elements (AREs) with increased mRNA stability. Overall, our study uncovers previously unrecognized connections between the Pol II CTD and CID, highlighting their influence on 3’ end processing and their contribution to abnormal cell growth in cancer.