Abstract B083: O-GlcNAcylation of RNA polymerase II regulates transcription initiation

Abstract Background and Objective: Prostate cancer (PC), the most commonly diagnosed cancer among men in the US, is driven by increased activity of androgen receptor (AR). We have previously shown that AR hyper-activates O-GlcNAc transferase (OGT) by enhancing flux through the hexosamine biosynthetic pathway. OGT forms a combinatorial lethal pair with transcriptional kinases, and it is enriched at transcription start sites (TSS). One of the major factors enriched at the TSS is RNA Polymerase II (RNA Pol II), and its O-GlcNAcylation was first described in 1993. However, due to the lack of techniques and tools the functional and mechanistic importance of RNA Pol II O-GlcNAcylation has remained enigmatic. Methods: To investigate how hypo- and hyper-O-GlcNAcylation affect RNA Pol II localization across the human genome, we performed ChIP-seq to map the distribution of total and differentially phosphorylated RNA Pol II. To directly assess how these O-GlcNAcylation states influence RNA Pol II-driven mRNA maturation, we employed nascent transcriptome profiling using SLAM-seq. Additionally, we obtained chemically synthesized, site-selectively O-GlcNAcylated CTD (gCTD) peptides and conducted mass spectrometry- based proteomics to systematically map the proteome. By integrating these data with time-resolved interactome analyses of RNA Pol II under conditions of altered OGT and OGA activity, we identified potential reader proteins of O-GlcNAcylated RNA Pol II. Results: Our data show that dynamic O-GlcNAcylation regulate RNA Pol II during transcription initiation and is also important later in the transcription process by recruiting appropriate mRNA-processing machinery. Mass spectrometry (MS)-based proteomics revealed that, similar to phosphorylation, site-selective O-GlcNAcylation serves to recruit distinct proteins to the polymerase. In particular, we identified a high number of mRNA binding proteins associated with O-GlcNAcylated RNA Pol II, implying that this modification coordinates with the splicing machinery. Conclusions: We propose that O-GlcNAcylation coordinates with phosphorylation to regulate RNA Pol II progression along the gene body. Defective mRNA processing and OGT activity are both significantly increased in aggressive prostate cancer, suggesting OGT is a viable target to control high transcription. However, because no clinical-grade OGT inhibitors are available, interference with metabolic flux or splicing offers an actionable immediate strategy. In short, this work elucidates the functional and mechanistic importance of RNA Pol II O-GlcNAcylation, identifies its reader proteins, and provides a rationale for targeting OGT-driven metabolic or splicing pathways in aggressive PC. Citation Format: Shivani Yalala, Harri M. Itkonen. O-GlcNAcylation of RNA polymerase II regulates transcription initiation [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Prostate Cancer Research and Treatment; 2026 Jan 20-22; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(2_Suppl):Abstract nr B083.