O-GlcNAc Transferase Activity is Essential for RNA Pol II Pausing in a Human Cell-Free Transcription System

Paused RNA polymerase II is the major regulated step of transcription in metazoans. We describe here a unique human cell-free transcription system that recapitulates RNA pol II pausing and assemble paused pol IIs on the human CMV IE, SV40, and heat shock promoters, all is the case in vivo. We then use the system to show that PARP-1 and CDK12/13 inhibitors directly affect pausing and elongation. We then show that O-GlcNAcylation is required for the establishment of a paused pol II: inhibition of OGT allowed pol II to bypass pausing and begin to elongate. Addback of rOGT or the pausing factor NELF re-established the pausing. In vivo nascent RNA measurements showed that OGA inhibition blocks elongation. These data show that cell-free systems can recapitulate RNA pol II pausing, that PARP-1 and CDK12/13 directly regulate RNA pol II elongation, and identify O-GlcNAc cycling regulating both pausing and pause release.