BackgroundPhosphorylation and O-GlcNAcylation are the key modifications regulating RNA Polymerase II (RNA Pol II)-driven transcription. Transcriptional kinases, cyclin-dependent kinase 7 (CDK7), CDK9 and CDK12 phosphorylate RNA Pol II, whereas O-GlcNAcylation is added by O-GlcNAc transferase (OGT) and removed by O-GlcNAcase (OGA). Currently, no study has systematically evaluated how inhibiting each of these enzyme activities impacts the assembly of the appropriate protein complexes on the polymerase and the maturation of mRNA.MethodsHere, we systematically evaluate remodeling of RNA Pol II interactome and effects on the nascent mRNA maturation by using mass spectrometry and SLAM-seq, respectively. For validation, we rely predominantly on analysis of intronic polyadenylation (IPA) sites, mitochondrial flux assays (Seahorse), western blotting and patient data.ResultsWe show that OGT / OGA inhibition reciprocally affect protein recruitment to RNA Pol II, and appropriate O-GlcNAcylation levels are required for optimal function of the RNA Pol II complex. These paradoxical effects are explained through IPA, because despite being prematurely poly-adenylated, these mRNAs are scored as mature in SLAM-seq. Unlike previously proposed, we show that, similar to inhibition of CDK12, also targeting CDK9 stimulates transcription of short genes at the cost of long genes. However, our systematic proteomic- and IPA-analysis revealed that these effects are mediated by distinct molecular mechanisms: CDK9 inhibition leads to a failure of recruiting Integrator complex to RNA Pol II, and we then show that depletion of Integrator subunits phenocopy the gene length-dependent effects. In contrast, CDK12 inhibition triggers IPA. Finally, we show that dynamic O-GlcNAcylation predominantly interplays with CDK9: OGT inhibition augments CDK9 inhibitor effects on mRNA maturation due to defects in transcription elongation, while OGA inhibition rescues mRNA maturation failure caused by targeting CDK9, but induces IPA.ConclusionWe show that dynamic O-GlcNAcylation is a negative regulator of mRNA biosynthesis and propose that the addition and removal of the modification serve as quality control-steps to ascertain successful generation of mature mRNAs. Our work identifies unprecedented redundancy in the regulation of RNA Pol II, which increases resilience towards transcriptional stress, and also underscores the difficulty of targeting transcription to control cancer.
