Gondane Aishwarya, Itkonen Harri M
Department of Biochemistry and Developmental Biology, Faculty of Medicine, University of Helsinki, 00014, Helsinki, Finland.
J Biomed Sci. 2025 Apr 4;32(1):39. doi: 10.1186/s12929-025-01135-9.
Phosphorylation 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.
Here, 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.
We 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.
We 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.
磷酸化和O-连接的N-乙酰葡糖胺化（O-GlcNAcylation）是调节RNA聚合酶II（RNA Pol II）驱动转录的关键修饰。转录激酶，细胞周期蛋白依赖性激酶7（CDK7）、CDK9和CDK12使RNA Pol II磷酸化，而O-GlcNAcylation由O-GlcNAc转移酶（OGT）添加，并由O-GlcNA酶（OGA）去除。目前，尚无研究系统评估抑制这些酶活性中的每一种如何影响聚合酶上适当蛋白质复合物的组装以及mRNA的成熟。
在这里，我们分别使用质谱和SLAM-seq系统评估RNA Pol II相互作用组的重塑以及对新生mRNA成熟的影响。为了进行验证，我们主要依赖于对内含子聚腺苷酸化（IPA）位点的分析、线粒体通量测定（海马）、蛋白质印迹和患者数据。
我们表明，OGT/OGA抑制相互影响蛋白质募集到RNA Pol II，并且适当的O-GlcNAcylation水平是RNA Pol II复合物最佳功能所必需的。这些矛盾的效应通过IPA得到解释，因为尽管这些mRNA过早地进行了多聚腺苷酸化，但在SLAM-seq中它们被判定为成熟。与先前提出的不同，我们表明，与抑制CDK12类似，靶向CDK9也会以长基因的转录为代价刺激短基因的转录。然而，我们的系统蛋白质组学和IPA分析表明，这些效应是由不同的分子机制介导的：抑制CDK9导致无法将整合子复合物募集到RNA Pol II，然后我们表明整合子亚基的缺失模拟了基因长度依赖性效应。相反，抑制CDK12会触发IPA。最后，我们表明动态O-GlcNAcylation主要与CDK9相互作用：由于转录延伸缺陷，OGT抑制增强了CDK9抑制剂对mRNA成熟的影响，而OGA抑制挽救了靶向CDK9导致的mRNA成熟失败，但诱导了IPA。
我们表明动态O-GlcNAcylation是mRNA生物合成的负调节因子，并提出该修饰的添加和去除作为质量控制步骤，以确保成功生成成熟的mRNA。我们的工作确定了RNA Pol II调节中前所未有的冗余，这增加了对转录应激的恢复力，也强调了靶向转录以控制癌症的难度。
