Dynamic O-GlcNAcylation and phosphorylation attract and expel proteins from RNA polymerase II to regulate mRNA maturation

Journal of Biomedical Science, Journal Year: 2025, Volume and Issue: 32(1)

Published: April 4, 2025

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

Language: Английский

---

The PNUTS phosphatase complex controls transcription pause release

Molecular Cell, Journal Year: 2024, Volume and Issue: 84(24), P. 4843 - 4861.e8

Published: Nov. 26, 2024

Language: Английский

---

The CDK9-SPT5 axis in control of transcription elongation by RNAPII

Journal of Molecular Biology, Journal Year: 2024, Volume and Issue: unknown, P. 168746 - 168746

Published: Aug. 1, 2024

Language: Английский

---

PP1/PNUTS Phosphatase Binds the Restrictor Complex and Stimulates RNA pol II Transcription Termination

Published: Jan. 1, 2025

The restrictor, ZC3H4/WDR82, terminates antisense transcription from bidirectional promoters, but its mechanism is poorly understood. We report that ZC3H4/WDR82 immunoprecipitate with PP1 phosphatase and nuclear targeting subunit, PNUTS, which binds to WDR82. AlphaFold predicts a complex of PP1/PNUTS restrictor where both PNUTS ZC3H4 contact A substrate trap, PP1H66K-PNUTS, comprising inactive fused the C-terminus antagonizes mediated termination whereas PP1WT-PNUTS has less effect suggesting activity required for termination. One implicated in by pol II CTD Ser5-P. PP1H66K-PNUTS induces Ser5-P hyperphosphorylation at 5' ends presumably inhibiting dephosphorylation. NET-seq analysis suggests Ser5 dephosphorylation would promote increasing pausing. Both inhibition require WDR82 binding domain mediates binding. In summary, associated via promotes efficient

Language: Английский

---

Multi-omics and biochemical reconstitution reveal CDK7-dependent mechanisms controlling RNA polymerase II function at gene 5'- and 3'-ends

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 9, 2025

CDK7 regulates RNA polymerase II (RNAPII) initiation, elongation, and termination through incompletely understood mechanisms. Because contaminating kinases precluded analysis with nuclear extracts, we completed biochemical assays purified factors. Reconstitution of RNAPII transcription initiation showed inhibition slowed and/or paused promoter-proximal transcription, which reduced re-initiation. These CDK7-regulatory functions were Mediator- TFIID-dependent. Similarly in human cells, by suppressing activity at promoters, consistent Moreover, widespread 3'-end readthrough was observed CDK7-inhibited cells; mechanistically, this occurred rapid depletion elongation factors, including high-confidence targets. Collectively, these results define how governs function gene 5'-ends 3'-ends, reveal that abundance factors is kinase-dependent. 3'-readthrough commonly induced during stress, our further suggest regulated suppression may enable transcriptional response.

Language: Английский

---

TFIIH kinase CDK7 drives cell proliferation through a common core transcription factor network

Science Advances, Journal Year: 2025, Volume and Issue: 11(9)

Published: Feb. 28, 2025

How cyclin-dependent kinase 7 (CDK7) coordinately regulates the cell cycle and RNA polymerase II transcription remains unclear. Here, high-resolution cryo–electron microscopy revealed how two clinically relevant inhibitors block CDK7 function. In cells, inhibition rapidly suppressed transcription, but constitutively active genes were disproportionately affected versus stimulus-responsive. Distinct factors (TFs) regulate constitutive stimulus-responsive genes. Accordingly, TFs refractory to whereas “core” repressed. Core (n = 78) are predominantly promoter associated control proliferative gene expression programs across types. Mechanistically, rapid suppression of core TF function can occur through CDK7-dependent phosphorylation changes in RB1. Moreover, depleted protein levels within hours, consistent with durable target suppression. Thus, a major unappreciated biological for is regulation cohort that drives proliferation, revealing an apparent universal mechanism by which coordinates RNAPII CDK regulation.

Language: Английский

---

PP1/PNUTS phosphatase binds the restrictor complex and stimulates RNA Pol II transcription termination

Cell Reports, Journal Year: 2025, Volume and Issue: 44(5), P. 115564 - 115564

Published: April 16, 2025

Language: Английский

---

Tripartite phosphorylation of SPT5 by CDK9 times pause release and tunes elongation rate of RNA polymerase II

Molecular Cell, Journal Year: 2025, Volume and Issue: unknown

Published: April 1, 2025

Language: Английский

---

Epstein-Barr Virus-Driven B-Cell Transformation under Germinal Center Hypoxia Requires External Unsaturated Fatty Acids

Research Square (Research Square), Journal Year: 2025, Volume and Issue: unknown

Published: April 24, 2025

Epstein-Barr virus (EBV) contributes to over 200,000 cancers annually, predominantly aggressive lymphomas originating from hypoxic germinal centers (< 1% O₂). However, conventional models fail recapitulate the physiologically relevant microenvironment which profoundly influences B-cell metabolic remodeling during transformation. Here, we establish an ex vivo model of EBV-driven transformation under O₂, demonstrating robust and super-enhancer activation oncogenic regulators, including MYC. Multi-omic analyses reveal distinct adaptations hypoxia. Unlike normoxic B-cells, rely on fatty acid desaturases oxidation mitigate lipotoxicity, hypoxically transformed B-cells suppress synthesis while upregulating glycerophospholipid metabolism lipid droplet formation buffer excess saturated lipids. Consequently, these cells exhibit heightened dependence external unsaturated acids support proliferation. Our findings provide first hypoxia, uncovering vulnerabilities that could inform targeted therapeutic strategies for EBV-associated malignancies.

Language: Английский

---

Pause Patrol: Negative Elongation Factor’s role in Promoter-Proximal Pausing and beyond

Journal of Molecular Biology, Journal Year: 2024, Volume and Issue: unknown, P. 168779 - 168779

Published: Sept. 1, 2024

Language: Английский

---