JMJD6 cleaves MePCE to release positive transcription elongation factor b (P-TEFb) in higher eukaryotes
- National Jewish Health, United States
- School of Medicine, Univeristy of Colorado at Anschutz Medical Center, United States
- Xiamen Univeristy, China
- Children Hospital, United States
- China Agriculture University, China
- Duke University Medical Center, United States
- Max-Planck-Society, Germany
- School of Medicine, University of Pennsylvannia, United States
- Howard Hughes Medical Institute, National Jewish Health, United States
- University of California, Berkeley, United States
- Xiamen University, China
- School of Medicine, University of Colorado at Anschutz Medical Center, United States
Abstract
More than 30% of genes in higher eukaryotes are regulated by promoter-proximal pausing of RNA polymerase II (Pol II). Phosphorylation of Pol II CTD by positive transcription elongation factor b (P-TEFb) is a necessary precursor event that enables productive transcription elongation. The exact mechanism on how the sequestered P-TEFb is released from the 7SK snRNP complex and recruited to Pol II CTD remains unknown. In this report, we utilize mouse and human models to reveal methylphosphate capping enzyme (MePCE), a core component of the 7SK snRNP complex, as the cognate substrate for Jumonji domain-containing 6 (JMJD6)’s novel proteolytic function. Our evidences consist of a crystal structure of JMJD6 bound to methyl-arginine, enzymatic assays of JMJD6 cleaving MePCE in vivo and in vitro, binding assays, and downstream effects of Jmjd6 knockout and overexpression on Pol II CTD phosphorylation. We propose that JMJD6 assists bromodomain containing 4 (BRD4) to recruit P-TEFb to Pol II CTD by disrupting the 7SK snRNP complex.
Data availability
Diffraction data have been deposited in PDB under the accession code 6mev,All data generated or analysed during this study are included in the manuscript and supporting files.
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Structure of JMJD6 bound to Mono-Methyl Arginine.Protein Data Bank, 6MEV.
Article and author information
Author details
Kathrin Maria Bernt
Funding
National Cancer Institute (CA201230)
- Kathrin Maria Bernt
National Institute of Allergy and Infectious Diseases (AI007405)
- Schuyler Lee
National Institute of Allergy and Infectious Diseases (AI074491)
- Haolin Liu
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2020, Lee et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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JMJD6 cleaves MePCE to release positive transcription elongation factor b (P-TEFb) in higher eukaryotes
eLife 9:e53930.
https://doi.org/10.7554/eLife.53930
Further reading
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- Biochemistry and Chemical Biology
N 6,2’-O-dimethyladenosine (m6Am) is a modified nucleotide located at the first transcribed position in mRNA and snRNA that is essential for diverse physiological processes. m6Am mapping methods assume each gene uses a single start nucleotide. However, gene transcription usually involves multiple start sites, generating numerous 5’ isoforms. Thus, gene-level annotations cannot capture the diversity of m6Am modification in the transcriptome. Here, we describe CROWN-seq, which simultaneously identifies transcription-start nucleotides and quantifies m6Am stoichiometry for each 5’ isoform that initiates with adenosine. Using CROWN-seq, we map the m6Am landscape in nine human cell lines. Our findings reveal that m6Am is nearly always a high stoichiometry modification, with only a small subset of cellular mRNAs showing lower m6Am stoichiometry. We find that m6Am is associated with increased transcript expression and provide evidence that m6Am may be linked to transcription initiation associated with specific promoter sequences and initiation mechanisms. These data suggest a potential new function for m6Am in influencing transcription.
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Glutamine synthetases (GS) are central enzymes essential for the nitrogen metabolism across all domains of life. Consequently, they have been extensively studied for more than half a century. Based on the ATP-dependent ammonium assimilation generating glutamine, GS expression and activity are strictly regulated in all organisms. In the methanogenic archaeon Methanosarcina mazei, it has been shown that the metabolite 2-oxoglutarate (2-OG) directly induces the GS activity. Besides, modulation of the activity by interaction with small proteins (GlnK1 and sP26) has been reported. Here, we show that the strong activation of M. mazei GS (GlnA1) by 2-OG is based on the 2-OG dependent dodecamer assembly of GlnA1 by using mass photometry (MP) and single particle cryo-electron microscopy (cryo-EM) analysis of purified strep-tagged GlnA1. The dodecamer assembly from dimers occurred without any detectable intermediate oligomeric state and was not affected in the presence of GlnK1. The 2.39 Å cryo-EM structure of the dodecameric complex in the presence of 12.5 mM 2-OG demonstrated that 2-OG is binding between two monomers. Thereby, 2-OG appears to induce the dodecameric assembly in a cooperative way. Furthermore, the active site is primed by an allosteric interaction cascade caused by 2-OG-binding towards an adaption of an open active state conformation. In the presence of additional glutamine, strong feedback inhibition of GS activity was observed. Since glutamine dependent disassembly of the dodecamer was excluded by MP, feedback inhibition most likely relies on the binding of glutamine to the catalytic site. Based on our findings, we propose that under nitrogen limitation the induction of M. mazei GS into a catalytically active dodecamer is not affected by GlnK1 and crucially depends on the presence of 2-OG.
