1. Biochemistry and Chemical Biology
  2. Chromosomes and Gene Expression

Highly efficient 5' capping of mitochondrial RNA with NAD+ and NADH by yeast and human mitochondrial RNA polymerase

  1. Jeremy G Bird
  2. Urmimala Basu
  3. David Kuster
  4. Aparna Ramachandran
  5. Ewa Grudzien-Nogalska
  6. Atif Towheed
  7. Douglas C Wallace
  8. Megerditch Kiledjian
  9. Dmitry Temiakov
  10. Smita S Patel  Is a corresponding author
  11. Richard H Ebright  Is a corresponding author
  12. Bryce E Nickels  Is a corresponding author
  1. Rutgers University, United States
  2. Heidelberg University, Germany
  3. The Children's Hospital of Philadelphia, United States
  4. University of Pennsylvania, United States
  5. Thomas Jefferson University, United States
https://doi.org/10.7554/eLife.42179
Share this article
Cite this article
  1. Jeremy G Bird
  2. Urmimala Basu
  3. David Kuster
  4. Aparna Ramachandran
  5. Ewa Grudzien-Nogalska
  6. Atif Towheed
  7. Douglas C Wallace
  8. Megerditch Kiledjian
  9. Dmitry Temiakov
  10. Smita S Patel
  11. Richard H Ebright
  12. Bryce E Nickels
(2018)
Highly efficient 5' capping of mitochondrial RNA with NAD+ and NADH by yeast and human mitochondrial RNA polymerase
eLife 7:e42179.
https://doi.org/10.7554/eLife.42179
  2. Download BibTeX
  3. Download .RIS

Abstract

Bacterial and eukaryotic nuclear RNA polymerases (RNAPs) cap RNA with the oxidized and reduced forms of the metabolic effector nicotinamide adenine dinucleotide, NAD+ and NADH, using NAD+ and NADH as non‑canonical initiating nucleotides for transcription initiation. Here, we show that mitochondrial RNAPs (mtRNAPs) cap RNA with NAD+ and NADH, and do so more efficiently than nuclear RNAPs. Direct quantitation of NAD+- and NADH-capped RNA demonstrates remarkably high levels of capping in vivo: up to ~60% NAD+ and NADH capping of yeast mitochondrial transcripts, and up to ~15% NAD+ capping of human mitochondrial transcripts. The capping efficiency is determined by promoter sequence at, and upstream of, the transcription start site and, in yeast and human cells, by intracellular NAD+ and NADH levels. Our findings indicate mtRNAPs serve as both sensors and actuators in coupling cellular metabolism to mitochondrial transcriptional outputs, sensing NAD+ and NADH levels and adjusting transcriptional outputs accordingly.

Data availability

All data generated or analysed during this study are included in the manuscript.

Article and author information

Author details

  1. Jeremy G Bird

    Department of Genetics, Rutgers University, Piscataway, United States
    Competing interests
    The authors declare that no competing interests exist.

  2. Urmimala Basu

    Department of Biochemistry and Molecular Biology, Rutgers University, Piscataway, United States
    Competing interests
    The authors declare that no competing interests exist.

  3. David Kuster

    Biochemistry Center (BZH), Heidelberg University, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8157-9223

  4. Aparna Ramachandran

    Department of Biochemistry and Molecular Biology, Rutgers University, Piscataway, United States
    Competing interests
    The authors declare that no competing interests exist.

  5. Ewa Grudzien-Nogalska

    Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, United States
    Competing interests
    The authors declare that no competing interests exist.

  6. Atif Towheed

    Center for Mitochondrial and Epigenomic Medicine, The Children's Hospital of Philadelphia, Philadelphia, United States
    Competing interests
    The authors declare that no competing interests exist.

  7. Douglas C Wallace

    Center for Mitochondrial and Epigenomic Medicine, University of Pennsylvania, Philadelphia, United States
    Competing interests
    The authors declare that no competing interests exist.

  8. Megerditch Kiledjian

    Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, United States
    Competing interests
    The authors declare that no competing interests exist.

  9. Dmitry Temiakov

    Department of Biochemistry & Molecular Biology, Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, United States
    Competing interests
    The authors declare that no competing interests exist.

  10. Smita S Patel

    Department of Biochemistry and Molecular Biology, Rutgers University, Piscataway, United States
    For correspondence
    patelss@rwjms.rutgers.edu
    Competing interests
    The authors declare that no competing interests exist.

  11. Richard H Ebright

    Department of Chemistry, Rutgers University, Piscataway, United States
    For correspondence
    ebright@waksman.rutgers.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8915-7140

  12. Bryce E Nickels

    Department of Genetics, Rutgers University, Piscataway, United States
    For correspondence
    bnickels@waksman.rutgers.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7449-8831

Funding

National Institutes of Health (GM126488)

  • Megerditch Kiledjian

American Heart Association (16PRE30400001)

  • Urmimala Basu

National Institutes of Health (GM118086)

  • Smita S Patel

National Institutes of Health (GM104231)

  • Dmitry Temiakov

National Institutes of Health (GM041376)

  • Richard H Ebright

National Institutes of Health (GM118059)

  • Bryce E Nickels

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

© 2018, Bird 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.

Metrics

  • 4,987
    views
  • 721
    downloads
  • 64
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Jeremy G Bird
  2. Urmimala Basu
  3. David Kuster
  4. Aparna Ramachandran
  5. Ewa Grudzien-Nogalska
  6. Atif Towheed
  7. Douglas C Wallace
  8. Megerditch Kiledjian
  9. Dmitry Temiakov
  10. Smita S Patel
  11. Richard H Ebright
  12. Bryce E Nickels
(2018)
Highly efficient 5' capping of mitochondrial RNA with NAD+ and NADH by yeast and human mitochondrial RNA polymerase
eLife 7:e42179.
https://doi.org/10.7554/eLife.42179

Share this article

https://doi.org/10.7554/eLife.42179

Categories and tags

Research organisms

Further reading

    1. Biochemistry and Chemical Biology
    2. Cancer Biology

    The deubiquitinase Ubp3/Usp10 constrains glucose-mediated mitochondrial repression via phosphate budgeting

    Vineeth Vengayil, Shreyas Niphadkar ... Sunil Laxman
    Research Article

    Many cells in high glucose repress mitochondrial respiration, as observed in the Crabtree and Warburg effects. Our understanding of biochemical constraints for mitochondrial activation is limited. Using a Saccharomyces cerevisiae screen, we identified the conserved deubiquitinase Ubp3 (Usp10), as necessary for mitochondrial repression. Ubp3 mutants have increased mitochondrial activity despite abundant glucose, along with decreased glycolytic enzymes, and a rewired glucose metabolic network with increased trehalose production. Utilizing ∆ubp3 cells, along with orthogonal approaches, we establish that the high glycolytic flux in glucose continuously consumes free Pi. This restricts mitochondrial access to inorganic phosphate (Pi), and prevents mitochondrial activation. Contrastingly, rewired glucose metabolism with enhanced trehalose production and reduced GAPDH (as in ∆ubp3 cells) restores Pi. This collectively results in increased mitochondrial Pi and derepression, while restricting mitochondrial Pi transport prevents activation. We therefore suggest that glycolytic flux-dependent intracellular Pi budgeting is a key constraint for mitochondrial repression.

    1. Biochemistry and Chemical Biology

    Immunotherapy: The power lies in the glycans

    Luca Unione, Jesús Jiménez-Barbero
    Insight

    Glycans play an important role in modulating the interactions between natural killer cells and antibodies to fight pathogens and harmful cells.

    1. Biochemistry and Chemical Biology
    2. Cell Biology

    Two-way Dispatched function in Sonic hedgehog shedding and transfer to high-density lipoproteins

    Kristina Ehring, Sophia Friederike Ehlers ... Kay Grobe
    Research Article

    The Sonic hedgehog (Shh) signaling pathway controls embryonic development and tissue homeostasis after birth. This requires regulated solubilization of dual-lipidated, firmly plasma membrane-associated Shh precursors from producing cells. Although it is firmly established that the resistance-nodulation-division transporter Dispatched (Disp) drives this process, it is less clear how lipidated Shh solubilization from the plasma membrane is achieved. We have previously shown that Disp promotes proteolytic solubilization of Shh from its lipidated terminal peptide anchors. This process, termed shedding, converts tightly membrane-associated hydrophobic Shh precursors into delipidated soluble proteins. We show here that Disp-mediated Shh shedding is modulated by a serum factor that we identify as high-density lipoprotein (HDL). In addition to serving as a soluble sink for free membrane cholesterol, HDLs also accept the cholesterol-modified Shh peptide from Disp. The cholesteroylated Shh peptide is necessary and sufficient for Disp-mediated transfer because artificially cholesteroylated mCherry associates with HDL in a Disp-dependent manner, whereas an N-palmitoylated Shh variant lacking C-cholesterol does not. Disp-mediated Shh transfer to HDL is completed by proteolytic processing of the palmitoylated N-terminal membrane anchor. In contrast to dual-processed soluble Shh with moderate bioactivity, HDL-associated N-processed Shh is highly bioactive. We propose that the purpose of generating different soluble forms of Shh from the dual-lipidated precursor is to tune cellular responses in a tissue-type and time-specific manner.