1. Biochemistry and Chemical Biology
  2. Cell Biology
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Puromycin reactivity does not accurately localize translation at the subcellular level

  1. Syed Usman Enam
  2. Boris Zinshteyn
  3. Daniel H Goldman
  4. Madeline Cassani
  5. Nathan M Livingston
  6. Geraldine Seydoux
  7. Rachel Green  Is a corresponding author
  1. Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, United States
  2. Johns Hopkins University School of Medicine, United States
Research Article
  • Cited 14
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Cite this article as: eLife 2020;9:e60303 doi: 10.7554/eLife.60303

Abstract

Puromycin is a tyrosyl-tRNA mimic that blocks translation by labeling and releasing elongating polypeptide chains from translating ribosomes. Puromycin has been used in molecular biology research for decades as a translation inhibitor. The development of puromycin antibodies and derivatized puromycin analogs has enabled the quantification of active translation in bulk and single-cell assays. More recently, in vivo puromycylation assays have become popular tools for localizing translating ribosomes in cells. These assays often use elongation inhibitors to purportedly inhibit the release of puromycin-labeled nascent peptides from ribosomes. Using in vitro and in vivo experiments in various eukaryotic systems, we demonstrate that, even in the presence of elongation inhibitors, puromycylated peptides are released and diffuse away from ribosomes. Puromycylation assays reveal subcellular sites, such as nuclei, where puromycylated peptides accumulate post-release and which do not necessarily coincide with sites of active translation. Our findings urge caution when interpreting puromycylation assays in vivo.

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Raw data for all plots have been deposited in accompanying excel files.

Article and author information

Author details

  1. Syed Usman Enam

    Department of Molecular Biology and Genetics, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8976-0660
  2. Boris Zinshteyn

    Department of Molecular Biology and Genetics, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, United States
    Competing interests
    No competing interests declared.
  3. Daniel H Goldman

    Department of Molecular Biology and Genetics, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, United States
    Competing interests
    No competing interests declared.
  4. Madeline Cassani

    Department of Molecular Biology and Genetics, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, United States
    Competing interests
    No competing interests declared.
  5. Nathan M Livingston

    Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4670-708X
  6. Geraldine Seydoux

    Department of Molecular Biology and Genetics, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8257-0493
  7. Rachel Green

    Department of Molecular Biology and Genetics, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, United States
    For correspondence
    ragreen@jhmi.edu
    Competing interests
    Rachel Green, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9337-2003

Funding

National Institutes of Health (2R37GM059425-14)

  • Rachel Green

National Institutes of Health (5R37HD037047-20)

  • Geraldine Seydoux

National Institutes of Health (5K99GM135450-02)

  • Boris Zinshteyn

Howard Hughes Medical Institute

  • Rachel Green

Howard Hughes Medical Institute

  • Geraldine Seydoux

Damon Runyon Cancer Research Foundation (DRG-2280-16)

  • Daniel H Goldman

Damon Runyon Cancer Research Foundation (DRG-2250-16)

  • Boris Zinshteyn

National Institutes of Health (T32 GM007445)

  • Madeline Cassani
  • Nathan M Livingston

National Science Foundation (DGE-1746891)

  • Madeline Cassani

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

Reviewing Editor

  1. Robert H Singer, Albert Einstein College of Medicine, United States

Publication history

  1. Received: June 22, 2020
  2. Accepted: August 22, 2020
  3. Accepted Manuscript published: August 26, 2020 (version 1)
  4. Version of Record published: September 14, 2020 (version 2)
  5. Version of Record updated: November 25, 2020 (version 3)

Copyright

© 2020, Enam 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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