Elongation inhibitors do not prevent the release of puromycylated nascent polypeptide chains from ribosomes

  1. Benjamin D Hobson
  2. Linghao Kong
  3. Erik W Hartwick
  4. Ruben L Gonzalez
  5. Peter A Sims  Is a corresponding author
  1. Columbia University Irving Medical Center, United States
  2. Columbia University, United States

Abstract

Puromycin is an amino-acyl transfer RNA analog widely employed in studies of protein synthesis. Since puromycin is covalently incorporated into nascent polypeptide chains, anti-puromycin immunofluorescence enables visualization of nascent protein synthesis. A common assumption in studies of local messenger RNA translation is that the anti-puromycin staining of puromycylated nascent polypeptides in fixed cells accurately reports on their original site of translation, particularly when ribosomes are stalled with elongation inhibitors prior to puromycin treatment. However, when we attempted to implement a proximity ligation assay to detect ribosome-puromycin complexes, we found no evidence to support this assumption. We further demonstrated, using biochemical assays and live cell imaging of nascent polypeptides in mammalian cells, that puromycylated nascent polypeptides rapidly dissociate from ribosomes even in the presence of elongation inhibitors. Our results suggest that attempts to define precise subcellular translation sites using anti-puromycin immunostaining may be confounded by release of puromycylated nascent polypeptide chains prior to fixation.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files. Source data files have been provided for all Figures.

The following previously published data sets were used

Article and author information

Author details

  1. Benjamin D Hobson

    Depart of Systems Biology, Medical Scientist Training Program, Columbia University Irving Medical Center, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2745-5318
  2. Linghao Kong

    Depart of Systems Biology, Columbia University Irving Medical Center, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Erik W Hartwick

    Department of Chemistry, Columbia University, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Ruben L Gonzalez

    Department of Chemistry, Columbia University, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1344-5581
  5. Peter A Sims

    Depart of Systems Biology, Department of Biochemistry & Molecular Biophysics, Columbia University Irving Medical Center, New York, United States
    For correspondence
    pas2182@cumc.columbia.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3921-4837

Funding

National Institutes of Health (R33CA202827)

  • Peter A Sims

National Institutes of Health (F30 DA047775)

  • Benjamin D Hobson

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 15, 2020
  2. Accepted: August 4, 2020
  3. Accepted Manuscript published: August 26, 2020 (version 1)
  4. Version of Record published: September 14, 2020 (version 2)

Copyright

© 2020, Hobson 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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  1. Benjamin D Hobson
  2. Linghao Kong
  3. Erik W Hartwick
  4. Ruben L Gonzalez
  5. Peter A Sims
(2020)
Elongation inhibitors do not prevent the release of puromycylated nascent polypeptide chains from ribosomes
eLife 9:e60048.
https://doi.org/10.7554/eLife.60048
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