Puromycin reactivity does not accurately localize translation at the subcellular level
- Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, United States
- Johns Hopkins University School of Medicine, United States
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.
Data availability
Raw data for all plots have been deposited in accompanying excel files.
Article and author information
Author details
Nathan M Livingston
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.
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.
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)
Puromycin reactivity does not accurately localize translation at the subcellular level
eLife 9:e60303.
https://doi.org/10.7554/eLife.60303
