Elongation inhibitors do not prevent the release of puromycylated nascent polypeptide chains from ribosomes
- Columbia University Irving Medical Center, United States
- 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.
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Rabbit 80S ribosome stalled on a poly(A) tailProtein Data Bank, 6SGC.
Article and author information
Author details
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.
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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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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