Puromycin reactivity does not accurately localize translation at the subcellular level.

Syed Usman Enam,Geraldine Seydoux,Madeline Cassani,Rachel Green,Daniel H Goldman,Nathan M Livingston,Boris Zinshteyn

doi:10.7554/elife.60303

Syed Usman Enam, Geraldine Seydoux + Show 5 more

Open Access

https://doi.org/10.7554/elife.60303

Copy DOI

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.

Highlights

Puromycin is a potent translational inhibitor that binds to ribosomes from all domains of life and has been used as a chemical probe and selectable marker for decades (Aviner, 2020; Yarmolinsky and Haba, 1959)
Puromycin mimics the 30 adenosine of a tRNA charged with a modified tyrosine, which binds in the ribosomal acceptor site (Figure 1A)
We have demonstrated that the puromycin method for visualizing localized translation does not faithfully detect nascent polypeptides at the site of their synthesis

Summary

Introduction

Puromycin is a potent translational inhibitor that binds to ribosomes from all domains of life and has been used as a chemical probe and selectable marker for decades (Aviner, 2020; Yarmolinsky and Haba, 1959). The development of anti-puromycin antibodies and of derivatized analogs of puromycin (Fujiwara et al, 1982; Liu et al, 2012) has led to the commonplace use of puromycin as a metabolic probe to measure the extent of active translation, replacing radioactive tracers such as S35 methionine. Subsequent development of the ribopuromycylation method (RPM) (David et al, 2012; Bastide et al, 2018) pushed the technique a step further, claiming to detect the subcellular localization of actively translating ribosomes using a puromycin-specific antibody. In these initial publications, the authors argued that the translation elongation inhibitors cycloheximide or emetine prevent dissociation of the puromycylated peptides from the ribosome. Previous work (Grollman, 1968; Colombo et al, 1965) established that these inhibitors prevent puromycin-induced splitting of ribosomes into individual subunits, but do not

Methods

Results

Conclusion