Puromycin reactivity does not accurately localize translation at the subcellular level

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. Here, using in vitro and in vivo experiments, 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 the in vivo context. Introduction:

The midbody and midbody remnant are assembly sites for RNA and active translation

“…The puromycin ring during mitosis was also observed in different cell types, including CHO, Retinal Pigmented Epithelial (RPE) cells, NeuroEpithelium Stem cells (NES) (Fig. S3B), We confirmed that this translation signal was indeed active using OPP-ClickIT and HPG-ClickIT (77). We observed that the HPG-ClickIT signal gave a hazy disk in the MB in early G1(Fig.…”

“…To determine whether MB mRNAs are translationally activated or silenced, we used two methods to quantify translation. We used the puromycin-based SUnSET technique to label nascent peptides and visualize sites of recent translation using anti-puromycin antibodies( 77 , 78 ), and OPP-ClickIT and HPG-ClickIT( 77 ), to determine whether active translation occurs in the MB and MBRs. Synchronized HeLa cells pulsed with puromycin for 4 minutes in early telophase (ET) showed little evidence of MB translation(~39% had rings at the ET stage)(Fig.3A, S3A).…”

A Quick Guide to Small-Molecule Inhibitors of Eukaryotic Protein Synthesis

“…Treating the cells with puromycin in a com bination with cycloheximide leads to the accumulation of ribosomes exclusively on start codons, which facilitates their identification by ribosome profiling [127]. On the other hand, the combined effect of puromycin and other antibiotics depends on the ratio and concentrations of these compounds and cannot always be predicted, which may lead to artifacts [128,129]. The activity of puromycin is unique; for example, structurally similar antibiotic A201A (see above) does not act as a peptide bond accep tor and only inhibits the peptidyl transferase reac tion [13,40].…”

Chloroplast-localized translation for protein targeting in Chlamydomonas reinhardtii

“…The RPM method takes advantage of the conjugation of puromycin to the nascent polypeptide when it terminates translation by imaging the IF signal from the resulting puromycin-conjugated nascent polypeptides as markers in situ for locations of translation in vivo ( 12 ). Routing of the puromycin-conjugated nascent polypeptides from sites of their synthesis, a concern when live cells are treated prior to fixation and IF-staining ( 20 ), is unlikely because we treated isolated chloroplasts with puromycin.…”