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

Elife. 2020 Aug 26:9:e60048. doi: 10.7554/eLife.60048.

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.

Keywords: biochemistry; cell biology; chemical biology; human; local translation; mouse; proximity ligation assay; puromycin; ribosome; translation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line, Tumor
  • Mice
  • Peptide Chain Elongation, Translational / drug effects*
  • Protein Synthesis Inhibitors* / metabolism
  • Protein Synthesis Inhibitors* / pharmacology
  • Proteins / chemistry
  • Proteins / metabolism
  • Puromycin* / metabolism
  • Puromycin* / pharmacology
  • RNA, Messenger / chemistry
  • RNA, Messenger / metabolism
  • RNA, Transfer, Amino Acyl / chemistry
  • RNA, Transfer, Amino Acyl / metabolism
  • Ribosomes* / drug effects
  • Ribosomes* / metabolism

Substances

  • Protein Synthesis Inhibitors
  • Proteins
  • RNA, Messenger
  • RNA, Transfer, Amino Acyl
  • Puromycin