Nuclear translation visualized by ribosome-bound nascent chain puromycylation.

TitleNuclear translation visualized by ribosome-bound nascent chain puromycylation.
Publication TypeJournal Article
Year of Publication2012
AuthorsDavid A, Dolan BP, Hickman HD, Knowlton JJ, Clavarino G, Pierre P, Bennink JR, Yewdell JW
JournalThe Journal of cell biology
Volume197
Issue1
Pagination45-57
Date Published2012 Apr 2
ISSN1540-8140
KeywordsAntibodies, Monoclonal, Catalysis, Cell Nucleolus, Cell Nucleus, Hela Cells, Humans, Microscopy, Fluorescence, Protein Biosynthesis, Puromycin, Ribosomes
Abstract

Whether protein translation occurs in the nucleus is contentious. To address this question, we developed the ribopuromycylation method (RPM), which visualizes translation in cells via standard immunofluorescence microscopy. The RPM is based on ribosome-catalyzed puromycylation of nascent chains immobilized on ribosomes by antibiotic chain elongation inhibitors followed by detection of puromycylated ribosome-bound nascent chains with a puromycin (PMY)-specific monoclonal antibody in fixed and permeabilized cells. The RPM correlates localized translation with myriad processes in cells and can be applied to any cell whose translation is sensitive to PMY. In this paper, we use the RPM to provide evidence for translation in the nucleoplasm and nucleolus, which is regulated by infectious and chemical stress.

DOI10.1083/jcb.201112145
Alternate JournalJ. Cell Biol.