Nuclear translation visualized by ribosome-bound nascent chain puromycylation

• Published in: The Journal of cell biology Vol. 197; no. 1; pp. 45 - 57
• Main Authors: David, Alexandre, Dolan, Brian P, Hickman, Heather D, Knowlton, Jonathan J, Clavarino, Giovanna, Pierre, Philippe, Bennink, Jack R, Yewdell, Jonathan W
• Format: Journal Article
• Language: English
• Published: United States Rockefeller University Press 02-04-2012; The Rockefeller University Press
• Subjects: Antibiotics; Antibodies, Monoclonal; Antibodies, Monoclonal - immunology; Catalysis; Cell Nucleolus; Cell Nucleolus - metabolism; Cell Nucleus; Cell Nucleus - metabolism; Cellular biology; HeLa Cells; Humans; Life Sciences; Microbiology and Parasitology; Microscopy; Microscopy, Fluorescence; Monoclonal antibodies; Protein Biosynthesis; Puromycin; Puromycin - analysis; Puromycin - immunology; Puromycin - metabolism; Ribonucleic acid; Ribosomes; Ribosomes - metabolism; RNA
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.201112145

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.