title: Nuclear translation visualized by ribosome-bound nascent chain puromycylation.
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.
authors.affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Alexandre
authors.initials: A
authors.lastname: David
authors.forename: Brian P
authors.initials: BP
authors.lastname: Dolan
authors.forename: Heather D
authors.initials: HD
authors.lastname: Hickman
authors.forename: Jonathan J
authors.initials: JJ
authors.lastname: Knowlton
authors.forename: Giovanna
authors.initials: G
authors.lastname: Clavarino
authors.forename: Philippe
authors.initials: P
authors.lastname: Pierre
authors.forename: Jack R
authors.initials: JR
authors.lastname: Bennink
authors.forename: Jonathan W
authors.initials: JW
authors.lastname: Yewdell
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D002384:Catalysis; D002466:Cell Nucleolus / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D014176:Protein Biosynthesis; D011691:Puromycin / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D011691:Puromycin