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Nuclear translation visualized by ribosome-bound nascent chain puromycylation.
|Title||Nuclear translation visualized by ribosome-bound nascent chain puromycylation.|
|Publication Type||Journal Article|
|Year of Publication||2012|
|Authors||David A, Dolan BP, Hickman HD, Knowlton JJ, Clavarino G, Pierre P, Bennink JR, Yewdell JW|
|Journal||The Journal of cell biology|
|Date Published||2012 Apr 2|
|Keywords||Antibodies, Monoclonal, Catalysis, Cell Nucleolus, Cell Nucleus, Hela Cells, Humans, Microscopy, Fluorescence, Protein Biosynthesis, Puromycin, Ribosomes|
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.
|Alternate Journal||J. Cell Biol.|