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Elongation inhibitors do not prevent the release of puromycylated nascent polypeptide chains from ribosomes
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 me...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
eLife Sciences Publications, Ltd
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7490010/ https://www.ncbi.nlm.nih.gov/pubmed/32844746 http://dx.doi.org/10.7554/eLife.60048 |
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author | Hobson, Benjamin D Kong, Linghao Hartwick, Erik W Gonzalez, Ruben L Sims, Peter A |
author_facet | Hobson, Benjamin D Kong, Linghao Hartwick, Erik W Gonzalez, Ruben L Sims, Peter A |
author_sort | Hobson, Benjamin D |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-7490010 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | eLife Sciences Publications, Ltd |
record_format | MEDLINE/PubMed |
spelling | pubmed-74900102020-09-16 Elongation inhibitors do not prevent the release of puromycylated nascent polypeptide chains from ribosomes Hobson, Benjamin D Kong, Linghao Hartwick, Erik W Gonzalez, Ruben L Sims, Peter A eLife Biochemistry and Chemical Biology 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. eLife Sciences Publications, Ltd 2020-08-26 /pmc/articles/PMC7490010/ /pubmed/32844746 http://dx.doi.org/10.7554/eLife.60048 Text en © 2020, Hobson et al http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/This article is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited. |
spellingShingle | Biochemistry and Chemical Biology Hobson, Benjamin D Kong, Linghao Hartwick, Erik W Gonzalez, Ruben L Sims, Peter A Elongation inhibitors do not prevent the release of puromycylated nascent polypeptide chains from ribosomes |
title | Elongation inhibitors do not prevent the release of puromycylated nascent polypeptide chains from ribosomes |
title_full | Elongation inhibitors do not prevent the release of puromycylated nascent polypeptide chains from ribosomes |
title_fullStr | Elongation inhibitors do not prevent the release of puromycylated nascent polypeptide chains from ribosomes |
title_full_unstemmed | Elongation inhibitors do not prevent the release of puromycylated nascent polypeptide chains from ribosomes |
title_short | Elongation inhibitors do not prevent the release of puromycylated nascent polypeptide chains from ribosomes |
title_sort | elongation inhibitors do not prevent the release of puromycylated nascent polypeptide chains from ribosomes |
topic | Biochemistry and Chemical Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7490010/ https://www.ncbi.nlm.nih.gov/pubmed/32844746 http://dx.doi.org/10.7554/eLife.60048 |
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