Puromycin reactivity does not accurately localize translation at the subcellular level
Puromycin is a tyrosyl-tRNA mimic that blocks translation by labeling and releasing elongating polypeptide chains from translating ribosomes. Puromycin has been used in molecular biology research for decades as a translation inhibitor. The development of puromycin antibodies and derivatized puromyci...
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2020-08-01
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| Series: | eLife |
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| Online Access: | https://elifesciences.org/articles/60303 |
| _version_ | 1811199320737710080 |
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| author | Syed Usman Enam Boris Zinshteyn Daniel H Goldman Madeline Cassani Nathan M Livingston Geraldine Seydoux Rachel Green |
| author_facet | Syed Usman Enam Boris Zinshteyn Daniel H Goldman Madeline Cassani Nathan M Livingston Geraldine Seydoux Rachel Green |
| author_sort | Syed Usman Enam |
| collection | DOAJ |
| description | Puromycin is a tyrosyl-tRNA mimic that blocks translation by labeling and releasing elongating polypeptide chains from translating ribosomes. Puromycin has been used in molecular biology research for decades as a translation inhibitor. The development of puromycin antibodies and derivatized puromycin analogs has enabled the quantification of active translation in bulk and single-cell assays. More recently, in vivo puromycylation assays have become popular tools for localizing translating ribosomes in cells. These assays often use elongation inhibitors to purportedly inhibit the release of puromycin-labeled nascent peptides from ribosomes. Using in vitro and in vivo experiments in various eukaryotic systems, we demonstrate that, even in the presence of elongation inhibitors, puromycylated peptides are released and diffuse away from ribosomes. Puromycylation assays reveal subcellular sites, such as nuclei, where puromycylated peptides accumulate post-release and which do not necessarily coincide with sites of active translation. Our findings urge caution when interpreting puromycylation assays in vivo. |
| first_indexed | 2024-04-12T01:47:15Z |
| format | Article |
| id | doaj.art-4f34ce7e063048d9bde53f7974f89f11 |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-04-12T01:47:15Z |
| publishDate | 2020-08-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-4f34ce7e063048d9bde53f7974f89f112022-12-22T03:53:03ZengeLife Sciences Publications LtdeLife2050-084X2020-08-01910.7554/eLife.60303Puromycin reactivity does not accurately localize translation at the subcellular levelSyed Usman Enam0https://orcid.org/0000-0001-8976-0660Boris Zinshteyn1https://orcid.org/0000-0003-0103-3501Daniel H Goldman2Madeline Cassani3Nathan M Livingston4https://orcid.org/0000-0003-4670-708XGeraldine Seydoux5https://orcid.org/0000-0001-8257-0493Rachel Green6https://orcid.org/0000-0001-9337-2003Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, United States; Howard Hughes Medical Institute, Baltimore, United StatesDepartment of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, United States; Howard Hughes Medical Institute, Baltimore, United StatesDepartment of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, United States; Howard Hughes Medical Institute, Baltimore, United StatesDepartment of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, United States; Howard Hughes Medical Institute, Baltimore, United StatesDepartment of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, United StatesDepartment of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, United States; Howard Hughes Medical Institute, Baltimore, United StatesDepartment of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, United States; Howard Hughes Medical Institute, Baltimore, United StatesPuromycin is a tyrosyl-tRNA mimic that blocks translation by labeling and releasing elongating polypeptide chains from translating ribosomes. Puromycin has been used in molecular biology research for decades as a translation inhibitor. The development of puromycin antibodies and derivatized puromycin analogs has enabled the quantification of active translation in bulk and single-cell assays. More recently, in vivo puromycylation assays have become popular tools for localizing translating ribosomes in cells. These assays often use elongation inhibitors to purportedly inhibit the release of puromycin-labeled nascent peptides from ribosomes. Using in vitro and in vivo experiments in various eukaryotic systems, we demonstrate that, even in the presence of elongation inhibitors, puromycylated peptides are released and diffuse away from ribosomes. Puromycylation assays reveal subcellular sites, such as nuclei, where puromycylated peptides accumulate post-release and which do not necessarily coincide with sites of active translation. Our findings urge caution when interpreting puromycylation assays in vivo.https://elifesciences.org/articles/60303ribosomepuromycinemetinepuromycylationop-puro |
| spellingShingle | Syed Usman Enam Boris Zinshteyn Daniel H Goldman Madeline Cassani Nathan M Livingston Geraldine Seydoux Rachel Green Puromycin reactivity does not accurately localize translation at the subcellular level eLife ribosome puromycin emetine puromycylation op-puro |
| title | Puromycin reactivity does not accurately localize translation at the subcellular level |
| title_full | Puromycin reactivity does not accurately localize translation at the subcellular level |
| title_fullStr | Puromycin reactivity does not accurately localize translation at the subcellular level |
| title_full_unstemmed | Puromycin reactivity does not accurately localize translation at the subcellular level |
| title_short | Puromycin reactivity does not accurately localize translation at the subcellular level |
| title_sort | puromycin reactivity does not accurately localize translation at the subcellular level |
| topic | ribosome puromycin emetine puromycylation op-puro |
| url | https://elifesciences.org/articles/60303 |
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