A fluorescence-based assay suitable for quantitative analysis of deadenylase enzyme activity
In eukaryotic cells, the shortening and removal of the poly(A) tail of cytoplasmic mRNA by deadenylase enzymes is a critical step in posttranscriptional gene regulation. The ribonuclease activity of deadenylase enzymes is attributed to either a DEDD (Asp-Glu-Asp-Asp) or an endonuclease–exonuclease–...
| Main Authors: | , , , , , , , |
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| Format: | Article |
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Oxford Journals
2013
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| Online Access: | https://eprints.nottingham.ac.uk/2323/ |
| _version_ | 1848790755037937664 |
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| author | Maryati, Marayti Kaur, Ishwinder Jadhav, Gopal P. Olotu-Umoren, Loyin Oveh, Blessing Hashmi, Lubna Fischer, Peter M. Winkler, G. Sebastiaan |
| author_facet | Maryati, Marayti Kaur, Ishwinder Jadhav, Gopal P. Olotu-Umoren, Loyin Oveh, Blessing Hashmi, Lubna Fischer, Peter M. Winkler, G. Sebastiaan |
| author_sort | Maryati, Marayti |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | In eukaryotic cells, the shortening and removal of the poly(A) tail of cytoplasmic mRNA by deadenylase enzymes is a critical step in posttranscriptional gene regulation. The ribonuclease activity of deadenylase enzymes is attributed
to either a DEDD (Asp-Glu-Asp-Asp) or an endonuclease–exonuclease–phosphatase domain. Both domains require the presence of two Mg2+ ions in the active site. To facilitate the biochemical analysis of deadenylase enzymes, we have
developed a fluorescence-based deadenylase assay. The assay is based on end-point measurement, suitable for quantitative analysis and can be adapted for 96- and 384-well microplate formats. We demonstrate the utility of the assay by screening a chemical compound library, resulting
in the identification of non-nucleoside inhibitors of the Caf1/CNOT7 enzyme, a catalytic subunit of the Ccr4–Not deadenylase complex. These compounds may be useful tools for the biochemical analysis of the Caf1/CNOT7 deadenylase
subunit of the Ccr4–Not complex and indicate the feasibility of developing selective inhibitors of deadenylase enzymes using the fluorescencebased assay. |
| first_indexed | 2025-11-14T18:17:39Z |
| format | Article |
| id | nottingham-2323 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| last_indexed | 2025-11-14T18:17:39Z |
| publishDate | 2013 |
| publisher | Oxford Journals |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-23232020-05-04T16:39:09Z https://eprints.nottingham.ac.uk/2323/ A fluorescence-based assay suitable for quantitative analysis of deadenylase enzyme activity Maryati, Marayti Kaur, Ishwinder Jadhav, Gopal P. Olotu-Umoren, Loyin Oveh, Blessing Hashmi, Lubna Fischer, Peter M. Winkler, G. Sebastiaan In eukaryotic cells, the shortening and removal of the poly(A) tail of cytoplasmic mRNA by deadenylase enzymes is a critical step in posttranscriptional gene regulation. The ribonuclease activity of deadenylase enzymes is attributed to either a DEDD (Asp-Glu-Asp-Asp) or an endonuclease–exonuclease–phosphatase domain. Both domains require the presence of two Mg2+ ions in the active site. To facilitate the biochemical analysis of deadenylase enzymes, we have developed a fluorescence-based deadenylase assay. The assay is based on end-point measurement, suitable for quantitative analysis and can be adapted for 96- and 384-well microplate formats. We demonstrate the utility of the assay by screening a chemical compound library, resulting in the identification of non-nucleoside inhibitors of the Caf1/CNOT7 enzyme, a catalytic subunit of the Ccr4–Not deadenylase complex. These compounds may be useful tools for the biochemical analysis of the Caf1/CNOT7 deadenylase subunit of the Ccr4–Not complex and indicate the feasibility of developing selective inhibitors of deadenylase enzymes using the fluorescencebased assay. Oxford Journals 2013-10-28 Article PeerReviewed Maryati, Marayti, Kaur, Ishwinder, Jadhav, Gopal P., Olotu-Umoren, Loyin, Oveh, Blessing, Hashmi, Lubna, Fischer, Peter M. and Winkler, G. Sebastiaan (2013) A fluorescence-based assay suitable for quantitative analysis of deadenylase enzyme activity. Nucleic Acids Research . pp. 1-10. ISSN 0305-1048 http://nar.oxfordjournals.org/content/early/2013/10/28/nar.gkt972.full doi:10.1093/nar/gkt972 doi:10.1093/nar/gkt972 |
| spellingShingle | Maryati, Marayti Kaur, Ishwinder Jadhav, Gopal P. Olotu-Umoren, Loyin Oveh, Blessing Hashmi, Lubna Fischer, Peter M. Winkler, G. Sebastiaan A fluorescence-based assay suitable for quantitative analysis of deadenylase enzyme activity |
| title | A fluorescence-based assay suitable for quantitative
analysis of deadenylase enzyme activity |
| title_full | A fluorescence-based assay suitable for quantitative
analysis of deadenylase enzyme activity |
| title_fullStr | A fluorescence-based assay suitable for quantitative
analysis of deadenylase enzyme activity |
| title_full_unstemmed | A fluorescence-based assay suitable for quantitative
analysis of deadenylase enzyme activity |
| title_short | A fluorescence-based assay suitable for quantitative
analysis of deadenylase enzyme activity |
| title_sort | fluorescence-based assay suitable for quantitative
analysis of deadenylase enzyme activity |
| url | https://eprints.nottingham.ac.uk/2323/ https://eprints.nottingham.ac.uk/2323/ https://eprints.nottingham.ac.uk/2323/ |