Marayti Maryati
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
Authors
Ishwinder Kaur
Gopal P. Jadhav
Loyin Olotu-Umoren
Blessing Oveh
Lubna Hashmi
Peter M. Fischer
G. Sebastiaan Winkler
Abstract
In eukaryotic cells, the shortening and removal of the poly(A) tail of cytoplasmic mRNA by deadenylase enzymes is a critical step in post-transcriptional 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 fluorescence-based assay. © The Author(s) 2013.
Citation
Maryati, M., Kaur, I., Jadhav, G. P., Olotu-Umoren, L., Oveh, B., Hashmi, L., …Winkler, G. S. (2014). A fluorescence-based assay suitable for quantitative analysis of deadenylase enzyme activity. Nucleic Acids Research, 42(5), https://doi.org/10.1093/nar/gkt972
Journal Article Type | Article |
---|---|
Online Publication Date | Oct 28, 2013 |
Publication Date | Mar 1, 2014 |
Deposit Date | Apr 16, 2014 |
Publicly Available Date | Mar 28, 2024 |
Journal | Nucleic Acids Research |
Print ISSN | 0305-1048 |
Electronic ISSN | 1362-4962 |
Publisher | Oxford University Press |
Peer Reviewed | Peer Reviewed |
Volume | 42 |
Issue | 5 |
DOI | https://doi.org/10.1093/nar/gkt972 |
Public URL | https://nottingham-repository.worktribe.com/output/718286 |
Publisher URL | https://academic.oup.com/nar/article/42/5/e30/1063553 |
Additional Information | Online version |
Files
Winkler_Fluorescence.pdf
(4.2 Mb)
PDF
Publisher Licence URL
https://creativecommons.org/licenses/by/3.0/
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