| Title |
A fluorescence-based assay suitable for quantitative analysis of deadenylase enzyme activity
|
|---|---|
| Published in |
Nucleic Acids Research, October 2013
|
| DOI | 10.1093/nar/gkt972 |
| Pubmed ID | |
| Authors |
Maryati Maryati, 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. |
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Geographical breakdown
| Country | Count | As % |
|---|---|---|
| United Kingdom | 1 | 50% |
| France | 1 | 50% |
Demographic breakdown
| Type | Count | As % |
|---|---|---|
| Scientists | 2 | 100% |
Mendeley demographics
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Germany | 1 | 1% |
| Unknown | 68 | 99% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Student > Ph. D. Student | 16 | 23% |
| Researcher | 12 | 17% |
| Student > Master | 9 | 13% |
| Student > Bachelor | 8 | 12% |
| Lecturer | 2 | 3% |
| Other | 5 | 7% |
| Unknown | 17 | 25% |
| Readers by discipline | Count | As % |
|---|---|---|
| Biochemistry, Genetics and Molecular Biology | 17 | 25% |
| Chemistry | 11 | 16% |
| Agricultural and Biological Sciences | 10 | 14% |
| Pharmacology, Toxicology and Pharmaceutical Science | 6 | 9% |
| Engineering | 3 | 4% |
| Other | 3 | 4% |
| Unknown | 19 | 28% |