Skip to main content

Research Repository

Advanced Search

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

Marayti 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. © 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 Apr 16, 2014
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 http://eprints.nottingham.ac.uk/id/eprint/2323
Publisher URL https://academic.oup.com/nar/article/42/5/e30/1063553
Copyright Statement Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0
Additional Information Online version

Files

Winkler_Fluorescence.pdf (4.2 Mb)
PDF

Publisher Licence URL
http://creativecommons.org/licenses/by/3.0/





You might also like



Downloadable Citations