A fluorescence-based assay suitable for quantitative analysis of deadenylase enzyme activity

Maryati Maryati, Ishwinder Kaur, Gopal Jadhav, Loyin Olotu-Umoren, Blessing Oveh, Lubna Hashmi, Peter M. Fischer, G. Sebastiaan Winkler

Research output: Contribution to journalArticle

10 Citations (Scopus)

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.

Original languageEnglish (US)
JournalNucleic Acids Research
Volume42
Issue number5
DOIs
StatePublished - Jan 1 2014
Externally publishedYes

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Fluorescence
Enzymes
Catalytic Domain
Small Molecule Libraries
Exonucleases
Endonucleases
Eukaryotic Cells
Enzyme Inhibitors
Ribonucleases
Phosphoric Monoester Hydrolases
Ions
Messenger RNA
Genes

All Science Journal Classification (ASJC) codes

  • Genetics

Cite this

A fluorescence-based assay suitable for quantitative analysis of deadenylase enzyme activity. / Maryati, Maryati; Kaur, Ishwinder; Jadhav, Gopal; Olotu-Umoren, Loyin; Oveh, Blessing; Hashmi, Lubna; Fischer, Peter M.; Winkler, G. Sebastiaan.

In: Nucleic Acids Research, Vol. 42, No. 5, 01.01.2014.

Research output: Contribution to journalArticle

Maryati, M, Kaur, I, Jadhav, G, Olotu-Umoren, L, Oveh, B, Hashmi, L, Fischer, PM & Winkler, GS 2014, 'A fluorescence-based assay suitable for quantitative analysis of deadenylase enzyme activity', Nucleic Acids Research, vol. 42, no. 5. https://doi.org/10.1093/nar/gkt972
Maryati, Maryati ; Kaur, Ishwinder ; Jadhav, Gopal ; 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. In: Nucleic Acids Research. 2014 ; Vol. 42, No. 5.
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AU - Oveh, Blessing

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AU - Fischer, Peter M.

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