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

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

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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)
Pages (from-to)e30
JournalNucleic Acids Research
Volume42
Issue number5
DOIs
StatePublished - 2014

All Science Journal Classification (ASJC) codes

  • Genetics

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    Maryati, M., Kaur, I., Jadhav, G. P., Olotu-Umoren, L., Oveh, B., Hashmi, L., Fischer, P. M., & Winkler, G. S. (2014). A fluorescence-based assay suitable for quantitative analysis of deadenylase enzyme activity. Nucleic Acids Research, 42(5), e30. https://doi.org/10.1093/nar/gkt972