title: A fluorescence-based assay suitable for quantitative analysis of deadenylase enzyme activity.
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.
authors.affiliation: School of Pharmacy and Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK.
authors.forename: Maryati
authors.initials: M
authors.lastname: Maryati
authors.forename: Ishwinder
authors.initials: I
authors.lastname: Kaur
authors.forename: Gopal P
authors.initials: GP
authors.lastname: Jadhav
authors.forename: Loyin
authors.initials: L
authors.lastname: Olotu-Umoren
authors.forename: Blessing
authors.initials: B
authors.lastname: Oveh
authors.forename: Lubna
authors.initials: L
authors.lastname: Hashmi
authors.forename: Peter M
authors.initials: PM
authors.lastname: Fischer
authors.forename: G Sebastiaan
authors.initials: GS
authors.lastname: Winkler
mesh_terms: D057075:Enzyme Assays / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005095:Exoribonucleases / Q000378:metabolism; D005453:Fluorescence; D006367:HeLa Cells; D006801:Humans; D012097:Repressor Proteins; D012260:Ribonucleases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D054852:Small Molecule Libraries; D014157:Transcription Factors / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: C489780:CNOT8 protein, human; D004791:Enzyme Inhibitors; D012097:Repressor Proteins; D054852:Small Molecule Libraries; D014157:Transcription Factors; C545829:CNOT7 protein, human; D005095:Exoribonucleases; D012260:Ribonucleases; C445084:mRNA deadenylase