A fluorescence-based assay suitable for quantitative analysis of deadenylase enzyme activity
Nucleic acids research, 2014•academic.oup.com
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 …
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 …
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.
Oxford University Press