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.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Maryati M,Kaur I,Jadhav GP,Olotu-Umoren L,Oveh B,Hashmi L,Fischer PM,Winkler GSdoi
10.1093/nar/gkt972subject
Has Abstractpub_date
2014-03-01 00:00:00pages
e30issue
5eissn
0305-1048issn
1362-4962pii
gkt972journal_volume
42pub_type
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