High-throughput assays for yeast RNA 5′ triphosphatase (Cet1p)

The 5′ cap on eukaryotic messenger RNA (mRNA) is critical for the stabilization, processing, nuclear transport, and translation of the transcript. Before capping can occur, the γ-phosphate from the 5′ end of newly synthesized RNA must be removed. In Saccharomyces cerevisiae, this reaction is catalyzed by Cet1p, an RNA triphosphatase. Because Cet1p is both essential for fungal growth and sufficiently different from its human counterpart in terms of three-dimensional structure and catalytic mechanism, it represents an unexplored target for antifungal drug discovery. To this end, we characterized the steady-state kinetics of Cet1p using both synthetic RNA oligos and nucleoside triphosphates. Nucleotide triphosphatase activity was measured in a scintillation proximity assay (SPA)-based high-throughput screen using [γ- 33P]biotin-11 GTP as substrate (GTP–SPA); the format is sensitive, accurate, robust, and compatible with automation. A charcoal absorption method was used to measure the release of free inorganic phosphate from an RNA substrate; the method was adapted to fit a 96-well plate format. The performance of the GTP–SPA and RNA assays was tested against a panel of commercially available compounds and found to be comparable. The charcoal absorption method run in the 96-well plate format has general utility for any phosphatase using nucleotides, nucleic acids, or proteins as substrate.