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

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• The 5' cap of eukaryotic messenger RNA (mRNA) is vital for RNA stability, processing, nuclear transport, and translation.
• Removal of the terminal gamma-phosphate from the 5' end of nascent RNA is a prerequisite for capping.
• Cet1p, an RNA triphosphatase in Saccharomyces cerevisiae, catalyzes this dephosphorylation reaction.

Purpose of the Study:

• To characterize the steady-state kinetics of the fungal RNA triphosphatase, Cet1p.
• To establish robust and scalable assay formats for Cet1p activity.
• To validate Cet1p as a potential antifungal drug target.

Main Methods:

• Characterization of Cet1p steady-state kinetics using synthetic RNA oligonucleotides and nucleoside triphosphates.
• Development of a scintillation proximity assay (SPA)-based high-throughput screen (GTP-SPA) utilizing [gamma-(33)P]biotin-11 GTP.
• Adaptation of a charcoal absorption method for 96-well plate format to measure inorganic phosphate release from RNA substrates.

Main Results:

• The GTP-SPA and 96-well charcoal absorption assays were optimized for sensitivity, accuracy, and robustness.
• Both assay formats demonstrated comparable performance when tested with known compounds.
• The developed assays are suitable for high-throughput screening and automation.

Conclusions:

• Cet1p is a validated target for antifungal drug discovery due to its essential role in fungal growth and distinct structural/catalytic properties compared to human counterparts.
• The developed GTP-SPA and charcoal absorption assays provide valuable tools for screening potential inhibitors of Cet1p.
• These methods have broader applicability for assaying various phosphatases involved in nucleotide, nucleic acid, or protein metabolism.