A real-time fluorescence assay for CPSF73, the nuclease for pre-mRNA 3′-end processing

CPSF73 is the endonuclease that catalyzes the cleavage reaction for 3'-end processing ofmRNAprecursors (pre-mRNAs) in two distinct machineries, a canonical machinery for the majority of pre-mRNAs and a U7 snRNP (U7 machinery) for replication-dependent histone pre-mRNAs in animal cells. CPSF73 also possesses 5'-3' exonuclease activity in the U7 machinery, degrading the downstream cleavage product after the endonucleolytic cleavage. Recent studies showthat CPSF73 is a potential target for developing anticancer, antimalarial, and antiprotozoal drugs, spurring interest in identifying new small-molecule inhibitors against this enzyme. CPSF73 nuclease activity has so far been demonstrated using a gel-based end-point assay, using radiolabeled or fluorescently labeled RNA substrates. By taking advantage of unique properties of the U7 machinery, we have developed a novel, real-time fluorescence assay for the nuclease activity of CPSF73. This assay is facile and high-throughput, and should also be helpful for the discovery of new CPSF73 inhibitors.

Automated summary

CPSF73 is an endonuclease that catalyzes the cleavage reaction for 3'-end processing of mRNA precursors in two distinct machineries. Recent studies suggest that CPSF73 is a potential target for developing anticancer, antimalarial, and antiprotozoal drugs. By developing a real-time fluorescence assay, the nuclease activity of CPSF73 can be efficiently measured.