A real-time fluorescence assay for CPSF73, the nuclease for pre-mRNA 3'-end processing.
Pedro A GutierrezKirk BaughmanYadong SunLiang TongPublished in: RNA (New York, N.Y.) (2021)
CPSF73 is the endonuclease that catalyzes the cleavage reaction for 3'-end processing of mRNA precursors (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 show that 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.
Keyphrases
- high throughput
- dna binding
- small molecule
- single cell
- induced apoptosis
- transcription factor
- single molecule
- binding protein
- computed tomography
- quantum dots
- energy transfer
- dna repair
- oxidative stress
- endoplasmic reticulum stress
- gene expression
- cell death
- positron emission tomography
- climate change
- case control
- nucleic acid