A real-time fluorescence assay for CPSF73, the nuclease for pre-mRNA 3'-end processing
Pedro A Gutierrez1, Kirk Baughman1, Yadong Sun1
1Department of Biological Sciences, Columbia University, New York, New York 10027, USA.
Abstract:
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.
Insights
We developed a new real-time fluorescence assay to measure the nuclease activity of CPSF73, an enzyme crucial for mRNA processing and a target for drug development.
Area of Science:
- Molecular Biology
- Biochemistry
Background:
- Cleavage and Polyadenylation Specificity Factor subunit 73 (CPSF73) is an endonuclease essential for 3'-end processing of pre-mRNAs.
- CPSF73 functions in both canonical and U7 snRNP-dependent machineries and exhibits 5'-3' exonuclease activity in the latter.
- CPSF73 is a promising drug target for anticancer, antimalarial, and antiprotozoal therapies.
Purpose of the Study:
- To develop a novel, real-time fluorescence assay for measuring CPSF73 nuclease activity.
- To facilitate the discovery of small-molecule inhibitors targeting CPSF73.
Main Methods:
- Utilized the unique properties of the U7 snRNP machinery.
- Developed a real-time fluorescence-based assay to monitor CPSF73 nuclease activity.
- The assay is designed to be facile and high-throughput.
Main Results:
- A novel real-time fluorescence assay for CPSF73 nuclease activity was successfully developed.
- The assay leverages the U7 machinery for enhanced specificity and efficiency.
- This new method offers an improvement over existing gel-based end-point assays.
Conclusions:
- The developed real-time fluorescence assay provides a facile and high-throughput method for assessing CPSF73 nuclease activity.
- This assay will be instrumental in the discovery and development of new CPSF73 inhibitors for therapeutic applications.
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