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.
Keywords:
U7 snRNP; endonuclease; exonuclease; histone pre-mRNA 3′-end processing.
© 2021 Gutierrez et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Biological Assay*
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Cell-Free System
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Cleavage And Polyadenylation Specificity Factor / chemistry
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Cleavage And Polyadenylation Specificity Factor / genetics
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Cleavage And Polyadenylation Specificity Factor / metabolism*
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Enzyme Assays
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Enzyme Inhibitors / chemistry
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Enzyme Inhibitors / pharmacology
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Fluorescence
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Fluorescent Dyes / chemistry
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Histones / chemistry
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Histones / genetics
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Histones / metabolism*
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Humans
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Models, Molecular
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Phenylalanine / analogs & derivatives
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Phenylalanine / chemistry
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Phenylalanine / pharmacology
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Piperazines / chemistry
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Piperazines / pharmacology
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Proteolysis
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RNA 3' End Processing*
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RNA Precursors / chemistry
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RNA Precursors / genetics
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RNA Precursors / metabolism*
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Rhodamines / chemistry
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Ribonucleoprotein, U7 Small Nuclear / chemistry
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Ribonucleoprotein, U7 Small Nuclear / genetics
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Ribonucleoprotein, U7 Small Nuclear / metabolism*
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Small Molecule Libraries / chemistry
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Small Molecule Libraries / pharmacology
Substances
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5-carboxytetramethylrhodamine succinimidyl ester
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CPSF3 protein, human
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Cleavage And Polyadenylation Specificity Factor
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Enzyme Inhibitors
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Fluorescent Dyes
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Histones
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JTE 607
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Piperazines
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RNA Precursors
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Rhodamines
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Ribonucleoprotein, U7 Small Nuclear
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Small Molecule Libraries
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Phenylalanine