A real-time fluorescence assay for CPSF73, the nuclease for pre-mRNA 3′-end processing
| Autor: | Pedro A Gutierrez, Liang Tong, Yadong Sun, Kirk Baughman |
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| Rok vydání: | 2021 |
| Předmět: |
Models
Molecular Exonuclease Phenylalanine Biology Cleavage (embryo) Fluorescence Piperazines Histones Small Molecule Libraries Endonuclease RNA Precursors Humans snRNP Enzyme Inhibitors Molecular Biology Enzyme Assays Fluorescent Dyes Messenger RNA Nuclease Cell-Free System Rhodamines Ribonucleoprotein U7 Small Nuclear Cleavage And Polyadenylation Specificity Factor RNA Biochemistry Proteolysis biology.protein Biological Assay RNA 3' End Processing Precursor mRNA |
| Zdroj: | RNA. 27:1148-1154 |
| ISSN: | 1469-9001 1355-8382 |
| DOI: | 10.1261/rna.078764.121 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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