A real-time fluorescence assay for CPSF73, the nuclease for pre-mRNA 3'-end processing.
| Autor: | Gutierrez PA; Department of Biological Sciences, Columbia University, New York, New York 10027, USA., Baughman K; Department of Biological Sciences, Columbia University, New York, New York 10027, USA., Sun Y; Department of Biological Sciences, Columbia University, New York, New York 10027, USA., Tong L; Department of Biological Sciences, Columbia University, New York, New York 10027, USA. |
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| Jazyk: | angličtina |
| Zdroj: | RNA (New York, N.Y.) [RNA] 2021 Oct; Vol. 27 (10), pp. 1148-1154. Date of Electronic Publication: 2021 Jul 06. |
| DOI: | 10.1261/rna.078764.121 |
| Abstrakt: | 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. (© 2021 Gutierrez et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.) |
| Databáze: | MEDLINE |
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