A real-time fluorescence assay for CPSF73, the nuclease for pre-mRNA 3'-end processing.
RNA
; 27(10): 1148-1154, 2021 10.
Article
em En
| MEDLINE
| ID: mdl-34230059
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.
Palavras-chave
Texto completo:
1
Bases de dados:
MEDLINE
Assunto principal:
Bioensaio
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Precursores de RNA
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Histonas
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Ribonucleoproteína Nuclear Pequena U7
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Processamento de Terminações 3' de RNA
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Fator de Especificidade de Clivagem e Poliadenilação
Tipo de estudo:
Prognostic_studies
Limite:
Humans
Idioma:
En
Revista:
RNA
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2021
Tipo de documento:
Article
País de afiliação:
Estados Unidos