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.

中文翻译：

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CPSF73 的实时荧光检测，用于前 mRNA 3' 端加工的核酸酶

CPSF73 是一种核酸内切酶，在两种不同的机器中催化 mRNA 前体（pre-mRNAs）的 3'-末端加工，一种用于大多数 pre-mRNAs 的规范机器和一种用于复制依赖的 U7 snRNP（U7 机器）动物细胞中的组蛋白前 mRNA。CPSF73 在 U7 机器中还具有 5'-3' 外切核酸酶活性，在核酸内切后降解下游的切割产物。最近的研究表明，CPSF73 是开发抗癌、抗疟疾和抗原虫药物的潜在靶点，激发了人们对识别针对这种酶的新小分子抑制剂的兴趣。迄今为止，CPSF73 核酸酶的活性已通过基于凝胶的终点测定法、放射性标记或荧光标记的 RNA 底物得到证实。利用 U7 机械的独特性能，我们开发了一种新颖的实时荧光检测方法，用于检测 CPSF73 的核酸酶活性。该检测方法简便、通量高，也有助于发现新的 CPSF73 抑制剂。