Substrate discrimination and quality control require each catalytic activity of TRAMP and the nuclear RNA exosome

Abstract: Quality control requires discrimination between functional and aberrant species to selectively target substrates for destruction. Nuclear RNA quality control in Saccharomyces cerevisiae includes the TRAMP complex that marks RNA for decay via polyadenylation and helicase-dependent 3′ to 5′ degradation by the RNA exosome. Using reconstitution biochemistry we show that polyadenylation and helicase activities of TRAMP cooperate with processive and distributive exoribonuclease activities of the nuclear RNA exosome to selectively target and degrade an unmodified tRNA while leaving native tRNA intact. Inactivation of the distributive exoribonuclease activity of Rrp6 results in loss of substrate discrimination, leading to degradation of all RNAs. These data suggest that the activities of the Mtr4 helicase and Rrp6 exoribonuclease endow the TRAMP-RNA exosome complex with the ability to protect stable RNA while degrading defective RNA species. Rrp6 and its 3′-5′ exonuclease activity have previously been shown to contribute to quality control and processing of various types of nuclear RNAs. Our sequencing analysis further confirms that Rrp6 contributes to this process.摘要：质量调控需区分功能性与异常性RNA物种，以选择性靶向底物并介导其降解。酿酒酵母（Saccharomyces cerevisiae）的细胞核RNA质量控制系统包含TRAMP复合物（TRAMP complex），该复合物可通过多聚腺苷酸化标记RNA，进而经由依赖解旋酶的RNA外切体（RNA exosome）介导的3′→5′降解途径实现RNA清除。本研究借助体外重建生化实验证实，TRAMP的多聚腺苷酸化与解旋酶活性，可与细胞核RNA外切体的持续型及分散型外切核糖核酸酶活性协同作用，选择性靶向并降解未修饰转运RNA（tRNA），同时保留天然转运RNA的完整性。失活Rrp6的分散型外切核糖核酸酶活性会导致底物识别特异性丧失，进而引发所有RNA的非选择性降解。上述数据表明，Mtr4解旋酶（Mtr4 helicase）与Rrp6外切核糖核酸酶的活性，赋予了TRAMP-RNA外切体复合物保护稳定RNA并降解缺陷RNA物种的能力。此前已有研究证实，Rrp6及其3′→5′外切核酸酶活性参与多种细胞核RNA的质量调控与加工过程。本研究的测序分析进一步验证了Rrp6在该过程中的作用。