Goldstrohm Aaron C, Hook Brad A, Wickens Marvin
Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Methods Enzymol. 2008;448:77-106. doi: 10.1016/S0076-6879(08)02605-0.
The 3'-poly(A) tail, found on virtually all mRNAs, is enzymatically shortened by a process referred to as "deadenylation." Deadenylation is a widespread means of controlling mRNA stability and translation. The enzymes involved-so-called deadenylases-are surprisingly diverse. They are controlled by RNA sequences commonly found in 3'-untranslated regions (UTRs), which bind regulatory factors. Both RNA-binding proteins and microRNAs accelerate deadenylation of specific mRNAs. In some cases, regulators enhance deadenylation by binding to and recruiting specific deadenylases to the target mRNA. The many hundreds of potential regulators encoded in mammalian genomes (both RNA-binding proteins and microRNAs) and the numerous deadenylases, coupled with the many potential regulatory sites represented in 3' UTRs of mRNAs, provide fertile ground for regulated deadenylation. Recent global studies of poly(A) regulation support this conclusion. Biochemical and genetic approaches will be essential for exploring regulated deadenylation. The methods we describe focus on the reconstruction in vitro of regulated deadenylation with purified components from yeast. We discuss broadly the strategies, problems, and history of in vitro deadenylation systems. We combine this with a more detailed discussion of the purification, activity, and regulation of the Saccharomyces cerevisiae Ccr4p-Pop2p deadenylase complex and its regulation by PUF (Pumilio and Fem-3 binding factor) RNA-binding proteins.
几乎所有信使核糖核酸（mRNA）上都存在的3' - 聚腺苷酸（poly(A)）尾，会通过一种称为“去腺苷酸化”的过程被酶促缩短。去腺苷酸化是控制mRNA稳定性和翻译的一种广泛方式。所涉及的酶——即所谓的去腺苷酸酶——种类惊人地多样。它们受3' - 非翻译区（UTR）中常见的RNA序列控制，这些序列会结合调控因子。RNA结合蛋白和微小RNA（miRNA）都会加速特定mRNA的去腺苷酸化。在某些情况下，调控因子通过与靶mRNA结合并招募特定的去腺苷酸酶来增强去腺苷酸化。哺乳动物基因组中编码的数百种潜在调控因子（包括RNA结合蛋白和miRNA）以及众多的去腺苷酸酶，再加上mRNA的3' UTR中存在的许多潜在调控位点，为受调控的去腺苷酸化提供了丰富的基础。最近对聚腺苷酸调控的全局性研究支持了这一结论。生化和遗传学方法对于探索受调控的去腺苷酸化至关重要。我们所描述的方法侧重于利用酵母中的纯化成分在体外重建受调控的去腺苷酸化。我们广泛讨论了体外去腺苷酸化系统的策略、问题和历史。我们还结合更详细地讨论酿酒酵母Ccr4p - Pop2p去腺苷酸酶复合物的纯化、活性和调控，以及它受PUF（Pumilio和Fem - 3结合因子）RNA结合蛋白的调控。
