He W, Parker R
Department of Molecular and Cellular Biology and Howard Hughes Medical Institute, University of Arizona, Tucson, Arizona 85721, USA.
Methods. 1999 Jan;17(1):3-10. doi: 10.1006/meth.1998.0701.
The analysis of mRNA turnover often requires a knowledge of the pathway by which a particular mRNA is being degraded. In this article we describe experimental procedures that can be used to determine the mechanism of degradation for yeast transcripts. These approaches include the insertion of strong secondary structures to block exonuclease cleavage and thereby trap decay intermediates. In addition, mRNA decay pathways can be analyzed by using regulatable promoters to perform transcriptional pulse-chase experiments, thereby allowing the determination of precursor-product relationships during the mRNA decay process. Finally, the mechanism of mRNA degradation can also now be determined by using trans-acting mutations specific for distinct mRNA turnover pathways. Most importantly, the combination of these three approaches can often clearly define the mechanism(s) by which a given transcript is degraded.
对mRNA周转的分析通常需要了解特定mRNA被降解的途径。在本文中，我们描述了可用于确定酵母转录本降解机制的实验程序。这些方法包括插入强二级结构以阻断核酸外切酶切割，从而捕获衰变中间体。此外，可以通过使用可调节启动子进行转录脉冲追踪实验来分析mRNA衰变途径，从而确定mRNA衰变过程中的前体-产物关系。最后，现在也可以通过使用针对不同mRNA周转途径的反式作用突变来确定mRNA降解的机制。最重要的是，这三种方法的结合通常可以清楚地定义给定转录本被降解的机制。
