The Signal Transduction Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, United States.
The Department of Biological Chemistry, University of Michigan, Ann Arbor, MI, United States.
Methods. 2019 Feb 15;155:77-87. doi: 10.1016/j.ymeth.2019.01.003. Epub 2019 Jan 6.
Decay of transcribed mRNA is a key determinant of steady state mRNA levels in cells. Global analysis of mRNA decay in cultured cells has revealed amazing heterogeneity in rates of decay under normal growth conditions, with calculated half-lives ranging from several minutes to many days. The factors that are responsible for this wide range of decay rates are largely unknown, although our knowledge of trans-acting RNA binding proteins and non-coding RNAs that can control decay rates is increasing. Many methods have been used to try to determine mRNA decay rates under various experimental conditions in cultured cells, and transcription inhibitors like actinomycin D have probably the longest history of any technique for this purpose. Despite this long history of use, the actinomycin D method has been criticized as prone to artifacts, and as ineffective for some promoters. With appropriate guidelines and controls, however, it can be a versatile, effective technique for measuring endogenous mRNA decay in cultured mammalian and insect cells, as well as the decay of exogenously-expressed transcripts. It can be used readily on a genome-wide level, and is remarkably cost-effective. In this short review, we will discuss our utilization of this approach in these cells; we hope that these methods will allow more investigators to apply this useful technique to study mRNA decay under the appropriate conditions.
mRNA 的降解是细胞中 mRNA 稳态水平的关键决定因素。对培养细胞中 mRNA 降解的全局分析显示，在正常生长条件下，降解率存在惊人的异质性，计算得到的半衰期从几分钟到几天不等。导致这种广泛降解率的因素在很大程度上尚不清楚，尽管我们对可以控制降解率的反式作用 RNA 结合蛋白和非编码 RNA 的了解正在不断增加。已经使用了许多方法来尝试在培养细胞中的各种实验条件下确定 mRNA 降解率，而放线菌素 D 等转录抑制剂可能是为此目的而使用的最古老的技术之一。尽管这种用途已有很长的历史，但放线菌素 D 法已被批评为容易产生假象，并且对某些启动子无效。然而，只要有适当的指南和对照，它就可以成为一种用于测量培养的哺乳动物和昆虫细胞中内源性 mRNA 降解以及外源表达转录物降解的多功能、有效技术。它可以在全基因组水平上方便地使用，并且具有很高的成本效益。在这篇简短的综述中，我们将讨论我们在这些细胞中使用这种方法的情况；我们希望这些方法将使更多的研究人员能够在适当的条件下将这种有用的技术应用于研究 mRNA 降解。
