Loflin P T, Chen C Y, Xu N, Shyu A B
Department of Biochemistry and Molecular Biology, University of Texas Houston Health Science Center Medical School, Houston, Texas 77030, USA.
Methods. 1999 Jan;17(1):11-20. doi: 10.1006/meth.1998.0702.
Modulation of mRNA stability provides a powerful means for controlling gene expression during the cell cycle, cell differentiation, the immune response, as well as many other physiological transitions. Through the years, many different methods have been developed for measuring mRNA stability. Frequently mRNA stability is studied indirectly by analyzing the steady-state level of mRNA. Therefore by inference, changes in mRNA abundance are thought to affect only the stability of the mRNA, an assumption that is not always correct. Alternatively, direct measurements of mRNA decay are performed in a number of ways, including kinetic labeling techniques and administration of transcriptional inhibitors. Due to the nature of these techniques, they either are technically demanding or introduce a significant change in cell physiology. In addition, many critical mechanistic issues as to deadenylation kinetics, decay intermediates, and precursor-product relationships cannot be readily addressed by these methods. Here, we describe and discuss in detail two different transcriptional pulsing methods based on the c-fos serum-inducible promoter and the tetracycline-regulated promoter systems as an effort to better elucidate the mechanistic steps and regulation underlying differential and selective mRNA turnover in mammalian cells. Both systems allow unequivocal monitoring of deadenylation and decay kinetics as well as determination of precursor-product relationship. In addition, decay rate constants and half-lives are determined and used in both methods to quantitatively denote the mRNA stability. Thus, they provide a reliable way to determine subtle yet physiologically meaningful changes in mRNA stability. Application of one method or the other covers the study of mRNA turnover in most mammalian cell types under a wide range of physiological conditions.
mRNA稳定性的调控为在细胞周期、细胞分化、免疫反应以及许多其他生理转变过程中控制基因表达提供了一种强有力的手段。多年来，已经开发出许多不同的方法来测量mRNA稳定性。通常，mRNA稳定性是通过分析mRNA的稳态水平来间接研究的。因此，通过推断，mRNA丰度的变化被认为仅影响mRNA的稳定性，而这一假设并不总是正确的。另外，mRNA降解的直接测量可以通过多种方式进行，包括动力学标记技术和转录抑制剂的施用。由于这些技术的性质，它们要么技术要求高，要么会在细胞生理学上引入显著变化。此外，关于去腺苷酸化动力学、降解中间体和前体-产物关系的许多关键机制问题不能通过这些方法轻易解决。在这里，我们详细描述和讨论基于c-fos血清诱导型启动子和四环素调控启动子系统的两种不同的转录脉冲方法，以更好地阐明哺乳动物细胞中差异和选择性mRNA周转的机制步骤和调控。这两种系统都允许明确监测去腺苷酸化和降解动力学以及确定前体-产物关系。此外，在两种方法中都确定了降解速率常数和半衰期，并用于定量表示mRNA的稳定性。因此，它们提供了一种可靠的方法来确定mRNA稳定性中细微但具有生理意义的变化。应用其中一种方法涵盖了在广泛的生理条件下对大多数哺乳动物细胞类型中mRNA周转的研究。
