Clement Sandra L, Lykke-Andersen Jens
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO, USA.
Methods Mol Biol. 2008;419:121-33. doi: 10.1007/978-1-59745-033-1_8.
The regulation of mRNA turnover occurs in part through the action of mRNA-binding proteins that recognize specific nucleotide sequences and either activate or inhibit the decay of transcripts to which they are bound. In many cases, multiple mRNA-binding proteins, including those with opposing functions, bind to the same RNA sequence. This can make the study of the function of any one of these proteins difficult. Furthermore, monitoring endogenous mRNA decay rates using drugs that inhibit transcription (e.g., actinomycin D) can introduce pleiotropic effects. One way to circumvent these problems is to tether the protein of interest (POI) through a heterologous RNA-binding domain to an inducible reporter mRNA and measure the effect of the bound protein on mRNA decay. In this chapter, we illustrate the use of the tethering technique to study the role of a particular mRNA-binding protein, TTP, on the decay of an otherwise stable mRNA to which it is tethered through a fusion to the bacteriophage MS2 coat protein.
mRNA 周转的调控部分是通过 mRNA 结合蛋白的作用实现的，这些蛋白识别特定的核苷酸序列，并激活或抑制它们所结合的转录本的降解。在许多情况下，多种 mRNA 结合蛋白，包括那些具有相反功能的蛋白，会结合到相同的 RNA 序列上。这可能会使研究这些蛋白中任何一种的功能变得困难。此外，使用抑制转录的药物（如放线菌素 D）监测内源性 mRNA 的降解速率可能会产生多效性影响。规避这些问题的一种方法是通过异源 RNA 结合结构域将感兴趣的蛋白（POI）与可诱导的报告 mRNA相连，并测量结合蛋白对 mRNA 降解的影响。在本章中，我们阐述了使用拴系技术来研究特定 mRNA 结合蛋白TTP对一种原本稳定的 mRNA 降解的作用，该蛋白通过与噬菌体 MS2 外壳蛋白融合而与该 mRNA 拴系在一起。
