Schilders Geurt, Pruijn Ger J M
Department of Biomolecular Chemistry, Nijmegen Center for Molecular Life Sciences, Institute for Molecules and Materials, Radboud University Nijmegen, Nijmegen, The Netherlands.
Methods Enzymol. 2008;448:211-26. doi: 10.1016/S0076-6879(08)02611-6.
A key component responsible for 3'- to 5'-RNA turnover in eukaryotic cells is the exosome, a multisubunit complex present in both the nucleus and cytoplasm of the cell. Here we describe several methods that can be applied to study the structure and function of the exosome in mammalian cell lines. The mammalian two-hybrid system has been successfully used to identify protein-protein interactions between exosome core components. Cell and glycerol gradient fractionation procedures are described that allow the identification and characterization of different exosome subsets. Finally, a protocol to study the function of the exosome in RNA turnover with RNA interference is presented.
负责真核细胞中3'至5'RNA周转的一个关键组分是外切体，它是一种存在于细胞核和细胞质中的多亚基复合体。在此，我们描述了几种可用于研究外切体在哺乳动物细胞系中的结构和功能的方法。哺乳动物双杂交系统已成功用于鉴定外切体核心组分之间的蛋白质-蛋白质相互作用。文中描述了细胞和甘油梯度分级分离程序，可用于鉴定和表征不同的外切体亚群。最后，介绍了一种利用RNA干扰研究外切体在RNA周转中功能的方案。
