Slobodin Boris, Gerst Jeffrey E
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.
Methods Mol Biol. 2011;714:387-406. doi: 10.1007/978-1-61779-005-8_24.
RNA metabolism involves regulatory processes, such as transcription, splicing, nuclear export, transport and localization, association with sites of RNA modification, silencing and decay, and necessitates a wide variety of diverse RNA-interacting proteins. These interactions can be direct via RNA-binding proteins (RBPs) or indirect via other proteins and RNAs that form ribonucleoprotein complexes that together control RNA fate. While pull-down methods for the isolation of known RBPs are commonly used, strategies have also been described for the direct isolation of messenger RNAs (mRNAs) and their associated factors. The latter techniques allow for the identification of interacting proteins and RNAs, but most suffer from problems of low sensitivity and high background. Here we describe a simple and highly effective method for RNA purification and identification (RaPID) that allows for the isolation of specific mRNAs of interest from yeast and mammalian cells, and subsequent analysis of the associated proteins and RNAs using mass spectrometry and reverse transcription-PCR, respectively. This method employs the MS2 coat RBP fused to both GFP and streptavidin-binding protein to precipitate MS2 aptamer-tagged RNAs using immobilized streptavidin.
RNA代谢涉及多种调控过程,如转录、剪接、核输出、转运与定位、与RNA修饰位点的结合、沉默与降解,并且需要各种各样不同的RNA相互作用蛋白。这些相互作用可以通过RNA结合蛋白(RBP)直接发生,或者通过形成核糖核蛋白复合体共同控制RNA命运的其他蛋白质和RNA间接发生。虽然常用于分离已知RBP的下拉方法很常见,但也有一些策略被描述用于直接分离信使RNA(mRNA)及其相关因子。后一种技术能够鉴定相互作用的蛋白质和RNA,但大多数都存在灵敏度低和背景高的问题。在这里,我们描述了一种简单且高效的RNA纯化与鉴定方法(RaPID),该方法能够从酵母和哺乳动物细胞中分离出感兴趣的特定mRNA,并分别使用质谱和逆转录PCR对相关蛋白质和RNA进行后续分析。该方法利用与绿色荧光蛋白(GFP)和链霉亲和素结合蛋白融合的MS2外壳RBP,通过固定化链霉亲和素来沉淀带有MS2适体标签的RNA。