RNA-蛋白质紫外交联分析
RNA-protein UV-crosslinking Assay.
作者信息
Poria Dipak Kumar, Ray Partho Sarothi
机构信息
Department of Biological Sciences, Indian Institute of Science Education and Research, Kolkata, India.
出版信息
Bio Protoc. 2017 Mar 20;7(6). doi: 10.21769/BioProtoc.2193.
Abstract
RNA-protein interactions play a crucial role in every aspect of RNA metabolism, and also plays a major role in post-transcriptional gene regulation. RNA-binding proteins have been implicated in viral gene expression (Ray and Das, 2002) and microRNA-mediated gene regulation (Poria ., 2016). Here we have described the protocol which (1) covalently links transiently interacting RNA-protein complexes by UV crosslinking, (2) removes the unprotected RNA by RNase digestion and (3) detects the RNA-protein complexes by SDS-PAGE analysis. This protocol provides a rapid and reliable means to directly assay RNA-protein interactions and their kinetics using purified proteins and also help in identifying novel RNA-protein interactions.
摘要
RNA-蛋白质相互作用在RNA代谢的各个方面都起着至关重要的作用，并且在转录后基因调控中也发挥着主要作用。RNA结合蛋白已被证明与病毒基因表达（Ray和Das，2002年）以及微小RNA介导的基因调控有关（Poria等人，2016年）。在此，我们描述了一种方法，该方法（1）通过紫外线交联将瞬时相互作用的RNA-蛋白质复合物共价连接，（2）通过核糖核酸酶消化去除未受保护的RNA，以及（3）通过SDS-PAGE分析检测RNA-蛋白质复合物。该方法提供了一种快速且可靠的手段，可使用纯化的蛋白质直接测定RNA-蛋白质相互作用及其动力学，同时也有助于鉴定新的RNA-蛋白质相互作用。
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