Tree Jai J, Gerdes Kenn, Tollervey David
School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, Australia.
Department of Biology, Centre for Bacterial Stress Response and Persistence, University of Copenhagen, Copenhagen, Denmark.
Methods Enzymol. 2018;612:467-488. doi: 10.1016/bs.mie.2018.08.009. Epub 2018 Sep 22.
Regulatory RNAs and RNA-binding proteins (RBPs) play critical roles in virulence gene expression in pathogenic bacteria. A wealth of regulatory RNAs have been identified in bacterial pathogens using RNA-seq and recent technical advances are uncovering their mRNA targets. UV-crosslinking is a powerful tool for identifying protein binding sites throughout the transcriptome providing base-pair resolution of sites in vivo. With minor modifications to the protocol, RNA-RNA interactions can also be captured by proximity-dependent ligation of RNA pairs on the protein. Here, we described a high-stringency UV-crosslinking method for recovery of both protein-RNA interactions (CRAC) and RNA-RNA interactions occurring on the bait protein (CLASH). These analyses provide complementary data that provide insights into RBP, and regulatory RNA function.
调控RNA和RNA结合蛋白（RBPs）在病原菌毒力基因表达中发挥着关键作用。利用RNA测序技术已在细菌病原体中鉴定出大量调控RNA，并且近期的技术进展正在揭示它们的mRNA靶标。紫外线交联是一种强大的工具，可用于识别整个转录组中的蛋白质结合位点，提供体内位点的碱基对分辨率。对该方案进行微小修改后，RNA-RNA相互作用也可通过蛋白质上RNA对的邻近依赖性连接来捕获。在此，我们描述了一种高严谨度的紫外线交联方法，用于回收蛋白质-RNA相互作用（CRAC）以及诱饵蛋白上发生的RNA-RNA相互作用（CLASH）。这些分析提供了互补数据，有助于深入了解RBP和调控RNA的功能。
