Sy Brandon, Wong Julia, Granneman Sander, Tollervey David, Gally David, Tree Jai J
School of Biotechnology and Biomolecular Sciences, University of New South Wales Sydney, Sydney, Australia.
Institute of Structural and Molecular Biology, Centre for Synthetic and Systems Biology (SynthSys), University of Edinburgh, Edinbugh, Scotland, UK.
Methods Mol Biol. 2018;1737:251-272. doi: 10.1007/978-1-4939-7634-8_15.
Small regulatory nonprotein-coding RNAs (sRNAs) have emerged as ubiquitous and abundant regulators of gene expression in a diverse cross section of bacteria. They play key roles in most aspects of bacterial physiology, including central metabolism, nutrient acquisition, virulence, biofilm formation, and outer membrane composition. RNA sequencing technologies have accelerated the identification of bacterial regulatory RNAs and are now being employed to understand their functions. Many regulatory RNAs require protein partners for activity, or modulate the activity of interacting proteins. Understanding how and where proteins interact with the transcriptome is essential to elucidate the functions of the many sRNAs. Here, we describe the implementation in bacteria of a UV-crosslinking technique termed CRAC that allows stringent, transcriptome-wide recovery of bacterial RNA-protein interaction sites in vivo and at base-pair resolution. We have used CRAC to map protein-RNA interaction sites for the RNA chaperone Hfq and ribonuclease RNase E in pathogenic E. coli, and toxins from toxin-antitoxin systems in Mycobacterium smegmatis, demonstrating the broad applicability of this technique.
小型调控非蛋白质编码RNA（sRNA）已成为细菌不同领域中普遍存在且丰富的基因表达调控因子。它们在细菌生理学的大多数方面发挥着关键作用，包括中心代谢、营养获取、毒力、生物膜形成和外膜组成。RNA测序技术加速了细菌调控RNA的鉴定，目前正被用于了解它们的功能。许多调控RNA需要蛋白质伴侣来发挥活性，或调节相互作用蛋白质的活性。了解蛋白质如何以及在何处与转录组相互作用对于阐明众多sRNA的功能至关重要。在这里，我们描述了一种称为CRAC的紫外线交联技术在细菌中的应用，该技术能够在体内以碱基对分辨率严格地、全转录组范围地回收细菌RNA-蛋白质相互作用位点。我们已使用CRAC来绘制致病性大肠杆菌中RNA伴侣Hfq和核糖核酸酶RNase E的蛋白质-RNA相互作用位点，以及耻垢分枝杆菌中毒素-抗毒素系统的毒素的蛋白质-RNA相互作用位点，证明了该技术的广泛适用性。
