Galgano Alessia, Gerber André P
Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, ETH Zurich, Zurich, Switzerland.
Methods Mol Biol. 2011;714:369-85. doi: 10.1007/978-1-61779-005-8_23.
Post-transcriptional gene regulation is largely mediated by RNA-binding proteins (RBPs) that modulate mRNA expression at multiple levels, from RNA processing to translation, localization, and degradation. Thereby, the genome-wide identification of mRNAs regulated by RBPs is crucial to uncover post--transcriptional gene regulatory networks. In this chapter, we provide a detailed protocol for one of the techniques that has been developed to systematically examine RNA targets for RBPs. This technique involves the purification of endogenously formed RBP-mRNA complexes with specific antibodies from cellular extracts, followed by the identification of associated RNAs using DNA microarrays. Such RNA-binding protein immunopurification-microarray profiling, also called RIP-Chip, has also been applied to identify mRNAs that are transported to distinct subcellular compartments by RNP-motor complexes. The application and further development of this method could provide global insights into the subcellular architecture of the RBP-RNA network, and how it is restructured upon changing environmental conditions, during development, and possibly in disease.
转录后基因调控主要由RNA结合蛋白（RBP）介导，这些蛋白在从RNA加工到翻译、定位和降解的多个水平上调节mRNA表达。因此，全基因组范围内鉴定受RBP调控的mRNA对于揭示转录后基因调控网络至关重要。在本章中，我们提供了一种已开发用于系统检测RBP的RNA靶标的技术的详细方案。该技术包括从细胞提取物中用特异性抗体纯化内源性形成的RBP-mRNA复合物，然后使用DNA微阵列鉴定相关RNA。这种RNA结合蛋白免疫纯化-微阵列分析，也称为RIP-Chip，也已用于鉴定由RNP运动复合物转运到不同亚细胞区室的mRNA。该方法的应用和进一步发展可以提供对RBP-RNA网络亚细胞结构的全局洞察，以及它在环境条件变化、发育过程中以及可能在疾病中是如何重组的。
