System-wide identification of RNA-binding proteins by interactome capture
System-wide identification of RNA-binding proteins by interactome capture
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DOI：
10.1038/nprot.2013.020
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发表时间：
2013-03-01
期刊：
影响因子：
14.8
通讯作者：
Hentze, Matthias W.
中科院分区：
文献类型：
--
作者：
Castello, Alfredo;Horos, Rastislav;Hentze, Matthias W.
由于其卓越的生物学功能，表达的RNA结合蛋白（RBP）的库和它们的活性状态是关于细胞系统的高度信息。我们已经开发了一种新的和公正的技术，称为相互作用组捕获，用于识别培养细胞的活性RBP。通过利用RBP与聚腺苷酸化RNA的体内紫外线交联，用寡聚（dT）磁珠捕获共价结合的蛋白质。严格洗涤后，通过定量质谱法（MS）测定mRNA相互作用组。该方案需要3个工作日通过蛋白质印迹法分析单个蛋白质，约2周通过MS测定完整的细胞mRNA相互作用组。相互作用组捕获相对于其他体外和计算机方法的最重要优点是，仅识别在生理环境中与RNA结合的RBP。当应用于HeLa细胞时，相互作用体捕获揭示了数百种新的RBP。相互作用组捕获也可广泛用于比较不同的生物状态，包括代谢应激、细胞周期、分化、发育或对药物的反应。
Owing to their preeminent biological functions, the repertoire of expressed RNA-binding proteins (RBPs) and their activity states are highly informative about cellular systems. We have developed a novel and unbiased technique, called interactome capture, for identifying the active RBPs of cultured cells. By making use of in vivo UV cross-linking of RBPs to polyadenylated RNAs, covalently bound proteins are captured with oligo(dT) magnetic beads. After stringent washes, the mRNA interactome is determined by quantitative mass spectrometry (MS). The protocol takes 3 working days for analysis of single proteins by western blotting, and about 2 weeks for the determination of complete cellular mRNA interactomes by MS. The most important advantage of interactome capture over other in vitro and in silico approaches is that only RBPs bound to RNA in a physiological environment are identified. When applied to HeLa cells, interactome capture revealed hundreds of novel RBPs. Interactome capture can also be broadly used to compare different biological states, including metabolic stress, cell cycle, differentiation, development or the response to drugs.