This protocol is an extension to: Nat. Protoc. 8, 491-500 (2013); doi:10.1038/nprot.2013.020; published online 14 February 2013RBDmap is a method for identifying, in a proteome-wide manner, the regions of RNA-binding proteins (RBPs) engaged in native interactions with RNA. In brief, cells are irradiated with UV light to induce protein-RNA cross-links. Following stringent denaturing washes, the resulting covalently linked protein-RNA complexes are purified with oligo(dT) magnetic beads. After elution, RBPs are subjected to partial proteolysis, in which the protein regions still bound to the RNA and those released to the supernatant are separated by a second oligo(dT) selection. After sample preparation and mass-spectrometric analysis, peptide intensity ratios between the RNA-bound and released fractions are used to determine the RNA-binding regions. As a Protocol Extension, this article describes an adaptation of an existing Protocol and offers additional applications. The earlier protocol (for the RNA interactome capture method) describes how to identify the active RBPs in cultured cells, whereas this Protocol Extension also enables the identification of the RNA-binding domains of RBPs. The experimental workflow takes 1 week plus 2 additional weeks for proteomics and data analysis. Notably, RBDmap presents numerous advantages over classic methods for determining RNA-binding domains: it produces proteome-wide, high-resolution maps of the protein regions contacting the RNA in a physiological context and can be adapted to different biological systems and conditions. Because RBDmap relies on the isolation of polyadenylated RNA via oligo(dT), it will not provide RNA-binding information on proteins interacting exclusively with nonpolyadenylated transcripts. Applied to HeLa cells, RBDmap uncovered 1,174 RNA-binding sites in 529 proteins, many of which were previously unknown.

中文翻译：

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使用RBDmap在系统范围内鉴定培养细胞中RNA结合蛋白的RNA结合结构域。

该协议是对以下内容的扩展：Nat。协议。8，491-500（2013）；doi：10.1038 / nprot.2013.020；2013年2月14日在线发布RBDmap是一种以蛋白质组学方式识别与RNA天然相互作用的RNA结合蛋白（RBP）区域的方法。简而言之，用紫外线照射细胞以诱导蛋白质-RNA交联。经过严格的变性洗涤后，用oligo（dT）磁珠纯化所得的共价连接的蛋白质-RNA复合物。洗脱后，将RBP进行部分蛋白水解，通过第二次oligo（dT）选择分离仍结合到RNA上的蛋白质区域和释放到上清液中的蛋白质区域。在样品制备和质谱分析之后，RNA结合的部分和释放的部分之间的肽强度比用于确定RNA结合区域。作为协议扩展，本文介绍了对现有协议的改编并提供了其他应用程序。较早的协议（用于RNA相互作用组捕获方法）描述了如何在培养的细胞中鉴定活性RBP，而此协议扩展也使能够鉴定RBP的RNA结合结构域。实验工作流程需要1周的时间，另加2周的时间进行蛋白质组学和数据分析。值得注意的是，与确定RNA结合结构域的经典方法相比，RBDmap具有许多优势：它可以在生理范围内生成蛋白质组范围内与RNA接触的蛋白质区域的高分辨率蛋白质图，并且可以适应不同的生物学系统和条件。由于RBDmap依赖于通过oligo（dT）分离多腺苷酸RNA，它不会提供仅与非聚腺苷酸转录物相互作用的蛋白质的RNA结合信息。应用于HeLa细胞后，RBDmap在529种蛋白质中发现了1,174个RNA结合位点，其中许多以前是未知的。