European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
Department of Biochemistry, University of Oxford, Oxford, UK.
Nat Protoc. 2017 Dec;12(12):2447-2464. doi: 10.1038/nprot.2017.106. Epub 2017 Nov 2.
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.
Nat. Protoc. 8, 491-500 (2013); doi:10.1038/nprot.2013.020; 在线发表于 2013 年 2 月 14 日；RBDmap 是一种在蛋白质组范围内鉴定 RNA 结合蛋白 (RBP) 与 RNA 发生天然相互作用的区域的方法。简而言之，用紫外线照射细胞以诱导蛋白-RNA 交联。经过严格的变性洗涤后，用 oligo(dT) 磁珠纯化形成的共价连接的蛋白-RNA 复合物。洗脱后，RBP 进行部分蛋白水解，其中仍与 RNA 结合的蛋白区域和释放到上清液中的蛋白区域通过第二次 oligo(dT) 选择分离。经过样品制备和质谱分析后，RNA 结合和释放部分之间的肽强度比用于确定 RNA 结合区域。作为方案扩展，本文描述了对现有方案的改编，并提供了其他应用。早期方案（用于 RNA 相互作用组捕获方法）描述了如何鉴定培养细胞中的活性 RBP，而本方案扩展还能够鉴定 RBP 的 RNA 结合结构域。实验工作流程需要 1 周加 2 周用于蛋白质组学和数据分析。值得注意的是，RBDmap 与确定 RNA 结合结构域的经典方法相比具有许多优势：它在生理环境中产生蛋白质与 RNA 相互作用的全蛋白质组、高分辨率图谱，并且可以适应不同的生物系统和条件。由于 RBDmap 依赖于通过 oligo(dT) 分离多聚腺苷酸化 RNA，因此它不会提供与非多聚腺苷酸化转录本特异性相互作用的蛋白质的 RNA 结合信息。在 HeLa 细胞中的应用，RBDmap 在 529 种蛋白质中发现了 1174 个 RNA 结合位点，其中许多是以前未知的。
