PAR-CliP--a method to identify transcriptome-wide the binding sites of RNA binding proteins.
PAR-CliP--a method to identify transcriptome-wide the binding sites of RNA binding proteins.
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DOI：
10.3791/2034
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发表时间：
2010-07-02
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影响因子：
--
通讯作者：
Tuschl, Thomas
中科院分区：
文献类型：
--
作者：
Hafner, Markus;Landthaler, Markus;Tuschl, Thomas
RNA转录本通过与数百种RNA结合蛋白（RBPs）以及含微小RNA的核糖核蛋白复合物（miRNPs）相互作用而受到转录后基因调控，这些蛋白和复合物通常以细胞类型依赖的方式表达。为了理解这些RNA结合因子的相互作用如何影响单个转录本的调控，体内蛋白质 - RNA相互作用的高分辨率图谱是必要的。通常会结合遗传学、生物化学和计算方法来鉴定RNA - RBP或RNA - RNP相互作用。与免疫纯化的RBPs相关的RNAs的微阵列分析（RIP - Chip）在转录组水平上确定靶标，但其应用局限于动力学稳定相互作用的表征，并且仅在极少数情况下能够识别长靶标RNA内的RBP识别元件（RRE）。通过将体内紫外线交联与免疫沉淀相结合，然后分离交联的RNA片段并进行cDNA测序（CLIP），可以获得更直接的RBP靶位点信息。CLIP已被用于鉴定许多RBPs的靶标。然而，CLIP受到254nm紫外线RNA - 蛋白质交联效率低的限制，并且在测序的交联片段中交联位置不易识别，这使得难以将紫外线交联的靶标RNA片段与样品中也存在的背景非交联RNA片段区分开来。我们开发了一种强大的基于细胞的交联方法，以高分辨率和在转录组范围内确定细胞RBPs和miRNPs的结合位点，我们将其称为PAR - CliP（光活化核糖核苷增强交联和免疫沉淀）（方法概述见图1A）。该方法依赖于活细胞将光反应性核糖核苷类似物，如4 - 硫尿苷（4 - SU）和6 - 硫鸟苷（6 - SG）掺入新生RNA转录本中。用365nm紫外线照射细胞可诱导光反应性核苷标记的细胞RNAs与相互作用的RBPs高效交联。对感兴趣的RBP进行免疫沉淀，然后分离交联的和共免疫沉淀的RNA。分离出的RNA被转化为cDNA文库，并使用Solexa技术进行深度测序。PAR - CliP制备的cDNA文库的一个特征是，通过测序cDNA中的突变可以确定交联的精确位置。当使用4 - SU时，交联序列会发生胸腺嘧啶核苷到胞嘧啶核苷的转变，而使用6 - SG则会导致鸟嘌呤核苷到腺嘌呤核苷的突变。交联序列中突变的存在使得将它们与来自丰富细胞RNAs的序列背景区分开来成为可能。Hafner等人报道了该方法在多种不同RNA结合蛋白上的应用。
RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary. A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip) defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking with immunoprecipitation followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP). CLIP was used to identify targets of a number of RBPs. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample. We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs. Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.