| Identification of Intrinsic RNA Binding Specificity of Purified Proteins by in vitro RNA Immunoprecipitation (vitRIP)
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Author:
Date:
2021-03-05
[Abstract] RNA-protein interactions are often mediated by dedicated canonical RNA binding domains. However, interactions through non-canonical domains with unknown specificity are increasingly observed, raising the question how RNA targets are recognized. Knowledge of the intrinsic RNA binding specificity contributes to the understanding of target selectivity and function of an individual protein.
The presented in vitro RNA immunoprecipitation assay (vitRIP) uncovers intrinsic RNA binding specificities of isolated proteins using the total cellular RNA pool as a library. Total RNA extracted from cells or tissues is incubated with purified recombinant proteins, RNA-protein complexes are immunoprecipitated and bound transcripts are identified by deep sequencing or quantitative RT-PCR. ...
[摘要] [摘要] RNA-蛋白质相互作用通常由专门的规范RNA结合域介导。然而，越来越多地观察到通过具有未知特异性的非经典结构域的相互作用，这提出了如何识别RNA靶标的问题。内在的RNA结合特异性的知识有助于理解单个蛋白质的靶标选择性和功能。
所呈现的体外RNA免疫沉淀测定法（vitRIP ）揭示固有RNA使用总细胞RNA池作为分离的蛋白质的结合特异性一个库。从细胞或组织中提取的总RNA与纯化的重组蛋白孵育，免疫沉淀RNA-蛋白复合物，并通过深度测序或定量RT-PCR鉴定结合的转录物。这些RNA中丰富的RNA类和核苷酸频率决定了重组蛋白的固有特异性。该简单而通用的方案可适用于任何细胞类型或组织的其他RNA结合蛋白和总RNA文库。
图形摘要：
图1.体外RNA免疫沉淀（vitRIP ）方案示意图
[背景]真核细胞包含许多不同的RNA类，具有成千上万的RNA种类以及与之相互作用的高度多样化的蛋白质。根据结合的RNA序列或结构的定义以及相互作用中涉及的蛋白质结构域的不同，RNA-蛋白质相互作用可分为特异性和非特异性（Jankowsky和Harris，2015）。越来越多地观察到通过未知特异性的非经典RNA结合结构域进行的RNA相互作用，这提出了如何识别专用RNA靶标的问题。 ...
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| RNA Degradation Assay Using RNA Exosome Complexes, Affinity-purified from HEK-293 Cells
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Author:
Date:
2017-04-20
[Abstract] The RNA exosome complex plays a central role in RNA processing and regulated turnover. Present both in cytoplasm and nucleus, the exosome functions through associations with ribonucleases and various adapter proteins (reviewed in [Kilchert et al., 2016]). The RNA exosome-associated EXOSC10 protein is a distributive, 3’-5’ exoribonuclease. The following protocol describes an approach to monitor the ribonucleolytic activity of affinity-purified EXOSC10-containing RNA exosomes, originating from HEK-293 cells, as reported in (Domanski et al., 2016) and further detailed in the companion bio-protocol to this one (Domanski and LaCava, 2017).
[摘要] RNA外植体复合物在RNA加工和调节营养中起核心作用。在细胞质和细胞核中存在，外来体通过与核糖核酸酶和各种衔接蛋白的关联起作用（参见[Kilchert等人，2016]）。 RNA外植体相关的EXOSC10蛋白是分布的3'-5'外核糖核酸酶。以下方案描述了监测源自HEK-293细胞的亲和纯化含EXOSC10的RNA外来体的核糖核酸裂解活性的方法，如（Domanski等人，2016）所报道的，并进一步详细描述这个配套生物协议（Domanski和LaCava，2017）。
在我们以前的工作中，我们在四环素诱导型CMV启动子（HEK-293 Flp-In T-REx-Thermo）的控制下，建立了表达C末端3xFLAG标记的外来体成分EXOSC10（RRP6）的同基因HEK-293细胞系费雪科学）。该系统允许我们以与内源WT蛋白质相当的水平表达标记的EXOSC10蛋白质，并且使用磁性抗FLAG亲和介质和来源于冷冻细胞粉末的蛋白质提取物来研究外来体纯化方案（Domanski等， / ...
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| UV Cross-linking Assay and Competition Assay
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Author:
Date:
2012-12-20
[Abstract] UV cross-linking assay is a standard method used to detect protein-RNA interaction. This method takes advantage of UV irradiation to trigger the formation of the covalently bonded RNP (ribonucleoprotein) complex that is more stable and makes it possible to be isolated in the denaturing conditions. Briefly, 32P-labeled RNA probe and proteins are incubated to form RNP complexes spontaneously; the mixture is then exposed to UV irradiation, followed by treatment with ribonuclease to remove RNA fragments not covalently bound to protein. The oligoribonucleotide-protein complexes are analyzed by SDS-PAGE, and the signals visualized by phosphorimaging. Competitive UV cross-linking assay is a method to determine the protein binding sites and specificity on the RNA substrate. In this ...
[摘要] UV交联测定是用于检测蛋白-RNA相互作用的标准方法。该方法利用UV辐射来触发更稳定的共价结合的RNP（核糖核蛋白）复合物的形成，并且使得可以在变性条件下分离。简言之，将32 P标记的RNA探针和蛋白质自发孵育以形成RNP复合物;然后将混合物暴露于UV辐射，随后用核糖核酸酶处理以除去未与蛋白质共价结合的RNA片段。通过SDS-PAGE分析寡核糖核苷酸 - 蛋白复合物，并通过磷光成像显现信号。竞争性UV交联测定是确定蛋白质结合位点和对RNA底物的特异性的方法。在该测定中，将过量的未标记的竞争剂RNA与蛋白质预孵育，然后加入32 P标记的RNA探针。如果竞争剂RNA包含蛋白质结合区，则蛋白质和RNA探针之间的结合将竞争并且放射性结合信号将降低。
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