R-DeeP: Proteome-wide and Quantitative Identification of RNA-Dependent Proteins by Density Gradient Ultracentrifugation
R-DeeP: Proteome-wide and Quantitative Identification of RNA-Dependent Proteins by Density Gradient Ultracentrifugation
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DOI：
10.1016/j.molcel.2019.04.018
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发表时间：
2019-07-11
期刊：
影响因子：
16
通讯作者：
Diederichs, Sven
中科院分区：
文献类型：
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作者：
Caudron-Herger, Maiwen;Rusin, Scott F.;Diederichs, Sven
RNA结合蛋白的全面而特异的鉴定以及RNA相关蛋白功能的发现仍然是RNA生物学的主要挑战。在这里，我们采用了RNA依赖性的概念，当蛋白质的相互作用组依赖于RNA时，将其定义为RNA依赖性。我们将这一概念转化为一种基于密度梯度超电泳的蛋白质组范围的、无偏见的、无富集的筛选，称为R-DeeP（RNA依赖性蛋白质）。定量质谱法鉴定了1,784种RNA依赖蛋白，其中537种缺乏已知的与RNA的连接。利用R-DeeP的定量性质，将蛋白质分类为不依赖、部分依赖或完全依赖RNA。R-DeeP鉴定了转录因子CTCF完全依赖于RNA，我们发现RNA是CTCF-染色质缔合所必需的。此外，R-DeeP允许基于共分离重建蛋白质复合物。整个数据集可在http：//R-DeeP上获得。dkfz。de，提供蛋白质组范围内的，特异性的，定量鉴定的蛋白质与RNA依赖的相互作用，并针对未来的功能发现的RNA-蛋白质复合物。
The comprehensive but specific identification of RNA-binding proteins as well as the discovery of RNA-associated protein functions remain major challenges in RNA biology. Here we adapt the concept of RNA dependence, defining a protein as RNA dependent when its interactome depends on RNA. We converted this concept into a proteome-wide, unbiased, and enrichment-free screen called R-DeeP (RNA-dependent proteins), based on density gradient ultracentrifugation. Quantitative mass spectrometry identified 1,784 RNA-dependent proteins, including 537 lacking known links to RNA. Exploiting the quantitative nature of R-DeeP, proteins were classified as not, partially, or completely RNA dependent. R-DeeP identified the transcription factor CTCF as completely RNA dependent, and we uncovered that RNA is required for the CTCF-chromatin association. Additionally, R-DeeP allows reconstruction of protein complexes based on co-segregation. The whole dataset is available at http://R-DeeP. dkfz. de, providing proteome-wide, specific, and quantitative identification of proteins with RNA-dependent interactions and aiming at future functional discovery of RNA-protein complexes.