Division of RNA Biology and Cancer, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany.
Department of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, NH 03755, USA.
Mol Cell. 2019 Jul 11;75(1):184-199.e10. doi: 10.1016/j.molcel.2019.04.018. Epub 2019 May 7.
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.
全面而具体地鉴定 RNA 结合蛋白，以及发现 RNA 相关蛋白的功能，仍然是 RNA 生物学的主要挑战。在这里，我们采用 RNA 依赖性的概念，将蛋白质定义为 RNA 依赖性，当其互作组依赖于 RNA 时。我们将这个概念转化为一种基于密度梯度超速离心的全蛋白质组、无偏倚和无富集的筛选方法，称为 R-DeeP（RNA 依赖性蛋白）。定量质谱分析鉴定了 1784 种 RNA 依赖性蛋白，其中 537 种蛋白与 RNA 缺乏已知联系。利用 R-DeeP 的定量性质，将蛋白质分为非、部分或完全 RNA 依赖性。R-DeeP 将转录因子 CTCF 鉴定为完全 RNA 依赖性，我们发现 RNA 是 CTCF-染色质结合所必需的。此外，R-DeeP 允许根据共分离来重建蛋白质复合物。整个数据集可在 http://R-DeeP.dkfz.de 上获得，该数据集提供了全蛋白质组、特异性和定量鉴定具有 RNA 依赖性相互作用的蛋白质，并旨在未来发现 RNA-蛋白质复合物的功能。
