R-DeeP: Proteome-wide and Quantitative Identification of RNA-Dependent Proteins by Density Gradient Ultracentrifugation

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. [Display omitted] •R-DeeP implements the RNA dependence concept using density gradient centrifugation•Proteome-wide, specific, and quantitative analysis of 1,784 RNA-dependent proteins•Comprehensive online resource, including 537 proteins never before linked to RNA•Quantitative RNA dependence uncovers RNA effect on CTCF recruitment to chromatin Caudron-Herger et al. developed a proteome-wide, specific, and quantitative analysis of RNA-dependent proteins based on density gradient ultracentrifugation. The R-DeeP resource is available online and guides the discovery of unexpected RNA functions by newly linking 537 proteins to RNA and enabling quantification of the RNA-dependent fraction of a protein.