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

• Published in: Molecular cell Vol. 75; no. 1; pp. 184 - 199.e10
• Main Authors: Caudron-Herger, Maiwen, Rusin, Scott F., Adamo, Mark E., Seiler, Jeanette, Schmid, Vera K., Barreau, Elsa, Kettenbach, Arminja N., Diederichs, Sven
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 11-07-2019
• Subjects: Centrifugation, Density Gradient - instrumentation; Centrifugation, Density Gradient - methods; Chromatin - chemistry; Chromatin - metabolism; CTCF; density gradient; Gene Expression Regulation; Gene Ontology; HeLa Cells; Humans; Information Dissemination; Internet; mass spectrometry; Molecular Sequence Annotation; Protein Binding; Protein Interaction Maps; Proteome - classification; Proteome - genetics; Proteome - metabolism; proteome-wide; proteomics; Proteomics - methods; R-DeeP; RNA; RNA - genetics; RNA - metabolism; RNA dependence; RNA-binding protein; RNA-Binding Proteins - classification; RNA-Binding Proteins - genetics; RNA-Binding Proteins - metabolism; RNase; Transcription Factors - classification; Transcription Factors - genetics; Transcription Factors - metabolism; R-DeeP; RNA-binding protein; proteome-wide; RNA; RNA dependence; proteomics; density gradient; mass spectrometry; CTCF; RNase
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2019.04.018

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.
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•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.