Other Publications (1)
Articles by Robert Warneford-Thomson in JoVE
Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions Robert Warneford-Thomson1,4, Chongsheng He1,2, Simone Sidoli1,3, Benjamin A. Garcia1,3, Roberto Bonasio1,2 1Epigenetics Institute, University of Pennsylvania Perelman School of Medicine, 2Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine, 3Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, 4Graduate Group in Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.
Other articles by Robert Warneford-Thomson on PubMed
High-Resolution Mapping of RNA-Binding Regions in the Nuclear Proteome of Embryonic Stem Cells Molecular Cell. Oct, 2016 | Pubmed ID: 27768875 Interactions between noncoding RNAs and chromatin proteins play important roles in gene regulation, but the molecular details of most of these interactions are unknown. Using protein-RNA photocrosslinking and mass spectrometry on embryonic stem cell nuclei, we identified and mapped, at peptide resolution, the RNA-binding regions in ∼800 known and previously unknown RNA-binding proteins, many of which are transcriptional regulators and chromatin modifiers. In addition to known RNA-binding motifs, we detected several protein domains previously unknown to function in RNA recognition, as well as non-annotated and/or disordered regions, suggesting that many functional protein-RNA contacts remain unexplored. We identified RNA-binding regions in several chromatin regulators, including TET2, and validated their ability to bind RNA. Thus, proteomic identification of RNA-binding regions (RBR-ID) is a powerful tool to map protein-RNA interactions and will allow rational design of mutants to dissect their function at a mechanistic level.