1. In Vivo Proximity Labeling for the Detection of Protein–Protein and Protein–RNA Interactions
- Author
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William J. Drury, Ryan Casey, Michiel Vermeulen, Roberto Bonasio, Zuo-Fei Yuan, Pascal W. T. C. Jansen, David B. Beck, Varun Narendra, and Benjamin A. Garcia
- Subjects
Streptavidin ,Models, Molecular ,Biotin ,RNA-binding protein ,Computational biology ,Plasma protein binding ,Biology ,Biochemistry ,Deep sequencing ,Protein–protein interaction ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Proximity labeling ,In vivo ,Tandem Mass Spectrometry ,Protein Interaction Mapping ,Technical Note ,Humans ,Enhancer of Zeste Homolog 2 Protein ,biotinylation ,030304 developmental biology ,0303 health sciences ,Molecular Structure ,Staining and Labeling ,Sequence Analysis, RNA ,Proteomics and Chromatin Biology ,Polycomb Repressive Complex 2 ,RNA ,RNA-Binding Proteins ,General Chemistry ,covalent tag ,Protein Structure, Tertiary ,HEK293 Cells ,chemistry ,protein−RNA interactions ,Biotinylation ,Nucleic Acid Conformation ,protein−protein interactions ,RNA-seq ,030217 neurology & neurosurgery ,Chromatography, Liquid ,Protein Binding - Abstract
Accurate and sensitive detection of protein–protein and protein–RNA interactions is key to understanding their biological functions. Traditional methods to identify these interactions require cell lysis and biochemical manipulations that exclude cellular compartments that cannot be solubilized under mild conditions. Here, we introduce an in vivo proximity labeling (IPL) technology that employs an affinity tag combined with a photoactivatable probe to label polypeptides and RNAs in the vicinity of a protein of interest in vivo. Using quantitative mass spectrometry and deep sequencing, we show that IPL correctly identifies known protein–protein and protein–RNA interactions in the nucleus of mammalian cells. Thus, IPL provides additional temporal and spatial information for the characterization of biological interactions in vivo.
- Published
- 2014