In vivo detection of RNA-binding protein interactions with cognate RNA sequences by fluorescence resonance energy transfer

• Published in: RNA (Cambridge) Vol. 15; no. 11; pp. 2063 - 2071
• Main Authors: Huranová, Martina, Jablonski, Joseph A, Benda, Ales, Hof, Martin, Stanek, David, Caputi, Massimo
• Format: Journal Article
• Language: English
• Published: United States Cold Spring Harbor Laboratory Press 01.11.2009
• Subjects: Base Sequence; Binding Sites; Fluorescence Resonance Energy Transfer - methods; Genetic Vectors - genetics; HeLa Cells; Humans; Method; Protein Binding; RNA - analysis; RNA - metabolism; RNA-Binding Proteins - genetics; RNA-Binding Proteins - metabolism; Substrate Specificity
• ISSN: 1355-8382; 1469-9001
• DOI: 10.1261/rna.1678209

Expression of the nascent RNA transcript is regulated by its interaction with a number of proteins. The misregulation of such interactions can often result in impaired cellular functions that can lead to cancer and a number of diseases. Thus, our understanding of RNA-protein interactions within the cellular context is essential for the development of novel diagnostic and therapeutic tools. While there are many in vitro methods that analyze RNA-protein interactions in vivo approaches are scarce. Here we established a method based on fluorescence resonance energy transfer (FRET), which we term RNA-binding mediated FRET (RB-FRET), which determines RNA-protein interaction inside cells and tested it on hnRNP H protein binding to its cognate RNA. Using two different approaches, we provide evidence that RB-FRET is sensitive enough to detect specific RNA-protein interactions in the cell, providing a powerful tool to study spatial and temporal localization of specific RNA-protein complexes.