In vivo detection of RNA-binding protein interactions with cognate RNA sequences by fluorescence resonance energy transfer.
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.
Huranová
Martina
M
Department of RNA Biology, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, CZ-14220 Prague 4, Czech Republic.
Jablonski
Joseph A
JA
Benda
Ales
A
Hof
Martin
M
Stanek
David
D
Caputi
Massimo
M
D001483:Base Sequence; D001665:Binding Sites; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D011485:Protein Binding; D012313:RNA / Q000032:analysis* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D016601:RNA-Binding Proteins; D012313:RNA