In vivo detection of RNA-binding protein interactions with cognate RNA sequences by fluorescence resonance energy transfer
Author: Martina Huranová,1 Joseph A. Jablonski,2 Aleš Benda,3 Martin Hof,3 David Staněk,1 and Massimo Caputi2
Affiliation:
1Department of RNA Biology, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, CZ-14220 Prague 4, Czech Republic 2Department of Basic Science, Florida Atlantic University, Boca Raton, Florida 33431, USA 3J. Heyrovsky Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, 182 23 Prague 8, Czech Republic Reprint requests to: Massimo Caputi, Department of Basic Science, Florida Atlantic University, Boca Raton, FL 33431, USA; e-mail: mcaputi@fau.edu; fax: (561) 297-2221.
Conference/Journal: RNA
Date published: 2009 Nov
Other:
Volume ID: 15 , Issue ID: 11 , Pages: 2063-2071 , Word Count: 161
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.
Keywords: FRET, FLIM, MS2, hnRNPH, protein–RNA interactions
PMCID: PMC2764471
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