A UV cross-linking method combined with infrared imaging to analyse RNA-protein interactions.

Abstract

Photo cross-linking of proteins with short RNA oligomers is a classical method to study RNA–protein interactions that are implicated in many aspects of RNA metabolism and function. Most commonly, this involves the use of [γ-32P]-labeled RNA probes. Although very sensitive, these procedures are complicated by the safety issues associated with the use of radioisotopes. Here, we describe a modified UV cross-linking method using oligonucleotide probes end labelled with the infrared dye IRDye®800. After UV cross-linking, proteins are separated by SDS-PAGE and cross-linked products are visualized with the Odyssey® Infrared Imaging system. This end labelling approach provides a streamlined alternative to random labelling which reduces the efficiency of in-vitro transcription. End labelling is also independent of the length of the probe, thus facilitating quantitative comparisons. To validate the method, we have confirmed the binding of HuD to the 3′-UTR of the mRNA for the microtubule-associated protein tau, implicated in the pathogenesis of Alzheimer’s disease. UV cross-linking of HuD with a labeled 21-mer probe was successfully performed using a recombinant purified glutathione-S-transferase–HuD fusion protein as well as with lysates from CHO cells transfected with HuD cDNA. UV cross-linking combined with infrared imaging offers a convenient and robust strategy to analyse RNA–protein interactions and their emerging importance in disease.