RNA-based affinity purification of ncRNA-protein-complexes

Recent studies have uncovered an enormous diversity of non-coding RNAs (ncRNAs), although most of them are not yet functionally described. To study the function of selected ncRNAs, we use an affinity purification system that is based on the RNA of interest. Most common methods for analyzing RNA-protein interactions, like RIP, are protein-based, thereby particular target proteins are used to identify unknown RNA binding partners. In contrast, we apply ncRNAs that we previously discovered in genome wide tiling and custom array analyzes to detect novel interacting protein-complexes. To this purpose, we use the MS2 affinity tag system, which involves a short RNA hairpin tag sequence that binds specifically to the MS2 coat protein. Thus, the bait ncRNAs are tagged by the MS2 hairpin RNA and the MS2 coat protein is expressed fused to a capture molecule. This approach is adaptive to large numbers of ncRNAs, allowing the identification of undiscovered ncRNP-complexes.