Nat. Methods15, 207–212 (2018)

Current approaches to studying RNA–protein interactions, such as the RNA electrophoretic mobility shift assay (EMSA) or UV or formaldehyde crosslinking combined with immunoprecipitation, are mostly performed in vitro, so they may not reflect physiologically relevant interactions in cells. Ramanathan et al. developed an approach called RaPID that enables detection of RNA–protein complexes without the need for crosslinking. RaPID utilizes a protein composed of the N terminus of Escherichia coli BirA*, a promiscuous biotin ligase, fused to the λN peptide, which specifically binds to BoxB stem loops. Incubation of the peptide with an RNA motif flanked by BoxB stem loops in cells in biotin-containing media enables biotinylation of proteins that are bound to the RNA motifs. These labeled proteins can then be detected with streptavidin capture and mass spectroscopy analysis. RaPID confirmed known RNA–protein interactions such as IREB2 binding to the iron-responsive element RNA motif and identified new interactions such as RC3H1 binding to the SM1v RNA motif, which is enriched in breast cancer patients. Sequence homology analysis guided the modification of Bacillus subtilis biotin ligase to BASU, which exhibited rapid biotin kinetics and improved signal-to-noise ratio compared to BirA*. Overall, the RaPID approach provides a useful alternative to EMSA and will enable detection of genetic or small-molecule-mediated disruption of RNA–protein interactions.

Credit: Nat. Methods