Detecting RNA–Protein Interactions by Photocross-Linking Using RNA Molecules Containing Uridine Analogs

Abstract

Various cross-linking techniques form covalent attachments between proteins and RNAs. Straight ultraviolet (UV) cross-linking using short-wavelength UV light is inherently very inefficient. Therefore, it is often desirable to increase the efficiency of cross-linking. The uridine analogs 4-thiouridine and halogenated uridine (5-bromouridine or 5-iodouridine) are much more photoreactive than uridine. Accordingly, RNAs substituted with these bases are quite effective for cross-linking applications. This protocol describes the detection of RNA–protein interactions by photocross-linking using RNA molecules containing 4-thiouridine, but identical procedures apply to using 5-bromouridine or 5-iodouridine. Fully substituted RNAs are useful in determining whether any proteins interact with the RNA of interest. However, it may be preferable to use site-specifically substituted RNA if one has foreknowledge of critical regions in the RNA of interest. For any cross-linking application using substituted RNA, it is essential to remember that the uridine analogs are activated at a wavelength >304 nm (312 nm) than is used for short-wavelength (254 nm) cross-linking.