title: 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.
authors: Timothy W
authors: TW
authors: Nilsen
mesh_terms: D008967:Molecular Biology / Q000379:methods*; D011485:Protein Binding; D012313:RNA / Q000032:analysis*; D016601:RNA-Binding Proteins / Q000032:analysis*; D013194:Staining and Labeling / Q000379:methods*; D014529:Uridine / Q000031:analogs & derivatives* / Q000378:metabolism*
chemical_list: D016601:RNA-Binding Proteins; D012313:RNA; D014529:Uridine