Biochemical purification of box H/ACA RNPs involved in pseudouridylation.

Box H/ACA RNPs, each consisting of four common core proteins and a single unique RNA, are the most complex pseudouridylases yet discovered. The RNA component serves as a guide that directs a target uridine for modification. To study the functions and mechanisms of RNA pseudouridylation, it is desirable to isolate the intact box H/ACA RNP complexes. Purified RNPs will allow further identification and characterization of the RNA component in each RNP complex and permit a systematic analysis of the mechanism by which the enzymes convert uridines to pseudouridines in a site-specific manner. Over the years, a number of purification techniques have been developed, providing important tools for RNA pseudouridylation research. Here, we describe three of these techniques, including biotin-streptavidin affinity purification by use of biotinylated 5-fluorouridine (5FU)-containing RNA, tandem affinity purification (TAP) by TAP-tagging one of the four core proteins in the complex, and immunoprecipitation by use of antibodies against one of the four core proteins.