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.
Karijolich
John
J
Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY, USA.
Stephenson
David
D
Yu
Yi-Tao
YT
D000818:Animals; D001710:Biotin; D002846:Chromatography, Affinity / Q000379:methods*; D006801:Humans; D011560:Pseudouridine / Q000378:metabolism*; D020718:Ribonucleoproteins, Small Nucleolar / Q000302:isolation & purification*; D019809:Streptavidin
C485802:GAR1 protein, human; D020718:Ribonucleoproteins, Small Nucleolar; D011560:Pseudouridine; D001710:Biotin; D019809:Streptavidin