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.
Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY, USA.
John
J
Karijolich
David
D
Stephenson
Yi-Tao
YT
Yu
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