During ribosome biogenesis, the RNA precursor to mature rRNAs undergoes numerous post-transcriptional chemical modifications of bases, including conversions of uridines to pseudouridines. In archaea and eukaryotes, these conversions are performed by box H/ACA small ribonucleoprotein particles (box H/ACA RNPs), which contain a small guide RNA responsible for the selection of substrate uridines and four proteins, including the pseudouridine synthase, Cbf5p. So far, no in vitro reconstitution of eukaryotic box H/ACA RNPs from purified components has been achieved, principally due to difficulties in purifying recombinant eukaryotic Cbf5p. In this study, we present the purification of a truncated derivative of yeast Cbf5p (Cbf5(Delta)p) that retains the highly conserved TRUB and PUA domains. We have used band retardation assays to show that Cbf5(Delta)p on its own binds to box H/ACA small nucleolar (sno)RNAs. We demonstrate that the conserved H and ACA boxes enhance the affinity of the protein for the snoRNA. Furthermore, like its archaeal homologs, Cbf5(Delta)p can bind to a single stem-loop-box ACA RNA. Finally, we report the first enzymatic footprinting analysis of a Cbf5-RNA complex. Our results are compatible with the view that two molecules of Cbf5p interact with a binding platform constituted by the 5' end of the RNA, the single-stranded hinge domain containing the conserved H box, and the 3' end of the molecule, including the conserved ACA box.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Annotation Extension||Reference|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Assay||Construct||Conditions||Strain Background||Reference|