title: Point mutations in yeast CBF5 can abolish in vivo pseudouridylation of rRNA.
abstract: In budding yeast (Saccharomyces cerevisiae), the majority of box H/ACA small nucleolar RNPs (snoRNPs) have been shown to direct site-specific pseudouridylation of rRNA. Among the known protein components of H/ACA snoRNPs, the essential nucleolar protein Cbf5p is the most likely pseudouridine (Psi) synthase. Cbf5p has considerable sequence similarity to Escherichia coli TruBp, a known Psi synthase, and shares the "KP" and "XLD" conserved sequence motifs found in the catalytic domains of three distinct families of known and putative Psi synthases. To gain additional evidence on the role of Cbf5p in rRNA biosynthesis, we have used in vitro mutagenesis techniques to introduce various alanine substitutions into the putative Psi synthase domain of Cbf5p. Yeast strains expressing these mutated cbf5 genes in a cbf5Delta null background are viable at 25 degrees C but display pronounced cold- and heat-sensitive growth phenotypes. Most of the mutants contain reduced levels of Psi in rRNA at extreme temperatures. Substitution of alanine for an aspartic acid residue in the conserved XLD motif of Cbf5p (mutant cbf5D95A) abolishes in vivo pseudouridylation of rRNA. Some of the mutants are temperature sensitive both for growth and for formation of Psi in the rRNA. In most cases, the impaired growth phenotypes are not relieved by transcription of the rRNA from a polymerase II-driven promoter, indicating the absence of polymerase I-related transcriptional defects. There is little or no abnormal accumulation of pre-rRNAs in these mutants, although preferential inhibition of 18S rRNA synthesis is seen in mutant cbf5D95A, which lacks Psi in rRNA. A subset of mutations in the Psi synthase domain impairs association of the altered Cbf5p proteins with selected box H/ACA snoRNAs, suggesting that the functional catalytic domain is essential for that interaction. Our results provide additional evidence that Cbf5p is the Psi synthase component of box H/ACA snoRNPs and suggest that the pseudouridylation of rRNA, although not absolutely required for cell survival, is essential for the formation of fully functional ribosomes.
authors.affiliation: Department of Molecular Biology, University of California, Santa Barbara, California 93106, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Zebarjadian
authors.forename: T
authors.initials: T
authors.lastname: King
authors.forename: M J
authors.initials: MJ
authors.lastname: Fournier
authors.forename: L
authors.initials: L
authors.lastname: Clarke
authors.forename: J
authors.initials: J
authors.lastname: Carbon
mesh_terms: D000595:Amino Acid Sequence; D017124:Conserved Sequence; D006836:Hydro-Lyases / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D017354:Point Mutation; D012319:RNA Polymerase II / Q000378:metabolism; D012322:RNA Precursors / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D012335:RNA, Ribosomal / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics*; D017411:Ribonucleoproteins, Small Nuclear; D020718:Ribonucleoproteins, Small Nucleolar / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014158:Transcription, Genetic; D014542:Uridine Monophosphate / Q000096:biosynthesis*
chemical_list: D008869:Microtubule-Associated Proteins; D012322:RNA Precursors; D012335:RNA, Ribosomal; D016601:RNA-Binding Proteins; D017411:Ribonucleoproteins, Small Nuclear; D020718:Ribonucleoproteins, Small Nucleolar; D029701:Saccharomyces cerevisiae Proteins; C100044:pseudouridylic acid; D014542:Uridine Monophosphate; D012319:RNA Polymerase II; D006836:Hydro-Lyases; C082278:CBF5 protein, S cerevisiae; C022479:pseudouridylate synthetase