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Molecular and Cellular Biology, November 1999, p. 7461-7472, Vol. 19, No. 11
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Point Mutations in Yeast CBF5 Can Abolish In Vivo Pseudouridylation of rRNA

Yeganeh Zebarjadian,1 Tom King,2 Maurille J. Fournier,2 Louise Clarke,1 and John Carbon1,*

Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, California 93106,1 and Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, Massachusetts 010032

Received 25 February 1999/Returned for modification 15 April 1999/Accepted 29 July 1999

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°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.

* Corresponding author. Mailing address: Department of Molecular, Cellular, and Developmental Biology, University of California at Santa Barbara, Santa Barbara, CA 93106. Phone: (805) 893-3163. Fax: (805) 893-4724. E-mail: carbon{at}lifesci.ucsb.edu.

Molecular and Cellular Biology, November 1999, p. 7461-7472, Vol. 19, No. 11
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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