Functional Mapping of the U3 Small Nucleolar RNA from the Yeast Saccharomyces cerevisiae

ABSTRACT

The U3 small nucleolar RNA participates in early events of eukaryotic pre-rRNA cleavage and is essential for formation of 18S rRNA. Using an in vivo system, we have developed a functional map of the U3 small nucleolar RNA from Saccharomyces cerevisiae. The test strain features a galactose-dependent U3 gene in the chromosome and a plasmid-encoded allele with a unique hybridization tag. Effects of mutations on U3 production were analyzed by evaluating RNA levels in cells grown on galactose medium, and effects on U3 function were assessed by growing cells on glucose medium. The major findings are as follows: (i) boxes C′ and D and flanking helices are critical for U3 accumulation; (ii) boxes B and C are not essential for U3 production but are important for function, most likely due to binding of a trans-acting factor(s); (iii) the 5′ portion of U3 is required for function but not stability; and, (iv) strikingly, the nonconserved hairpins 2, 3, and 4, which account for 50% of the molecule, are not required for accumulation or function.

ACKNOWLEDGMENTS

We thank Nandita Sharma for help in preparing box B and box C substitution mutations, Matthew Huang and Xiwei Wang for sharing unpublished data, Andrey Balakin for valuable discussions, and Elizabeth Furter-Graves for expert editorial work. We also thank Susan Baserga, Michael Terns, and anonymous reviewers for critical reading of the manuscript prior to publication and for helpful suggestions.

This research was supported by National Institutes of Health grant GM19351.