Identification of Genes That Function in the Biogenesis and Localization of Small Nucleolar RNAs in Saccharomyces cerevisiae

Abstract

Small nucleolar RNAs (snoRNAs) orchestrate the modification and cleavage of pre-rRNA and are essential for ribosome biogenesis. Recent data suggest that after nucleoplasmic synthesis, snoRNAs transiently localize to the Cajal body (in plant and animal cells) or the homologous nucleolar body (in budding yeast) for maturation and assembly into snoRNPs prior to accumulation in their primary functional site, the nucleolus. However, little is known about the trans-acting factors important for the intranuclear trafficking and nucleolar localization of snoRNAs. Here, we describe a large-scale genetic screen to identify proteins important for snoRNA transport in Saccharomyces cerevisiae. We performed fluorescence in situ hybridization analysis to visualize U3 snoRNA localization in a collection of temperature-sensitive yeast mutants. We have identified Nop4, Prp21, Tao3, Sec14, and Htl1 as proteins important for the proper localization of U3 snoRNA. Mutations in genes encoding these proteins lead to specific defects in the targeting or retention of the snoRNA to either the nucleolar body or the nucleolus. Additional characterization of the mutants revealed impairment in specific steps of U3 snoRNA processing, demonstrating that snoRNA maturation and trafficking are linked processes.

ACKNOWLEDGMENTS

We thank Charles Cole (Dartmouth Medical School) for the temperature-sensitive mutant yeast collection, Anita Hopper (Penn State University) for the yeast genomic libraries, Edouard Bertrand (CNRS, France) for the pG14-U14/MS2X2 plasmid, Peter Novick (Yale University) for the Tao3 plasmid, David Levin (Johns Hopkins University) for the Htl1 plasmid, John P. Aris (University of Florida) for the anti-Nop1 and anti-Nsr1 antibodies, and Guillaume Chanfreau (University of California, Los Angeles) for the U14-specific probes.

This work was supported by NIH grant GM54682 to M.T. and R.T. and NIH grant GM58728 to A.C. Research in the laboratory of D.L.J.L. was supported by the Fonds National de la Recherche Scientifique, Université Libre de Bruxelles, Communauté Française de Belgique (ARC convention 06/11-345), the Fonds Alice & David van Buuren, the Fonds Brachet, the Fonds Defay, and the International Brachet Stiftung.