Synthetic Lethality with Conditional dbp6 Alleles Identifies Rsa1p, a Nucleoplasmic Protein Involved in the Assembly of 60S Ribosomal Subunits

Abstract

Dbp6p is an essential putative ATP-dependent RNA helicase that is required for 60S-ribosomal-subunit assembly in the yeast Saccharomyces cerevisiae (D. Kressler, J. de la Cruz, M. Rojo, and P. Linder, Mol. Cell. Biol. 18:1855–1865, 1998). To identify factors that are functionally interacting with Dbp6p, we have performed a synthetic lethal screen with conditional dbp6 mutants. Here, we describe the cloning and the phenotypic analysis of the previously uncharacterized open reading frame YPL193W, which we renamed RSA1 (ribosome assembly 1). Rsa1p is not essential for cell viability; however, rsa1 null mutant strains display a slow-growth phenotype, which is exacerbated at elevated temperatures. The rsa1 null allele synthetically enhances the mild growth defect of weak dbp6 alleles and confers synthetic lethality when combined with stronger dbp6 alleles. Polysome profile analysis shows that the absence of Rsa1p results in the accumulation of half-mer polysomes. However, the pool of free 60S ribosomal subunits is only moderately decreased; this is reminiscent of polysome profiles from mutants defective in 60S-to-40S subunit joining. Pulse-chase labeling of pre-rRNA in the rsa1 null mutant strain indicates that formation of the mature 25S rRNA is decreased at the nonpermissive temperature. Interestingly, free 60S ribosomal subunits of a rsa1 null mutant strain that was grown for two generations at 37°C are practically devoid of the 60S-ribosomal-subunit protein Qsr1p/Rpl10p, which is required for joining of 60S and 40S subunits (D. P. Eisinger, F. A. Dick, and B. L. Trumpower, Mol. Cell. Biol. 17:5136–5145, 1997). Moreover, the combination of the Δrsa1 and qsr1-1 mutations leads to a strong synthetic growth inhibition. Finally, a hemagglutinin epitope-tagged Rsa1p localizes predominantly to the nucleoplasm. Together, these results point towards a function for Rsa1p in a late nucleoplasmic step of 60S-ribosomal-subunit assembly.

ACKNOWLEDGMENTS

We are indebted to the following colleagues for their kind gift of material used in this study: J. de la Cruz (Universidad de Sevilla) for strain JDY50-1A and plasmid YCplac33-SPB4; B. L. Trumpower (Dartmouth Medical School) for strain MMY3-3B and polyclonal rabbit anti-Qsr1p antibodies; J. Venema (Vrije Universiteit Amsterdam) for plasmid pHT4467; J. R. Warner (Albert Einstein College) for monoclonal mouse anti-Rpl3p antibodies; J. L. Woolford, Jr. (Carnegie Mellon University) for strains JWY4917, JWY5081, and JWY5082; and N. I. T. Zanchin (University of Rochester) for strain DG130. We are grateful to J. de la Cruz, M.-C. Daugeron, K. Tanner, and C. Tu for fruitful discussions. We thank R. Boeck and K. Tanner for critical reading of the manuscript. We gratefully acknowledge C. Georgopoulos for support.

This work was supported by a grant from the Swiss National Science Foundation to P.L.