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Molecular and Cellular Biology, December 1999, p. 8633-8645, Vol. 19, No. 12
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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

Dieter Kressler,^1,* Monique Doère,¹ Manuel Rojo,^2, and Patrick Linder^1,*

Département de Biochimie Médicale, Centre Médical Universitaire,¹ and Département de Biochimie, Sciences II,² Université de Genève, 1211 Geneva 4, Switzerland

Received 17 June 1999/Returned for modification 4 August 1999/Accepted 2 September 1999

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 rsa1 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.

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^* Corresponding author. Mailing address: Département de Biochimie Médicale, Centre Médical Universitaire, Université de Genève, 1 rue Michel-Servet, CH-1211 Geneva 4, Switzerland. Phone: 41 22 702 54 84. Fax: 41 22 702 55 02. E-mail for Dieter Kressler: dieter.kressler{at}medecine.unige.ch. E-mail for Patrick Linder: patrick.linder{at}medecine.unige.ch.

Present address: INSERM U523-Institut de Myologie, Groupe Hospitalier Pitié-Salpêtrière, 75651 Paris Cedex 13, France.

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Molecular and Cellular Biology, December 1999, p. 8633-8645, Vol. 19, No. 12
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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