Synthetic lethality with conditional dbp6 alleles identifies Rsa1p, a nucleoplasmic protein involved in the assembly of 60S ribosomal subunits

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 Db p6p, we have performed a synthetic lethal screen with conditional dbp6 muta nts. Here, we describe the cloning and the phenotypic analysis of the previ ously uncharacterized open reading frame YPL193W, which we renamed RSAI (ri bosome assembly 1). Rsa1p is not essential for cell viability; however, rsa 1 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 whe n combined with stronger dbp6 alleles. Polysome profile analysis shows that the absence of Rsa1p results in the accumulation of half-mer polysomes. Ho wever, 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 mut ant strain indicates that formation of the mature 25S rRNA is decreased at the nonpermissive temperature. Interestingly, free 60S ribosomal subunits o f a rsa1 null mutant strain that was grown for two generations at 37 degree s C are practically devoid of the 60S-ribosomal-subunit protein Qsr1p/Rpl10 p, which is required for joining of 608 and 40S subunits (D. P. Eisinger, F . A. Dick, and B. L. Trumpower, Mol. Cell. Biol. 17:5136-5145, 1997). Moreo ver, the combination of the Delta rsa1 and qsr1-1 mutations leads to a stro ng synthetic growth inhibition. Finally, a hemagglutinin epitope-tagged Rsa 1p localizes predominantly to the nucleoplasm. Together, these results poin t towards a function for Rsa1p in a late nucleoplasmic step of 60S-ribosoma l-subunit assembly.