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 ⌬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.The synthesis of ribosomes is one of the major cellular activities, which, in eukaryotes, takes place primarily, although not exclusively, in a specialized subnuclear compartment termed the nucleolus (33, 39). There, the ribosomal DNA is transcribed as precursors (pre-rRNAs), which undergo processing and covalent modification. Maturation of pre-rRNAs and their concomitant assembly with the ribosomal proteins (r-proteins) are dependent on various cis-acting elements and require a large number of nonribosomal trans-acting factors. Experimental evidence suggests that the basic outline of ribosome synthesis is conserved throughout eukaryotes. However, most of our knowledge comes from the combination of molecular genetics and biochemical approaches applied to the yeast Saccharomyces cerevisiae (reviewed in references 14, 46, 55, and 62).In S. cerevisiae, the large 60S ribosomal subunits are composed of 46 r-proteins and three rRNA species (5S, 5.8S, and 25S), while the small 40S ribosomal subunits contain 32 rproteins and the 18S rRNA (34, 62). Three of the four rRNAs (18S, 5.8S, and 25S) are transc...

Npa1p is an essential trans-acting factor required for an early step in the assembly of 60S ribosomal subunits in Saccharomyces cerevisiae

“…We have previously described the function of Dbp6p, a putative RNA helicase that is required for an early step during the assembly of 60S r-subunits (Kressler et al 1998). We have elucidated the functional environment of Dbp6p by establishing a genetic interaction network with the trans-acting factors Dbp7p, Dbp9p, Nop8p, Rsa1p, and Rsa3p and the 60S r-protein Rpl3p (Kressler et al 1999a;de la Cruz et al 2004b). All of these proteins have been implicated in similar early nucleolar steps during the assembly of 60S r-subunits, with the exception of Rsa1p, which localizes to the nucleoplasm.…”

“…The sl phenotype of the sl1-1, sl1-3, sl1-5, sl1-7, sl2-2, sl3-3, and sl4-3 mutants was complemented by the RPL3 gene. Strain sl1-4, which has an sg phenotype at any temperature tested, was transformed with a YCplac111-based yeast genomic library (Kressler et al 1999a) and about 15,000 transformants were screened for wild-type growth on SD-Leu plates at 37°C. One plasmid, pIV222, containing an 8.4-kb insert, complemented both the sg and sl phenotypes of the sl1-4 mutant.…”

Nascent 60S ribosomal subunits enter the free pool bound by Nmd3p

“…Considering the role of Nmd3p in 60S subunit export (Ho et al+, 2000), the genetic interaction between NMD3 and RPL10 (Karl et al+, 1999;Zuk et al+, 1999) could be due to effects on nuclear transport of 60S subunits+ Although Rpl10p exchanges on the 60S subunit in the cytoplasm (Dick et al+, 1997;Nguyen et al+, 1998), it is possible that Rpl10p is initially loaded in the nucleus and that it is required on the nascent 60S subunit for efficient export+ QM protein, the human homolog of Rpl10, is reported to be restricted to the cytoplasm (Nguyen et al+, 1998)+ However, a transient presence of QM protein in the nucleus, perhaps in the nucleoplasm, could not be ruled out+ Nmd3p, which also appears restricted to the cytoplasm at steady state, does, in fact, bind to nascent 60S subunits in the nucleus (Ho et al+, 2000)+ It should also be noted that Rpl10p has a putative NLS and that the nucleoplasmic protein Rsa1p is required for efficient Rpl10p loading onto the 60S subunit (Kressler et al+, 1999a), supporting the notion that Rpl10p may enter the nucleus+ If Rpl10p does initially load onto the nascent 60S subunit in the nucleus and is required for efficient export, suppression by NMD3 may be due to increasing the rate of transport+ We are currently carrying out experiments to distinguish between these models for Nmd3p function+…”

RNA Helicase Prp43 and Its Co-factor Pfa1 Promote 20 to 18 S rRNA Processing Catalyzed by the Endonuclease Nob1

“…Deletion disruption and C-terminal tagging at the genomic locus were performed as described previously (25)(26)(27). The synthetic lethal screen with the ltv1⌬ strain based on red/white colony sectoring was performed at 30°C as described previously (28). Site-directed mutagenesis of Nob1 was performed by fusion PCR.…”

“…7, B and C). Longer exposure revealed two additional cleavage products, corresponding to cleavage at A 28 and A 31 (Fig. 7, B, D, E, and F).…”