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. 1999 Dec;19(12):8633-45.
doi: 10.1128/MCB.19.12.8633.

Synthetic lethality with conditional dbp6 alleles identifies rsa1p, a nucleoplasmic protein involved in the assembly of 60S ribosomal subunits

D Kressler  1 , M Doère, M Rojo, P Linder
Affiliations

Synthetic lethality with conditional dbp6 alleles identifies rsa1p, a nucleoplasmic protein involved in the assembly of 60S ribosomal subunits

D Kressler et al. Mol Cell Biol. 1999 Dec.

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 degrees 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 Deltarsa1 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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Figures

FIG. 1
FIG. 1
Pre-rRNA processing and ribosome assembly in S. cerevisiae. The RNA polymerase I-transcribed pre-rRNA contains the sequences for the mature 18S, 5.8S, and 25S rRNAs that are separated by two internal transcribed spacer sequences, ITS1 and ITS2, and flanked by two external transcribed spacer sequences, 5′ ETS and 3′ ETS. The mature rRNA species are shown as bars, and the transcribed spacer sequences are shown as lines. The processing sites and their locations are indicated. The primary RNA pol I transcript undergoes covalent modifications (2′-O-ribose methylation and pseudouridylation), and it is processed at its 3′ end to yield the 35S pre-rRNA, which is the longest detectable precursor. Early-associating 40S and 60S r-proteins as well as trans-acting factors (proteins and snoRNAs) assemble on this precursor to form a 90S preribosomal particle (90S RNP). The 35S pre-rRNA is first cleaved at the U3 snoRNP-dependent site A0 to generate the 33S pre-rRNA. This molecule is subsequently processed at sites A1 and A2; the latter cleavage results in the separation of the pre-rRNAs destined for the small and large ribosomal subunits and allows the 90S RNP to separate into a 43S RNP and a 66S RNP. The early pre-rRNA cleavages at A0 to A2 require snoRNP components, Rrp5p, and the putative ATP-dependent RNA helicases Dbp4p, Fal1p, Rok1p, and Rrp3p. Additional nucleolar assembly reactions probably occur concomitantly to the early cleavages and include incorporation of the Rpl5p-5S RNP and of later-associating r-proteins. The structural rearrangements within early or intermediate preribosomal particles are likely to require the putative ATP-dependent RNA helicases Dbp6p, Dbp7p, and Drs1p. The 43S RNP is exported to the cytoplasm, where endonucleolytic cleavage of the 20S precursor at site D yields the mature 18S rRNA. Then, the newly formed 40S subunits associate with translation initiation factors and are recruited to capped mRNAs, which they search for the first start codon. The 27SA2 precursor within the 66S RNP is processed by two alternative pathways that both lead to the formation of nuclear pre-60S particles containing the mature 5.8S and 25S rRNAs. In the major pathway, the 27SA2 precursor is cleaved at site A3 by the RNase MRP complex. Rrp5p and the putative ATP-dependent RNA helicase Dpb3p assist in this processing step. The 27SA3 precursor is exonucleolytically digested 5′→3′ up to site B1S to yield the 27SBS precursor, a reaction requiring the exonucleases Xrn1p and Rat1p. A minor pathway processes the 27SA2 molecule at site B1L, producing the 27SBL pre-rRNA. While processing at site B1 is being completed, the 3′ end of mature 25S rRNA is generated by processing at site B2. The subsequent ITS2 processing of both 27SB species appears to be identical. Cleavage at sites C1 and C2 releases the mature 25S rRNA and the 7S pre-rRNA. The putative ATP-dependent RNA helicase Spb4p is a good candidate for assisting cleavage at site C1 or C2. The 7S pre-rRNA undergoes exosome-dependent 3′→5′ exonuclease digestion to the 3′ end of the mature 5.8S rRNA. It has been proposed that Dob1p/Mtr4p, a putative ATP-dependent RNA helicase, assists the exosome activity. The data presented in this study suggest that Rsa1p is involved in a nucleoplasmic assembly step of pre-60S ribosomal subunits, which is required for the efficient recruitment of the exchangeable 60S r-protein Qsr1p/Rpl10p. Cytoplasmic assembly of Qsr1p/Rpl10p and formation of 60S ribosomal subunits that are competent for 60S-to-40S subunit joining are likely to require the trans-acting factors Sqt1p and Nmd3p. Mature 60S subunits containing Qsr1p/Rpl10p can bind to 40S subunits to form 80S monosomes that can then engage in translation elongation. The 90S, 66S, and 43S RNPs, as well as the pre-60S and the mature 40S and 60S subunits, are shown as ovals. The nuclear envelope is represented by the stippled bars.
FIG. 2
FIG. 2
Amino acid sequence of Rsa1p. A putative seven-amino-acid pattern nuclear localization signal is present near the C terminus of the protein (amino acids 363 to 369; boldface and underlined). The amino acids that are changed in the rsa1-1 (K) and rsa1-2 (M) mutants are in boldface. The rsa1-1 mutation changes the lysine codon (AAG) at amino acid position 225 to a premature stop codon (TAG), and the rsa1-2 mutation changes the ATG start codon to TTG. The asterisk defines the stop codon of the RSA1 ORF.
FIG. 3
FIG. 3
The rsa1 null mutation confers slow growth and temperature sensitivity, and it synthetically enhances the weak slow-growth phenotype of the dbp6-3 mutant. (A) YDK44 (MATa/MATα rsa1::kanMX4/RSA1) was sporulated, and tetrads were dissected. A wild-type and a Δrsa1 spore clone from a representative tetrad are shown on YPD plates that were incubated for 48 h at 30 and 37°C, respectively. (B) Wild-type (YDK46-7B pRS414-HA-DBP6), Δrsa1 (YDK46-7A pRS414-HA-DBP6), dbp6-3 (YDK46-7B pRS414-dbp6-3), and Δrsa1/dbp6-3 (YDK46-7A pRS414-dbp6-3) strains are shown on a YPD plate that was incubated for 60 h at 30°C.
FIG. 4
FIG. 4
Absence of Rsa1p leads to the accumulation of half-mer polysomes. (A) YDK44-1A (wild type) grown at 30°C. YDK44-1B (Δrsa1) was grown at 30°C (B) or shifted to 37°C for 5 h (C). Cells were grown in YPD and harvested at an OD600 of around 0.8. Cell extracts were resolved in 7-to-50% sucrose gradients. Gradients were analyzed by continuous monitoring at A254. Sedimentation is from left to right. The peaks of free 40S and 60S subunits, 80S ribosomes (free couples and monosomes), and polysomes are indicated. Half-mers are indicated by arrows.
FIG. 5
FIG. 5
Absence of Rsa1p leads to reduced synthesis of the mature 25S rRNA. Strains YDK44-1A (RSA1) and YDK44-1B (Δrsa1) were grown in SD-Met medium at 30°C (A) or shifted for 6 h to 37°C (B). Cells were pulsed-labeled (p) for 1 min with [methyl-3H]methionine and then chased (c) for 2, 5, and 15 min with an excess of unlabeled methionine. Total RNA was extracted, and 20,000 cpm was loaded and separated on a 1.2% agarose–formaldehyde gel, transferred to a nylon membrane, and visualized by fluorography. The positions of the different pre-rRNAs and mature rRNAs are indicated.
FIG. 6
FIG. 6
The rsa1 null mutation enhances the polysome profile phenotype of the dbp6-3 mutant. Polysome profiles are shown for the following strains: wild type (YDK46-7B pRS414-HA-DBP6) (A), dbp6-3 strain (YDK46-7B pRS414-dbp6-3) (B), Δrsa1 strain (YDK46-7A pRS414-HA-DBP6) (C), and Δrsa1/dbp6-3 strain (YDK46-7A pRS414-dbp6-3). Strains were grown in YPD at 30°C, and cells were harvested at an OD600 of around 0.8. Cell extracts were resolved in 7-to-50% sucrose gradients. Gradients were analyzed by continuous monitoring at A254. Sedimentation is from left to right. The peaks of free 40S and 60S subunits, 80S ribosomes (free couples and monosomes), and polysomes are indicated. Half-mers are indicated by arrows.
FIG. 7
FIG. 7
Synthetic enhancement of the slow-growth phenotype of the qsr1-1 mutant by the rsa1 null mutation. The strains MMY3-3B (qsr1-1), carrying the plasmid YCplac33-RSA1, and YDK45-11A (Δrsa1) were crossed, the resulting diploid was sporulated, and tetrads were dissected. Complete tetrads were restreaked on 5-FOA-containing plates to counter-select YCplac33-RSA1. A representative tetratype tetrad after 5-FOA counter-selection is shown on a YPD plate that was incubated for 72 h at 30°C.
FIG. 8
FIG. 8
Absence of Rsa1p leads to decreased levels of Qsr1p on free 60S ribosomal subunits. Shown are a polysome profile analysis and fractionation followed by Western blotting to detect the 60S r-proteins Qsr1p and Rpl3p. (A) YDK44-1A (wild type) grown at 30°C. YDK44-1B (Δrsa1) was grown at 30°C (B) or shifted to 37°C for 4 h (C). Cells were grown in YPD and harvested at an OD600 of around 0.8. Cell extracts were resolved in 7-to-50% sucrose gradients. Gradients were analyzed by continuous monitoring at A254. Sedimentation is from left to right. The peaks of free 40S and 60S subunits, 80S ribosomes (free couples and monosomes), and polysomes are indicated. Half-mers are indicated by vertical arrows. A total of 13 fractions were collected, proteins were concentrated by trichloroacetic acid precipitation, and equal volumes were resolved on sodium dodecyl sulfate–12% polyacrylamide gels and subjected to Western blotting. T, total extract. Numbers correspond to fraction numbers. The same blot was decorated simultaneously with polyclonal rabbit anti-Qsr1p and monoclonal mouse anti-Rpl3p antibodies to detect the 60S r-proteins Qsr1p and Rpl3p, respectively. The Qsr1p and Rpl3p signals are indicated.
FIG. 9
FIG. 9
HA-Rsa1p localizes to the nucleoplasm. Indirect immunofluorescence was performed with cells expressing HA-Rsa1p from the RSA1 promoter (YDK44-1B YCplac111-HA-RSA1). (A) Nop1p was detected by polyclonal rabbit anti-Nop1p antibodies, followed by decoration with a goat anti-rabbit fluorescein-conjugated antibody. (B) HA-Rsa1p was detected by the monoclonal mouse anti-HA 16B12 antibody, followed by decoration with a goat anti-mouse rhodamine-conjugated antibody. (C) Chromatin DNA was stained with DAPI. Pseudocolors were assigned to the digitized micrographs (A to C), and images were merged. The overlapping distributions are revealed in yellow for Nop1p and HA-Rsa1p colocalization (D), magenta for HA-Rsa1p and chromatin DNA colocalization (E), and cyan for Nop1p and chromatin DNA colocalization (F).
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