Genetics, Vol. 166, 1687-1699, April 2004, Copyright © 2004

The Putative RNA Helicase Dbp6p Functionally Interacts With Rpl3p, Nop8p and the Novel trans-acting Factor Rsa3p During Biogenesis of 60S Ribosomal Subunits in Saccharomyces cerevisiae

Jesús de la Cruz1,a, Thierry Lacombeb, Olivier Delocheb, Patrick Linderb, and Dieter Kressler1,3,b
a Departamento de Genética, Facultad de Biología, Universidad de Sevilla, E-41012 Sevilla, Spain
b Département de Biochimie Médicale, Centre Médical Universitaire, Université de Genève, CH-1211 Genève 4, Switzerland

Corresponding author: Jesús de la Cruz, Facultad de Biología, Universidad de Sevilla, Avda. Reina Mercedes, 6, E-41012 Sevilla, Spain., jdlcd{at}us.es (E-mail)

Communicating editor: M. SACHS


*  ABSTRACT
*TOP
 *ABSTRACT
*MATERIALS AND METHODS
 *RESULTS
 *DISCUSSION
 *LITERATURE CITED

Ribosome biogenesis requires at least 18 putative ATP-dependent RNA helicases in Saccharomyces cerevisiae. To explore the functional environment of one of these putative RNA helicases, Dbp6p, we have performed a synthetic lethal screen with dbp6 alleles. We have previously characterized the nonessential Rsa1p, whose null allele is synthetically lethal with dbp6 alleles. Here, we report on the characterization of the four remaining synthetic lethal mutants, which reveals that Dbp6p also functionally interacts with Rpl3p, Nop8p, and the so-far-uncharacterized Rsa3p (ribosome assembly 3). The nonessential Rsa3p is a predominantly nucleolar protein required for optimal biogenesis of 60S ribosomal subunits. Both Dbp6p and Rsa3p are associated with complexes that most likely correspond to early pre-60S ribosomal particles. Moreover, Rsa3p is co-immunoprecipitated with protA-tagged Dbp6p under low salt conditions. In addition, we have established a synthetic interaction network among factors involved in different aspects of 60S-ribosomal-subunit biogenesis. This extensive genetic analysis reveals that the rsa3 null mutant displays some specificity by being synthetically lethal with dbp6 alleles and by showing some synthetic enhancement with the nop8-101 and the rsa1 null allele.

THE synthesis of ribosomes is a major cellular activity that, in eukaryotes, takes place primarily in a specialized subnuclear compartment termed the nucleolus (OLSON et al. 2000 Down). Ribosome synthesis is evolutionarily conserved throughout eukaryotes (EICHLER and CRAIG 1994 Down), and so far most of our knowledge concerning this highly complex and dynamic process comes from studies with Saccharomyces cerevisiae. In yeast nucleoli, the ribosomal RNA (rRNA) genes are transcribed as precursors (pre-rRNAs), which undergo processing and covalent modification (KRESSLER et al. 1999B Down; VENEMA and TOLLERVEY 1999 Down; for a simplified pre-rRNA processing scheme, see Fig 4A). Three of the four rRNAs (18S, 5.8S, and 25S) are transcribed as a single large pre-rRNA by RNA polymerase I, whereas the fourth rRNA (5S) is transcribed by RNA polymerase III. The maturation of pre-rRNAs is intimately linked to their assembly with the 78 ribosomal proteins (r-proteins). The large 60S ribosomal subunits (r-subunits) are composed of 46 r-proteins and three rRNA species (5.8S, 25S, and 5S), while the small 40S r-subunits contain 32 r-proteins and the 18S rRNA (PLANTA and MAGER 1998 Down).



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  		Figure 1. Synthetic-lethal mutant strains with dbp6 alleles show a deficit in free 60S r-subunits and an accumulation of half-mer polysomes. (A) sl263210 (rsa3-1), (B) sl264205 (rpl3-102), and (C) sl264409-42D (nop8-101) were grown in YPD at 30° up to an OD600 of ~0.8. Cell extracts were resolved in 7–50% 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.



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  		Figure 2. The rsa3 null mutation confers a mild temperature-sensitive slow-growth phenotype; the rsa3 null, rpl3-101, and nop8-101 mutations are synthetically lethal with the dbp6-2 allele. (A) YMD3-2A (RSA3) and YMD3-2D ({Delta}rsa3) were grown on YPD plates at 30° (top) or 37° (bottom). (B) Wild type (DBP6/RSA3), {Delta}rsa3 (DBP6/{Delta}rsa3), dbp6-2 (dbp6-2/RSA3), and dbp6-2 {Delta}rsa3 are shown on a 5-FOA plate that was incubated at 30°. (C) Wild-type (DBP6/RPL3), rpl3-101 (DBP6/rpl3-101), dbp6-2 (dbp6-2/RPL3), and dbp6-2 rpl3-101 are shown on a 5-FOA plate that was incubated at 30°. (D) Wild type (DBP6/NOP8), nop8-101 (DBP6/nop8-101), dbp6-2 (dbp6-2 nop8-101), and dbp6-2 nop8-101 are shown on a 5-FOA plate that was incubated at 30°. See also RESULTS and MATERIALS AND METHODS for details.



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  		Figure 3. The {Delta}rsa3 mutant has a deficit in free 60S r-subunits and accumulates half-mer polysomes. (A) YMD3-1A pHAC33-RSA3 (wild type) and (B) YMD3-1A YCplac33 ({Delta}rsa3) were grown in SD-Ura at 30° and then shifted for 4 hr to 37° (OD600 ~0.8). Cell extracts were resolved in 7–50% 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.



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  		Figure 4. Absence of Rsa3p leads to a mild reduction in the synthesis of the mature 25S rRNA. (A) Simplified pre-rRNA processing scheme. The early cleavage reactions A0, A1, and A2 process the 35S pre-rRNA in which the mature rRNA sequences are separated by two internal transcribed spacer sequences, ITS1 and ITS2, and are flanked by two external transcribed spacer sequences, 5'ETS and 3'ETS, into the 20S and 27SA2 pre-rRNAs. Endonucleolytic cleavage of the 20S pre-rRNA at site D in the cytoplasm yields the mature 18S rRNA. The 27SA2 precursor is processed by two alternative pathways: in the major pathway, the 27SA2 pre-rRNA is first cleaved at site A3 and is then 5'–3' exonucleolytically digested up to site B1S to yield the 27SBS precursor; a minor pathway processes the 27SA2 pre-rRNA at site B1L, thus producing the 27SBL precursor. The subsequent ITS2 processing of both 27SB species appears to be identical and leads to the formation of the mature 5.8S and 25S rRNAs. For details see VENEMA and TOLLERVEY 1999 Down and KRESSLER et al. 1999B Down. (B) Strains YMD3-2A (RSA3) and YMD3-2D ({Delta}rsa3) were first grown in SD-Met at 30° and then for 14 hr at 37° up to an OD600 of ~0.8. Cells were pulse labeled (p) for 1 min with [methyl-3H]methionine and then chased (c) for 2, 5, and 15 min with an excess of cold 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.

While the processing steps, as well as the protein trans-acting factors involved therein, that lead to the pre-rRNA intermediates and the mature rRNAs are fairly well characterized, less is known about the assembly, intranuclear transport, and export of preribosomal particles. Studies performed in the 1970s outlined a ribosome assembly pathway through identification of a 90S preribosomal particle, containing the 35S pre-rRNA, that is matured into 43S and 66S preribosomes, which contain the 20S and the 27S precursors to the mature 18S and 25S/5.8S rRNAs, respectively (TRAPMAN et al. 1975 Down; KRESSLER et al. 1999B Down). Only recently, the application of proteomic approaches allowed a more detailed insight (FATICA and TOLLERVEY 2002 Down; FROMONT-RACINE et al. 2003 Down). The 90S preribosomal particles contain, in addition to the 35S pre-rRNA, the U3 small nucleolar ribonucleoprotein particle, protein trans-acting factors implicated in the biogenesis of 40S r-subunits and small subunit r-proteins; however, they predominantly lack 60S biogenesis protein trans-acting factors and large subunit r-proteins (DRAGON et al. 2002 Down; GRANDI et al. 2002 Down). This has led to the proposal of a biphasic model in which the machinery required for 40S r-subunit synthesis almost exclusively assembles onto the 35S pre-rRNA, whereas the 60S r-subunit assembly factors are recruited only to form an early 66S preribosomal particle after the separation of the 20S and the 27SA2 pre-rRNAs by cleavage at site A2 (FATICA and TOLLERVEY 2002 Down; GRANDI et al. 2002 Down). The early 66S particle is found in the nucleolus and exchange of the Noc1p-Noc2p complex by the Noc2p-Noc3p complex has been suggested to trigger its intranuclear transport to the nucleoplasm (MILKEREIT et al. 2001 Down). Several distinct pre-60S complexes have been identified (BASSLER et al. 2001 Down; HARNPICHARNCHAI et al. 2001 Down; SAVEANU et al. 2001 Down; FATICA et al. 2002 Down; NISSAN et al. 2002 Down) and they can be placed in a tentative pre-60S assembly pathway (FATICA and TOLLERVEY 2002 Down). In addition to providing a refined framework for the course of ribosome assembly, the above proteomic analyses identified additional protein trans-acting factors, thus raising the inventory to ~150 proteins. Some of these protein trans-acting factors can be grouped according to their physical association with snoRNAs or their proposed enzymatic function (KRESSLER et al. 1999B Down). In agreement with the dynamic nature of the process, several putative GTPases (BASSLER et al. 2001 Down; GELPERIN et al. 2001 Down; SAVEANU et al. 2001 Down; WEGIERSKI et al. 2001 Down), two AAA-type ATPases (BASSLER et al. 2001 Down; GADAL et al. 2001A Down), and 18 putative ATP-dependent RNA helicases of the DEAD-box and related protein families (KRESSLER et al. 1999B Down; BOND et al. 2001 Down; EMERY et al. 2004 Down) are implicated in ribosome assembly. These proteins likely promote or monitor structural rearrangements of rRNA:rRNA, rRNA:protein, or protein:protein interactions within preribosomal particles, which finally lead to the generation of export and translation-competent r-subunits.

Despite the success of the proteomic approach in identifying preribosomal particles and their components, not all protein trans-acting factors implicated in ribosome assembly have been found in the identified preribosomal particles. One such factor is the putative RNA helicase Dbp6p, which is an essential nucleolar protein that is required for 60S r-subunit assembly and has been proposed to act at an early step during this process (KRESSLER et al. 1998 Down). As for almost all of the putative RNA helicases involved in ribosome biogenesis, the functional environment of Dbp6p is not well established. To gain more insight into the cellular role of Dbp6p, we performed a synthetic lethal (sl) screen with conditional dbp6 alleles (KRESSLER et al. 1999A Down). We previously reported the isolation and characterization of RSA1 (ribosome assembly 1), whose null allele is synthetically lethal with dbp6 alleles (KRESSLER et al. 1999A Down). Rsa1p is a nonessential nucleoplasmic protein that is likely involved in a nucleoplasmic assembly step of 60S r-subunits and their subsequent nuclear export (KRESSLER et al. 1999A Down; GADAL et al. 2001B Down). Here we describe the cloning and the genetic analysis of three additional sl genes: the large subunit r-protein gene RPL3, the known protein trans-acting factor encoding gene NOP8 (ZANCHIN and GOLDFARB 1999 Down), and the so-far-uncharacterized gene RSA3 (ribosome assembly 3). We provide evidence that the predominantly nucleolar Rsa3p is required for optimal biogenesis of 60S r-subunits. Furthermore, we show that Rsa3p and Dbp6p cosediment on sucrose gradients and that Rsa3p is efficiently co-immunoprecipitated with Dbp6p, suggesting that these two proteins functionally interact within the same early pre-60S ribosomal particles.


*  MATERIALS AND METHODS
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
*RESULTS
 *DISCUSSION
 *LITERATURE CITED

Strains, media, and genetic methods:
Most S. cerevisiae strains used in this study (Table 1) are derivatives of the diploid strain W303 (MATa/MAT{alpha} ade2-1/ade2-1 his3-11,15/his3-11,15 leu2-3,112/leu2-3,112 trp1-1/trp1-1 ura3-1/ura3-1). Preparation of standard media and genetic manipulations were according to established procedures (AUSUBEL et al. 1994 Down; KAISER et al. 1994 Down). Yeast cells were transformed by a lithium acetate method (GIETZ et al. 1992 Down). Deletion disruptions with the heterologous kanMX4 or HIS3MX6 marker modules were carried out as previously described (KRESSLER et al. 1999A Down). For tetrad dissection, a Singer MSM micromanipulator was used.


 
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  		Table 1. Yeast strains used in this study

Plasmids:
All recombinant DNA techniques were done according to established procedures using Escherichia coli DH10B and DH5{alpha} for cloning and plasmid propagation (SAMBROOK et al. 1989 Down). Relevant plasmids used in this study are listed in Table 2. More information on the plasmids is available on request.


 
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  		Table 2. Plasmids used in this study

Cloning of RSA3 and rsa3-1:
The original sl-mutant strain sl263210 was outcrossed once with a haploid W303-derived wild-type strain to yield strain sl263210-6B, which was transformed with both a pSEY18 (gift of M. N. Hall)- and a pFL44L (gift of F. Lacroute)-based yeast genomic library and screened for clones complementing the slow-growth (sg) phenotype at 37°. Candidate plasmids were isolated from yeast, amplified in E. coli, and retransformed into sl263210-6B. Sequence analysis of three clones revealed that all had the YLR221C (RSA3) open reading frame (ORF) in common.

To determine whether sl263210 indeed had the RSA3 gene mutated, this gene was amplified by PCR from genomic DNA prepared from a wild-type control strain and sl263210. The wild-type PCR product was digested and cloned into pHAC33 and pHAC111 (gift of M. N. Hall). Sequencing of the rsa3-1 PCR product as well as of the cloned RSA3 revealed that the rsa3-1 allele contains two mutations: first, a C-to-T transition at nucleotide position +329, which changes S110(TCT) to F(TtT), and second, an insertion of one A at nucleotide position +331, which causes a frameshift followed by a premature stop codon leading to a truncated protein of 144 amino acids (aa).

Cloning of NOP8 and nop8-101:
The original sl-mutant strain sl264409 was outcrossed once with a haploid W303-derived wild-type strain to yield strain sl264409-42D, which was transformed with both a YCplac111- and a YEplac181-based yeast genomic library and screened for clones complementing the sg and cold-sensitive phenotype. Candidate plasmids were isolated from yeast, amplified in E. coli, and retransformed into sl264409-42D. Sequence analysis of six clones revealed that all clones had the NOP8 ORF in common. To confirm that the NOP8 ORF indeed harbored the complementing activity, YCplac111-NOP8, which contains only the NOP8 gene, was constructed. This construct complements the sg phenotype and the polysome defect of sl264409-42D to the wild-type extent.

To determine whether sl264409-42D indeed had the NOP8 gene mutated, this gene was amplified by PCR from genomic DNA prepared from a wild-type control strain and sl264409-42D. The PCR products were digested and cloned into pGEM-4 (Promega, Madison, WI). Sequencing revealed that the nop8-101 mutation is an insertion of one A at nucleotide position +765, which causes a frameshift followed by a premature stop codon leading to a truncated protein of 257 aa.

Cloning of RPL3, rpl3-101, and rpl3-102:
The sl strains sl263309 and sl264205 were transformed with a YCplac111-based yeast genomic library and screened for clones complementing the sg and sl phenotypes. Candidate plasmids were isolated from yeast, amplified in E. coli, and retransformed into sl264205 and sl263309. Sequence analysis of two clones revealed that both contained the RPL3 gene. To confirm that the RPL3 ORF harbored the complementing activity, YCplac111-RPL3, which contains only the RPL3 gene, was constructed. This construct complemented the sl phenotype of sl264205 as well as the inviability of the rpl3-null-mutant strain to the wild-type extent.

To determine whether sl263309 (rpl3-101) and sl264205 (rpl3-102) indeed had the RPL3 gene mutated, this gene was amplified by PCR from genomic DNA prepared from a wild-type control strain and the two sl strains. The PCR products were digested and cloned into YCplac111 to yield the plasmids YCplac111-RPL3, YCplac111-rpl3-101, and YCplac111-rpl3-102. Sequencing revealed that the rpl3-101 mutation is an A-to-C transversion at nucleotide position +1113, which changes Q371(CAA) to H(CAc), and that the rpl3-102 mutation corresponds to a simultaneous A-to-G and G-to-A transition at nucleotide positions +88 and +90, respectively, which changes K30(AAG) to E(gAa).

More information on the cloning of RSA3, NOP8, RPL3, and their respective mutations can be found on the Linder laboratory website (http://www.medecine.unige.ch/linder/S1_delacruz_2003.pdf).

Synthetic interaction crosses:
To determine if different mutants affecting assembly of 60S r-subunits were showing synthetic enhancement phenotypes, the crosses described below were performed.

dbp6 rsa3::HIS3MX6: YDK8-1A pRS416-DBP6 was crossed to YMD3-2D pRS413, the resulting diploid was sporulated, and tetrads were dissected. Spore clones from two complete tetratype tetrads were transformed with pRS414, pRS414-HA-DBP6, pRS414-dbp6-2, and pRS414-dbp6-4. Transformants were restreaked on SD-Trp plates and subjected to plasmid shuffling on 5-fluoroorotic (5-FOA)-containing plates. No viable dbp6-2 rsa3::HIS3MX6 and dbp6-4 rsa3::HIS3MX6 double mutants could be recovered.

dbp6 nop8-101: YDK8-2A pRS416-DBP6 was crossed to sl264409-42D pRS413, the resulting diploid was sporulated, and tetrads were dissected. Spore clones from two complete tetratype tetrads were transformed with pRS414, pRS414-HA-DBP6, pRS414-dbp6-2, and pRS414-dbp6-4. Transformants were restreaked on SD-Trp plates and subjected to plasmid shuffling on 5-FOA-containing plates. No viable dbp6-2 nop8-101 and dbp6-4 nop8-101 double mutants could be recovered.

dbp6 rpl3: YDK8-1A pRS416-DBP6 was crossed to JDY319 YCplac111-RPL3, the resulting diploid was sporulated, and tetrads were dissected. Two complete tetratype tetrads were selected, and two G418R, His+, Ura+, and Leu+ spore clones were transformed with the plasmids pRS414-HA-DBP6, pRS414-dbp6-2, and pRS414-dbp6-4. Transformants were restreaked on SD-Trp plates, subjected to plasmid shuffling on 5-FOA-containing plates, and then transformed with YCplac33-RPL3. Transformants were restreaked on SD-Ura, subjected to segregation of YCplac111-RPL3 and then they were transformed with YCplac111, YCplac111-RPL3, YCplac111-rpl3-101, and YCplac111-rpl3-102. Transformants were restreaked on SD-Leu plates and subjected to plasmid shuffling on 5-FOA-containing plates. No viable dbp6-2rpl3-101, dbp6-2 rpl3-102, dbp6-4 rpl3-101, and dbp6-4 rpl3-102 double mutants could be recovered.

Information on the crosses for the analysis of the synthetic enhancement phenotypes of other double-mutant combinations can be found on the Linder laboratory website (http://www.medecine.unige.ch/linder/S1_delacruz_2003.pdf).

Sucrose gradient analyses:
Polysome and r-subunit preparations and their analyses were as previously described (KRESSLER et al. 1997 Down). Gradient analysis was performed with an ISCO UA-6 gradient UV detection and fraction collection system with continuous monitoring at A254.

For fractionation analyses, extract preparations and gradient centrifugation conditions were identical to those used for r-subunit analysis, except that 10 A254 units of cell extract was layered onto the gradients. Fractions of ~500 µl were collected. The gradient position of the 40S and 60S r-subunits was determined from the UV profile. To analyze the gradient position of protA-Dbp6p, Rsa3-HAp, and Rpl3p, fractions (200 µl) were processed exactly as described (KRESSLER et al. 1999A Down) and subjected to Western blot analysis. Polyclonal rabbit anti-protein A (at 5 ng/ml, Sigma, St. Louis), monoclonal mouse 16B12 (1:10,000 dilution, BAbCo), and monoclonal mouse anti-Rpl3p antibodies (1:10,000 dilution, gift of J. R. Warner) were used as primary antibodies. Blots were decorated with goat anti-mouse or anti-rabbit IgG horseradish-peroxidase-conjugated secondary antibodies (1:15,000 dilution, Bio-Rad, Richmond, CA) and developed with the SuperSignal West Pico detection kit (Pierce, Rockford, IL). To analyze the gradient position of pre-rRNAs, fractions (250 µl) were processed and subjected to Northern blot analysis exactly as described (DE LA CRUZ et al. 1998 Down).

The yeast strain used for the sucrose gradient fractionation and co-immunoprecipitation analyses was obtained by crossing YDK8-1A pRS415-protA-DBP6-T7 (this plasmid expresses N-terminally protA- and C-terminally T7-tagged Dbp6p from its cognate promoter) to YMD3-2D pHAC33-RSA3. The resulting diploid was sporulated and tetrads were dissected to yield the meiotic segregant JDY139 pRS415-protA-DBP6-T7 pHAC33-RSA3.

Co-immunoprecipitation:
For immunoprecipitation experiments, strains JDY139 pRS415-protA-DBP6-T7 pHAC33-RSA3 and YMD3-1A pHAC33-RSA3 were grown to an OD600 of ~1 in 100 ml SD-Ura medium at 30°. Cells were first washed with 50 ml ice-cold water and then with 50 ml ice-cold lysis buffer (50 mM Tris-HCl pH 7.5, 50 mM NaCl, 2 mM MgCl2, 1 mM 2-mercaptoethanol, 0.1% NP-40) and cell pellets were finally resuspended in 0.7 ml of ice-cold lysis buffer containing 1 mM phenylmethylsulfonyl fluoride. Cells were lysed by vortexing eight times for 30 sec in the presence of an equal volume of glass beads (425–600 µm, Sigma) at 4°. Aliquots of cleared lysates (0.35 ml) were diluted with 0.7 ml lysis buffer and incubated with 50 µl IgG Sepharose 6 Fast Flow beads (Amersham, Buckinghamshire, UK) for 1.5 hr at 4° with end-over-end tube rotation. The beads were first washed five times with 2 ml lysis buffer, three times with 2 ml lysis buffer containing different salt concentrations (50–500 mM NaCl), and finally another three times with 2 ml lysis buffer. Bound proteins were eluted with 200 µl 0.5 M acetic acid and they were concentrated by lyophilization. Total, nonbound, and bound proteins were subjected to Western blot analysis using polyclonal rabbit anti-protein A (at 5 ng/ml) and monoclonal mouse 16B12 (1:10,000 dilution) antibodies.

Pulse-chase labeling of pre-rRNA:
Strains YMD3-2A (RSA3) and YMD3-2D ({Delta}rsa3) were first grown in SD-Met medium at 30° and then for 14 hr at 37° up to an OD600 of ~0.8. A total of 30 OD600 units of cells were harvested by centrifugation and the pre-rRNA was labeled with [methyl-3H]methionine and analyzed as previously described (KRESSLER et al. 1999A Down).

Indirect immunofluorescence:
YMD3-1A pHAC33-RSA3 pUN100-NOP1-GFP was grown to an OD600 of ~0.8 in SD-Ura-Leu medium at 30°, and 3 OD600 units were prepared for immunofluorescence as previously described (CHUANG and SCHEKMAN 1996 Down). Primary monoclonal mouse anti-hemagglutinin (HA) antibodies 12CA5 (1:1000 dilution, BAbCo) and secondary goat anti-mouse Cy3-conjugated antibodies (1:400 dilution, Jackson ImmunoResearch, West Grove, PA) were used to detect Rsa3-HAp. Images were acquired with a Zeiss Axiophot fluorescence microscope equipped with an Axiocam color CCD camera that was controlled by a PentiumIII computer using the AxioVision software.


*  RESULTS
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
 *RESULTS
*DISCUSSION
 *LITERATURE CITED

Synthetic lethality with dbp6 alleles identifies RPL3, NOP8, and the novel RSA3:
To dissect the functional environment of Dbp6p, we carried out an sl screen with dbp6 alleles that led to the isolation of six sl strains (KRESSLER et al. 1999A Down). We previously described the cloning and functional characterization of RSA1, which complemented two of the six sl strains (KRESSLER et al. 1999A Down). Rsa1p is a nonessential nucleoplasmic protein involved in the assembly and nuclear export of 60S r-subunits (KRESSLER et al. 1999A Down; GADAL et al. 2001B Down). To extend the above study, we subjected the remaining four sl strains to polysome profile analysis, which revealed that, compared to a wild-type strain, they all had a deficit in free 60S r-subunits and had accumulated half-mer polysomes (Fig 1, A–C; data not shown and see also Fig 3A). To clone the genes that complemented the four sl strains, we transformed the original sl strains or outcrossed mutant strains with yeast genomic libraries (see MATERIALS AND METHODS). Sequencing into the library plasmids indicated that sl263210-6B was complemented by the nonessential, uncharacterized ORF YLR221C, which we named RSA3, sl263309 and sl264205 were complemented by the 60S r-protein gene RPL3, and sl264409-42D by the protein trans-acting factor encoding gene NOP8, whose product is required for assembly of 60S r-subunits (ZANCHIN and GOLDFARB 1999 Down). To verify that these genes, when mutated, were responsible for the sl phenotype with dbp6 alleles, we first determined whether the mutant strains indeed had mutations in the respective genes. To this end, we amplified these ORFs by PCR from the mutant strains as well as from the corresponding wild-type strains. Sequencing revealed that all mutant strains harbored mutations in their complementing genes (see MATERIALS AND METHODS); we therefore refer to the mutations as rsa3-1 (sl263210), rpl3-101 (sl263309), rpl3-102 (sl264205), and nop8-101 (sl264409). Second, we determined whether the rsa3 null, rpl3-101, rpl3-102, or nop8-101 mutant alleles were synthetically lethal with the mildly affected dbp6-2 mutant or the more strongly affected dbp6-4 mutant (see MATERIALS AND METHODS). In agreement with their isolation in the sl screen with dbp6 alleles, no viable dbp6 {Delta}rsa3, dbp6 rpl3, and dbp6 nop8-101 double mutants could be recovered (for dbp6-2, see Fig 2, B–D; data not shown for dbp6-4).

RSA3 encodes a relatively short protein of 220 aa with a calculated molecular mass of 24.7 kD, which, on the basis of a codon adaptation index of 0.17, is predicted to be of low cellular abundance. Furthermore, Rsa3p is predicted to be a rather acidic protein (pI 4.83) and to localize to the nucleus (PSORTII program) due to the presence of four overlapping "4 residue pattern" nuclear localization signals located between aa 18 and aa 24. Rsa3p is likely a yeast-specific protein since no homologous proteins could be identified by sequence comparison searches in Drosophila, Caenorhabditis, or mammalian databases. However, there is a significant homology (38% identity and 50% similarity) between Rsa3p and the Candida albicans ORF IPF3878. A more detailed sequence analysis revealed a region (aa 29–45) within the N-terminal part of Rsa3p that contains almost exclusively serine, aspartic acid, and glutamic acid residues (S/D/E-rich region); similar S/D/E-rich regions are found in other nucleolar proteins and in the case of Nsr1p it is much more extended (YAN and MELESE 1993 Down; KRESSLER et al. 1998 Down). Moreover, aa 129–150 of Rsa3p may adopt a coiled-coil conformation (COILS program; LUPAS et al. 1991 Down), which could serve as a homo- or heterodimerization surface (see DISCUSSION). This coiled-coil region is likely to be important for the function of Rsa3p since the rsa3-1 mutation, which leads to a frameshift after aa 110, confers the same phenotypes as the rsa3 null mutation does.

Absence of Rsa3p leads to a deficit in free 60S r-subunits, an accumulation of half-mer polysomes, and a slightly impaired production of 25S rRNA:
To analyze the function of Rsa3p, we first constructed a rsa3-null-mutant strain by replacing one copy of the RSA3 ORF in the diploid strain W303 with the HIS3MX6 marker module. Tetrad dissection and restreaking of spore clones on YPD plates at 18°, 30°, and 37° showed that the rsa3-null-mutant strain had a mild slow-growth phenotype at 37° (Fig 2A). Doubling times of 1.5 hr for the wild-type and of 1.9 hr for the rsa3-null-mutant strain were obtained in liquid YPD medium at 37°. As a first step to the functional analysis, we performed polysome profile analysis in a rsa3-null-mutant strain (YMD3-1A YCplac33) and a wild-type control strain (YMD3-1A pHAC33-RSA3) that were grown in SD-Ura at 30° and then shifted for 4 hr to 37°. The rsa3-null-mutant strain displayed a reduction in the amount of free 60S r-subunits as well as an accumulation of half-mer polysomes (Fig 3B). In contrast, the {Delta}rsa3 strain transformed with pHAC33-RSA3 had normal "wild-type" polysome profiles (Fig 3A). Quantification of total r-subunits in cell extracts from strains grown at 37° in YPD by sucrose gradient centrifugation under low-Mg2+ conditions confirmed the imbalance between 40S and 60S r-subunits. An A254 60S to 40S subunit ratio of 2.1 was observed for the wild-type control strain (YMD3-2A) whereas the ratio decreased to 1.7 for the {Delta}rsa3-null-mutant strain (YMD3-2D). We conclude that the absence of Rsa3p leads to a slight underaccumulation of 60S r-subunits, which likely accounts for the mild growth defect associated with the rsa3 null mutation.

Next, we attempted to study the role of Rsa3p during 60S r-subunit metabolism in more detail. To this end, we analyzed the effects of the rsa3 null mutation on the synthesis and processing of pre-rRNA by [methyl-3H]methionine pulse-chase labeling experiments. The wild-type control strain YMD3-2A (RSA3) and the rsa3-null-mutant strain YMD3-2D ({Delta}rsa3) were first grown in SD-Met medium at 30° and then for 14 hr at 37° up to an OD600 of ~0.8. Cells were harvested by centrifugation, pulse-labeled for 1 min with [methyl-3H]methionine, and chased for 2, 5, and 15 min with an excess of cold methionine. Total RNA was extracted, resolved on an agarose-formaldehyde gel, transferred to a nylon membrane, and finally subjected to fluorography. Compared to the wild-type strain, the {Delta}rsa3 mutant displayed a mild decrease in the synthesis of the mature 25S rRNA while the formation of 18S rRNA was not affected (Fig 4B). We could not observe a reduced synthesis of the 27S precursors to the mature 25S rRNA (Fig 4B, lanes 1 and 5); however, this might be due to the fact that there is only a mild growth defect associated with the rsa3-null-mutant strain. Also, the loading of equal counts (20,000 cpm) in all lanes might mask some of the effects of the absence of Rsa3p on the production of the 27S pre-rRNAs.

We conclude that the absence of Rsa3p causes a mild deficit in 60S r-subunit levels and therefore leads to a slight reduction in the levels of mature 25S rRNA.

Rsa3p localizes predominantly to the nucleolus:
To distinguish between a cytoplasmic, nuclear, or nucleolar role for Rsa3p in maintenance of 60S r-subunit levels, we analyzed the subcellular localization of a C-terminally HA-tagged version of Rsa3p that was expressed from the cognate RSA3 promoter (plasmid pHAC33-RSA3). The Rsa3-HAp fusion protein was fully functional since it complemented the deficit in 60S r-subunits of the {Delta}rsa3 mutant (see Fig 3A) and the synthetic lethality of a dbp6-2 {Delta}rsa3 double mutant (data not shown). Immunofluorescence was performed on cells of the rsa3 null strain YMD3-1A transformed with pHAC33-RSA3 and pUN100-GFP-NOP1 (TEIXEIRA et al. 2002 Down). The HA-tagged Rsa3p was detected by anti-HA antibodies, followed by decoration with goat anti-mouse Cy3-conjugated antibodies (Fig 5B). For precise subnuclear localization, the nucleoplasm was visualized by staining the DNA with 4',6-diamidino-2-phenylindole (DAPI; Fig 5C) and the nucleolus was revealed by the green fluorescent protein (GFP)-tagged Nop1p (Fig 5D). Rsa3-HAp localized to a cap-like structure that was mostly excluded from the DAPI-stained area (Fig 5E) but overlapped with the GFP-Nop1p signal (Fig 5F). Only a weak background staining was obtained with anti-HA antibodies on the control strain containing untagged Rsa3p (data not shown). We conclude that Rsa3p is localized predominantly in the nucleolus. This localization is consistent with a direct role of Rsa3p in 60S r-subunit biogenesis as a trans-acting factor.



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  		Figure 5. Rsa3-HAp localizes predominantly to the nucleolus. Indirect immunofluorescence was performed with cells expressing Rsa3-HAp from its cognate promoter (YMD3-1A pHAC33-RSA3 pUN100-GFP-NOP1). (A) Phase-contrast acquisition (Nomarski) of the same field as in B–F. (B) Rsa3-HAp was detected by the monoclonal mouse anti-HA 12CA5 antibody, followed by decoration with a goat anti-mouse Cy3-conjugated antibody. (C) Chromatin DNA was stained with DAPI. (D) GFP-Nop1p. Pseudocolors were assigned to the digitized micrographs (B–D) and images were merged. (E) Colocalization of Rsa3-HAp and chromatin DNA. (F) Colocalization of Rsa3-HAp and GFP-Nop1p.

Synthetic lethality network with dbp6 alleles:
To assess the specificity of the sl interactions observed between dbp6 alleles and the nop8, rpl3, and rsa3 mutants, we tested the synthetic interaction relationship among mutants of genes encoding 60S r-subunit proteins or protein trans-acting factors that have been shown to genetically interact with Dbp6p (see Fig 6). In addition to Dbp6p, Nop8p, Rpl3p, and Rsa3p, these factors included two DEAD-box proteins (Dbp7p and Dbp9p), Rsa1p, and the large r-subunit protein Rpl10p/Qsr1p. Like Dbp6p, Dbp7p and Dbp9p are likely involved in early 60S r-subunit assembly steps since their inactivation leads to reduced steady-state levels and decreased synthesis of the 27S precursors to mature 25S and 5.8S rRNAs (DAUGERON and LINDER 1998 Down; DAUGERON et al. 2001 Down). In addition, the combination of certain dbp6 and dbp9 alleles leads to synthetic lethality and elevated dosage of Dbp9p has been shown to suppress the growth defect of certain dbp6 mutants (DAUGERON et al. 2001 Down). Rsa1p, also identified in the sl screen with conditional dbp6 alleles, is important for efficient export of 60S r-subunits and has been suggested to assist the assembly of the large subunit r-protein Rpl10p/Qsr1p (EISINGER et al. 1997 Down) onto 60S r-subunits in the nucleoplasm (KRESSLER et al. 1999A Down; GADAL et al. 2001B Down). Rpl10p/Qsr1p is the docking site for the nuclear export adaptor Nmd3p, which contains a nuclear export sequence and mediates export of 60S r-subunits (HO et al. 2000 Down; GADAL et al. 2001B Down). As a specificity control, we selected the DEAD-box protein Spb4p since we had previously shown that there is no synthetic interaction between the spb4-1 mutant and the dbp6 alleles, the dbp7 null, or the rsa1 null allele (DAUGERON and LINDER 1998 Down; KRESSLER et al. 1999A Down). Spb4p is most likely involved in late pre-60S maturation steps since its inactivation entails an accumulation of the 27SB pre-rRNAs (DE LA CRUZ et al. 1998 Down). As depicted in Fig 6, there is a clear network of synthetic interactions between mutant alleles of dbp6, dbp7, dbp9, nop8, and rpl3. In addition, certain combinations of the above mutant alleles with the rsa1 null allele show synthetic lethality or enhancement (see Fig 6). In contrast, any combination of the tested mutants with the spb4-1 allele does not lead to a synthetic interaction phenotype. This clearly demonstrates that the genetic interactions described above are specific and not due solely to an implication of the analyzed factors in the same cellular process, that is, 60S r-subunit biogenesis. Interestingly, this extensive genetic analysis also revealed that the {Delta}rsa3 mutant displays some specificity by being synthetically lethal only with dbp6 alleles (see also Fig 2B) and by showing some mild synthetic enhancement with the nop8-101 and the rsa1 null mutant (data not shown). Importantly, the {Delta}rsa3 null mutant, which displays only a minor growth phenotype, was already synthetically lethal with very mild dbp6 alleles (see Fig 2A and Fig B; data not shown). The specific genetic interaction between Dbp6p and Rsa3p strongly suggests that these two proteins functionally interact during assembly of 60S r-subunits.



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  		Figure 6. Synthetic enhancement interaction network of protein trans-acting factor and ribosomal protein encoding genes involved in the biogenesis of 60S ribosomal subunits. Solid arrows represent synthetic lethal interactions and dashed arrows represent synthetic enhancement. Putative RNA helicases are inside shaded ovals. Note that the synthetic enhancement phenotypes of the {Delta}dbp7 rpl10, dbp9 rpl10, dbp9 spb4-1, nop8-101 rpl10, nop8-101 spb4-1, and rpl3 spb4-1 double mutants have not been analyzed. The phenotypes of the dbp6 {Delta}dbp7, dbp6 dbp9, dbp6 rpl10, dbp6 {Delta}rsa1, dbp6 spb4-1, {Delta}dbp7 spb4-1, rpl10 spb4-1, and {Delta}rsa1 spb4-1 double mutants have been previously described (DAUGERON and LINDER 1998 Down; KRESSLER et al. 1999A Down; DAUGERON et al. 2001 Down). See also RESULTS and MATERIALS AND METHODS for details.

Dbp6p and Rsa3p are associated with preribosomal particles:
To address whether the genetic interaction between Dbp6p and Rsa3p may represent a functional interaction within pre-60S ribosomal particles, we first investigated the migration of Dbp6p and Rsa3p in sucrose gradients under polysome runoff and low Mg2+ concentration conditions that are normally used for the analysis of r-subunits. To this end, strain JDY139, which coexpresses protA-Dbp6p and Rsa3-HAp from centromeric plasmids, was constructed (see Table 1). Both the Rsa3-HAp (see above) and the protA-Dbp6p fusion protein were fully functional, and the latter complemented the lethality of the dbp6 null mutant to the wild-type extent (data not shown). Under the above-mentioned experimental conditions, the majority of protein-A-tagged Dbp6p and HA-tagged Rsa3p were present in large complexes. These complexes had a sedimentation behavior similar to that of 60S r-subunits (Fig 7), as evidenced by the absorption at A254 and the migration of the large subunit r-protein Rpl3p (see Fig 7, bottom). Moreover, a substantial amount of both protA-Dbp6p and Rsa3-HAp sedimented faster, which is most likely due to an association with preribosomal particles since the 27SA, 27SB, and, to a lesser extent, the 35S pre-rRNAs were found in these regions of the gradients. Taking into account that both Dbp6p and Rsa3p are mainly nucleolar proteins, these data are consistent with the direct association of both Dbp6p and Rsa3p with nucleolar preribosomal particles to mature 60S r-subunits.



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  		Figure 7. Dbp6p and Rsa3p are associated with pre-60S ribosomal particles. Strain JDY139 pRS415-protA-DBP6-T7 pHAC33-RSA3 was grown at 30° up to an OD600 of ~0.8. Cell extracts were prepared in a buffer containing 50 mM NaCl, 50 mM Tris-HCl pH 7.4, and 1 mM dithiothreitol. Then, 10 A254 units of cell extract were resolved in 7–50% sucrose gradients containing a low concentration of Mg2+ to dissociate ribosomes into subunits and fractions of 500 µl were collected. Sedimentation is from left to right and numbers (1–16) indicate the fractions. Proteins were extracted from each fraction (200 µl) and equal volumes were resolved on a 12% SDS-PAGE and subjected to Western blotting using polyclonal rabbit anti-protein A antibodies to detect protA-Dbp6p (top), monoclonal mouse 16B12 antibodies to detect Rsa3-HAp (middle), and monoclonal mouse anti-Rpl3p antibodies to detect Rpl3p (bottom). The gradient position of the 40S and 60S ribosomal subunits was determined from the A254 profile by continuous monitoring. To analyze the gradient position of pre-rRNAs, fractions (250 µl) were processed and subjected to Northern blot analysis with probes revealing the 20S, 27SA/B, and 35S pre-rRNAs. As a control, a total extract (T) was also subjected to Western and Northern blot analyses.

To assess whether Dbp6p and Rsa3p are present within the same cellular complexes, we also subjected extracts obtained from cells of strain JDY139 to immunoprecipitation with IgG Sepharose beads at the ionic strength of 50 mM NaCl. Then, equivalent aliquots of total cell extracts, unbound fractions, and immunoprecipitates were subjected to Western blotting. As shown in Fig 8, these analyses revealed that Rsa3-HAp was efficiently co-immunoprecipitated with protA-Dbp6p. Rsa3-HAp was not detected in immunoprecipitates of extracts from control cells (strain YMD3-1A pHAC33-RSA3) that coexpress Rsa3-HAp and untagged wild-type Dbp6p. These results indicate that Rsa3p interacts in vivo with Dbp6p-containing complexes. However, Rsa3-HAp could not be co-immunoprecipitated with protA-Dbp6p when we increased the salt concentration in the wash buffer from 50 to 500 mM NaCl (data not shown); therefore, the association of Rsa3p with Dbp6p-containing complexes or the Dbp6p-containing complexes themselves seem not to be very stable. Indeed, when we explored further the immunoprecipitates of the protA-Dbp6p strain, we were unable to specifically coprecipitate Rpl3p or any pre-rRNA, despite efficient protA-Dbp6p precipitation (data not shown).



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  		Figure 8. Dbp6p and Rsa3p are associated with each other. Strains JDY139 pRS415-protA-DBP6-T7 pHAC33-RSA3 and YMD3-1A pHAC33-RSA3 were grown to an OD600 of ~1 in SD-Ura medium at 30°. Cell extracts were prepared in a buffer containing 50 mM Tris-HCl pH 7.5, 50 mM NaCl, 2 mM MgCl2, 1 mM 2-mercaptoethanol, and 0.1% NP-40 and subjected to immunoprecipitation with IgG Sepharose beads. Total extracts (T), unbound proteins (U), and bound proteins (B) were resolved on a 12% SDS-PAGE and subjected to Western blotting using polyclonal rabbit anti-protein A antibodies to detect protA-Dbp6p (top) and monoclonal mouse 16B12 antibodies to detect Rsa3-HAp (bottom).

Altogether, these results suggest that a substantial amount of Dbp6p and Rsa3p is associated with the same cellular complexes, which most likely correspond to nucleolar preribosomal particles to mature 60S r-subunits. However, Dbp6p might be loosely bound to these preribosomal particles and therefore dissociated from them following purification by immunoprecipitation.


*  DISCUSSION
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
 *RESULTS
 *DISCUSSION
*LITERATURE CITED

In this study, we have further dissected the functional environment of the putative ATP-dependent RNA helicase Dbp6p by cloning the complementing genes of four previously isolated mutants that were synthetically lethal with dbp6 alleles (KRESSLER et al. 1999A Down). These genes encode the large subunit r-protein Rpl3p (PLANTA and MAGER 1998 Down), the protein trans-acting factor Nop8p, which is involved in 60S r-subunit biogenesis (ZANCHIN and GOLDFARB 1999 Down), and the so-far-uncharacterized YLR221Cp protein, which we named Rsa3p. Several lines of evidence indicate that the nonessential Rsa3p is required for efficient biogenesis of 60S r-subunits:

  1. The absence of Rsa3p leads to a slight deficit in 60S r-subunits (Fig 3).

  2. The {Delta}rsa3 null mutant interacts synthetically with selected factors involved in the biogenesis of 60S r-subunits (Dbp6p, Nop8p, and Rsa1p).

  3. Rsa3-HAp localizes predominantly to the nucleolus (Fig 5).

  4. Rsa3-HAp is likely associated with preribosomal complexes since a majority of the cellular Rsa3-HAp sediments below the cytoplasmic 60S r-subunits in sucrose gradients (Fig 7). Consistent with these data, Rsa3p has been found in a Noc1p-containing complex (GAVIN et al. 2002 Down; MILKEREIT et al. 2003 Down), which represents either a late 90S or an early 66S preribosomal particle.

  5. Finally, RSA3 shows an expression profile similar to many ribosome synthesis genes at the mRNA level (GASCH et al. 2000 Down).

Our initial phenotypic analysis of Dbp6p suggested that the predominantly nucleolar Dbp6p is required for early assembly steps within pre-60S ribosomal particles (KRESSLER et al. 1998 Down). Here we show that a substantial amount of protA-tagged Dbp6p sediments faster than cytoplasmic 60S r-subunits (Fig 7), which likely represents an association with early pre-60S ribosomal particles. The observed synthetic lethality between dbp6 alleles and dbp9, nop8, and rpl3 mutants also supports a function of Dbp6p during early pre-60S r-subunit assembly reactions. Dbp9p is found in the so-called early E1 66S preribosomal particle and its genetic depletion leads to reduced formation and steady-state levels of all 27S species, especially the 27SB pre-rRNAs (DAUGERON et al. 2001 Down; FATICA and TOLLERVEY 2002 Down; GAVIN et al. 2002 Down). Nop8p is a mainly nucleolar protein whose genetic depletion leads to a reduced synthesis and/or stability of 27S pre-rRNAs (ZANCHIN and GOLDFARB 1999 Down), and Rpl3p belongs to a group of large subunit r-proteins that associate early with preribosomal particles (KRUISWIJK et al. 1978 Down). The fact that there is an intimate sl network among dbp6, dbp9, nop8, and rpl3 alleles (note that all combinations of double mutants are sl; see Fig 2 and Fig 6) opens up the possibility that all the corresponding proteins operate in the same environment within early pre-60S ribosomal particles. In agreement with this model, we have previously revealed a physical interaction between Dbp6p and Dbp9p by using the yeast two-hybrid system (DAUGERON et al. 2001 Down).

Importantly, the rsa3 null mutant displays some specificity by being synthetically lethal only with dbp6 alleles (Fig 2 and Fig 6). In addition to this specific genetic interaction, the similar sedimentation pattern of Dbp6p and Rsa3p in sucrose gradients (Fig 7), as well as the relatively efficient co-immunoprecipitation of Rsa3-HAp with protA-Dbp6p (Fig 8), strongly suggest that the two proteins functionally interact within the same 66S preribosomal particles. Unfortunately, attempts to explore these preribosomal particles by co-immunoprecipitation with protA-Dbp6p failed, suggesting that Dbp6p might only loosely and transiently interact with preribosomal particles and dissociate during immunoprecipitation. We also found that HA-Dbp6p is not mislocalized in rsa3-null-mutant cells and overexpression of Dbp6p from a multicopy plasmid neither restores the underaccumulation of 60S r-subunits associated with the rsa3 null mutation nor exacerbates the slow-growth phenotype of the rsa3 null mutant (data not shown). Moreover, overexpression of Rsa3p does not suppress the growth defect of the dbp6-2 or the dbp6-4 allele (data not shown). A classical model to explain the synthetic lethality between dbp6 alleles and the {Delta}rsa3 null mutant would be the synergistic destabilization of the Dbp6p- and Rsa3p-containing preribosomal particle or subcomplex when both factors are mutated. However, we prefer to propose a model in which Rsa3p acts as a specific cofactor of Dbp6p that either stimulates the enzymatic activity of Dbp6p or facilitates its substrate recognition. Even though we have so far not gathered any evidence for a direct physical interaction between Dbp6p and Rsa3p, it is intriguing that regions in both Dbp6p [aa 288–309; COILS program (LUPAS et al. 1991 Down)] and Rsa3p (aa 129–150; see RESULTS) may adopt coiled-coil conformations, which could serve as heterodimerization surfaces. Moreover, the potential coiled-coil region of Rsa3p is likely to be important for its function since the rsa3-1 mutation, which leads to a frameshift after aa 110, confers the same phenotypes as the rsa3 null mutation confers. The rsa3 null mutant also mildly exacerbates the slow-growth phenotype of the nop8-101 and the rsa1 null allele (Fig 6 and data not shown). The fact that Rsa3p functionally interacts with Dbp6p, together with the above-mentioned possibility that both Dbp6p and Nop8p might work in the same precise environment within early 60S preribosomal particles, could fully explain the genetic interaction between the rsa3 null and the nop8-101 allele. Previous results have shown that the rsa1 null allele is sl with dbp6 alleles (KRESSLER et al. 1999A Down). At present, we cannot provide a simple explanation for the sl interaction between dbp6 alleles and the rsa1 null mutant; we can speculate only that the absence of nucleoplasmic Rsa1p leads to a synergistic destabilization of late pre-60S r-subunits that are qualitatively altered in dbp6 mutant strains (KRESSLER et al. 1999A Down). The fact that the rsa3 null mutant also genetically interacts with the rsa1 null allele reinforces the model in which Rsa3p and Dbp6p work together in the same complex. In this sense, it is worthwhile to mention that Rsa3p seems not to be required for export of pre-60S r-subunits since, and in contrast to the rsa1 null mutant, there is no nuclear accumulation of the Rpl25p-eGFP large subunit reporter (GADAL et al. 2001B Down) in rsa3-null-mutant cells at either 30° or 37° (data not shown).

Intriguingly, there are no readily discernible homologs of Dbp6p and not even potential homologs of Nop8p, Rsa1p, and Rsa3p in higher eukaryotes. At least and unlike Rsa1p, Dbp6p (CaDBP6; 38% identity and 58% similarity) and Rsa3p (IPF3878; 33% identity and 50% similarity) have likely homologs in C. albicans whereas Nop8p has weakly conserved homologs in Schizosaccharomyces pombe (accession NP_595780; 20% identity and 40% similarity) and C. albicans (IPF16935; 24% identity and 39% similarity). Therefore, it seems that certain aspects of eukaryotic ribosome biogenesis are yeast specific or even restricted to S. cerevisiae. An appealing model that may explain why Dbp6p and its functionally interacting partners Rsa1p, Rsa3p, and Nop8p are not evolutionarily conserved is based on the finding that increased dosage of Dbp9p, which has clear homologs in higher eukaryotes (ZIRWES et al. 2000 Down), suppresses the sg phenotype of dbp6 alleles (DAUGERON et al. 2001 Down). Accordingly, the Dbp9p homologs may have taken over the yeast-specific function of Dbp6p in higher eukaryotes. The sl screen with dbp6 alleles also identified the evolutionarily conserved large subunit r-protein Rpl3p. Given that both the rpl3-101 and the rpl3-102 mutation conferred synthetic lethality to dbp6, dbp9, and nop8 alleles but not to the slow-growing dbp7 null allele (see Fig 6), we conclude that there is some specificity associated with the functional interaction between the r-protein Rpl3p and the protein trans-acting factors Dbp6p, Dbp9p, and Nop8p. Both the rpl3-101 and rpl3-102 mutants strongly underaccumulate cytoplasmic 60S r-subunits (Fig 1B and data not shown), indicating that these mutations affect proper assembly of 60S r-subunits. Importantly, this assembly defect is not due to instability of the mutant Rpl3 proteins, since both Rpl3-101p (Q371H) and Rpl3-102p (K30E) are expressed at the same level as wild-type Rpl3p (D. KRESSLER, unpublished results). Simple interpretations of the sl interaction between the rpl3-101 and rpl3-102 mutants and the dbp6, dbp9, and nop8 alleles are either that these protein trans-acting factors help the efficient recruitment of Rpl3p or that proper assembly of Rpl3p is a prerequisite for Dbp6p, Dbp9p, and Nop8p to function. It has been proposed that the protein trans-acting factor Rrb1p functions as the assembler of Rpl3p onto early 66S preribosomal particles (IOUK et al. 2001 Down; SCHAPER et al. 2001 Down). In agreement with the above-mentioned interpretations, the rrb1-TAP allele, which displays a sg and temperature-sensitive phenotype (SCHAPER et al. 2001 Down), is synthetically lethal with the rpl3-102 and dbp6-4 alleles and synthetically enhances the growth defect of the dbp6-2 and the rsa3 null strain (data not shown).

We have started to explore the functional environment of putative RNA helicases of the DEAD-box protein family involved in ribosome synthesis by genetic means. Since not all protein trans-acting factors, as, for example, Dbp6p and Nop8p, that have been implicated in ribosome assembly have been found in the so-far-identified preribosomal particles (GAVIN et al. 2002 Down; NISSAN et al. 2002 Down; FROMONT-RACINE et al. 2003 Down; MILKEREIT et al. 2003 Down), sl screens, among other different genetic tools, represent a necessary complementary strategy not only to identify all protein trans-acting factors involved in ribosome biogenesis, but also to reveal functional interaction networks between these factors. For instance, this study clearly supports the idea that at least Dbp6p, Dbp9p, Nop8p, Rpl3p, and Rsa3p all functionally interact. Future studies that combine proteomic and genetic approaches are required to address the exciting issues of identifying the precise substrates and the timing of action of the energy-consuming enzymes, such as putative RNA helicases, involved in ribosome biogenesis.


*  FOOTNOTES

1 These authors contributed equally to this work. Back
3 Present address: Biochemie-Zentrum Heidelberg, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany. Back


*  ACKNOWLEDGMENTS

We thank V. Doye for the kind gift of pUN100-GFP-NOP1 and O. Gadal for the Rpl25p-eGFP construct. We are grateful to M. Doère and R. Bisig for excellent technical assistance. This work was funded by a grant from the Spanish Ministry of Science and Technology (BMC2001-2660) to J.d.l.C. and by a grant from the Swiss National Science Foundation to P.L. P.L. and J.d.l.C. gratefully acknowledge the Societé Académique de Genève for support. J.d.l.C. also acknowledges the Andalusian Regional Government for support (CVI271).

Manuscript received July 21, 2003; Accepted for publication January 2, 2004.


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