Genes Encoding Ribosomal Proteins Rps0A/B of Saccharomyces cerevisiae Interact With TOM1 Mutants Defective in Ribosome Synthesis

Corresponding author: Steven R. Ellis, Department of Biochemistry and Molecular Biology, University of Louisville, Louisville, KY 40292., srellis{at}louisville.edu (E-mail)

Communicating editor: M. JOHNSTON

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The Saccharomyces cerevisiae RPS0A/B genes encode proteins of the 40S ribosomal subunit that are required for the maturation of 18S rRNA. We show here that the RPS0 genes interact genetically with TOM1. TOM1 encodes a member of the hect-domain-containing E3 ubiquitin-protein ligase family that is required for growth at elevated temperatures. Mutant alleles of the RPS0 and TOM1 genes have synergistic effects on cell growth at temperatures permissive for TOM1 mutants. Moreover, the growth arrest of TOM1 mutants at elevated temperatures is partially suppressed by overexpression of RPS0A/B. Strains with mutant alleles of TOM1 are defective in multiple steps in rRNA processing, and interactions between RPS0A/B and TOM1 stem, in part, from their roles in the maturation of ribosomal subunits. Ribosome synthesis is therefore included among the cellular processes governed by members of the hect-domain-containing E3 ubiquitin-protein ligase family.

RPS0A and RPS0B are duplicated genes of Saccharomyces cerevisiae that encode protein components of the 40S ribosomal subunit (DEMIANOVA et al. 1996 ). Deletion of either RPS0 gene reduces growth rate, and deletion of both genes is lethal. Recent studies have shown that the Rps0 proteins are required for processing the 20S rRNA precursor to mature 18S rRNA, a late event in the maturation of 40S subunits (FORD et al. 1999 ).

The Rps0 proteins have >60% sequence identity with the human p40/37-kD laminin-binding protein (37-LBP). The p40/37-LBP gene is overexpressed in a wide range of human tumors (MAFUNE et al. 1990 ; D'ERRICO et al. 1991 ; CASTRONOVO 1993 ; VACCA et al. 1993 ; PEI et al. 1996 ; HALATSCH et al. 1997 ; DAHERON et al. 1998 ). The link between the overexpression of p40/37-LBP and tumorigenesis is frequently interpreted in terms of the putative role for p40/37-LBP as a precursor for the 67-kD high affinity laminin receptor (LIOTTA 1986 ; CASTRONOVO 1993 ). This relationship is controversial, however, and it is now known that, like the Rps0 proteins, p40/37-LBP is a component of the 40S ribosomal subunit (ARDINI et al. 1998 ). It is therefore important to determine if changes in expression of members of this family can influence cell growth and proliferation in their roles as components of the translational machinery.

In an effort to understand how the level of expression of the RPS0 genes could impact other genes and influence yeast cell growth, we undertook a synthetic lethal screen to identify genes that are critically sensitive to the level of Rps0 protein. A gene identified in this screen was TOM1, which encodes a 3268-amino-acid protein containing a hect (homologous to E6-AP C terminus) domain. Hect domains are characteristic of a family of E3 ubiquitin-protein ligases that include the human E6-AP protein and the yeast Rsp5 (HUIBREGTSE et al. 1995 ). Some of these proteins, including Tom1, interact with transcription coactivator complexes (SALEH et al. 1998 ). Tom1 is required for full induction of the general stress and heat shock responses and is also required for efficient mRNA export (UTSUGI et al. 1999 ; DUNCAN et al. 2000 ; SASAKI et al. 2000 ). Cells that have the hect domain of TOM1 deleted display temperature sensitivity and arrest growth at the G2/M transition of the cell cycle (UTSUGI et al. 1999 ). In addition to the synthetic lethal interaction between RPS0A/B and TOM1, growth arrest in TOM1 mutants can be partially suppressed by overexpression of RPS0 genes. This observation is particularly intriguing in light of the link between overexpression of p40/37-LBP proteins and cancer.

Here, we show that mutant alleles of TOM1 affect the steady-state level of both large and small ribosomal subunits. Analysis of rRNA processing in TOM1 mutants revealed that the Tom1 protein influences the maturation of 18S rRNA by affecting several early steps in the rRNA processing pathway. Therefore, the synthetic interaction between TOM1 and RPS0 mutants resides, in part, on a combined effect on the level of 40S subunits. TOM1 mutants also affect the level of 60S subunits, indicating that the Tom1 protein has one or more additional roles in maturation/stability of ribosomal subunits. These data add ribosome synthesis to the growing list of processes governed by members of the hect-domain-containing E3 ubiquitin ligase family. Moreover, these data show that overexpression of a member of the p40/37-LBP family of proteins can influence cell cycle progression in certain genetic backgrounds.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Yeast and bacterial strains:
The yeast strains used in this work are listed in Table 1. Media used in cultivating yeast were YPD (1% w/v yeast extract, 2% w/v peptone, and 2% w/v glucose) and synthetic (0.67% w/v yeast nitrogen base without amino acids and 2% w/v glucose). Where appropriate, nutrients were added to synthetic media in amounts specified by SHERMAN 1991 . Diploids were sporulated on solid sporulation media (1% w/v potassium acetate, 0.1% w/v yeast extract, 0.05% w/v glucose, and 2% w/v agar). Where appropriate, nutrients were added to sporulation media in 25% of the amounts used in synthetic media. The Escherichia coli strain used in this study was XL1-Blue (Stratagene, La Jolla, CA).

DNA construction and sectoring assay:
The RPS0A gene was inserted into the plasmid pTSV30A for use in a synthetic lethal screen for genes critically sensitive to RPS0 gene dosage. The plasmid pTSV30A contains the selectable marker LEU2 and a wild-type ADE3 gene for use in a color sectoring assay (BENDER and PRINGLE 1991 ). The RPS0A gene was excised from plasmid pJM9 as a 2.7-kb BamHI/SalI fragment (DEMIANOVA et al. 1996 ) and inserted into the unique BamHI and SalI sites of plasmid pTSV30A. This plasmid was transformed into the strain WYY5-2, which contains a disrupted allele of RPS0B and mutant alleles of ade2 and ade3. The WYY5-2 strain was created by mating Y505D with 8-3dy and selecting for slow-growing white colonies. Disruption of the RPS0B gene was confirmed by Northern analysis (data not shown). Transformation of WYY5-2 with plasmids containing wild-type ADE3 resulted in red colonies because of a pigment that accumulated in the ade2 mutants. Cells that lost the ADE3-containing plasmid did not accumulate the red pigment since the ADE3 gene product functions earlier in the adenine biosynthetic pathway than ADE2. Consequently, these cells were white.

WYY5-2 cells containing pTSV30A(RPS0A) were mutagenized with ethyl methanesulfonate to 10% survival and then plated on nonselective (YPD) media at 37°. Cells that lost the pTSV30A(RPS0A) plasmid formed red colonies with white sectors. Cells that did not sector and remained red could have acquired a mutation that made the plasmid-borne RPS0A gene essential. Other mechanisms could also give a nonsectoring phenotype such as integration of the plasmid into the chromosome or gene conversion between the wild-type ADE3 on the plasmid with the mutant ade3 locus (BENDER and PRINGLE 1991 ). However, these latter mechanisms would not be expected to give a sectoring phenotype that was temperature sensitive. Therefore, only cells that did not sector at 37° but did sector at 30° were studied further. Two independent strains were characterized that exhibited a temperature-sensitive sectoring phenotype. Each of these strains exhibited temperature-sensitive growth in the absence of the pTSV30A(RPS0A) plasmid, which could be complemented by RPS0A or RPS0B carried on plasmids other than pTSV30A. These strains were labeled DOR for dependent on RPS0.

Genetic procedures:
DOR1-111 and DOR1-113 were crossed to the 8-3dy strain, which has a disrupted allele of RPS0B. The resulting diploids were tested for temperature-sensitive growth to determine if the loci responsible for the synthetic interactions with the RPS0B deletion were recessive. Both loci were shown to be recessive (data not shown). Crosses were also made to determine if the loci involved in the synthetic interactions with the RPS0B deletion formed a single complementation group. Since DOR1-111 and DOR1-113 are the same mating type, DOR1-111 was outcrossed with 8-3dy to obtain a strain of opposite mating type that exhibited the synthetic interaction with disrupted RPS0B. A haploid strain from this cross, DOR1-111C, was crossed to DOR1-113 and the resulting diploids were tested for temperature-sensitive growth. The diploids were temperature sensitive, demonstrating that loci involved in the synthetic interactions with the RPS0B deletion formed a single complementation group (data not shown).

The synthetic interactions with the RPS0B deletion in the DOR strains could arise from mutations in the chromosomal RPS0A gene. To determine if the synthetic loci were alleles of RSP0A, they were mapped relative to ADE3, which is within 10 kb of RPS0A on chromosome VII. Mapping the synthetic locus relative to ADE3 was carried out by linkage analysis of progeny from the cross of 8-3dy, which is wild type for ADE3, with DOR1-111, which has a mutant allele of ade3. By determining the color of the cells exhibiting the synthetic temperature-sensitive interaction it was possible to show that the locus exhibiting the synthetic interaction was unlinked to ADE3 and thereby to RPS0A (data not shown).

Polysome analysis:
Polysomes were prepared and fractionated on 7–47% sucrose gradients as described by BAIM et al. 1985 . Centrifugation was generally carried out at 21,000 rpm for 15 hr in an SW28.1 rotor (Beckman Instruments, Fullerton, CA). Sucrose gradients were fractionated and absorbance at 254 nm was monitored using an ISCO model 185 gradient fractionator and a UA-5 absorbance detector. Data were collected directly using Systems Gold software (Beckman Instruments) or digitized using UN-SCAN-IT software (Silk Scientific, Orem, UT) and analyzed using Slide Write Plus.

Northern analysis:
Total RNA was prepared from yeast cells by the hot phenol method (SCHMITT et al. 1990 ). RNA was fractionated on 1.5% formaldehyde-agarose gels and transferred to Zeta-probe (Bio-Rad, Hercules, CA) membrane. Membranes were hybridized with the following oligonucleotides: (1) 5'-CCAGATAACTATCTTAAAAG-3'; (2) 5'-GAAATCTCTCACCGTTTGGAATAGC-3'; (3) 5'-ATGAAAACTCCACAGTG-3'; (4) 5'-CCAGTTACGAAAATTCTTG-3'; and (5) 5'-GGCCAGCAATTTCAAGTTA-3'. The regions of the rRNA repeat that hybridize with these oligonucleotides are shown in Fig 6. Membranes were also hybridized with an oligonucleotide complementary to U3 snoRNA, 5'-GGATTGCGGACCAAGCTAA-3'. Oligonucleotide probes were labeled at their 5' ends with [-³²P]ATP (New England Nuclear, Boston) using T4 polynucleotide kinase (New England Biolabs, Beverly, MA). Membranes were subjected to phosphorimage analysis (Phosphoimager SF; Molecular Dynamics, Sunnyvale, CA) and exposed to X-ray film.

View larger version (60K): In this window In a new window Download PPT slide   Figure 1. (A) Plasmid-borne TOM1 complements growth arrest of DOR1-111 and DOR1-113 at 37°. DOR1-111 and DOR1-113 cells were transformed with either pRS316 or pRS316 containing TOM1. The pRS316(TOM1) plasmid was described previously (UTSUGI et al. 1999 ). Exponentially growing cultures were diluted to an OD600nm of 0.1, and 3 µl of 10-fold serial dilutions were spotted onto selective plates and incubated at 37°. Strains used were as follows: lane 1, DOR1-111/pRS316; lane 2, DOR1-111/pRS316(TOM1); lane 3, DOR1-113/pRS316; lane 4, DOR1-113/pRS316(TOM1). (B) The tom1-2 allele interacts synthetically with rps0A::URA3. W8-3dx and RAY3 strains were crossed and the resulting diploids were sporulated and tetrads dissected. The colonies shown are meiotic progeny from a representative tetrad grown on YPD media at 30°.

View larger version (77K): In this window In a new window Download PPT slide   Figure 2. RPS0A is a multicopy suppressor of the tom1-111- dependent growth arrest. The DOR1-111 strain was outcrossed to W303-1B to create strain 1W29. 1W29 contains the tom1-111 allele in a wild-type RPS0A/B background. (A) Photographs of 1W29 cells transformed with pTSV30A (top) or pTSV30A(RPS0A) (bottom) grown at 35°. Cells were stained with 0.25% trypan blue. (B) Exponentially growing 1W29 cells transformed with either pTSV30a or pTSV30a(RPS0A) were diluted to an OD600nm of 0.1, spotted in 1:10 serial dilutions onto selective media, and incubated at the given temperature.

View larger version (20K): In this window In a new window Download PPT slide   Figure 3. Polysome profiles from strains with different combinations of wild-type and mutant alleles of RPS0B and TOM1. Cells were grown at the permissive temperature (30°) and shifted to the nonpermissive temperature (37°). Seven hours after the temperature shift, cells were harvested, and extracts were prepared and fractionated by sucrose gradient centrifugation as described by BAIM et al. 1985 . Gradients were fractionated and the absorbance at 254 nm was monitored using an ISCO model 185 density gradient fractionator and UA-5 absorbance detector. Relevant alleles in the strains are as follows: (A) - - -, TOM1/RPS0B and —, TOM1/rps0B::HIS3; (B) - - -, TOM1/RPS0B and —, tom1-113/RPS0B; (C) - - -, TOM1/RPS0B and —, tom1-113/rps0B::HIS3.

View larger version (18K): In this window In a new window Download PPT slide   Figure 4. Polysome profiles from strains harboring the tom1-2 allele. The RAY5 strain was transformed with either pRS316 or pRS316 containing TOM1. Cells were grown at 30° and then either shifted to 37° or maintained at 30°. After 6 hr, cells were harvested and extracts prepared and fractionated as described in Fig 3. A and B contain polysome profiles from RAY5/pRS316 and RAY5/pRS316(TOM1) cells, respectively, after 6 hr at 30° (- - -) or 37° (—). The upward arrow shows half-mer polysomes.

View larger version (56K): In this window In a new window Download PPT slide   Figure 5. Northern blot analysis of pre-rRNA processing in wild-type and mutant TOM1 strains. RAY5 strains described in Fig 4 were grown at 30° and shifted to 37° or maintained at 30° for 6 hr. After 6 hr, cells were harvested and total RNA was prepared as described by SCHMITT et al. 1990 . RNA was fractionated by electrophoresis on a 1.5% agarose-formaldehyde gel, transferred to Zeta-probe (Bio-Rad), and hybridized with oligonucleotides shown in Fig 6A or to U3 snoRNA. (A) oligonucleotide 2; (B) oligonucleotide 3; (C) oligonucleotide 4; (D) oligonucleotide 1; (E) oligonucleotide 5; (F) oligonucleotide to U3 snoRNA. The strong signal at the bottom of C is from cross-hybridization of oligonucleotide 4 with 18S rRNA. Relevant TOM1 alleles and growth temperatures of cells used for RNA isolation are as follows: lane 1, tom1-2 (30°); lane 2, TOM1 (30°); lane 3, tom1-2 (37°); lane 4, TOM1 (37°).

View larger version (26K): In this window In a new window Download PPT slide   Figure 6. The pre-rRNA processing pathway in S. cerevisiae. (A) Structure of the 35S rRNA precursor. Mature rRNA species are represented by boxes. External and internal transcribed sequences are shown as thin lines. Numbered letters indicate processing sites and lines numbered under the 35S precursor represent oligonucleotide probes defined in MATERIALS AND METHODS. (B) The major rRNA processing pathway in yeast. Horizontal arrows represent exonucleolytic cleavages; other cleavage steps are endonucleolytic. (C) Alternative rRNA processing pathway in tom1-2 mutants at nonpermissive temperatures. In this pathway the 35S rRNA precursor is either cleaved directly at the A3 site to give the 23S dead-end intermediate or cleaved at A0/A1 and A3 sites to generate the 21S rRNA species. Only early steps in the rRNA pathway are shown.

Pulse-chase labeling of pre-rRNA:
Pulse-chase labeling of pre-rRNA was carried out as described in FORD et al. 1999 . RAY5 cells transformed with pRS316 or pRS316(TOM1) were grown at 30° in synthetic media lacking uracil. Cells were split into equal amounts with one fraction maintained at 30° and the other shifted to 37°. After the temperature shift, cells were grown for 6 hr to an OD_600nm of 0.5. Forty milliliters of each culture was concentrated to a total of 1 ml in synthetic media lacking uracil and methionine. Each culture was pulse-labeled for 1 min with 250 µCi [methyl-³H]methionine. Labeled cells were split in three aliquots of 250 µl each. Each aliquot was diluted into synthetic media containing 1 mg/ml methionine and chased for 0, 5, and 15 min after which time total RNA was isolated. Total RNA was fractionated on 1.5% agarose-formaldehyde gels and transferred to Zeta-probe membrane. Membranes were baked for 2 hr at 80° and exposed to Biomax MS film at -70° using a Biomax LE intensifying screen (Eastman Kodak Company, Rochester, NY).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Mutant alleles of TOM1 interact synthetically with RPS0 null alleles:
We undertook a synthetic lethal screen to identify mutants critically sensitive to RPS0 gene dosage in an effort to gain a better understanding of the role of the Rps0 proteins in yeast cell growth. An ade2 ade3 strain with an intact copy of RPS0A and a deleted allele of RPS0B was transformed with a multicopy plasmid containing wild-type RPS0A and ADE3. Cells containing the plasmid were red, whereas those that lost the plasmid were white because of the interplay between mutant and wild-type alleles of the ADE2 and ADE3 genes (BENDER and PRINGLE 1991 ). Cells were mutagenized with ethyl methanesulfonate and colonies that did not sector at 37° were isolated. Three independent colonies were identified that could sector at 30° but not at 37°, indicating that they might harbor temperature-sensitive alleles of a gene or genes sensitive to RPS0 gene dosage. Two mutants formed a single complementation group that was analyzed further. Both alleles were recessive to wild type (data not shown), and one mutant had a pronounced growth defect at 30°, indicating that the two strains arose independently.

To identify the gene that exhibited a synthetic interaction with an RPS0B deletion, the two mutants were transformed with either low or high copy number yeast genomic libraries. Surprisingly, only RPS0A and RPS0B could support growth arrest of the mutants at nonpermissive temperatures. However, genetic mapping studies showed that the gene responsible for the synthetic interaction with RPS0B was unlinked to RPS0A (data not shown). This gene apparently cannot be readily recovered from available libraries.

Other studies (see below) had revealed a genetic link between the RPS0 genes and TOM1, a gene encoding a 3268-amino-acid protein containing a hect domain. TOM1 was previously shown to interact with ADA/Spt3-Ada-Gen5-acetyltransferase (SAGA) transcriptional coactivator complexes, and to be required for progression through the G2/M transition of the cell cycle at elevated temperatures (SALEH et al. 1998 ; UTSUGI et al. 1999 ). Fig 1A shows that plasmid-borne TOM1 can complement the growth defect of our mutants at 37°. A mutation, tom1-2, that deletes the hect domain Tom1 (UTSUGI et al. 1999 ) fails to complement our mutants (data not shown). To confirm that TOM1 was responsible for the synthetic interaction, we dissected tetrads from diploids containing tom1-2 and rps0::URA3 alleles. Spores corresponding to the double mutants germinate but divide very slowly at 30° and exhibit a number of morphological abnormalities (Fig 1B). Similar results were obtained when the tom1-2 allele was combined with the RPS0B disruption (data not shown), indicating that null mutations in either RPS0 gene exhibit synthetic interactions with TOM1 mutants.

Overexpression of RPS0 genes partially suppresses growth arrest in TOM1 mutants:
Strains containing the tom1-111 allele alone are osmosensitive and arrest growth with a single bud at nonpermissive temperatures (Fig 2A, top). This phenotype, which differs from the synthetic interaction described previously, is suppressed by multicopy RPS0A (Fig 2B) and RPS0B (data not shown). The suppression of phenotypes linked to the tom1-111 allele by multiple copies of the RPS0 genes was incomplete, since suppression was more efficient at 35° than 37°, and even at 35° suppressed cells grew slowly and exhibited osmosensitivity and morphological abnormalities (Fig 2A, bottom). In this context, it is also worth noting that the RPS0 genes were also identified in a screen for multicopy suppressors of the tom1-2 allele (data not shown).

Steady-state levels of ribosomal subunits are decreased in TOM1 mutants:
To determine if the basis for the genetic interactions between TOM1 and RPS0 genes resulted from the involvement of the Tom1 protein in some aspect of ribosome synthesis or function, we examined polysome profiles from the tom1-113 strain containing different combinations of plasmid-borne TOM1 and RPS0B alleles. The tom1-113 mutant was used in these experiments because the synthetic interaction between RPS0B mutations and tom1-2 or tom1-111 alleles caused pronounced growth defects at all temperatures tested, but the synthetic interaction observed for tom1-113 was evident only at 37°. Relative to wild type, the strain containing the TOM1 and rps0B::HIS3 alleles had a reduction in the amount of large polysomes, a reduced level of free 40S subunits, and a large increase in free 60S subunits (Fig 3A), all of which are consistent with previous observations that 40S subunits are limiting in RPS0 mutants (DEMIANOVA et al. 1996 ). The strain containing the tom1-113 and RPS0B alleles had a reduction in polysomes and decreased levels of free 60S subunits relative to wild type (Fig 3B). The shoulder present on the 80S and first polysome peak may represent half-mer polysomes, indicating that 60S subunits may have been reduced to a greater extent than 40S subunits in the TOM1 mutant. Double mutants containing tom1-113 and rps0B::HIS3 alleles show a further reduction in polysomes and again have limiting amounts of free 40S subunits (Fig 3C). A combined effect of the two mutant alleles on the level of 40S subunits presumably plays a major role in the synthetic interaction observed between the TOM1 and RPS0B genes.

These results suggest that TOM1 mutants may have decreased levels of 40S and 60S subunits with a preferential effect on 60S subunits. Since cells with the tom1-113 allele alone do not exhibit a pronounced growth defect at elevated temperatures, we examined polysome profiles of strains with the Tom1 hect domain deleted. Fig 4 shows polysome profiles of a strain containing a chromosomal tom1-2 allele that was either transformed with the plasmid pRS316 or pRS316 containing wild-type TOM1 (pRS316-TOM1). Relative to 30°, the tom1-2 mutant at 37° had a pronounced reduction of polysomes with the appearance of half-mer polysomes, indicating that 60S subunits were limiting (Fig 4A). Consistent with this observation, the peak corresponding to free 60S subunits also showed a reduction relative to 40S subunits in mutants grown at 37° compared with 30°. Plasmid-borne TOM1 increased polysome levels and decreased half-mer polysomes at 37° (compare solid lines in Fig 4A and Fig B) but did not completely reverse the 60S subunit deficit in the mutant strain, indicating that complementation by plasmid-borne TOM1 may have been incomplete. Nevertheless, taken together, these data demonstrate that TOM1 mutants have reduced levels of 40S and 60S ribosomal subunits and that at nonpermissive temperatures 60S subunits become limiting for protein synthesis.

Multiple steps in rRNA processing are disrupted in TOM1 mutants:
To understand more about the role of Tom1 in ribosome synthesis we examined rRNA processing in strains harboring the tom1-2 allele. Strains transformed with pRS316 or pRS316(TOM1) were grown as described previously, except that, after the temperature shift, total RNA was isolated, blotted to a solid support, and hybridized with oligonucleotide probes against mature and precursor rRNAs. At 37° the tom1-2 strain had elevated levels of both the 35S and 23S rRNA precursors and reduced amounts of 20S rRNA relative to the other cultures (Fig 5A). The 35S precursor is an early intermediate in the rRNA processing pathway that retains the 5' external transcribed sequence (5'-ETS) upstream of the 18S rRNA coding region (Fig 6B and Fig C). The 23S rRNA species retains the 5' ETS but is cleaved at either the A2 or A3 site within the internal transcribed sequence 1 (ITS1). To determine how far the 23S species extended into the ITS1 region, the blot was hybridized with oligonucleotides complementary to regions between the A2 and A3 cleavage sites (Fig 6A, oligonucleotide 3) or downstream of A3 (Fig 6A, oligonucleotide 4). The 23S species hybridized to oligonucleotide 3 (Fig 5B) but not to oligonucleotide 4 (Fig 5C), indicating that this species likely terminates at the A3 site. Accumulation of the 35S and 23S in TOM1 mutants is consistent with inefficient cleavage at A0/A1 sites within the 5' ETS and at site A2 within ITS1 (Fig 6C). Several studies have shown that cleavage at these three sites is coupled (VENEMA and TOLLERVEY 1999 ). Inefficient cleavage at these sites preferentially affects the pathway leading to mature 18S rRNA. Therefore, the decrease in 20S rRNA observed in TOM1 mutants is consistent with a role for Tom1 in these early processing reactions.

Interestingly, another species migrating just above 18S rRNA is also elevated in TOM1 mutants. This species is observed with oligonucleotide 3 (Fig 5B) but is not detected with oligonucleotide 1 complementary to the 5' ETS (Fig 5D) or oligonucleotide 4 (Fig 5C). This species presumably represents a 21S pre-rRNA cleaved at A1 but extending to A3 (Fig 6C). The increase in 21S rRNA linked to the tom1-2 allele is temperature-independent, indicating that the defect in A2 cleavage in TOM1 mutants can be uncoupled from effects on A0 and A1 cleavage at certain temperatures.

Inefficient cleavage at the A0, A1, and A2 sites cannot, however, account for the preferential reduction in 60S subunits observed in TOM1 mutants, since other genes that exclusively affect cleavage at these sites show a preferential reduction in the steady-state level of 40S subunits. Fig 5E shows complex effects of the tom1-2 mutation on the pathway leading to the mature 5.8S and 25S rRNAs of the 60S ribosomal subunit. At 30°, both 27S and 7S rRNAs were increased by 25% in tom1-2 mutants relative to wild type when normalized to U3 snoRNA (Fig 5E and Fig F, lanes 1 and 2). At 37° (Fig 5E and Fig F, lanes 3 and 4), 27S and 7S rRNAs in the tom1-2 mutant were decreased by 50% relative to wild type when normalized to U3 snoRNA. The more dramatic decrease in 27S and 7S rRNAs in mutants relative to wild type after the temperature shift is consistent with a role for Tom1 in the maturation of large subunit rRNAs.

We used pulse-chase labeling of rRNA precursors as another means to analyze the effects of the tom1-2 mutation on the rRNA processing pathway. Fig 7 shows delayed processing of the 35S precursor in the tom1-2 mutant at 37°. This delay was accompanied by the appearance of the 23S rRNA precursor, consistent with an effect of the tom1-2 mutation on cleavage at A0, A1, and A2 sites. The tom1-2 mutant also showed a delay in processing of the 21S/20S precursor at both 30° and 37°. This delay presumably reflects inefficient processing of the 21S rRNA precursor, which is consistent with the temperature-independent increase in the steady-state level of this precursor observed by Northern analysis. The tom1-2 mutant at 37° also showed a delay in processing of 27S rRNA precursors consistent with a role for Tom1 in the maturation of large subunit rRNAs.

View larger version (58K): In this window In a new window Download PPT slide   Figure 7. Pulse-chase analysis of rRNA processing in wild-type and mutant TOM1 strains. RAY5 strains described in Fig 4 were grown at 30° and shifted to 37° or maintained at 30° for 6 hr. After 6 hr, cells were harvested, concentrated, and pulse-labeled with [3H]methionine as described in MATERIALS AND METHODS. After pulse labeling, cells were chased for 0, 5, or 15 min in media containing cold methionine. Total RNA was isolated from cells at each time point, fractionated on a 1.5% agarose-formaldehyde gel, and transferred to Zetaprobe. The filter was baked for 2 hr at 80° and exposed to Kodak BioMax MS film at -70° with a Kodak LE intensifying screen. Chase periods are listed below each lane and relevant TOM1 alleles and temperatures used for growth and pulse-chase labeling are at the top.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The studies reported here show that the yeast RPS0 genes, which encode small subunit ribosomal proteins, interact genetically with TOM1, a gene encoding a hect-domain-containing E3 ubiquitin-protein ligase. These genes exhibit two types of genetic interactions. The first is a synthetic interaction, where TOM1/RPS0 double mutants have a more pronounced effect on yeast cell growth than either single mutant alone. In the second interaction, overexpression of RPS0 genes partially suppresses phenotypes linked to TOM1 mutants. These relationships pointed to a role for Tom1 in ribosome synthesis.

TOM1 encodes a 3268-amino-acid protein that contains a hect domain at its carboxyl terminus. The hect domain is found in a family of E3 ubiquitin-protein ligases that have diverse cellular functions (SCHWARZ et al. 1998 ; HARVEY and KUMAR 1999 ). These functions include regulation of cell cycle progression, membrane channels and permeases, and transcription. Some of the hect-domain-containing proteins, such as the human E6-associated protein (E6-AP) and the yeast Rsp5 protein, have multiple functions. E6-AP, which is defective in Angelman syndrome, associates with the papilloma virus E6 protein and together they interact with the tumor suppressor p53 to promote p53 degradation by the proteasome (SCHEFFNER et al. 1993 ). In addition, E6-AP regulates transcription as a coactivator for the nuclear hormone receptor superfamily (NAWAZ et al. 1999 ). The yeast Rsp5 protein functions in regulating the steady-state level of membrane permeases through ubiquitin-dependent degradation (HEIN et al. 1995 ). Rsp5 can also function as a transcriptional coactivator for the nuclear hormone receptor superfamily when these receptors are expressed in yeast (IMHOF and MCDONNELL 1996 ). Another role for Rsp5 in transcription is suggested by the observation that the largest subunit of RNA polymerase II, Rpb1, is a substrate for Rsp5-mediated ubiquitination (HUIBREGTSE et al. 1997 ). Like other hect-domain-containing proteins, Tom1 also appears to have multiple functions. SALEH et al. 1998 have shown that Tom1 influences transcriptional activation through the ADA/SAGA coactivator complex. Tom1 has also been shown to be required for full induction of the general stress and heat shock responses (SASAKI et al. 2000 ). In addition, other studies have shown that, at 37°, TOM1 mutants arrest growth near the G2/M transition of the cell cycle and have pleiotropic defects in nuclear structure and function including fragmentation of the nucleolus and impaired mRNA transport (UTSUGI et al. 1999 ; DUNCAN et al. 2000 ). Here, we extend the known processes affected by members of the hect-domain-containing E3 ubiquitin-protein ligase family by demonstrating that TOM1 mutants are defective in ribosome synthesis.

Strains deleted for the ubiquitin ligase domain of Tom1 exhibit multiple defects in the rRNA processing pathway. Cleavage of rRNA at sites A0, A1, and A2 is reduced in the TOM1 mutant at nonpermissive temperatures. A number of studies have shown that cleavage at these three sites, particularly A1 and A2, may be coupled in vivo (VENEMA and TOLLERVEY 1999 ). In most cases cis- and trans-acting mutations that affect A2 also affect A1 cleavage. Interestingly, the temperature-independent accumulation of 21S rRNA in the tom1-2 mutant indicates a selective effect on A2 cleavage. A similar result has also been reported for cells depleted of the yeast Rrp7 protein, which is thought to be required for the incorporation of ribosomal protein S27 into 40S subunits (BAUDIN-BAILLIEU et al. 1997 ). Cells defective in A0, A1, and A2 cleavage have reduced levels of 20S rRNA and 18S rRNA which, in turn, reduces the level of 40S subunits (VENEMA and TOLLERVEY 1999 ). Cells depleted of Rps0 proteins also have reduced amounts of 40S subunits; however, in this case, 20S rRNA accumulates. This indicates that the primary processing step affected by Rps0 depletion is cleavage at site D, which forms the mature 3' end of 18S rRNA (FORD et al. 1999 ). Disruption of 40S subunit maturation at multiple steps in the rRNA processing pathway leading to 18S rRNA presumably plays a major role in the synthetic interactions between TOM1 and RPS0 mutants.

While inefficient cleavage at early sites in the rRNA processing pathway explains the reduced levels of 40S subunits in the tom1-2 mutant, it does not account for the observation that 60S subunits are also decreased and appear to be limiting for protein synthesis at nonpermissive temperatures. Pulse-chase analysis showed that, at nonpermissive temperatures, 27S rRNA processing was reduced in the tom1-2 mutant relative to wild type. Inefficient processing of 27S rRNA is consistent with the decrease in 7S rRNA in mutants at 37° observed by Northern analysis. The observation that 27S rRNA levels also decrease in tom1-2 mutants at 37° is a reflection of delayed processing of the 35S rRNA. Further understanding of the role of Tom1 in the production of 60S subunits may also provide insight into its role in the maturation of 40S subunits, since there appears to be a link between the level of 60S subunits and early events in the rRNA processing pathway (VENEMA and TOLLERVEY 1999 ; ZANCHIN and GOLDFARB 1999 ).

Ubiquitination of one or more ribosomal proteins by Tom1 could provide an explanation for the role of this putative ubiquitin-protein ligase in ribosome synthesis. Ubiquitin has been shown to facilitate the incorporation of ribosomal protein S31 into 40S subunits (FINLEY et al. 1989 ). Ubiquitin, in this case, is encoded as a fusion to the amino terminus of Rps31 in the hybrid gene UBI3. Other ribosomal proteins, not encoded as ubiquitin fusions, may also need to be ubiquitinated for their efficient incorporation into ribosomal subunits. These proteins would need to be ubiquitinated post-translationally, presumably by a ubiquitin-protein ligase. A role for ubiquitination of ribosomal proteins in both the assembly and degradation of subunits may represent a quality control mechanism to eliminate immature subunits that may not be assembled properly and may not have their ubiquitin moieties removed efficiently.

Alternatively, the defects in ribosome synthesis in TOM1 mutants may be the consequence of alterations in other cellular processes influenced by Tom1. Tom1 plays a role in transcriptional activation by modifying properties of the ADA/SAGA transcriptional coactivator complex. Consequently, it is possible that expression of a gene product under control of this complex may be involved in one or more of the rRNA processing reactions defective in TOM1 mutants. Interestingly, it has recently been shown that Ada5, a component of the ADA/SAGA complex, functions in RNA processing (WELIHINDA et al. 2000 ). The Ada5 protein is required for splicing of HAC1 mRNA as part of the unfolded protein response. However, the role of the Ada5 protein in splicing appears to be separate from its role as a transcriptional coactivator, indicating that components of the coactivator complex may have distinct roles in transcription and RNA processing. Thus, further study is needed to determine whether the ADA/SAGA complex plays a role in any of the rRNA processing steps defective in TOM1 mutants or if Tom1 affects rRNA processing independently of this complex.

Some of the rRNA processing defects observed in the tom1-2 mutant at nonpermissive temperatures may be the result of pleiotropic defects in nuclear structure and function. At nonpermissive temperatures, TOM1 mutants appear to have fragmented nucleoli and accumulate poly(A⁺) RNA in the nucleus (UTSUGI et al. 1999 ; DUNCAN et al. 2000 ). Defects in nucleolar structure could, in principle, affect rRNA processing and subunit assembly. However, studies have shown that an organized nucleolus is not required for efficient rRNA processing or subunit assembly (NIERRAS et al. 1997 ). In contrast, defects in nuclear transport have been shown to disrupt various steps in the rRNA processing pathway (KADOWAKI et al. 1995 ; MOY and SILVER 1999 ). Mutations in genes involved in transport of ribosomal subunits from the nucleolus to the cytoplasm can interfere with rRNA processing. The step in rRNA processing that is most sensitive to defects in nuclear export of ribosomes is cleavage D, which occurs in the cytoplasm (UDEM and WARNER 1973 ; KADOWAKI et al. 1995 ). Since tom1-2 mutants are clearly defective in multiple steps in the rRNA processing pathway known to occur in the nucleolus, it seems unlikely that defects in subunit export are responsible for the effects of this mutant on rRNA processing. Mutations that reduce the import of ribosomal proteins into the nucleus, on the other hand, have been shown to disrupt subunit assembly and various rRNA processing reactions, including some of the early steps in rRNA processing affected by the tom1-2 mutation at nonpermissive temperatures (MOY and SILVER 1999 ). Whether the import of ribosomal proteins into the nucleus is defective in the tom1-2 mutant has not been determined. Therefore, we cannot rule out the possibility that some of the effects of the TOM1 mutants on rRNA processing at nonpermissive temperatures could be the result of defects in nuclear transport. Nonetheless, some of the effects of the tom1-2 mutation on ribosome synthesis, such as inefficient cleavage at A2, are observed at 30° where poly(A⁺) RNA does not accumulate in the nucleus. Thus, pleiotropic defects in nuclear structure and function cannot account for all of the effects of TOM1 mutants on rRNA processing.

The partial suppression of the growth arrest of TOM1 mutants at nonpermissive temperatures by overexpression of RPS0 genes is also most readily explained through a more specific role for Tom1 in rRNA processing. The ability of Rps0 proteins to partially compensate for the loss of Tom1 function suggests that these proteins may have some overlap in function. While the major effect of Rps0 proteins on rRNA processing is on the D cleavage step, recent results indicate that the Rps0 proteins also influence A2 cleavage (A. TABB, unpublished experiments). Thus, cleavage at the A2 site, a point of overlap between Rps0 and Tom1 function, may lie at the heart of the genetic interactions between their respective genes.

Whether there is a hect-domain-containing E3 ubiquitin ligase in mammalian cells that plays a role in ribosome synthesis analogous to that of Tom1 remains to be determined. Sequence analysis has identified a minimum of 20 hect-domain-containing proteins in the human genome, many of which have unknown functions (SCHWARZ et al. 1998 ). A protein similar to Tom1 could regulate multiple cellular processes through its ubiquitin-protein ligase activity. Loss of these regulatory functions could lead to cell cycle arrest, such as that observed in TOM1 mutants (UTSUGI et al. 1999 ). Overexpression of proteins like Rps0, which offsets one of these functions but not others, can potentially lead to growth of cells that should be arrested in the cell cycle. Growth under these conditions could result in cells that are genetically unstable and promote other mutations that undermine growth control.

Ribosome synthesis is clearly an important part of cell growth and proliferation (POLYMENIS and SCHMIDT 1999 ; SCHMIDT 1999 ; VOLAREVIC et al. 2000 ). Mitogen signaling increases ribosome synthesis through a variety of mechanisms, and an increase in ribosome synthesis, in turn, can potentially regulate decisions made at cell cycle checkpoints (POLYMENIS and SCHMIDT 1997 ; VOLAREVIC et al. 2000 ). Thus, ribosome synthesis is tightly integrated with other processes needed for coordinating cell growth and division. Overexpression of certain proteins could potentially subvert these controls, leading to cell division under unfavorable conditions, which could then promote tumorigenesis. Much remains to be learned regarding the role of p40/37-LBP in ribosome synthesis in mammalian cells. Further, it remains to be seen whether a hect-domain-containing E3 ubiquitin-protein ligase like Tom1 is involved in ribosome synthesis. Nonetheless, these studies provide a new perspective from which to consider how overexpression of p40/37-LBP, in its role as a component of the translational machinery, could compromise growth control and therefore create conditions favorable for tumor formation.

	ACKNOWLEDGMENTS

We thank Drs. Lisa R. Williams, Robert Gray, and Thomas Geoghegan for critically reading the manuscript. This work was supported by National Institutes of Health grant RR11803, research funding from the University of Louisville and Kentucky EPSCoR, an intramural research incentive grant from the vice president for research at the University of Louisville, and the University of Louisville Medical School research committee.

Manuscript received September 15, 2000; Accepted for publication November 22, 2000.

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