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and Jonathan R. Warner*
Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461
Received 30 August 2002/ Returned for modification 11 October 2002/ Accepted 16 October 2002
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ABSTRACT |
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INTRODUCTION |
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These three pathways, and perhaps many more, in some way coordinately regulate the transcription both of rRNA genes by RNA polymerase I and of the 137 genes encoding ribosomal proteins (RPs) by RNA polymerase II (38). However, the downstream events by which the regulatory pathways interface with the ribosomal genes remain almost totally obscure. It is not known, for instance, whether they act through the same or different effectors of transcription, or even whether transcription of RP genes depends on concomitant transcription of rRNA genes or vice versa.
In an attempt to approach this question, we searched for mutants that interfered with the repression of ribosome biosynthesis in response to a defect in the secretory pathway. Surprisingly, one such mutant inactivated one of the two genes encoding RP L1 of the 60S ribosomal subunit. The primary effect of such a mutant is to cause an imbalance in the synthesis of the two ribosomal subunits, leading to the accumulation of excess 40S subunits. Further experiments showed that limiting production of 60S subunits by any of several means has the same effect. (During the preparation of this report, another report of this phenomenon appeared [18].) Limiting the production of 40S ribosomal subunits does not. Several lines of evidence suggest that it is not a simple deficiency of 60S subunits that is responsible for this phenotype, but rather it is the degradation of improperly assembled 60S subunits.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Plasmids and primers. The integrative plasmid pRS306-G418 carrying URA3 and an RPL24-G418r expression cassette was constructed as follows. An open reading frame encoding the bacterial G418r gene was amplified from pFA-kanMX2 (37) with primers P1 (5'-ATGGGTAAGGAAAAGACT-3') and P2 (5'-TTAGAAAAACTCATCGAG-3'). The promoter and transcriptional terminator of RP RPL24A were also amplified from pRS-L24L30 (15) by using two primer sets, P3 (5'-GTGCACCACATATTTTTG-3') and P4 (5'-AGTCTTTTCCTTACCCATTTTATCACTATATTA-3') and P5 (5'-CTCGATGAGTTTTTCTAAGATTTATGCTCGAAC-3') and P6 (5'-AAGCTTTAGATGCGGACA-3'). Secondary PCR was done with primers P3 and P6 from the mixture of the first PCR products with two consecutively overlapped regions. The PCR-amplified G418r expression cassette was inserted into pRS306 (30). pRS306-G418 was transformed into W303(SLY1) and 312XX(sly1-1) after linearization at the unique EcoRV site in URA3. Genomic PCR of RPL1A and RPL1B from the wild type and sm9 was performed with primers JW1394 (5'-AACAGTCAACCAGTCGTCCA-3') and JW1395 (5'-CTATGAAAACACGTAATTATGC-3') for RPL1A and primers JW1396 (5'-GGAAGACTAATTACATATCAT-3') and JW1397 (5'-GTATTAATCGCTCGGAAGTGA-3') for RPL1B.
Mutant selection. A temperature-sensitive secretion defect mutant 312XX, carrying sly1-1 (19), was used as a parental strain to identify factors involved in the regulatory interconnection between the secretory pathway and ribosome synthesis. 312XX grows normally at a temperature of <30°C but grows at a reduced rate between 32 and 34°C, possibly due to a partial limitation of ribosome synthesis as a result of the secretion defect (Fig. 1B). We developed a window of selection for the cells with reduced repression of RP synthesis in the presence of a secretion defect by using semipermissive temperatures between 30 and 34°C and cells carrying a G418 selection marker under the control of the RPL24A promoter. Strain 312XX was first integratively transformed with EcoRV-digested pRS306-G418 containing the G418 resistance gene under the control of the RPL24A promoter and selected in synthetic complete minimal medium lacking uracil. A single colony was further tested for resistance to G418 on YPD plates with concentrations of G418 ranging from 0 to 200 µg/ml as a function of temperature (Fig. 1B). For the selection of spontaneous mutants that can grow under the conditions of nonpermissive temperature and G418 concentration, 5 x 108 cells were plated on a YPD plate with 100 µg of G418/ml and incubated at 32°C for 3 days.
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Polyribosome profiles. Yeast strains were grown in 50 ml of YPD medium at 30°C to an optical density at 600 nm of 0.5 to 1.0. Cycloheximide was added to a 50-µg/ml final concentration, and the culture was chilled immediately on ice. The cells were harvested and washed twice with LHB solution (10 mM Tris [pH 7.4], 100 mM NaCl, 30 mM MgCl2, 50 µg of cycloheximide/ml, 200 µg of heparin/ml). The cell pellet was resuspended in 0.5 ml of LHB, glass beads were added, and the sample was vortexed twice for 30 s at 4°C. One milliliter of LHB was added, and the sample was centrifuged twice at 10,000 x g for 10 min. The supernatant was layered onto a 10 to 35% sucrose gradient in TMN buffer (50 mM acetyl Tris [pH 7.0], 50 mM NH4Cl, 12 mM MgCl2) and centrifuged at 38,000 rpm for 4 h in a Beckman SW41 rotor. All gradients were scanned at 254 nm from the top with an Isco gradient collector.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As a reporter for the synthesis of RPs, the G418 resistance gene was placed under the control of the promoter of the RP gene RPL24A (Fig. 1A) and integrated into strain 312XX, carrying a temperature-sensitive allele of SLY1, a component of the secretory pathway (see Materials and Methods). Titration of growth as a function both of temperature and of G418 concentration suggested that our supposition may be correct; the strain carrying sly1-1 was more sensitive to G418 even at a temperature at which its growth without drug was hardly compromised (Fig. 1B). To carry out the screen, we spread 5 x 108 cells on plates containing 100 µg of G418/ml and incubated them for 3 days at 32°C. Ten colonies grew up.
At least three different types of mutants could be predicted using such a screen: mutants that suppress the sly1-1 mutation, spontaneous mutants providing G418 resistance, and our goal, mutants that compromise the signaling pathway. To distinguish among these alternatives we first tested the cells for growth at 37°C to rule out reversion or suppression of the sly1-1 mutation. Secondary screening was then carried out with tunicamycin, which inhibits protein glycosylation within the endoplasmic reticulum and induces a secretory defect, as shown by induction of the unfolded protein response (3). Treatment with tunicamycin also represses the transcription of RP genes, with the same signaling as the temperature-sensitive signal mutant (23). Therefore, a true signal mutant should render RP transcription insensitive to tunicamycin. One mutant strain, sm9, also passed this test.
The critical phenotype of strain sm9 is shown in Fig. 2A. As we have found previously, upon a shift to 37°C in a wild-type cell, there is a temporary inhibition of transcription of RPL30, representing the RP genes (Fig. 2A, lane 2) (15). There is no such effect on the ACT1 mRNA. In cells carrying the temperature-sensitive sly1-1 allele, transcription of the RP gene declines precipitously and does not recover (Fig. 2A, lanes 5 to 8). Actin remains unaffected. The induced transcription of KAR2, encoding a chaperone that is induced by the unfolded protein response, serves as a positive control for the distress of the secretory pathway. In strain sm9, it is clear that the repression of RP synthesis that usually accompanies a shift to the nonpermissive temperature in a sly1-1 strain no longer occurs (Fig. 2A, lanes 9 to 12). Note, however, that the temporary heat shock-induced inhibition of transcription of RPL30, apparent at the 30-min time point, is not affected by the mutation. Furthermore, the decline in the RP mRNA in Fig. 2A, lane 10, shows that the mutation in strain sm9 does not alter the half-life (t1/2) of the RP mRNA.
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Strain sm9 with a plasmid carrying an intact RPL1B gene or an RPL1A gene grows at a normal rate and shows the usual repression of RP gene expression upon transfer to the nonpermissive temperature (Fig. 2, lanes 14 to 19), confirming that the original phenotype is due to a deficiency of RP L1.
Effects on rRNA transcription. The major cost of ribosome synthesis is the transcription of rRNA. A defect in the secretory pathway leads to repression not only of RP genes but also of rRNA genes (19). We asked whether the limitation of 60S biosynthesis due to L1 deficiency also prevented the repression of rRNA transcription. Figure 3 shows the precursor and mature rRNAs labeled with a 3-min pulse of [C3H3]methionine. Disturbance of the secretory pathway, in this case with tunicamycin, brings about a severe repression of rRNA transcription in a wild-type cell, evident by 90 min and almost complete by 150 min. However, in strain sm9, carrying rpl1b-1, tunicamycin has relatively little effect on rRNA transcription, although rRNA processing is slowed somewhat. Thus, limitation of 60S subunits interferes with the repression of ribosome synthesis at the levels of both RNA polymerase I and RNA polymerase II.
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We then asked whether limitation of ribosomal subunits in general could counteract the repression of ribosome synthesis due to a defect in the secretory pathway, by using cells with a deletion either of a gene encoding a small subunit protein, RPS6A, or of a gene encoding a different large subunit protein, RPL11B. In each case, cell growth is limited by the loss of one of a pair of RP genes, with doubling times of 160 and 180 min, respectively, compared to 90 min for the wild type. In cells lacking RPS6A, large amounts of free 60S subunits accumulate; in cells lacking RPL11B, 40S subunits and half-mers accumulate to a substantially greater extent than in sm9 cells (data not shown). After cells are shifted to the nonpermissive temperature, the repression of RP gene transcription in cells bearing sly1-1 is independent of the presence of RPS6A, and it is almost abolished in cells lacking RPL11B (Fig. 4A). Note that the transcriptional response of a gene encoding a 40S protein, RPS30A, is the same as that of genes encoding 60S proteins. The deletion of RPL11B also protects transcription of rRNA from repression by tunicamycin (Fig. 3), showing the generality of the phenomenon.
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To test the possibility that the effect of deficient 60S subunits is mediated through the imbalance of the 40S and 60S subunits, we generated two sets of double-mutant strains missing genes encoding both RPL11B and RPS6A or RPS23A. The growth rate of these strains differs little from that of the single mutants, and the level of half-mers is reduced, though not eliminated. Nevertheless, the RP genes are still protected from repression by tunicamycin (Fig. 4B, four right lanes, and Table 2, compare the last two lines). Thus, we conclude that an imbalance in subunit concentration is not responsible for this effect.
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strains grow at nearly the same rate, with a doubling time of about 4 h. Thus, L11 is no longer limiting for growth, although it is limiting for translation, as half-mers occur to the same extent as in YPD medium (data not shown). Nevertheless, treatment of the two strains with tunicamycin leads to repression of RP mRNA in wild-type cells but not in rpl11b cells (Fig. 6). This result demonstrates that a partial block in the synthesis of 60S ribosomal subunits can influence the control of ribosome synthesis even when they are not limiting for growth.
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Deletion of RPL11B does not lead to an increase in the mRNA level of other RPs.
Cells might compensate for the deficiency of 60S subunits by increasing the overall synthesis of ribosomes. However, comparison of the level of RP mRNAs in mutant and wild-type strains revealed no such stimulation (Table 3). The level of RP mRNA, compared to that of the stable U3 snoRNA, generally reflects the growth rate of the cells. In cells growing more slowly due to a less favorable carbon source, the overall level of RP mRNA is reduced. In cells growing more slowly due to deletion of RPL11B, the level of RP mRNA is also reduced. In YPGE medium, where the growth rates of the two strains are similar, the levels of the RP mRNAs are also similar. Thus, the phenotype of the strain carrying rpl11b, in reducing the effect of a defect in the secretory pathway on ribosome synthesis, is not due to a general increase in ribosome synthesis. Furthermore, since much of the response to the carbon source is mediated through the ras-PKA pathway (22), these data suggest that this pathway is unaffected by a deficiency of 60S subunits.
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Perhaps some aspect of the generation of defective 60S subunits, or their degradation, e.g., the accumulation of some intermediate breakdown product, influences the regulation of ribosome synthesis. To try to distinguish whether it is the generation of defective 60S particles or their attendant degradation that contributes to the phenotype that we observe, we have examined the effects of tunicamycin on strains carrying mutations in three genes encoding proteins implicated in late steps of 60S production. Indeed, each has been linked circumstantially with the export pathway. Tif6p (28, 29) and Nmd3p (11) appear to be shuttling proteins that accompany the newly formed 60S particle as it is exported to the cytoplasm. Kap120p is one of the karyopherins involved in 60S export (31). Mutations in the genes encoding these three proteins all bring about a diminution of 60S synthesis, an imbalance in ribosomal subunits, and the appearance of half-mers. However, the immediate phenotypes differ. In cells lacking eIF-6p, newly formed 25S rRNA is not observed, either because the 27S pre-rRNA is degraded or because the 25S rRNA is degraded with a vanishingly short t1/2 (2). In cells with temperature-sensitive Nmd3p, 25S rRNA is formed but decays with a t1/2 of 3 to 4 min (10). In the absence of the karyopherin Kap120p, cells generate apparently mature 25S rRNA that is stable for the period of a 10- to 15-min pulse-chase, although eventually it must be degraded since the steady-state molar ratio of 25S/18S is about 0.5 (31).
As seen in Fig. 5, depletion of eIF-6p protects RP genes from the repression caused by tunicamycin. Temperature-sensitive nmd3-4 cells grown at a semipermissive temperature limit the cells for 60S subunits and cause the accumulation of half-mers. These cells appear even less responsive to tunicamycin than the nop4ts cells or the rpl11b cells (Fig. 8). However, kap120 cells, with the same growth rate and accumulation of half-mers, are substantially more responsive to tunicamycin, though less so than wild-type cells (Fig. 8). We suggest that the difference between the kap120 strain and the nmd3-4 strain is due to a difference in the rate, and perhaps the mechanism, of the degradation of defective (or untransportable) 60S subunits.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have endeavored to determine which of the many manifestations of a deficiency of 60S subunits is responsible for the observed effect.
A deficiency of 60S subunits reduces the rate of protein synthesis, as evident from the slower growth rate of the initial sm9 mutant. Supplementation of the cell with another copy of either RPL1A or RPL1B overcomes the slow-growth phenotype, demonstrating that insufficient L1 protein is limiting for cell growth. As an alternative way of reducing protein synthesis and growth rate, we used temperature-sensitive mutants affecting two proteins of the translational initiation machinery, Prt1p, which is part of eIF-3 and acts at a relatively early stage of initiation (21), and Tif5p, a GTPase-activating protein for the GTPase of eIF-2p that induces GTP hydrolysis when the 43S initiation complex reaches the initiator AUG of the mRNA at a relatively late stage (17). In each case, the strains were grown at a semipermissive temperature at which translation initiation is limiting and the growth rate was approximately the same as that of sm9 cells. For both the translation initiation mutants, tunicamycin brought about the normal repression of ribosome synthesis (Fig. 7). These results, together with the observation that deficient 40S subunits have no effect on regulation (Fig. 4), suggest that neither reduced protein synthesis nor reduced growth rate is the cause of the suppression of repression that we observe.
A deficiency of 60S subunits brings about the formation of half-mers, where 43S initiation complexes are stalled at the initiator AUG, waiting for limiting 60S subunits (27). While this reduces the rate of initiation, as dealt with in the previous paragraph, it also disrupts the dynamic stoichiometry of initiation factors and 40S subunits. The initiation factors are present at only 5 to 10% of the level of ribosomes (based on mRNA levels) (12), a situation that is efficient if most of the ribosomes are elongating, as is usually the case. However, if a substantial fraction of the 40S subunits are tied up as half-mers, then their associated initiation factors may become limiting, a situation that could send a signal to restore ribosome synthesis. Therefore, we considered whether the accumulation of half-mers could overcome the repressive signal. However, this appears not to be the case. When cells in which deletion of RPL11B is combined with deletion of RPS6A or RPS23B, the balance of subunits is largely, though not completely, restored, and the level of half-mers is reduced. Nevertheless, the lack of a 60S protein appears dominant, as ribosome synthesis in these cells is resistant to repression by a defective secretory pathway (Fig. 4B and Table 2). Furthermore, although the level of half-mers is consistently higher in cells carrying rpl11b than in sm9 cells or in cells carrying rpl1b, the effect of tunicamycin is suppressed to almost the same degree in all of these strains. Therefore, we conclude that the formation of half-mers, per se, is not responsible for the effects of a deficiency of 60S subunits on ribosome synthesis.
The manufacture of a 60S ribosomal subunit is extraordinarily complex, involving nearly 200 auxiliary protein and RNA molecules and many serial steps of processing the rRNA together with assembly and disassembly of RPs and non-RPs (1, 8, 14). The data presented above show that improper assembly of 60S subunits, whether due to insufficiency of L1, L11, L20, or L34, to a lack of eIF-6p, to a temperature-sensitive Nop4p, or to a temperature-sensitive Nmd3p, substantially prevents the repression of ribosome synthesis in response to a defect in the secretory pathway. These results suggest that it is not a specific step of assembly that impacts the regulation of ribosome synthesis but rather the existence of malformed 60S subunits and/or their degradation.
Since the RNA components of 60S and 40S subunits, except for 5S RNA, derive from a common 35S pre-rRNA precursor, they start their lives as equimolar. Furthermore, many measurements suggest that the synthesis of RPs is roughly equimolar (reviewed in reference 39). Therefore, cells in which half the supply of one essential RP is eliminated have a serious disposal problem. Cells carrying rpl1b-1 destroy more than half of their 27S pre-rRNA and more than half of their 60S RPs, representing some 15% of the total transcription and 5% of the total translation of the cell. Little is known of this process, but it must be efficient because little if any accumulation of partially assembled ribosomal subunits or of RPs has been detected. We wondered if some aspect of the degradation process could be responsible for the effects on the transcription of ribosomal components.
Results with the strain carrying kap120 support this view. In that case, assembly of the 60S subunits appears to be essentially complete, but the subunits are exported only inefficiently and accumulate within the nucleus (31). As a result, cytoplasmic polysomes have abundant half-mers, and protein synthesis is slowed. However, these cells are substantially more responsive to tunicamycin than are cells carrying other mutants affecting the synthesis of 60S subunits (Fig. 8). By contrast, cells limited for a different export factor, Nmd3p, produce apparently mature 25S rRNA but degrade it rapidly (10). Ribosome synthesis in these cells is resistant to the effects of tunicamycin. Thus, we conclude that it is more likely the degradation rather than the malassembly of 60S subunits that blocks the signal from PKC that a defective secretory pathway is endangering the cell and that ribosome synthesis should be shut down. Why a similar effect is not seen for malassembled 40S subunits is not clear. One possibility is that since the final steps of 40S maturation take place in the cytoplasm (20, 34), perhaps the quality control of 40S subunits is a cytoplasmic phenomenon while that of 60S subunits is a nuclear phenomenon that can more readily be utilized for regulation. However, since there remains some ambiguity regarding the cellular compartment in which Nmd3p and Kap120p function, one should be cautious with such an interpretation.
As pointed out above, little is known about the turnover of malassembled ribosomal subunits. However, we can draw two conclusions. First, Fig. 2A and 5 provide striking evidence that the components of 60S subunits lacking an essential protein, or improperly assembled, are not recycled. Any excess of the RP L30 can bind to the unspliced transcript of its own gene and prevent its splicing, leading to accumulation of unspliced transcript (5, 36). Recycling of L30 derived from improperly assembled subunits would lead to an excess of L30 and consequently to the accumulation of unspliced RPL30 transcript. Since little, if any, such accumulation is evident in Fig. 2A or 5 (pre-RPL30), we conclude that malassembled subunits are degraded in their entirety. Second, the growth of cells missing an RP gene seems to be roughly proportional to the availability of that protein. This suggests that the turnover of malassembled subunits does not include the associated proteins involved in ribosome maturation (1, 8). Since these are present in much smaller amounts (as estimated from transcriptome data) (12), their degradation would bring ribosome synthesis to a halt.
In summary, we suggest that the influence of mutations that reduce the biosynthesis of 60S subunits on the transcription of ribosomal genes is not due to the reduction of protein synthesis or growth rate caused by insufficient 60S subunits, nor to some interference with translational initiation mediated through the formation of half-mers, nor to the malassembly of 60S subunits per se, but is brought about by the process or the products of the degradation of malformed 60S subunits.
We know embarrassingly little about the interface between the signal transduction pathways regulating ribosome synthesis, the heat shock, the PKA, the PKC, or the TOR pathway, with the apparatus modulating the transcription of rRNA genes by polymerase I and of RP genes by polymerase II. The interesting aspect of our observations is that problems with the synthesis of 60S subunits affect only the repression of ribosome biosynthesis that is regulated through the PKC pathway. Cells with a defect in 60S production are as sensitive as wild-type cells to heat shock (Fig. 2) and to rapamycin (data not shown) while their RP mRNA level has a normal ras-PKA-mediated response to the carbon source (Table 3). Moreover, deficient and/or defective 60S subunits do not lead to a generalized stimulation of ribosome synthesis. These considerations suggest that the regulation of ribosome synthesis is even more subtle and more attuned to the needs of the cell than we had originally imagined.
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ACKNOWLEDGMENTS |
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This work was supported in part by grant GM25532 to J.R.W. and grant CA13330 to the Albert Einstein Cancer Center and by a Human Frontiers grant to J.R.W.
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FOOTNOTES |
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Present address: Microbial Genomics Laboratory, Korea Research Institute of Bioscience and Biotechnology, Yusong, Taejon 305-806, Korea.
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REFERENCES |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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