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Previous Article | Next Article 
Molecular and Cellular Biology, April 2004, p. 2998-3010, Vol. 24, No. 7
0270-7306/04/$08.00+0 DOI: 10.1128/MCB.24.7.2998-3010.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology, Manchester M60 1QD, United Kingdom,1 Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 947202
Received 8 October 2003/ Returned for modification 12 November 2003/ Accepted 15 January 2004
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ABSTRACT |
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kinase-dependent amino acid starvation pathway in yeast has been used to evaluate this highly conserved stress response in all eukaryotic cells. Using a similar approach we have found that even though the mRNA decay mutants maintain high levels of general translation, they exhibit many of the hallmarks of amino acid starvation, including increased eIF2 phosphorylation and activated GCN4 mRNA translation. Therefore, these mutants appear translationally oblivious to decreased ternary complex abundance, and we propose that this is due to higher rates of mRNA recruitment to the 40S ribosomal subunit. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A precedent for this type of regulation exists in the general control pathway of yeast (17). Here global protein synthesis is rapidly inhibited upon severe amino acid starvation (41). It is thought that a specific protein kinase (Gcn2p) senses the levels of amino acids indirectly via the level of uncharged tRNA in the cell. An accumulation of uncharged tRNA stimulates the phosphorylation of the eIF2 subunit at serine 51 by Gcn2p, leading to the sequestration of a guanine nucleotide exchange factor, eIF2B. The eIF2B complex normally catalyzes the recycling of eIF2-GDP to the translationally active form, eIF2-GTP. eIF2-GTP is capable of interacting with the methionyl initiator tRNA (
) to form the ternary complex (eIF2-GTP ). Therefore, inhibition of eIF2B (following severe starvation for amino acids and the subsequent phosphorylation of eIF2) ultimately leads to decreased levels of the ternary complex (17). Recently it has been suggested that the ternary complex might interact with other translation initiation factors (eIFs 1, 5, and 3) to form a multifactor complex (MFC) prior to interaction with the 40S ribosomal subunit (43).
As well as inhibiting global protein synthesis, starvation for amino acids induces the expression of a transcription factor, Gcn4p (17). The activation of this transcription factor at the translational level serves to overcome the amino acid stress by inducing the transcription of amino acid biosynthetic genes (18). The GCN4 mRNA contains four short upstream open reading frames (uORFs) that are involved in the translational activation via a complex mechanism involving translational reinitiation at downstream ORFs. The translation of this mRNA is exquisitely sensitive to the levels of ternary complex in the cell. Therefore, following translation of the first uORF (in the presence of high levels of ternary complex), reinitiation at downstream uORFs and subsequent translation termination is favored. In contrast, when ternary complex levels are low (for instance, following amino acid starvation), there is a greater chance that ternary complex will be acquired after ribosomes pass the uORFs, thus allowing GCN4 ORF translation (17).
A variety of other stresses have also been shown to activate GCN4 translation initiation. These include purine starvation, glucose starvation, fusel alcohol addition, salt stress, and rapamycin addition (4, 8, 15, 22, 30, 44, 48). Although the recently described effects of rapamycin on GCN4 do rely on increases in eIF2 phosphorylation (8, 22, 44), at least some of these stress responses do not appear to require an induction of eIF2 phosphorylation. Therefore, it seems plausible that these responses might decrease levels of ternary complex via alternative mechanisms. For example, we have proposed that fusel alcohols directly regulate the activity of eIF2B and thus lead to decreased ternary complex levels (4).
Starvation for glucose has a complex effect on translation initiation. Immediately following glucose removal (i.e., within minutes) there is a rapid inhibition of translation initiation (3, 23, 42). This inhibition still occurs in mutants that are incapable of phosphorylating eIF2. In wild-type cells, however, activation of GCN4 translation at later time points following glucose starvation has been described and this activation is Gcn2p dependent (48). The precise mechanisms for these effects on global and GCN4 translation initiation remain to be elucidated.
Two general pathways of mRNA decay have been described for yeast. In both of these pathways, mRNAs are first deadenylated in a process thought to involve the Ccr4p/Pop2p deadenylase (40). In the major or 5' to 3' pathway, the 5' cap structure is then removed by the Dcp1p/Dcp2p decapping complex and this facilitates the degradation of the body of the mRNA via Xrn1p, the 5' to 3' exoribonuclease (36, 39). In the exosome-mediated pathway, the deadenylated mRNA is degraded by the cytoplasmic exosome complex (which includes proteins with 3' to 5' exoribonuclease activity and putative RNA helicase activity) (26). As well as these general pathways for mRNA decay, pathways exist to degrade mRNAs containing premature stop codons. These nonsense-mediated decay pathways utilize many of the same components described for the general pathways of mRNA decay; however, these mRNAs are targeted for decay via a specific complex containing the Upf proteins (14, 27).
In this study we have analyzed a catalogue of mutants in these mRNA decay processes and assessed translational control in response to stress. As a result, we have found that major pathway mRNA decay mutants acting at stages downstream of deadenylation are globally resistant to stress-induced translational inhibition. At least in the case of severe amino acid starvation, these strains are translationally resistant to the decrease in ternary complex levels that occurs as a result of this stress. Thus, this study provides an example of the intimate relationship that exists between protein synthesis and mRNA turnover in the cytoplasm of cells and the interdependence of mechanisms involved in their regulation.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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[35S]methionine incorporation assay. Yeast strains were grown to an OD600 of 0.4 in SCD medium lacking methionine, and two 7.5-ml aliquots were pelleted and then resuspended in 20 ml of SCD-Met or SC-Met. Methionine was added to achieve a final concentration of 60 ng/ml, of which 0.5 ng/ml was [35S]methionine (New England Nuclear, Boston, Mass.) (cell-labeling grade; 1,175 Ci/mmol). Samples (1 ml) were taken at the indicated times and processed as described previously (3).
Assays of GCN4-lacZ reporter expression. Standard methods for measuring the ß-galactosidase activity for strains bearing GCN4-lacZ fusions were used (24). ß-Galactosidase levels are expressed as nanomoles of o-nitrophenol-ß-D-galactopyranoside (ONPG) hydrolyzed per minute per microgram of total protein.
Measurement of capped mRNA with the Xrn1p assay. Yeast strains were grown to an OD600 of 0.7 in YPD and divided into three aliquots, and cells were pelleted. One of the cell pellets was immediately frozen in liquid nitrogen. The other two were resuspended in YPD or YP, incubated, pelleted, and frozen in liquid nitrogen such that the total incubation time was 10 min. RNA was extracted from the frozen pellets and used for Northern blot analysis (7). The Xrn1p assay was performed as described previously (7). Total RNA (5 µg) was incubated with 400 ng of purified Xrn1p (a gift from A. Johnson, University of Texas, Austin) in a final volume of 5 µl in 33 mM Tris-HCl (pH 8.0)-50 mM NaCl-2.5 mM MgCl2-0.2 mM dithiothreitol in the presence or absence of 5 mM EDTA. Reactions were incubated at 37°C for 30 min and stopped using RNA loading buffer.
Immunoblotting of eIF2 and phosphoserine 51 eIF2.
Yeast strains were grown to an OD600 of 0.7 in SCD-His. Three 15-ml aliquots were taken per strain, the cells were pelleted, and SCD-His medium, SCD minus all amino acids, or SCD-His plus 50 mM 3-aminotriazole (3-AT) was added to each of the three aliquots, respectively. The cells were incubated, pelleted at 30°C, and rapidly frozen in liquid N2 such that the total incubation time was 15 min. The cells were lysed and protein samples were prepared, electrophoretically separated, and subjected to immunoblot analysis as described previously (3). The phosphospecific eIF2 (Biosource International) and eIF2 (a gift from G. Pavitt, University of Manchester Institute of Science and Technology, Manchester, United Kingdom) antibodies were used for the detection.
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RESULTS |
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and lsm1 mutants maintain translation following glucose removal.
Previously we have found that the removal of glucose from yeast growth medium results in a rapid inhibition of translation initiation (3). This inhibition is characterized by a loss of polysomes and a concomitant increase in 80S monosomes (Fig. 1A). A candidate gene strategy was adopted to test a whole range of strains (either mutated in or overexpressing translation initiation factor and mRNA decay genes) (data not shown). Intriguingly, strains lacking either Dcp1p, a key component of the mRNA decapping enzyme, or Lsm1p, a member of the mRNA decay Lsm complex, maintain polysomal levels following glucose removal (Fig. 1A).
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mutant strain has a doubling time approximately twofold higher than its isogenic wild type (data not shown). In consistency with this observation, the rate of protein synthesis in the presence of glucose for the dcp1 mutant is lower than that seen with the wild type. For both the lsm1 and dcp1 strains, however, it is clear that (following a switch to medium with no glucose) the level of [35S]methionine incorporation is significantly higher than that seen with the appropriate wild-type strains. Indeed the dcp1 mutant strain continues to translate at a rate typical of that seen with glucose-containing medium. This demonstrates that the observed maintenance of polysomes following glucose removal from these mutants is due to sustained protein synthesis and does not occur due to slowed rates of elongation or termination in these strains.
Loss of decapping activity in DCP1 mutants allows the maintenance of translation initiation following glucose removal.
Strains lacking the decapping component Dcp1p have been characterized by their lack of decapping function and a resulting inability to decay mRNAs in a 5' to 3' direction (6). In addition, the original loss-of-function mutation in LSM1 (SPB8) was shown to be partially inhibited for mRNA decapping (7). This is consistent with the partial maintenance of protein synthesis observed following glucose removal in an lsm1 mutant (Fig. 1). To investigate whether a decrease in decapping function was responsible for continued translation following glucose removal, a well-characterized series of DCP1 mutants was assessed (37). Strains with mutations in DCP1 that either have little effect (dcp1-13, dcp1-14, and dcp1-21) or are partial-loss-of-function mutations (dcp1-4) in terms of mRNA decapping (37) show a redistribution of polysomes upon glucose withdrawal typical of that observed for wild-type strains (Fig. 2A). However, dcp1-7 and dcp1-34 mutants, which are known to have a strong loss of mRNA decapping function (37), maintain protein synthesis following glucose withdrawal (Fig. 2A). Hence, where decapping activity is low in the DCP1 mutants, translation is maintained following glucose removal.
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As the inhibition mechanism following glucose removal is as yet unknown, polysome profiles of a dcp1 mutant were examined following amino acid removal. Severe amino acid starvation results in polysomal runoff indicative of an inhibition of translation initiation (41). The translational inhibition following amino acid starvation occurs via a well-described pathway that has not been shown to involve mRNA decay (17). Therefore, polysomal runoff in response to severe amino acid starvation involves eIF2 phosphorylation via Gcn2p kinase; thus, the gcn2 mutant is translationally resistant to this stress (Fig. 2C). Surprisingly, polysomal profiling of a dcp1 mutant shows that in contrast to the results seen with the wild-type strain, active translation is maintained following amino acid removal as well as glucose removal (Fig. 2C). Therefore, rather than a global decapping of mRNA upon either amino acid starvation or glucose removal accounting for the polysomal runoff and hence the translational resistance of the dcp1 strain, it seems more likely that the dcp1 mutant is generally resistant to translational control.
An analysis of mRNA decay mutants following either glucose or amino acid starvation. To further investigate the connection between mRNA decay and the regulation of translation initiation in response to nutritional stress, we analyzed the translational phenotypes of a series of mRNA decay mutants. The mutants used harbored deficiencies in the nonsense-mediated, exosome-mediated, or major 5' to 3' pathway of mRNA decay. Mutants were resuspended in rich medium and medium lacking either glucose or amino acids and were analyzed by polysomal profiling (Fig. 3A). Figure 3B depicts a graph summarizing these data in which polysome maintenance is a measure of the level of polysomes remaining after each stress relative to the level of polysomes present in unstressed cells.
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For mutants specifically deficient in the exosome (e.g., ski2, ski3, and ski6-2) or nonsense-mediated mRNA decay pathways (e.g., upf1), translation was inhibited following either glucose or amino acid withdrawal in a manner similar to that seen with wild-type strains (Fig. 3). However, one exception to this was noted: that of the rrp4-1 mutant. This strain has multiple phenotypes (26) and is analyzed further below.
The lsm1 and dcp1 mutants thus far identified are deficient in the decapping step of the major 5' to 3' pathway of mRNA decay. As deadenylation is a prerequisite to decapping in this decay pathway, deadenylation mutants were also analyzed. For mutants such as the poly(A) nuclease pan2 pan3 mutant or ccr4, the deadenylation mutant, translation was largely inhibited following either glucose or amino acid removal (Fig. 3). Pop2p has also been found as part of the deadenylase complex. In this mutant, partial resistance to the nutritional stresses was observed. This agrees with previously published observations and probably relates more to another function of Pop2p in glucose repression (42). Indeed, previously we have found that mutants which are constitutively derepressed for glucose repression genes are resistant to these stress-induced translational controls (3). Other mRNA decay mutants, affecting steps downstream of deadenylation (such as lsm mutants, pat1, edc1, and dhh1), are at least partially resistant to the translational effects of glucose or amino acid removal. Mutants which directly affect either decapping (e.g., dcp1 and dcp2) or 5' to 3' exonucleolytic digestion of the RNA (e.g., xrn1) are almost completely resistant to these translational stresses. However, mutation of the nucleus-specific LSM8 gene (lsm8-1) showed wild-type levels of translational inhibition. The majority of the mutants that have been identified as resistant or partially resistant to translational stress influence the decapping step of the major mRNA decay pathway. As Xrn1p acts downstream of this step by degrading the body of the mRNA, it is intriguing that an xrn1 mutant is resistant to stress-induced translational regulation. Recently, it has been shown that Xrn1p is found associated with both the Lsm complex and the decapping complex (13). This correlates with recent evidence suggesting that many of the mRNA decay factors colocalize to large complexes called cytoplasmic processing bodies (35). Therefore, mutation of XRN1 is likely to have more complex effects than previously anticipated and may influence upstream stages of the mRNA decay process.
All of the mRNA decay mutants described above that show resistance to translational stress actively deadenylate mRNAs. Therefore, these mutants accumulate mRNAs with reduced lengths of 3' poly(A) tail (19, 38). The poly(A) tail impacts upon translation initiation via Pab1p in the closed loop model of translation initiation (31), possibly by facilitating the recruitment of the 60S large ribosomal subunit (32, 34). Therefore, it is plausible that the deadenylated mRNAs that accumulate in mRNA decay mutants might influence the control of translation initiation. We tested this model in a number of ways. We made use of the poly(A) polymerase mutant pap1-1, which at the nonpermissive temperature should accumulate mRNAs, without poly(A) tails, that get incorporated into polysomes in the cytoplasm (29). However, the translation rate for this mutant following a switch to nonpermissive temperature was already too low to allow assessment of whether this mutant is capable of responding to stress in terms of translation (data not shown). Another approach was to attempt to rescue the control of translation in the mRNA decay mutants by inactivating the deadenylation process. Therefore, we generated deadenylation mutants in the dcp1 background. Both the dcp1 ccr4 mutant strains and the dcp1 pan2 pan3 mutant strains were as resistant to the stress-induced translational inhibition as the dcp1 strain (Fig. 3B). However, it is unclear whether disruption of these nucleases is sufficient to reverse the poly(A) tail deficiency in a dcp1 mutant.
A further argument against the poly(A) deficiency model comes from studies of Pab1p. It seems likely that if there is a function for the poly(A) tail in maintaining the capacity for translational control, then this function would be mediated by Pab1p. Deletion of PAB1 is lethal for S. cerevisiae, but viability can be maintained by a number of suppressor mutations: for example, deletion of pbp1 (spb9) (25). Translation in both the pbp1 strains and pbp1 pab1 strains is regulated normally following glucose and amino acid removal (Fig. 3B). This shows that Pab1p is not required for translational regulation and suggests that the resistance of mRNA decay mutants does not result from an accumulation of deadenylated mRNA transcripts.
Therefore, this comprehensive analysis of many mRNA decay mutants shows that mutants affecting the major pathway of mRNA decay at stages downstream of deadenylation are resistant to certain translational stresses.
Mutation of DCP1 generates resistance to a host of translational controls.
A number of stress conditions have been shown to elicit effects on translational initiation. To further examine how compromised mRNA decay pathways might influence these processes, the dcp1 mutant was subjected to a host of these translational inhibitory stresses. Figure 4A shows that following the addition of rapamycin or lithium (in galactose medium) or the removal of amino acids or glucose, a redistribution of polysomes into the monosome peak is observed for the wild-type strain. In contrast, when the dcp1 mutant was subjected to these stresses no change in the polysome profile was observed.
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subunit of eIF2B. Therefore, we disrupted the DCP1 gene in a butanol-sensitive (BUTS) strain background. Addition of 1-butanol to the parental strain for as little as 10 min leads to a dramatic inhibition of translation initiation, as shown by the change in polysome profile (Fig. 4B). However, in the dcp1 mutant the same stress had little effect on the polysome profile, suggesting that translation is maintained in this mutant (Fig. 4B). Indeed, we have now shown that DCP1 deletion in three different strain backgrounds leads to resistance to translational inhibition. In terms of growth this disruption has background-dependent effects, resulting in quite different growth rates for individual dcp1 strains. However, the translational regulation characteristics and shapes of the polysome profiles across these different backgrounds were indistinguishable (data not shown). Therefore, mutation of the DCP1 gene of the major mRNA decay pathway causes resistance to many different forms of translational stress.
Phosphorylation of eIF2 and decreased ternary complex levels do not inhibit translation initiation in the dcp1 mutant.
Amino acid starvation activates the Gcn2p protein kinase and eIF2 phosphorylation, inhibiting general translation initiation and yet activating the translation of Gcn4p via four upstream ORFs (17). The translational resistance of the dcp1 mutant to amino acid starvation is similar to that of mutants in this pathway with Gcn- phenotypes (17). For example, the gcn2 mutant cannot phosphorylate eIF2 and maintains polysomes following severe amino acid starvation (12) (Fig. 2C). Surprisingly, the dcp1 mutant was capable of phosphorylating eIF2 to an extent similar to that seen with the wild-type strain in response to either severe amino acid starvation or addition of the competitive inhibitor of histidine biosynthesis 3-AT for 15 min (Fig. 5A). The rapid phosphorylation of eIF2 by Gcn2p following amino acid starvation inhibits eIF2B, leading to lower levels of eIF2 GTP , the ternary complex. Translation of the GCN4 mRNA is exquisitely sensitive to changes in ternary complex abundance. Over a period of 6 h following amino acid starvation, there is a gradual increase in Gcn4p protein levels which relies upon this decrease in ternary complex abundance (1). As the level of ternary complex has never been directly assayed, the GCN4-lacZ reporter (containing the GCN4 5' leader that includes the four uORFs) has therefore become an accepted indirect in vivo predictor for ternary complex levels (17). A dcp1 mutant maintained polysomes and protein synthesis for more than 1 h following amino acid starvation (data not shown); therefore, we used a 1-h time point to evaluate GCN4-lacZ levels. In keeping with the results seen with previous analyses, it is clear that amino acid starvation induces GCN4-lacZ approximately two- to threefold in the wild-type strain over this time frame (Fig. 5B). This induction was not observed when a gcn2 mutant in the same strain background was used (data not shown). However, even though in terms of general translation the dcp1 mutant behaved like a gcn mutant, GCN4-lacZ was still induced upon amino acid starvation (Fig. 5B). This induction relies upon the four upstream ORFs, as no induction was observed in a mutant lacking these (Fig. 5B).
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strain than in the wild type (Fig. 5B). The results of Northern analysis of GCN4 mRNA levels suggest that this difference is due to higher levels of the GCN4 mRNA in this mutant, probably resulting from deficient mRNA decay (data not shown).
The induction of GCN4 reporter activity following amino acid starvation in the dcp1 mutant correlates with the fact that unlike the gcn2 mutant, the dcp1 strain is not sensitive to 3-AT, the histidine starvation inducer, in terms of growth (Fig. 5C). Under conditions of severe starvation for amino acids, therefore, a dcp1 mutant strain responds by increasing the phosphorylation of eIF2 and the subsequent levels of Gcn4p. As eIF2 phosphorylation is known to decrease ternary complex levels (leading to an induction of GCN4 translation), these results suggest that the ternary complex levels decrease in a dcp1 mutant strain following amino acid starvation and yet that global protein synthesis is sustained.
Both 60S ribosomal subunit biogenesis mutant and eIF4G overexpression strains are translationally resistant to nutritional stress.
Given the apparent normal response to amino acid starvation in the dcp1 strain (as measured using the Gcn4p regulatory system), the obvious question is why do global rates of translation initiation not decrease following translational stress? It seems that this mutant strain background is somehow capable of maintaining translation even though the levels of ternary complex decrease. This further indicates that in this mutant strain background, the rate of translation initiation is less dependent upon the rate of ternary complex recruitment to the 40S ribosomal subunit.
One possible explanation is that increased levels of other components in the translation initiation process (such as the 40S ribosomal subunit or the eIF4F-bound mRNA) might overcome the decreased ternary complex and thus the translational inhibition by a mass action mechanism. To test this idea we have taken two broad approaches.
First, we have used mutants in the 60S ribosomal biogenesis pathway, reasoning that the excess of 40S ribosomal subunits in these strains could facilitate a level of translational resistance similar to that observed in the mRNA decay mutants. More specifically, Dob1p is a putative ATP-dependent RNA helicase that is involved in biogenesis of the 5.8S rRNA. Mutation of this gene leads to a deficiency in 60S ribosomal subunits and an increase in both the free 40S ribosomal subunits and "half-mers" (10). Half-mers are characteristic peaks which sediment just after each ribosomal peak and represent mRNAs with a 40S subunit that has not yet complexed with the 60S subunit. In contrast to the results seen with a wild-type strain, polysomes are maintained or partially maintained in the dob1-1 mutant following amino acid starvation or glucose starvation, respectively (Fig. 6A). Interestingly, Dob1p interacts with components of the nuclear exosome (including Rrp4p) and is thought to facilitate 5.8S rRNA biogenesis without affecting cytoplasmic 3' to 5' mRNA decay (2, 10). This suggests that the explanation for the translational resistance of the rrp4-1 mutant (Fig. 3B and 6A) lies more in the 60S ribosomal subunit biogenesis defect for this strain rather than in a 3' to 5' mRNA decay defect.
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mutant (which has a 60S subunit joining deficiency) was completely inhibited for translation initiation following glucose removal (data not shown). Therefore it seems more likely that an accumulation of 40S ribosomal subunits can overcome translational inhibition. It is also possible that general alterations to the relative levels of ribosomal subunits could allow resistance to translational stress. To investigate this possibility we analyzed a 40S subunit biogenesis mutant, fal1-1, which accumulates free 60S subunits (21). In contrast to the results seen with 60S biogenesis mutants, fal1-1 was largely inhibited following either amino acid or glucose withdrawal (Fig. 6B). Therefore, the accumulation of 60S subunits (in 40S biogenesis mutants) did not give the same resistance to translational stress as the accumulation of 40S subunits (in 60S biogenesis mutants).
In a second approach designed to test whether excess downstream components can increase the level of preinitiation complex formation (especially under conditions of limiting ternary complex), we overexpressed TIF4632. This encodes eIF4G, the least abundant translation initiation factor in the eIF4F cap binding complex (46). We reasoned that accumulation of the eIF4F complex in a wild-type strain might alter the existing cap competition between Dcp1p and eIF4F, thereby allowing ultimately greater concentrations of mRNA/eIF4F complexes. Therefore, this scenario may mimic the results seen with a dcp1 mutant. As shown in Fig. 6C and in contrast to the results seen with a strain containing plasmid vector alone, overexpression of eIF4G from a plasmid vector allows continued translation following both amino acid and glucose starvation. Clearly this effect is less substantial than that seen with the dcp1 mutant, but it does suggest a plausible explanation for the continued synthesis of proteins in mRNA decay mutants under stress conditions. That is, an excess of downstream components of the translation pathway can counter the stress-dependent inhibition of translation initiation. Ultimately, these results suggest that there is a delicate stoichiometrical balance of translational, ribosomal, and mRNA decay complexes which gives cells the capacity to rapidly control translation initiation.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this paper, we describe studies that approach the problem of translation and mRNA decay coordination from a different angle. Our goal was to determine how the various pathways of mRNA decay might impact upon the control of translation initiation. We show that specific mutants in the major 5' to 3' pathway of mRNA decay (most notably a dcp1 mutant strain) are almost completely resistant to stress-induced translational inhibitory pathways. To further evaluate the mechanism involved in this connection between translational control and mRNA decay, we studied the well-characterized amino acid starvation pathway of global translational inhibition and Gcn4p induction. We show that even though the dcp1 strain is translationally resistant to severe amino acid starvation, this mutant does not bear other hallmarks of a gcn mutant. For instance, this mutant is capable of phosphorylating eIF2 and activating GCN4 translation. As eIF2 phosphorylation is known to decrease ternary complex levels and consequently activate GCN4 translation, these results suggest that (in terms of global translation initiation) dcp1 mutants are resistant to decreases in ternary complex abundance.
The fact that we have identified mutants such as dcp1 that are resistant to almost all forms of translational stress in yeast might mean that these translational regulatory responses have a common mechanism. For amino acid starvation, the pathway of translational regulation is well characterized and ultimately impacts upon eIF2B activity and ternary complex levels (17). It seems likely that fusel alcohols affect translation initiation via a similar mechanism except that this regulation does not rely on eIF2 kinases but instead probably affects the activity of eIF2B more directly (4). Rapamycin has recently been shown to inhibit translation initiation by activating the yeast eIF2 kinase, Gcn2p, and therefore also inhibits eIF2B activity, leading to lower ternary complex levels (8). All three of these translational stresses lead to increased translation of GCN4, which demonstrates the reduction in ternary complex levels (4, 8, 17, 22, 44). Glucose starvation also leads to increased translation of GCN4 (48). However, the kinetics of this increased GCN4 translation are relatively slow compared with the results seen with other stresses such as amino acid starvation and fusel alcohol addition (data not shown). This suggests that the primary cause of the translational inhibition following glucose removal may not be the decreased ternary complex levels. The other stress that we have used in this study was the addition of lithium when yeast was grown in galactose medium. For this stress it has been found that overexpression of the gene for eIF4A suppresses the translational inhibitory effects (28). In addition, we have measured GCN4-lacZ reporter activity; according to the results seen with this indirect assay, lithium addition does not decrease ternary complex levels (data not shown). Overall it seems that even though mRNA decay mutants are resistant to many forms of translational stress, it is likely that these stresses affect translation via distinct mechanisms.
Translational control via eIF2 kinases is an evolutionarily conserved response to stress. For example, four eIF2 kinases have been identified in mammalian cells: HRI (which responds to the depletion of heme), PKR (which is activated during viral infection), PEK/PERK (which responds to misfolded proteins within the lumen of the endoplasmic reticulum), and mGCN2 (which responds to nutrient availability). All of these kinases target the guanine nucleotide exchange function of eIF2B, highlighting the role of eIF2B and the subsequent regulation of ternary complex levels as a major control point of protein synthesis (reviewed in reference 11). Furthermore, inherited mutations in eIF2B have recently been shown to cause the fatal human brain disease childhood ataxia with central nervous system hypomyelination (45). Therefore, the ternary complex level within eukaryotic cells is considered a key rate-limiting determinant for translation initiation. In this study we have identified specific yeast mutants where cells can maintain protein synthesis even though ternary complex levels are decreased. In addition, these mutants are resistant to translational stresses that have no obvious impact upon ternary complex levels (as described above). One possible explanation for these observations is that a specific mRNA(s) might be upregulated in mRNA decay mutants and have direct effects upon translational regulation. However, it seems unlikely that changes in expression of specific mRNAs can generate resistance to so many divergent translational stresses that are thought to act via distinct mechanisms. Therefore, we favor a model in which the mRNA decay mutants have global effects on the gene expression profile of cells. Indeed, these mutants have globally relevant characteristics which may affect translation initiation and prevent translation regulation. These include an increased abundance of mRNA and accumulation of mRNA decay intermediates (19).
A model that has been proposed to explain the connection between translation initiation and mRNA decay is the cap competition model. In this scenario, the lack of decapping in a dcp1 mutant would allow an accumulation of translationally competent or eIF4F-bound mRNA (33). Here we have shown that stabilization of the eIF4F complex in a wild-type strain reduces the extent of translational regulation following stress. It seems that decreasing either the activity of Dcp1p (Fig. 2) or its access to the 5' cap (Fig. 6C) allows resistance to reduced levels of ternary complex and to translational stresses considered to act via alternate mechanisms. We favor a model whereby an accumulation of eIF4F bound mRNA might globally increase the recruitment rate of 43S complexes by mass action, therefore negating the effect of changes in ternary complex level (Fig. 7).
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The results presented in this study graphically demonstrate the intimate relationship that exists between protein synthesis, mRNA decay, and ribosomal biogenesis. Although these connections have been implicated previously in a number of studies, this study represents the first analysis in which the global control of translation in response to stress has been added to the equation. Surprisingly, mRNA decay mutants can maintain translation under stress even though ternary complex levels appear to decrease normally. As regulation of ternary complex is a highly conserved mechanism of translational control, this may have wide ranging implications. Our interpretation of these results is that there is a delicate balance in the stoichiometry of different translation initiation components (such as MFC, 40S ribosomes, and mRNA) and that the maintenance of this equilibrium is critical in preserving the cell's capacity to globally control protein synthesis.
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ACKNOWLEDGMENTS |
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We thank G. Pavitt, C. Grant, A. Johnson, C. Wilutz, S. Peltz, S. Butler, and P. Linder for reagents and advice. We especially thank R. Parker for his generous donation of yeast strains and plasmids, without which this work would not have been possible.
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FOOTNOTES |
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