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Corresponding author: Stewart Shuman, Molecular Biology Program, Sloan-Kettering Institute, 1275 York Ave., New York, NY 10021., s-shuman{at}ski.mskcc.org (E-mail)
Communicating editor: A. P. MITCHELL
 | ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Here we present a genetic analysis of the yeast cap-methylating enzyme Abd1p. To identify individual amino acids required for Abd1p function, we introduced alanine mutations at 35 positions of the 436-amino acid yeast protein. Two new recessive lethal mutations, F256A and Y330A, were identified. Alleles F256L and Y256L were viable, suggesting that hydrophobic residues at these positions sufficed for Abd1p function. Conservative mutations of Asp-178 established that an acidic moiety is essential at this position (i.e., D178E was viable whereas D178N was not). Phe-256, Tyr-330, and Asp-178 are conserved in all known cellular cap methyltransferases. We isolated temperature-sensitive abd1 alleles and found that abd1-ts cells display a rapid shut-off of protein synthesis upon shift to the restrictive temperature, without wholesale reduction in steady-state mRNA levels. These in vivo results are consistent with classical biochemical studies showing a requirement for the cap methyl group in cap-dependent translation. We explored the issue of how cap methylation might be regulated in vivo by conducting a genetic screen for high-copy suppressors of the ts growth defect of abd1 mutants. The identification of the yeast genes SAM2 and SAM1, which encode AdoMet synthase, as abd1 suppressors suggests that Abd1p function can be modulated by changes in the concentration of its substrate AdoMet. We also identified the ubiquitin conjugating enzyme Cdc34p as a high-copy abd1 suppressor. We show that mutations of Cdc34p that affect its ubiquitin conjugation activity or its capacity to interact with the E3-SCF complex abrogate its abd1 suppressor function. Moreover, the growth defect of abd1 mutants is exacerbated by cdc34-2. These findings suggest a novel role for Cdc34p in gene expression and engender a model whereby cap methylation or cap utilization is negatively regulated by a factor that is degraded when Cdc34p is overexpressed.
THE m7GpppN cap of eukaryotic mRNA is synthesized by three enzymatic reactions: (i) the 5' triphosphate end of nascent pre-mRNA is hydrolyzed to a diphosphate by RNA triphosphatase; (ii) the diphosphate end is capped with GMP by GTP:RNA guanylyltransferase; and (iii) the GpppN structure is methylated by AdoMet:RNA-(guanine-N7)-methyltransferase. The capping apparatus of the budding yeast Saccharomyces cerevisiae consists of separate triphosphatase (Cet1p), guanylyltransferase (Ceg1p), and methyltransferase (Abd1p) gene products (
The role of the cap in yeast mRNA metabolism has been addressed by studying the consequences of genetic inactivation of the cap guanylyltransferase using temperature-sensitive ceg1 mutants. Failure to guanylate yeast mRNAs results in their accelerated decay through the agency of the 5' exoribonuclease Xrn1p (
We have initiated a genetic analysis of the cap-methylating enzyme Abd1p. Abd1p is a 436-amino acid polypeptide that catalyzes the transfer of a methyl group from AdoMet to the GpppN terminus of RNA to produce the m7GpppN-terminated RNA and AdoHcy. Deletion analysis has shown that the N-terminal 109 amino acids and the C-terminal 10 amino acids are dispensable for Abd1p function in vivo. Six individual amino acid side chains required for methyltransferase activity in vitro and in vivo have been identified through alanine scanning of 26 positions (
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Delineation of structure-activity relationships, although valuable, does not instruct us regarding the role of Abd1p in gene expression and whether cap methylation may be regulated in vivo. To address these issues, we have isolated a collection of temperature-sensitive (ts) abd1 alleles and found that abd1-ts cells display a rapid shut-off of protein synthesis at the restrictive temperature, without a global decline in mRNA levels. These genetic findings are consistent with the classic biochemical studies of Shatkin and colleagues showing a requirement for the cap methyl group in cap-dependent translation in vitro (
| MATERIALS AND METHODS |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Site-directed mutagenesis and test of ABD1 function by plasmid shuffle:
Missense mutations in the ABD1 gene were programmed by synthetic oligonucleotides as described (
yeast strain YBS10 (MAT
leu2 ura3 lys2 trp1 his3 abd1::LEU2 p360-ABD1). YBS10 contains an extrachromosomal copy of ABD1 on a CEN URA3 plasmid (p360-ABD1). Trp+ transformants were selected at 30° on medium lacking tryptophan. Individual colonies were patched on medium lacking tryptophan. Cells from each patch were then streaked on medium containing 0.75 mg/ml of 5-fluoroorotic acid (5-FOA). The plates were incubated at 30°. Alleles that supported the formation of wild-type-sized colonies after 3 days were scored as +++. Lethal mutations were those that formed no colonies after 7 days on 5-FOA (scored as -).
Isolation of abd1-ts mutants:
The ABD1 gene was amplified in vitro by Taq DNA polymerase. The standard PCR reaction mixture was modified to contain a reduced concentration of dATP (0.2 mM) relative to the other three dNTPs (each at 1 mM). Plasmid pET-His-ABD1 (. After amplification in vivo, a pooled plasmid library was prepared from ~24,000 ampicillin-resistant colonies harvested directly from the agar plates. This DNA library was transformed into the abd1 strain YBS6 (MAT leu2 ura3 lys2 trp1 his3 abd1::hisG p360-ABD1; , and retested for the conditional growth phenotype by plasmid shuffle. In this way, we obtained a collection of 22 abd1-ts mutants. Of these mutant abd1 clones, 10 were mapped at the nucleotide level by DNA sequencing. Multiple missense mutations in the abd1 gene were noted in every case (see Fig 2). The 10 CEN TRP1 abd1-ts plasmids were transformed into YBS10. Trp+ transformants were plated on 5-FOA to eliminate the CEN URA3 ABD1 plasmid.
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Temperature shift and measurement of protein synthesis by pulse-labeling:
Yeast cells were grown in supplemented minimal medium lacking methionine (SC-Met) at 25°. One-half of each culture was removed and shifted to 37° by rapid addition of an equal volume of SC-Met medium preheated to 52°. Incubation was continued thereafter at 37°. The remaining half of each culture was mixed with an equal volume of SC-Met medium at 25°. At 0, 20, 40, 60, and 120 min post-shift, 2-ml aliquots of the cultures were removed, mixed with 8 µCi of [35S]methionine (1175 Ci/mmol; Dupont NEN), and incubated for 10 min at 37° or 25°. Incorporation of labeled methionine was measured by hot trichloroacetic acid (TCA) precipitation (
SDS-PAGE analysis of pulse-labeled polypeptides:
Pulse-labeling was performed as described above. After quenching the culture and heating in TCA, the acid-insoluble material was recovered by centrifugation at 13,000 rpm for 20 min at 4°. The pellets were washed with 1% TCA, followed by three cycles of washing with ethyl ether. The samples were dried in a vacuum centrifuge and then resuspended in SDS-PAGE sample buffer. The samples were heated for 5 min at 95° and then electrophoresed through a 10% polyacrylamide gel containing 0.1% SDS. The gels were dried and autoradiographed.
RNA analysis:
Yeast cells were grown in SC-Met at 25° to an A600 of 0.2–0.4. After withdrawing an aliquot (time zero), an equal volume of SC-Met medium preheated to 52° was added to the rest of the culture to adjust the temperature abruptly to 37°. The cultures were transferred to a 37° incubator. Aliquots were removed at 1, 2, and 3 hr after temperature shift. The cells were harvested by centrifugation and stored as cell pellets at -80°. RNA was isolated from thawed resuspended cells by extraction with hot phenol (
RNase protection:
RNase protection analysis of poly(A)+ ABD1, SAM2, and CDC34 RNA was performed using an RNase protection kit according to the instructions of the vendor (Boehringer Mannheim). [32P]GMP-labeled antisense RNA probes were synthesized in vitro by T7 or SP6 RNA polymerase from the following pTOPO-Blunt-based plasmid templates: (i) pAbd1 containing a 137-bp ABD1 insert from nucleotides +44 to +151 [+1 being the start of the ABD1 open reading frame (ORF)], (ii) pSam2 containing a 139-bp SAM2 insert from nucleotides +39 to +169, and (iii) pCdc34 containing a 163-bp CDC34 insert from nucleotides +51 to +213. Total RNA was isolated from cells grown at 30° in SC-Ura medium to an A600 of 0.7–0.8. Poly(A)+ RNA was isolated by oligo(dT) affinity chromatography using Dynabeads-Oligo(dT)25 according to the instructions of the vendor (Dynal). Poly(A)+ RNA samples (1 µg) were annealed in separate reactions to the 207-nucleotide ABD1 probe, the 242-nucleotide SAM2 probe, or the 261-nucleotide CDC34 probe. The RNase digests were analyzed by electrophoresis through a 6% polyacrylamide gel containing 7 M urea in TBE. 32P-Labeled DNA restriction fragments (pBR322 MspI digest) served as size markers.
| RESULTS |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Alanine-scanning mutagenesis of yeast cap methyltransferase:
Prior mutational analysis of S. cerevisiae Abd1p by alanine scanning led to the identification of six amino acid residues that are essential for Abd1p function in vivo; these are denoted by arrows above the Abd1p sequence in Fig 1. Here, we introduced alanine substitutions at 35 additional positions of Abd1p (indicated by dots in Fig 1). The residues to be mutated were chosen on the basis of one or more of the following criteria: (i) conservation in other cellular cap methyltransferases; (ii) proximity to residues known to be essential for Abd1p function, especially those within the putative AdoMet-binding motif I (Fig 1); and (iii) the potential for ionic, polar, or hydrophobic side-chain interactions with AdoMet or the GpppN cap substrates. We particularly targeted conserved aromatic residues (e.g., Tyr-207, Phe-250, Phe-256, Phe-279, Trp-305, Phe-314, Tyr-330, Tyr-416, Phe-419, and Phe-421) in light of recent crystallographic findings for several proteins involved in cap formation or cap recognition that the protein-bound cap guanine is stabilized by base-stacking interactions with aromatic amino acids (
The 33 singly mutated ABD1-Ala alleles and 1 doubly mutated allele (ABD1-N150A/N151A) were cloned into CEN TRP1 vectors under the control of the ABD1 promoter, transformed into an abd1 strain containing a CEN ABD1 URA3 plasmid, and then tested for function by plasmid shuffle. Of the mutants, 32 were functional in vivo; i.e., the Trp+ transformants formed wild-type-sized colonies on medium containing 5-FOA (Table 1). All of the 31 viable single Ala mutants formed wild-type-sized colonies on YPD medium at 18°, 25°, 30°, and 37° (not shown). The N150A-N151A double mutant displayed a slow growth phenotype at 18° (not shown). Two of the mutants, F256A and Y330A, failed to grow on 5-FOA (Table 1). The lethal abd1-F256A or abd1-Y330A alleles did not exert a dominant negative effect on cell growth when introduced into a wild-type ABD1 strain. The F256A and Y330A proteins were completely insoluble when expressed in bacteria (not shown); thus, we were unable to assess the mutational effects on cap methyltransferase activity in vitro. The equivalent alanine mutants of human cap methyltransferase Hcm1p (F291A and F354A) are defective in cap methylation in vitro (
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Structure-activity relationships at essential residues of Abd1p:
Alanine substitution, which eliminates the side chain beyond the ß-carbon, provides an indication of the importance of the side chain for protein function, but does not reveal the properties of the missing side chain that are important. This was addressed by introducing conservative substitutions at the newly defined essential residues, Phe-256 and Tyr-330, and at position Asp-178, which was shown previously by alanine scanning to be important for Abd1p function (
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Temperature-sensitive mutants of yeast cap methyltransferase:
To examine the role of the cap methyl group in vivo, we isolated a collection of temperature-sensitive abd1 alleles from a pool of mutagenized ABD1 clones. To do this, we performed PCR amplification of the ABD1 gene under reaction conditions designed to promote nucleotide misincorporation by Taq polymerase. The PCR products were restricted and cloned into a CEN TRP1 expression plasmid, so as to replace the wild-type ABD1 gene (under the control of its natural promoter) with the PCR-mutagenized DNA. After amplification in vivo in E. coli, a pooled library of CEN TRP1 DNAs was transformed into yeast containing wild-type ABD1 on a URA3 plasmid. After selection for Trp+ growth at 25°, followed by selection on 5-FOA to eliminate the wild-type allele, individual isolates were screened for growth at 37°. We obtained a collection of abd1-ts isolates that were temperature sensitive, i.e., they readily formed colonies at 25°, but not at 37°. Plasmid DNA was recovered from all conditional isolates and retested by plasmid shuffle for the ts phenotype.
A total of 10 abd1 mutant DNA clones were sequenced. Multiple missense mutations in the abd1 gene were noted in every case (Fig 2A). Although several of the missense changes were encountered more than once, the 10 abd1-ts alleles represented independent isolates, insofar as each mutant clone contains unique mutations not found in the other mutant clones. The 67 missense mutations were unevenly distributed within the 436-amino acid Abd1 polypeptide (Fig 2B). Mutations were underrepresented in the N-terminal segment, which is nonessential for cap methyltransferase activity. Mutations were absent from the essential AdoMet binding motif (Fig 2B). Most of the mutations were located in the carboxyl portion of the enzyme. We surmise that none of the 60 amino acids that were mutated can be considered strictly essential for Abd1p function in vivo, because every allele supported cell growth at 25°. No effort was made to determine which, if any, of the many mutations might be singly responsible for the conditional phenotypes. All 10 abd1-ts alleles were recessive to wild-type ABD1; i.e., growth at 37° could be restored to the abd1-ts strains by transforming them with a CEN URA3 plasmid bearing the wild-type ABD1 gene (not shown). We observed that the abd1-ts alleles had different thresholds for thermal inactivation in vivo. Most of the mutants were sick or dead even at 34° (see Fig 6).
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Conditional inactivation of ABD1 inhibits protein synthesis in vivo:
Although the cap methyltransferase activity of Abd1p is required for cell viability, the specific roles played by the cap methyl group in vivo are still unclear. We therefore conducted a preliminary phenotypic characterization of two abd1-ts mutants, abd1-8 and abd1-5, both of which grow normally at 25°, but display a tight growth arrest at 37°. We first analyzed the rate of protein synthesis. In the experiment shown in Fig 3, yeast cells were grown in methionine-free medium at 25°. The cultures were then split such that one-half was mixed with an equal volume of 25° medium and maintained thereafter at permissive temperature, whereas the other half of the culture was adjusted immediately to 37° and maintained thereafter at nonpermissive temperature. Aliquots of the 25° and 37° cultures were exposed to [35S]methionine for a 10-min pulse at the time the cultures were split (time zero) and at various time intervals thereafter. Protein synthesis was quantitated by the incorporation of [35S]methionine into hot-TCA-insoluble material during the 10-min pulse. The level of incorporation at each time point was normalized relative to the incorporation at time zero and plotted as a function of the time of initiation of the pulse. The data shown in the figure represent the average of three separate experiments (with error bars shown). It can be readily appreciated that the rates of protein synthesis by ABD1, abd1-8, and abd1-5 cells were unaffected by splitting the cultures at 25° (Fig 3B). Protein synthesis by the wild-type cells was unperturbed after shift to 37°, whereas the rates of protein synthesis by abd1-8 and abd1-5 cells declined sharply to 28% of the time zero values by 20 min post-shift to 37°. At 40–120 min post-shift, methionine incorporation was reduced to 17% of the time zero value (or 12% of the incorporation of ABD1 cells after 2 hr at 37°; Fig 3A). The distribution of pulse-labeled polypeptides was analyzed by SDS-PAGE (Fig 3C). The polypeptide profile was qualitatively and quantitatively unchanged when wild-type ABD1 cells were shifted to 37°. In contrast, abd1-8 and abd1-5 cells experienced a progressive and general inhibition of polypeptide synthesis over 2 hr at 37°.
We next performed a Northern analysis of RNA isolated from ABD1 and abd1-5 cells before and after shift to 37°. The results indicated that steady-state levels of four different mRNAs were either unaffected or modestly reduced at the restrictive temperature (Fig 4). GCN4 mRNA abundance was unaffected; i.e., the level of GCN4 transcript at 3 hr post-shift (as percentage of the initial level at time zero) was 83% for wild-type ABD1 cells compared to 93% for abd1-5 cells. ACT1 and PGK1 mRNA levels were stable after shift of ABD1 cells to 37°, whereas in abd1-5 cells, ACT1 and PGK1 RNA levels at 3 hr post-shift were 39 and 31%, respectively, of the pre-shift values. U3 snRNA levels were not altered significantly in abd1-5 cells after the shift to 37°.
With respect to pre-mRNA processing, we did not detect an increase in the level of unspliced ACT1 precursor in the abd1-5 mutant after shift to 37°. However, we noted consistently that the relative amounts of unspliced pre-CYH2 transcript vs. mature CYH2 mRNA [expressed as pre-CYH2/(pre-CYH2 + CYH2)] was increased in abd1-5 cells at the permissive temperature. In ABD1 cells grown at 25°, pre-CYH2 RNA constituted 9% of the total, whereas in abd1-5 cells at 25°, the unspliced precursor accounted for 27% of total CYH2 transcript (Fig 4). The distribution of spliced vs. unspliced CYH2 RNA did not change significantly after shift to 37°. In ABD1 cells at 37°, pre-CYH2 was 12% of the total. In abd1-5 cells at 1, 2, and 3 hr post-shift to 37°, pre-CYH2 was 28–32% of the total. Similar increases in pre-CYH2 at permissive and restrictive temperatures were observed for abd1-8 cells (not shown). Thus, the increase in pre-CYH2 in abd1 cells at permissive temperature (relative to ABD1 cells) was not exacerbated by the shift to the restrictive temperature. This suggests that defective splicing of CYH2 RNA is not the cause of the ts growth defect in the abd1 mutants. Rather, our experiments point to defective protein synthesis as the limiting transaction. This is consistent with long-held views about the requirement for the cap methyl group in cap-dependent translation initiation (
Isolation of multicopy suppressors of ceg1-ts mutations:
We exploited the newly isolated conditional mutants of yeast cap methyltransferase to identify multicopy suppressors of the abd1-ts growth defect, the rationale being that such suppressor genes might encode proteins that either interact physically or functionally with the cap methyltransferase or else impact on cap-dependent RNA transactions in vivo.
We tested whether the CEG1 gene encoding the guanylyltransferase component of the yeast capping apparatus could act as a dosage-dependent abd1 suppressor. A 2µ URA3 plasmid containing CEG1 was transformed into three abd1 mutant strains (abd1-42, abd1-16, and abd1-37) and the Ura+ cells selected at 25° were tested for growth at 34° and 37°. We found that none of these abd1-ts alleles was suppressed by 2µ CEG1 (not shown). These findings are consistent with the earlier observations that Abd1p isolated from yeast cell extracts is a monomeric enzyme (
In light of the above findings suggesting that genetic interactions of the triphosphatase and guanylyltransferase do not overlap with the cap methyltransferase, we initiated a screen for novel genes that would act as high-copy suppressors of conditional abd1 mutations. The screen entailed transformation of several of the abd1-ts strains with a 2µ plasmid-based wild-type genomic DNA library and selection for Ura+ colonies that grew at 34°. We analyzed at least 10 positives for each of the abd1-ts strains that were transformed with the 2µ library. Plasmid DNA was recovered from individual yeast colonies and transformed into E. coli. Diagnostic restriction enzyme digestion of the plasmids recovered from bacteria revealed whether wild-type ABD1 had been selected. Candidate suppressors that did not contain the ABD1 gene were retransformed into the ts strain from which they were originally isolated and tested for growth at 34°. Four genomic clones retested faithfully. Restriction mapping of the genomic inserts revealed that these four clones derived from two distinct genetic loci, which we provisionally named CMS1 and CMS2 (CMS = cap methyltransferase suppressor). CMS1 was isolated in abd1-15. CMS2 was recovered three times, twice in abd1-37 and once in abd1-35. Analysis of the two suppressor genes is presented below.
Suppression of cap methyltransferase mutations by AdoMet synthase:
DNA sequencing of the borders of the genomic insert in the 2µ CMS1 plasmid revealed that the suppressor gene was located on chromosome IV between coordinates 1452295 and 1461657. This 9.4-kbp region includes three known genes, SAM2 (AdoMet synthase), LPP1 (lipid phosphate synthase), and PSP1 (a high-copy suppressor of mutations in DNA polymerase), along with two ORFs of unknown function (YDR504C and YDR506C) and a partial ORF of unknown function (YDR501W). Our attention focused immediately on SAM2, which is one of two yeast genes that encodes AdoMet synthase, the enzyme that forms AdoMet from methionine and ATP (
Final identification of the suppressor locus was achieved by cloning the SAM2 ORF into a 2µ URA3 yeast vector in which SAM2 expression is driven by the constitutive TPI1 promoter. Suppression of the abd1-8 mutation by the 2µ TPI-SAM2 construct is shown in Fig 5. Serial 10-fold dilutions of abd1-8 cells were plated at 25° and 34°. abd1-8 cells transformed with the 2µ vector grew at 25°, but not at 34°; cells transformed with a wild-type ABD1 gene grew well at both temperatures. The 2µ SAM2 clone restored growth at 34° (Fig 5) and also improved growth at 37° (not shown). The efficacy of high-copy suppression by SAM2 was somewhat greater when expression was driven by the TPI1 promoter than when its expression was under the control of its natural promoter (not shown); we presume this reflects the strength of the TPI1 promoter.
The 2µ URA3 TPI-SAM2 plasmid did not display strict allele specificity, but rather was capable of suppressing all of the abd1-ts alleles, albeit not to the same extent. Some of the abd1 alleles were restored to growth at 34° and 37°, whereas others (e.g., the most severely affected allele abd1-20) were suppressed only at the lower restrictive temperatures of 30°, 32°, or 34° (not shown). Thus, the strength of suppression correlated roughly with the severity of the ts phenotype.
To exclude the possibility that overexpression of AdoMet synthase was genetically bypassing ABD1, we constructed a 2µ TRP1 TPI-SAM2 plasmid and transformed it into a yeast strain in which the chromosomal ABD1 gene is deleted and growth is contingent on maintenance of a CEN URA3 ABD1 plasmid. The 2µ TRP1 TPI-SAM2 transformants were unable to form colonies on medium containing 5-FOA (not shown); hence SAM2 overexpression is not bypassing the requirement for cap methyltransferase for yeast cell growth.
The S. cerevisiae SAM1 gene encodes a second isozyme of AdoMet synthase; the amino acid sequence of Sam1p is 92% identical to that of Sam2p (
Suppression of cap methyltransferase mutations by exogenous AdoMet:
The simplest explanation for suppression of abd1 mutations by overexpression of AdoMet synthase is that elevated intracellular levels of the methyl donor either stimulate the activity of the mutant Abd1p enzymes or stabilize the mutant Abd1p proteins at the restrictive temperature. A more elaborate explanation would be that AdoMet synthase interacts directly with Abd1p. If the former model is correct, then it should be possible to suppress the abd1-ts mutations by an alternative route that provides AdoMet without increasing the levels of AdoMet synthase. S. cerevisiae can actively take up AdoMet from the medium; moreover, it has been reported that exogenous AdoMet lowers the expression of SAM1 and SAM2 in strains that are wild type for both AdoMet synthase loci (
Suppression of cap methyltransferase mutations by CDC34:
CMS2 was isolated three times in the high-copy suppressor screen. Sequencing of the margins of the genomic inserts in the three different 2µ CMS2 plasmids revealed that the suppressor gene was located on chromosome IV. The three inserts extended from genome coordinates 554800 to 567450, 560860 to 567450, and 551880 to 567081, respectively. The 6.2-kbp overlap region includes one known gene, CDC34 (an essential E2 ubiquitin conjugating enzyme), along with two ORFs of unknown function (YDR055W and YDR056C) and a partial ORF of unknown function (YDR057W). Restriction fragments of the inserts were subcloned into YEp24 and retested for abd1 suppression. Suppression was observed with a 2.44-kbp fragment that contained CDC34 (not shown).
Definitive identification of the suppressor locus was accomplished by cloning the 295-amino acid CDC34 ORF into a 2µ URA3 vector in which CDC34 expression is driven by the TPI1 promoter. Suppression of the abd1-8 mutation by CDC34(1-295) is shown in Fig 7. abd1-8 cells transformed with the 2µ vector grew at 25°, but not at 34°; cells transformed with a wild-type ABD1 gene grew well at both temperatures. The 2µ CDC34(1-295) clone restored growth at 34° (Fig 7) and also at 37° (not shown).
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CDC34 was capable in high copy of suppressing all of the abd1-ts alleles. As with SAM2, the strength of suppression by CDC34 correlated with the severity of the ts phenotype of the individual mutants. A 2µ TRP1 TPI-CDC34 plasmid was unable to confer growth of an abd1 strain on medium containing 5-FOA. Thus, CDC34 overexpression is not bypassing the requirement for ABD1.
The ubiquitin conjugating activity of Cdc34p is essential for abd1 suppression:
Cdc34p is an E2 ubiquitin conjugating enzyme that is critical for proper cell cycle progression. Cdc34p catalyzes the ubiquitination of Sic1p, which is an inhibitor of the Cdc28p-cyclin protein kinase complexes that are required for the G1-S transition. Sic1p ubiquitination results in its degradation and consequent activation of the Cdc28p-cyclin protein kinase. Cdc34p functions in conjunction with an E3 complex, SCF, containing Cdc53p, Skp1p, and one of several F-box proteins (e.g., Cdc4p, Grr1p, or Met30p;
The biochemical properties and domain structure of Cdc34p have been analyzed in considerable detail. Ubiquitin transfer by E2 enzymes entails the formation of a covalent thiol ester intermediate between an E2 cysteine (Cys-95 in the case of Cdc34p) and the C-terminal carboxyl moiety of ubiquitin. Replacement of the cysteine by alanine abolishes activity, whereas replacement by serine permits the formation of an E3 ubiquitin acyl intermediate, but inhibits subsequent transfer of ubiquitin to the target protein (
Cdc34p catalyzes both intramolecular and intermolecular ubiquitin transfer reactions (
An N-terminal segment from residues 1 to 209 is sufficient for Cdc34p function in vivo (
Suppression of abd1 by AdoMet synthase and Cdc34p is independent of the ABD1 promoter:
Analyses of mutational effects on Abd1p function in vivo and in vitro have indicated that yeast cell growth is dependent on a threshold level of cap methyltransferase activity (
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This hypothesis is supported by RNase protection analysis of the steady-state levels of poly(A)+ ABD1 mRNA in cells that overexpress AdoMet synthase or Cdc34p (Fig 9). Whereas the basal level of ABD1 transcript (Fig 9A, lane 3) was increased dramatically by the introduction of ABD1 on a multicopy plasmid (Fig 9A, lane 2), the introduction of multicopy plasmids containing SAM2 (Fig 9A, lane 4) or CDC34 (Fig 9A, lane 5) elicited no change in the abundance of ABD1 mRNA. Control RNase protection experiments confirmed that the steady-state levels of CDC34 and SAM2 mRNAs were themselves increased by increased CDC34 and SAM2 gene dosage (Fig 9B, lane 5, and Fig 9C, lane 4).
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The results in Fig 9 also reveal a lack of crosstalk at the transcriptional level between AdoMet synthase and Cdc34p. Overexpression of AdoMet synthase had no effect on the steady-state level of CDC34 mRNA (Fig 9B, compare lanes 3 and 4). More important, the overexpression of CDC34 did not increase the level of mRNA encoding AdoMet synthase (Fig 9C, compare lanes 3 and 5). (The SAM2 antisense probe will detect both SAM1 and SAM2 mRNAs.) We infer that the suppression of abd1 mutations by overexpression of CDC34 does not occur via upregulation of SAM gene expression.
Phenotypic synergy of abd1 and cdc34 mutations:
If abd1 suppression by Cdc34p overexpression reflects a role of Cdc34p in cap methylation or m7G cap-dependent pathways, then we might expect a partial loss-of-function mutation of Cdc34p to exacerbate the growth phenotype of abd1 cells. To test for mutational synergy, we constructed isogenic abd1 CDC34 and abd1 cdc34-2 strains containing CEN plasmid-borne ABD1 or abd1 alleles. cdc34-2 cells display a temperature-sensitive growth phenotype caused by a single Cdc34p missense mutation of Gly-58 to Arg (
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| DISCUSSION |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Structure-function relationships in cellular cap methyltransferases:
Alanine mutations have now been targeted to 61 positions of Abd1p located within the C-terminal catalytic domain. The eight residues found to be essential for Abd1p function (Glu-170, Gly-174, Asp-178, Asp-194, Arg-206, Tyr-254, Phe-256, and Tyr-330) are conserved in the cap methyltransferases from other fungi, including S. pombe Pcm1p and C. albicans Ccm1p (Fig 1). cDNAs encoding the human cap methyltransferase have been identified recently ( mutation (
What does mutational analysis tell us about cap methyltransferase function? It is remarkable that alanine substitutions at 53/61 positions had no effect on Abd1p function in vivo, even though most of the nonessential residues are conserved among the cellular cap methyltransferases (Fig 1). The eight essential amino acid residues are candidates to comprise the active site. But what is their role in substrate recognition or catalysis? Essential residues Glu-170, Gly-174, and Asp-178 are located within or immediately flanking a putative AdoMet binding motif VL(D/E) x G x G x G, which is conserved among AdoMet-requiring methyltransferases (
A database search by
Sequence gazing indicates that essential Abd1p residue Asp-178 immediately flanking motif I is found in only 3 out of 31 Abd1p-like proteins (
Inactivation of Abd1p affects yeast protein synthesis:
The shut-off of protein synthesis in abd1-ts cells is detectable at 20 min post-shift to 37° and fully manifest at 40 min post-shift. Inactivation of Ceg1p elicits the same decrement in the rate of yeast protein synthesis (
The in vivo half-lives of "stable" yeast mRNAs such as PGK1, ACT1, and CYH2 are on the order of 25–45 min at 37° (
We consider it unlikely that defective mRNA transport was responsible for the protein synthesis defect seen in abd1 cells at the restrictive temperature, insofar as abd1-8 and abd1-5 cells did not accumulate poly(A)+ RNA in the nucleus after shift to 37°; the intracellular distribution of poly(A)+ RNA in the abd1 mutants at the restrictive temperature was indistinguishable from that of ABD1 cells (C. SAAVEDRA and C. COLE, personal communication). Note also that
Potential for regulation of cap methylation and cap-dependent transactions:
The issue of whether (and how) cap methylation might be regulated has received scant attention. Potential regulatory events include: (i) those that affect the transcription or stability of the mRNA encoding cap methyltransferase, (ii) those that influence the targeting of the cap methyltransferase to the transcription apparatus, and (iii) those that affect the activity of the cap methyltransferase.
Genome-wide analyses of mRNA transcript levels in S. cerevisiae cells have shown that ABD1 mRNA does not vary during the mitotic cell cycle (
Cap methyltransferase is targeted to nascent pre-mRNAs by direct binding of Abd1p to the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (
The catalytic activity of Abd1p could conceivably be modulated by: (i) changes in the concentrations of the substrate AdoMet or the inhibitory reaction product AdoHcy or (ii) protein-protein interactions that affect enzyme activity. There is precedent for protein-mediated stimulation of cap methylation by vaccinia capping enzyme, whereby the low basal methyltransferase activity of the catalytic D1 subunit is stimulated 50- to 100-fold by its association with the regulatory D12 subunit (
This study suggests the plausibility of regulation by substrate concentration, i.e., via our finding that abd1-ts mutations are suppressed by overexpression of AdoMet synthase and by provision of exogenous AdoMet. Suppression by AdoMet synthase in turn suggests plausible explanations for the conditional defects of the Abd1-ts proteins: that they either have a decreased binding affinity for AdoMet at the restrictive temperature or that AdoMet stabilizes the mutant proteins against thermal inactivation. Studies of the vaccinia capping enzyme indicate that AdoMet allosterically stimulates binding of the RNA cap to the methyl acceptor site of the enzyme (
A novel mode of regulation is suggested by the findings that abd1-ts mutations are suppressed by overexpression of the ubiquitin conjugating enzyme Cdc34p and that Cdc34p overexpression has no effect on the steady-state levels of mRNAs encoding Abd1p or AdoMet synthase. Given that Cdc34p-mediated ubiquitination is known to target yeast proteins for intracellular proteolysis, we posit a model whereby cap methylation or cap utilization is negatively regulated by a factor that is degraded when Cdc34p is overexpressed. This implies a balance between the normal function of Abd1p and a putative antagonist of cap methylation or m7G-dependent transactions that is deranged in favor of the antagonist in abd1-ts cells at the restrictive temperature. The ts growth defect of abd1 cells is exacerbated in a cdc34-2 background, presumably because the antagonist is stabilized when Cdc34p function is diminished. A more elaborate alternative model, which we do not exclude, is that ubiquitination by Cdc34p positively regulates Abd1p function, either by ubiquitination of Abd1p itself or ubiquitination of a putative Abd1p activator, such that the Ub modification leads to a gain of function for the target protein rather than proteolysis.
The key issues raised by the inhibitor model are: (i) the identity of the target protein that, when ubiquitinated by Cdc34p, relieves the abd1-ts phenotype and (ii) whether it is cap methylation or m7G cap utilization that is affected by the target protein. We initially considered the possibility that alterations in the level or function of certain translation initiation factors might affect the abd1-ts phenotype. Yeast p20 (Caf20p) is a repressor of cap-dependent translation initiation that competes with eIF4G for binding to the m7G cap-binding protein eIF4E (Cdc33p;
