INTRODUCTION

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Starvation of yeast cells for amino acids or purines leads to increased expression of GCN4, a transcriptional activator of amino acid biosynthetic enzymes (general amino acid control). GCN4 expression is stimulated at the translational level by a mechanism involving four short upstream open reading frames (uORFs) in its mRNA leader. During growth on amino acid-replete medium, scanning ribosomes translate the first uORF (uORF1) and reinitiate downstream at uORF2, uORF3, or uORF4 but cannot reinitiate again at the GCN4 start codon. In amino acid-starved cells, eukaryotic translation initiation factor 2 (eIF2) is phosphorylated on its  subunit by protein kinase GCN2, and the phosphorylated eIF2 inhibits the guanine nucleotide exchange factor for eIF2, known as eIF2B. Consequently, formation of the ternary complex containing eIF2, GTP, and initiator methionyl-tRNA (Met-tRNA_i^Met) is reduced, impairing delivery of tRNA_i^Met to the ribosome. In GCN4 mRNA, the ensuing delay in rebinding of ternary complex to 40S ribosomes which have translated uORF1 allows them to scan past uORF2 to uORF4 and reinitiate downstream at the GCN4 start codon instead (25, 26). Thus, GCN4 translation is induced under conditions of diminished ternary-complex formation.

It is thought that GCN2 is activated in amino acid-starved cells by uncharged tRNAs (34, 42, 43, 54) which accumulate under these conditions and bind to a regulatory domain in GCN2 homologous to histidyl-tRNA synthetases (55, 57, 58). Because starvation for any of several amino acids elicits activation of GCN2 and attendant derepression of GCN4 (34, 57), it is probable that most uncharged tRNAs can bind to GCN2 and activate its kinase function. Two positive regulators of GCN2, encoded by GCN1 and GCN20 (41, 53), show sequence similarity to translation elongation factor eEF3 and have ribosome binding activities (40). It has been proposed that the GCN1-GCN20 complex functions at the ribosome in promoting activation of GCN2 by uncharged tRNAs which have entered the decoding site (40).

There are several instances where GCN4 translation is stimulated in a manner dependent on the uORFs but independent of GCN2 and eIF2 phosphorylation. Mutations in subunits of eIF2 or eIF2B appear to reduce the functions of these two factors and mimic the effects of eIF2 phosphorylation in restricting ternary-complex formation. These mutations constitutively derepress GCN4 translation and the amino acid biosynthetic enzymes subject to general amino acid control (Gcd phenotype). The same phenotype is observed for deletions that reduce the number of IMT genes encoding tRNA_i^Met (14) and thereby decrease the steady-state level of this component of the ternary complex. Mutations in GCD10 (18, 22) and GCD14 (10, 13), whose products are required for methylation of adenosine-58 in tRNA_i^Met (1), also have GCN2-independent Gcd phenotypes. Lack of m¹A58 specifically impairs 5'-end processing and stability of tRNA_i^Met. In these instances, the Gcd phenotype can be explained by a reduction in the ternary-complex level independently of eIF2 phosphorylation by GCN2.

Previously, we observed GCN2-independent derepression of GCN4 translation in cells overexpressing tRNAs under conditions where it was presumed that the excess tRNA was not aminoacylated efficiently. This occurred most notably with a mutant form of tRNA_AAC^Val harboring an A-to-G substitution in the 3'-terminal nucleotide (tRNA^Val*), which is expected to impair aminoacylation by valyl-tRNA synthetase. Overexpression of tRNA^Val* did not lead to eIF2 phosphorylation in strains containing GCN2; however, it exacerbated the growth defect of a GCN2^c mutant (expressing a constitutively active kinase) in which eIF2 is hyperphosphorylated and thus impaired in general translation initiation. The latter findings suggested that excess tRNA^Val* leads to reduced eIF2 function by a mechanism independent of eIF2 phosphorylation (54). To explain these findings, we proposed that yeast cells have a second sensor of uncharged tRNA besides GCN2 that also constrains eIF2 activity. Moreover, because tRNA^Val* overexpression did not activate GCN2, it seemed possible that this defective tRNA was physically sequestered from GCN2 (54).

GCN2-independent derepression of GCN4 translation also was elicited by overexpression of NME1 (51), encoding the RNA component of RNase MRP (48). RNase MRP is involved in processing rRNA, and it was suggested that defects in ribosome biogenesis caused by NME1 overexpression could impair GCN4 translational control. Partial derepression of GCN4 translation additionally occurred during growth on rich medium in mutant strains with constitutively high levels of protein kinase A (PKA) function (RAS2^Val19 and bcy1 mutants) (16). It is unknown how elevated PKA function impairs translational control of GCN4.

In this report we show that overexpression of the tRNA pseudouridine 55 synthase encoded by PUS4 (7) stimulates GCN4 translation independently of GCN2 and its phosphorylation site on eIF2. We present several lines of evidence that PUS4 overexpression does not reduce the amount of functional tRNA_i^Met as the means of limiting ternary-complex formation or its utilization in translation initiation. Instead, it appears that excess PUS4 impedes the 5'-end processing and export of certain tRNAs in the nucleus. The same mechanism seems to apply to overexpressed NME1; moreover, overproduction of a mutant pre-tRNA that cannot be processed by RNase P elicits a GCN2-independent Gcd phenotype. These and other findings strongly suggest that yeast contains a surveillance system that perceives defects in tRNA processing or transport in the nucleus and reduces the efficiency of translation initiation in the cytoplasm in response.

	MATERIALS AND METHODS

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Identification of PUS4 as a high-copy-number suppressor of gcn2-1. Plasmid pAH14 was isolated previously as a high-copy-number suppressor of a gcn2-1 mutant (27). Sequencing the ends of the DNA insert and comparison with the complete yeast genome sequence showed that the insert is ~5 kb and contains three genes from chromosome XIV: RFC3 (36), MID1 (30), and PUS4 (7). Subclones pHQ536 carrying MID1 and PUS4, pHQ537 carrying PUS4, and pHQ538 carrying RFC3 and MID1 were constructed from pAH14 (see below) and introduced into gcn2-1 mutant H113, and the resulting transformants were tested for growth on medium containing 3-aminotriazole (3-AT). Only plasmids pHQ536 and pHQ537 conferred 3-AT resistance, indicating that PUS4 is a high-copy-number suppressor of gcn2-1. To confirm this conclusion, 5' and 3' deletions (pHQ546 and pHQ545, respectively) and an internal frameshift mutation (pHQ575) in PUS4 were constructed (see below). None of these plasmids conferred 3-AT resistance in strain H113, and neither did the single-copy-number PUS4 plasmid pHQ543 that we constructed.

Yeast strains and plasmid construction. All yeast strains except HQY316 used in this study were described previously and are listed in Table 1. HQY316 was constructed from H1895 by replacing LOS1 with the los1::hisG::URA3::hisG allele using a ~4.8-kb EcoRI-BglII fragment from plasmid pHQ871. The resulting los1 strain was identified by PCR and further confirmed by complementation of its Gcd phenotype by LOS1 plasmid pHQ860. The plasmids used in this work were constructed as follows. Plasmid pHQ536 containing MID1 (30) and PUS4 was constructed by inserting an ~4-kb SalI fragment from pAH14 into YEplac181 (20) at the SalI site. An ~2.0-kb BglII-Asp718 fragment containing PUS4 (7) from pHQ536 was inserted into YEplac181 between the BamHI and Asp718 sites to produce plasmid pHQ537. An ~3.8-kb NheI-XbaI fragment containing RFC3 (36) and MID1 from pAH14 was inserted into YEplac181 at the XbaI site to produce pHQ538. To construct the C-terminal deletion of PUS4, an ~1.5-kb XbaI fragment from pHQ537 was subcloned into YEplac181 at the XbaI site to produce pHQ545, encoding PUS4 amino acids 1 to 342. The N-terminal deletion of PUS4 was constructed by removing the BamHI fragment from pHQ537 to produce plasmid pHQ546, in which the 5' noncoding region and first 74 codons of PUS4 were deleted. A frameshift mutation in PUS4 was constructed by digesting pAH14 with BamHI, filling in the ends, and religating to produce pHQ575. Single-copy-number plasmid pHQ543 bearing PUS4 was constructed by inserting an ~1.8-kb BglII-NaeI fragment from pHQ536 into YCplac111 (20). High-copy-number PUS4 plasmid pHQ547 was constructed by inserting the BglII-SphI fragment containing PUS4 from pAH14 into YEp24 between the BamHI and SphI sites. The single-copy-number GCN2 plasmid pHQ548 was created by inserting the XbaI-SalI fragment from p722 (56) into YCplac111. To add the hemagglutinin (HA) epitope to PUS4, NruI and MluI sites were first introduced into pHQ537 immediately 5' to the PUS4 stop codon by site-directed mutagenesis, using the Quik-Change site-directed mutagenesis kit (Stratagene), producing plasmid pHQ732. An ~100-bp PCR fragment encoding three copies of HA with Ecl136II and MluI ends was then inserted between the NruI-MluI sites of pHQ732 to produce plasmid pHQ753 encoding PUS4-HA. An ~2-kb SalI fragment encoding PUS4-HA from pHQ753 was inserted into YCplac111 and pHQ583 to produce single-copy-number and high-copy-number plasmids pHQ771 and pHQ839, respectively. pHQ583 is a derivative of YEplac181 in which the polycloning sites SacI to BamHI were deleted by filling in the EcoRI and XbaI sites and religating. pHQ853 and pHQ857, encoding pus4-1-HA and pus4-2-HA, respectively, were derived from pHQ839 by site-directed mutagenesis using the Quik-Change site-directed mutagenesis kit.

Assay of HIS4-lacZ and GCN4-lacZ fusions. Assays were conducted using cell extracts prepared from cultures grown in SD medium containing only the required supplements as described previously (37). For repression conditions, saturated cultures were diluted 1:50 into fresh medium and harvested in mid-logarithmic phase after 6 h of growth. For derepression conditions, cultures were grown for 2 h under repression conditions and then for 6 h after adding 3-AT to 10 mM, 5-methyltryptophan (5-MT) to 2 mM, or sulfometuron methyl (SM) to 0.5 µg/ml.

Assay of yeast tRNA pseudouridine 55 synthase. Synthesis of pseudouridine is accompanied by the release of a proton from carbon 5 in the pyrimidine ring of the uridine base (12); therefore, release of tritium from [5-³H]uridine-labeled tRNA can be used as a measure of pseudouridine 55 synthase activity (46). PUS4, the S. cerevisiae enzyme, can catalyze the formation of pseudouridine 55 in a model substrate corresponding to the acceptor stem and TC stem-loop of tRNA^Asp (mut#2 minihelix) (8). Accordingly, we assayed PUS4 activity in cell extracts by measuring the release of tritium (46) from mut#2 minihelix RNA synthesized in vitro in the presence of [5-³H]UTP. tRNA^Asp mut#2 RNA labeled with [5-³H]uridine was synthesized in vitro as previously described (46). Briefly, 10 µg of MvaI-digested pHQ731 was mixed with 100 µCi of [5-³H]UTP (14.5 Ci/mmol; Amersham), dried under vacuum, and resuspended in 100 µl of a reaction mixture containing 40 mM Tris-HCl (pH 7.9), 6 mM MgCl₂, 2 mM spermidine, 10 mM NaCl, 10 mM dithiothreitol, 10 mM GMP, 1 mM each GTP, CTP, and ATP, 250 µM UTP, and 100 U of RNasin (Promega). The reaction was initiated by adding 100 U of T7 RNA polymerase (Promega), and the mixture was incubated at 37°C for 2 h. Afterwards, the mixture was extracted once with phenol-chloroform (1:1) and the [³H]RNA was ethanol precipitated and resuspended in water pretreated with diethylpyrocarbonate.

Analysis of tRNA modification and aminoacylation in vivo. The primer extension assay used for mapping pseudouridine residues was conducted as described previously (5-7). In this assay, RNA is treated with 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate (CMCT), a chemical that reacts with both pseudouridine and uridine residues. The presence of CMCT-coupled nucleosides in tRNA impedes reverse transcription primed by an oligonucleotide annealed 3' to the CMCT-coupled base. Because CMCT-pseudouridine is more resistant than CMCT-uridine to alkali treatment, the locations of pseudouridine residues in a tRNA molecule can be deduced from the presence of alkali-insensitive blocks to reverse transcription (5, 6). Chromatography of aminoacylated tRNA_i^Met and elongator tRNA^Met (tRNA_e^Met) on RPC-5 resin was carried out as described previously (1). For Northern analysis of in vivo-aminoacylated tRNAs, total RNA was prepared under acidic conditions and resolved by electrophoresis on acid-urea gels as described previously (52). The following oligonucleotides were used to probe the Northern blots: 5'-TGGTAGCGCCGCTCGGTTTCGAATCC-3' (tRNA_i^Met), 5'-TGCTCCAGGGGAGGTTCGAACTCTCGACC-3' (tRNA_e^Met), 5'-CACTCACGATGGGGGTCGAA-3' (tRNA_UCU^Arg), 5'-TGCTCGAGGTGGGGA/TTTGAACCCACGACGG-3' (tRNA_UAU^Ile), 5'-GATTGCAGCACCTGAGTTTCGCGTTATGG-3' (5S rRNA), and 5'-GGTGGGAGACTTTCAACCCAAAGC-3' (NME1).

Fluorescence in situ hybridization. The fluorescence in situ hybridization procedure was conducted as described previously (47), except that transformants carrying plasmids were grown at 30°C to log phase. The oligonucleotides used were probe 04 (47), to detect tRNA_UAU^Ile, and 5'-CGCCCAGGATCGAACTG GGGACGTTCTGCGTGTTAAGCAGATGCCATAACCGACTAGACC-3', to detect tRNA_AAC^Val.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Overexpression of PUS4, encoding tRNA pseudouridine 55 synthase, derepresses GCN4 translation in the absence of eIF2 kinase GCN2. In an effort to identify a novel regulator of GCN2, we analyzed a previously described high-copy-number plasmid, pAH14, which suppresses the 3-AT-sensitive (3-AT^s) phenotype of a gcn2-1 mutant (27). 3-AT is a competitive inhibitor of the histidine biosynthetic enzyme encoded by HIS3, and GCN4-mediated derepression of HIS3 transcription is required for growth in the presence of this inhibitor. Accordingly, gcn2 mutants are 3-AT^s because they fail to derepress GCN4 translation in response to histidine starvation. Suppression of the 3-AT^s phenotype of gcn2-1 by pAH14 suggested that HIS3 derepression had been restored independently of GCN2. Sequencing the ends of the genomic DNA insert in pAH14 revealed that it contains three genes from chromosome XIV: RFC3 (36), MID1 (30), and PUS4, of which the last encodes tRNA pseudouridine 55 synthase (7). By analyzing subclones of pAH14, we determined that high-copy-number PUS4 was sufficient for suppression of gcn2-1 and that deletions removing the 5' or 3' end of the PUS4 ORF or introduction of a frameshift mutation in PUS4 abolished suppression (see Materials and Methods). Moreover, PUS4 on a low-copy-number plasmid failed to suppress the gcn2-1 allele (data not shown). Thus, we concluded that PUS4 is a high-copy-number suppressor of gcn2-1.

View larger version (58K): [in this window] [in a new window]   FIG. 1.   High-copy-number plasmid encoding PUS4 derepresses histidine biosynthetic genes in the absence of GCN2 and Ser-51 of eIF2. Isogenic strains H1816 (gcn2 SUI2), H1897 (GCN2 sui2-S51A), and H1817 (gcn2 sui2-S51A) were transformed with the indicated plasmids, replica-plated to SD medium or to SD medium containing 30 mM 3-AT, and incubated for 3 days at 30°C. pHQ547 is a high-copy-number (h.c.) plasmid containing PUS4; pC102-2 is a low-copy-number (l.c.) plasmid containing GCN2, and p919 is a low-copy-number (l.c.) plasmid containing SUI2.

View this table: [in this window] [in a new window]   TABLE 2.   Effect of hcPUS4 on HIS4-lacZ expression in a gcn2 strain starved for different amino acids

View this table: [in this window] [in a new window]   TABLE 3.   Derepression of GCN4-lacZ expression by hcPUS4 in a gcn2 strain requires uORFs in GCN4 mRNA

View this table: [in this window] [in a new window]   TABLE 4.   Nonadditive effects of high-copy-number plasmids carrying tRNAVal* and PUS4 on derepression of GCN4-lacZ expression in a gcn2 strain

hcPUS4 leads to elevated pseudouridine 55 synthase activity in vivo. To show that cells bearing hcPUS4 contain increased amounts of PUS4 protein, we assayed pseudouridine 55 synthase activity in cell extracts (see Materials and Methods). As shown in Table 5, extracts of transformants containing hcPUS4 or a functional HA-tagged form of hcPUS4 contained 15 to 16 times as much pseudouridine 55 synthase activity than did the corresponding extract from the vector transformant. This increase in enzyme activity was similar in magnitude to the increase in PUS4 protein levels measured in extracts from transformants bearing the HA-tagged PUS4 allele (PUS4-HA) on high-copy-number versus single-copy-number plasmids, as judged by immunoblot analysis with anti-HA antibodies (Table 5). Based on these results, we conclude that hcPUS4 leads to a large increase in the level of PUS4 enzyme activity in vivo.

Increased pseudouridine 55 synthase activity is not required for suppression of gcn2 mutations by hcPUS4. The observations that overexpressing the mutant tRNA^Val* derepressed GCN4 translation independently of GCN2 (54), that hctRNA^Val* and hcPUS4 had nonadditive effects on GCN4 expression, and that PUS4 is a tRNA modification enzyme led us to consider that overexpression of PUS4 might lead to aberrant pseudouridine formation in tRNAs and impede aminoacylation by their cognate aminoacyl-tRNA synthetases. If this occurred with tRNA_i^Met, the only known tRNA in Saccharomyces cerevisiae that normally lacks this modification (49), it would lower ternary-complex levels and thereby derepress GCN4 translation in gcn2 cells.

View larger version (33K): [in this window] [in a new window]   FIG. 2.   High-copy-number PUS4 does not alter base modification of tRNAiMet. (A) Samples of total tRNA (10 µg) prepared from gcn2 strain H1894 carrying empty vector (YEplac181) or hcPUS4 plasmid (pHQ537) were treated (+) or not treated () with CMCT, and 1 µg was reverse transcribed using end-labeled primers complementary to tRNAiMet or tRNAeMet (nucleotides 60 to 76). Reverse transcription products were resolved in an 8% sequencing gel. The strong stops in reverse transcription of CMCT-treated tRNA correspond to pseudouridine-55 (55), as indicated by the arrow. On the left of the gel is a sequence ladder of initiator tRNAMet. The strong stops at position 52 for tRNAiMet observed independently of CMCT presumably arise from strong secondary structure. (B) The same tRNA samples as in panel A were aminoacylated with [3H]methionine or [35S]methionine, and ca. 500,000 cpm was resolved on an RPC-5 column. Radioactivity in each fraction (2 ml) was measured by liquid scintillation and plotted against the fraction number. The elution positions of the methionine-accepting tRNAs are indicated at the appropriate positions.

Evidence that hcPUS4 elicits derepression of GCN4 partly by interfering with 5'-end processing of tRNA by RNase P. Although the pseudouridine 55 synthase activity of PUS4 is not required for its suppressor activity, it was possible that increased binding of overexpressed PUS4 to one or more tRNAs would restrict the access of other enzymes involved in modification or processing of these tRNAs. As indicated above, we were particularly interested in possible differences in the structure or function of tRNA_i^Met that could reduce ternary-complex formation. To investigate this last possibility, we first aminoacylated total tRNA from transformants containing hcPUS4 or vector alone with [³⁵S]methionine or [³H]methionine, respectively, and resolved the labeled tRNAs by RPC-5 column chromatography (31). tRNAs that differ by only a single methyl group can be resolved by RPC-5 chromatography (15). The results in Fig. 2B show that the elution positions of [³⁵S]methionine-charged tRNA_i^Met and tRNA_e^Met were identical between gcn2 transformants bearing hcPUS4 and vector alone. These results suggest that mature methionine-accepting tRNAs are modified identically in cells overexpressing PUS4 and wild-type cells; however, it is possible that certain modifications would not alter the behavior of methionyl-tRNAs on RPC-5 chromatography.

View larger version (42K): [in this window] [in a new window]   FIG. 3.   Evidence that hcPUS4 does not reduce in vivo aminoacylation of various tRNAs. Total RNAs prepared under acidic conditions (52) from strain H1894 (gcn2) carrying empty vector (YEplac181) or high-copy-number plasmids carrying PUS4 (pHQ537) or PUS4-HA (pHQ839) were resolved by electrophoresis on an acid-urea polyacrylamide gel and subjected to Northern blot analysis. The same blot was probed with radiolabeled oligonucleotides that specifically hybridized to the indicated tRNAs by stripping one probe from the blot before using the next. An aliquot of tRNAiMet was deacylated in 2 M Tris-HCl (pH 8.0) and loaded in lane 1. The intensities of the hybridization signals corresponding to charged and uncharged tRNAs were quantified by phosphorimaging analysis, and the ratios of charged to uncharged tRNA signals are listed below each lane.

View larger version (64K): [in this window] [in a new window]   FIG. 4.   Overexpressing PUS4 or NME1 leads to accumulation of untrimmed tRNA precursors. Total RNA (9 µg) prepared from gcn2 strains carrying empty vector YEplac181 or high-copy-number plasmids pHQ839 (h.c.PUS4-HA), pHQ864 (h.c.PUS4-HA/RPR1), (Vector) pHQ862 (h.c.NME1), and pHQ863 (h.c.NME1/RPR1) were subjected to Northern blot analysis and probed with a radiolabeled oligonucleotide complementary to tRNAiMet. The same blots were stripped and reprobed with radiolabeled oligonucleotides specific for tRNAUAUIle or 5S rRNA (see Materials and Methods). The positions of pre-tRNAiMet, mature tRNAiMet, pre-tRNAUAUIle, mature tRNAUAUIle, and 5S rRNA are indicated on the left. The primary transcript (upper band) and the 5'- and 3'-end-processed intron-containing pre-tRNAUAUIle (lower band) are indicated on the right by the letters a and b, respectively.

View larger version (33K): [in this window] [in a new window]   FIG. 5.   Overexpression of initiator tRNAMet does not suppress the Gcd phenotype of hcPUS4. (A) Transformants of strain H1894 (gcn2) bearing high-copy-number plasmids YEplac181 and YEp24 (Vectors), pHQ839 and YEp24 (h.c.PUS4-HA/vector), or pHQ839 and pC50 (h.c.PUS4-HA/h.c.IMT4) were replica-plated to SD medium containing 30 mM 3-AT and incubated for 3 days at 30°C. (B) Total RNA (6 µg) isolated from strains carrying the indicated high-copy-number plasmids were subjected to Northern blot analysis and probed with an oligonucleotide specific for initiator tRNAMet.

View larger version (32K): [in this window] [in a new window]   FIG. 6.   Overexpression of RPR1 reduces the Gcd phenotype of hcPUS4. (A) Transformants of strain H1895 (gcn2) bearing high-copy-number plasmids YEplac181 (Vector), pHQ839 (h.c.PUS4-HA), pHQ864 (h.c.PUS4-HA/RPR1), or pHQ682 (h.c.RPR1) were replica-plated to SC medium containing 30 mM 3-AT and incubated for 3 days at 30°C (left panel). Extracts from the same transformants grown under repressing (nonstarvation) conditions were assayed for -galactosidase activity, and the results shown in the right panel are the means and standard deviations from three individual transformants. (B) Expression of PUS4-HA in transformants carrying high-copy-number plasmids pHQ839 (h.c.PUS4-HA) or pHQ864 (h.c.PUS4-HA/RPR1) measured by Western blot analysis. PUS4-HA was detected by anti-HA antibody and visualized by enhanced chemiluminescence. The intensities of bands were calculated with a scanner (Silverscanner III) and NIH image software (version 1.61). The relative levels were calculated by averaging the band intensities from two independent extract preparations for each transformant (lanes 1 to 4, pHQ839 transformant; lanes 5 to 8, pHQ864 transformant).

View larger version (42K): [in this window] [in a new window]   FIG. 7.   Overexpression of RPR1 reduces the Gcd phenotype of hcNME1. (A) Transformants of strain H1895 (gcn2) bearing high-copy-number plasmids YEplac181 (Vector), pHQ862 (h.c.NME1), pHQ863 (h.c.NME1/RPR1), or pHQ682 (h.c.RPR1) were replica-plated to SC medium containing 30 mM 3-AT and incubated for 3 days at 30°C (left panel). Extracts from the same transformants grown under repressing (nonstarvation) conditions were assayed for -galactosidase activity, and the results shown in the right panel are the means and standard deviations of activities from three individual transformants. (B) Expression of NME1 in transformants carrying high-copy-number plasmids YEplac181 (Vector), pHQ862 (h.c.NME1), pHQ863 (h.c.NME1/RPR1), and pHQ682 (h.c.RPR1) was measured by Northern blot analysis using radiolabeled oligonucleotide specific to NME1. (C) Transformants of strain H1895 (gcn2) bearing high-copy-number plasmids YEplac181 (Vector), pHQ982 (h.c. wild-type pre-tRNATyrGUA) and pHQ985 (h.c. mutant pre-tRNATyrGUA) were replica-plated to SC medium containing 30 mM 3-AT and incubated for 3 days at 30°C.

Evidence that PUS4 overexpression elicits derepression of GCN4 partly by interfering with nuclear export of tRNAs. It is thought that tRNA export in mammalian cells requires exportin-t (Xpo-t), which binds tRNA directly with high affinity (33). It also requires the GTP-bound form of Ran (RanGTP), which forms a complex with Xpo-t and tRNA (2, 33) involving extensive interactions with the backbone of the TC and acceptor arms of the tRNA (3). LOS1 is a yeast homolog of Xpo-t (2, 33), and the nuclear accumulation of tRNA observed in a los1 mutant (47) plus the ability of LOS1 to interact with Ran-GTP in a tRNA-dependent fashion (23) have implicated LOS1 in tRNA export from the yeast nucleus. If LOS1 resembles Xpo-t in binding to the TC and acceptor arms of tRNA, overexpressed PUS4 might compete with LOS1 for tRNA binding and interfere with tRNA export. This possibility is consistent with the findings that PUS4 can form stable complexes with tRNA in vitro (45), that a minimal substrate for enzymatic formation of pseudouridine 55 by PUS4 is a TC stem-loop structure (8), and that pseudouridine 55 synthase in Escherichia coli requires the TC stem-loop to catalyze pseudouridine formation (21). The accumulation of mature tRNA in the nucleus resulting from inhibition of LOS1 function by PUS4 might be a signal for derepression of GCN4 translation. According to this hypothesis, overexpression of LOS1 should reduce the derepression of GCN4 elicited by hcPUS4.

View larger version (53K): [in this window] [in a new window]   FIG. 8.   Overexpression of LOS1 reduces the Gcd phenotype of hcPUS4. (A) Transformants of strain H1895 (gcn2) bearing high-copy-number plasmids YEplac181/YEp24 (Vectors), pHQ839/YEp24 (h.c.PUS4-HA/Vector), pHQ839/YEpLOS1 (h.c.PUS4-HA/h.c.LOS1), p856/YEp24 (h.c.tRNAVal*/Vector), and p856/YEpLOS1 (h.c.tRNAVal*/h.c.LOS1) were replica-plated to SC medium containing 30 mM 3-AT and incubated for 3 days at 30°C (left panel). Extracts from the same transformants grown under repressing (nonstarvation) conditions were assayed for -galactosidase activity, and the results shown in the right panel are the means and standard deviations from three individual transformants. (B) Expression of PUS4-HA in transformants carrying high-copy-number plasmids pHQ839/Vector (Vector + h.c.PUS4-HA) or pHQ839/YEpLOS1 (h.c.LOS1 + h.c.PUS4-HA) measured by Western blot analysis. PUS4-HA was detected with an anti-HA antibody and visualized by enhanced chemiluminescence. Intensities of bands were calculated with a scanner (Silverscanner III) and NIH image software (version 1.61). The relative levels were calculated by averaging the band intensities from two independent extract preparations for each transformant (lanes 1 to 4, pHQ839/Vector transformant; lanes 5 to 8, pHQ839/YEpLOS1 transformant). (C) Transformants of strain H1895 (gcn2) bearing high-copy-number plasmids YEplac181/YEp24 (Vectors), pHQ862/YEp24 (h.c.NME1/Vector), pHQ862/YEpLOS1 (h.c.NME1/h.c.LOS1), or YEplac181/YEpLOS1 (Vector/h.c.LOS1) were replica-plated to SC medium containing 30 mM 3-AT and incubated for 3 days at 30°C. (D) Deletion of LOS1 has a Gcd phenotype. Transformants of strain HQY316 (gcn2los1) bearing plasmids YEplac181 (vector), pHQ860 (h.c.LOS1), and pHQ839 (h.c.PUS4-HA) were replica-plated to SD medium containing the required supplements and 30 mM 3-AT and incubated for 3 days at 30°C.

los1 gcn2

View larger version (16K): [in this window] [in a new window]   FIG. 9.   hcPUS4-HA leads to nuclear accumulation of tRNAUAUIle detected by fluorescence in situ hybridization. Cells of transformants of strain H1895 (gcn2) bearing high-copy-number plasmids YEplac181/YEp24 (vectors) (A and a), pHQ839/YEp24 (h.c.PUS4-HA/vector) (B and b), or pHQ839/YEpLOS1 (h.c.PUS4-HA/h.c.LOS1) (C and c) were subjected to fluorescence in situ hybridization using a probe specific for tRNAUAUIle (panels A, B, and C) or stained with 4',6-diamidino-2-phenylindole (DAPI) to visualize nuclei (panels a, b, and c).

View larger version (2534K): [in this window] [in a new window]   FIG. 10.   Evidence that mutant tRNAVal* is defective for aminoacylation and processing and is retained in the nucleus. (A to C) Cells of transformants of strain H1937 (gcn2) bearing empty vector YEp24 (A and a), p1362 (tRNAVal*) (B and b), or p1308 (wild-type tRNAVal) (C and c) were subjected to fluorescence in situ hybridization using a probe specific for tRNAAACVal (A, B, and C) or stained with DAPI to visualize nuclei (a, b, and c). (D) Total RNAs prepared under acidic conditions from the strains analyzed in panels A to C were resolved by electrophoresis on an acid-urea polyacrylamide gel and subjected to Northern blot analysis using a probe specific for tRNAAACVal. In lanes 1, 3, and 5, the RNA samples were deacylated in 2 M Tris-HCl (pH 8.0) prior to electrophoresis.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Evidence that unprocessed tRNAs in the nucleus elicit derepression of GCN4 translation independently of eIF2 phosphorylation. GCN4 translation can be stimulated independently of GCN2 in mutants with lesions in subunits of eIF2 or eIF2B, in the genes encoding tRNA_i^Met, or in the GCD10- or GCD14-encoded proteins required for methylation of adenosine-58 in tRNA_i^Met. It is thought that all of these mutations mimic the effects of GCN2-mediated eIF2 phosphorylation by lowering the concentration of ternary complexes in the cytoplasm. It was suggested that a defect in ribosome biogenesis was responsible for derepressing GCN4 translation in gcn2 cells overexpressing NME1 (51). Our finding that the Gcd phenotype of hcNME1 was suppressed by overexpressing RPR1 points to a reduction in RNase P levels and diminished tRNA 5'-end processing as the cause of derepression. We propose that overexpression of NME1 reduces RNase P levels by titrating from RPR1 one or more protein subunits shared between RNases MRP and P. Consistent with this hypothesis, we detected an ca. twofold increase in the precursor/mature ratio for tRNA_i^Met in the hcNME1 strain versus the wild type, and this phenotype was partially reversed by hcRPR1. The small reduction in mature initiator tRNA^Met abundance caused by hcNME1 cannot account for its Gcd phenotype, because it was not suppressed by hcIMT4. Instead, we propose that an increase in the levels of unprocessed tRNAs in the nucleus activates a regulatory mechanism that down-regulates ternary complex binding to 40S ribosomes by an amount sufficient to derepress GCN4 translation. We cannot exclude the possibility that a defect in ribosome biogenesis also contributes to the derepression of GCN4 conferred by hcNME1.

Evidence that overexpression of PUS4 elicits GCN2-independent derepression of GCN4 by impeding nuclear export and 5'-end processing of tRNAs. The derepression of GCN4 translation in cells overexpressing PUS4 also seems to be triggered partly by the accumulation of pre-tRNAs in the nucleus. The Gcd phenotype of hcPUS4 was partially reversed by hcRPR1, suggesting that overexpressed PUS4 interferes with 5'-end processing by RNase P. Consistent with this model, we observed a ca. twofold increase in the precursor/mature ratio for tRNA_i^Met in the hcPUS4 transformant, which was reversed by cooverexpressing RPR1. The postulated interference with RNase P exerted by overexpressed PUS4 could involve direct competition between these two enzymes for binding to a subset of tRNA precursors. This idea is ostensibly at odds with the fact that 5'-end processing of pre-tRNA_i^Met was impaired by hcPUS4 even though this tRNA is not a substrate for PUS4. When overexpressed 15-fold, however, PUS4 may bind tightly to pre-tRNA_i^Met and block access of RNase P even though it fails to synthesize pseudouridine-55. Alternatively, PUS4 and RNase P may interact with a common tRNA chaperone that facilitates the activities of both enzymes, and overexpression of PUS4 could reduce the availability of this hypothetical chaperone for 5'-end processing of pre-tRNA_i^Met by RNase P.