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1 Institute for Biochemistry and Molecular Biology, University of Berne, CH-3012 Bern, Switzerland
2 Institut National des Sciences Appliquées (INSA), Complexe Scientifique de Rangueil, 31077 Toulouse Cédex 4, France
Reprint requests to: Michael Altmann, Institute for Biochemistry and Molecular Biology, University of Berne, Bühlstrasse 28, CH-3012 Bern, Switzerland; e-mail: michael.altmann{at}mci.unibe.ch; fax: 0041-31-631-37-37.
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ABSTRACT |
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Keywords: yeast; cell-free translation; internal initiation; RACE; eIF4G
Abbreviations: eIF, eukaryotic initiation factor; SDS, sodium dodecyl sulfate; 5-FOA, 5-fluoro-orotic acid; IRES, internal ribosome entry site; Ac, acetate; RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction; RT, reverse transcription; RLU, relative luminescence units; ORF, open reading frame; nt, nucleotide; DTT, dithiothreitol; EGTA, ethylene glycol-bis-(2-aminoethyl)-N,N,N',N'-tetraacetic acid; EDTA, ethylenedioxy-diethylene-dinitrilo-tetraacetic acid; BSA; bovine serum albumin; DEPC, diethylpyrocarbonate
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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; Hershey and Merrick 2000; Dever 2002). This pathway involves recognition of the cap structure m7GpppN (where N is any nucleotide and m is a methyl group) at the 5'-end of mRNA by the cap-binding protein eIF4E (eukaryotic initiation factor 4E) bound to the anchor protein eIF4G (for reviews, see Sachs et al. 1997; Gingras et al. 1999) and recruitment of eIF4A and eIF4B and ATP-hydrolysis-dependent melting of RNA secondary structure in the 5'-untranslated region (UTR) by the DEAD-box helicase eIF4A. This creates a binding site for the 40S ribosomal subunit, which carries the ternary complex eIF2–GTP–Met-tRNAi and the initiation factors eIF1, eIF1A, eIF3, and eIF5 (43S initiation complex). After binding to mRNA near the cap structure, the 43S complex scans the mRNA in the 3' direction in search of the initiator AUG codon (Kozak 1989). AUG recognition by base-pairing with the anticodon of Met-tRNAi (Cigan et al. 1988) triggers the hydrolysis of GTP bound to eIF2, release of initiation factors from the 40S ribosomal subunit, and binding of a 60S ribosomal subunit to form a 80S initiation complex competent to translate the open reading frame (ORF) of the mRNA.
An alternative initiation pathway by which a number of viral and some eukaryotic cellular mRNAs are translated is internal initiation mediated by an internal ribosome entry site (IRES; for reviews, see Jackson and Kaminski 1995; Belsham and Jackson 2000; Carter et al. 2000; Jackson 2000; Hellen and Sarnow 2001). The initiation factor requirement for internal initiation depends on IRES type and structure. Translation initiation on most of the viral IRESs does not require either cap-binding factor eIF4E or intact eIF4G (Belsham and Sonenberg 1996; Belsham and Jackson 2000; Jackson 2000) except for the IRES element of hepatitis A virus, which is known to require intact initiation factor eIF4G (Borman and Kean 1997). In contrast, recruitment of 40S ribosomes to hepatitis C virus and swine fever virus IRES does not require the initiation factors of the eIF4 group (Pestova et al. 1998), and cricket paralysis virus IRES directs initiation without requirement for any of the canonical initiation factors (Wilson et al. 2000).
The yeast Saccharomyces cerevisiae is a powerful system to study the mechanism and regulation of translation initiation; however, rather little is known about internal initiation in this system (Altmann et al. 1990; Iizuka et al. 1994; Paz et al. 1999). Reported examples of internal initiation in yeast include reporter mRNAs carrying the poliovirus 5'-UTR (Altmann et al. 1990), a 148-nucleotide IRES element located in the 5'-UTR of the coat protein gene of crucifer-infecting tobamovirus (crTMV; Dorokhov et al. 2002); the cricket paralysis viral RNA (Thompson et al. 2001); the mRNAs coding for the transcription factors TFIID, HAP4, and YAP1 (Iizuka et al. 1994); the mRNA coding for URE2 (Komar et al. 2003); and the mRNA encoding translation initiation factor eIF4G1. The latter was reported to be very potent (Zhou et al. 2001).
Yeast eIF4G is represented by two proteins that are 50% identical at the amino acid level, namely, eIF4G1 (encoded by the gene TIF4631) and eIF4G2 (encoded by the gene TIF4633; Goyer et al. 1993). Internal initiation of translation of mRNA encoding eIF4G seems plausible because synthesis of eIF4G protein may have to occur under conditions in which eIF4G is limiting in cells. Like in most cases of internal initiation, the presence of an IRES in eIF4G1 mRNA was demonstrated by placing the 5'-UTR in a dicistronic reporter construct between two ORFs. The IRES then supported second ORF translation (Zhou et al. 2001).
We attempted to study internal initiation in yeast extracts with dicistronic mRNAs and chose the 5'-UTR of TIF4631 mRNA for our experiments. To our surprise, we found a promoter in this region of the TIF4631 gene.
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RESULTS |
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). We inserted this sequence into DNA constructs containing one or two reporter genes (Fig. 1A), obtained the corresponding mono- or dicistronic mRNAs by in vitro transcription and tested them in a yeast in vitro translation system (strain CWO4; Fig. 1B; Table 1). This system supports both cap-dependent and internal initiation as shown with a reporter construct carrying the IRES of yeast URE2 mRNA (Komar et al. 2003). Capped dicistronic mRNA (RNA 1) yielded strong first ORF (Renilla luciferase) and lower second ORF (Photinus luciferase) expression. Second ORF expression from this mRNA was in the order of 10% of what was obtained when this ORF was expressed from a monocistronic mRNA (RNA 3). The rather high expression from the second ORF may be due to reinitiation and/or leaky scanning because it was partially sensitive to cap analog, although less sensitive than first ORF expression. Reinitiation appears more plausible than leaky scanning (scanning through 32 AUG codons before reaching the Photinus initiation codon) because the intergenic region is very short (13 nt). Insertion of 505 nt of the sequence preceding the start codon of the TIF4631 ORF (referred to here as the TIF4631 5'-UTR) significantly inhibited second ORF translation in dicistronic mRNA (RNA 2) while having little effect on first ORF translation. This indicates that this sequence interferes with reinitiation and/or leaky scanning and does not support internal initiation. Inhibition of reinitiation and scanning by the TIF4631 5'-UTR is expected because this nucleotide sequence contains several AUG codons and short ORFs (Goyer et al. 1993).
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The 5'-UTR of the TIF4631 gene contains a promoter
To understand the discrepancy between our in vitro and the in vivo translation experiments reported earlier (Zhou et al. 2001), we analyzed Renilla and Photinus luciferase expression in vivo in our strains CWO4 and 334 (Table 1
) from the vectors pGal-R.P, pGal-R.4G(-508/-3), pGal-R.4G(-250/-3).P (Table 2) encoding dicistronic mRNAs (Fig. 2, top) and compared the expression levels with those previously described by Zhou et al. (2001) with strain EGY48. Using these vectors we were able to reproduce their results: upon induction of transcription of the dicistronic mRNAs with galactose in all three strains (Fig. 2, bottom, +) the low second ORF expression from construct 1 was stimulated 100–1000-fold when the TIF4631 5'-UTR (from position -508 to -3 in construct 2 or from position -250 to -3 relative to the translation initiation codon in construct 3) was inserted between the two ORFs. Surprisingly, second ORF expression was about the same in all strains analyzed when the transcription of the dicistronic mRNA was not induced (Fig. 2, bottom, -). Under these conditions, dicistronic mRNA levels should be at least 20-fold reduced as shown by the reduction of first ORF translation. This control (which had not been mentioned by Zhou et al. 2001) indicates the presence of a promoter in the TIF4631 5'-UTR between positions -250 and -3, which leads to the synthesis of a monocistronic mRNA encoding Photinus luciferase.
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). Reverse transcription was primed with the oligonucleotide P', PCR with the reverse primer P' and the forward primer P (to detect transcripts encoding the second ORF, P-fragment) or forward primer R (to detect transcripts encoding both ORFs, R-fragment). Control experiments omitting reverse transcriptase did not give any signals indicating the absence of contaminating DNA (Fig. 3B, lanes 2,4,6,8,10,12,14,16). PCR reactions with the plasmids pGal-R.P (minus TIF4631 5'-UTR; Fig. 3B, lanes 17,18) and pGal-R.4G(-508/-3) (plus TIF4631 5'-UTR; Fig. 3B, lanes 19,20) were performed to optimize PCR conditions. Induction with galactose of transcription from the GAL1/10 promoter on plasmid pGal-R.P (lanes 13–16) led to the synthesis of dicistronic mRNA, increasing its concentration strongly above the level found in the presence of glucose (Fig. 3B, cf. lanes 9 and 11 with lanes 13 and 15). Induction of transcription on plasmid pGal-R.4G(-508/-3) generated a dicistronic mRNA with a longer intercistronic region (Fig. 3B, lanes 5,7). In the presence of glucose, we could not detect the dicistronic mRNA (Fig. 3B, lane 1) but a monocistronic mRNA containing the Photinus luciferase ORF (Fig. 3B, lane 3). This result confirms our hypothesis that a promoter in the 5'-UTR of TIF4631 generates a monocistronic mRNA. We assume that transcription from this promoter may inhibit residual transcription from the GAL1/10 promoter under repressing conditions through steric hinderance and thus lower the amount of the full-length dicistronic mRNA, rendering it undetectable (Fig. 3B, lane 1).
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) lacking both chromosomal copies of TIF4631 and TIF4632. This strain carries the wild-type TIF4631 gene on a URA3 plasmid and is therefore viable. By plating transformants on 5-FOA (5-fluoro-orotic acid) plates (selection for loss of URA3 plasmid), cells carrying a truncated TIF4631 gene but still able to express eIF4G1 could be selected (Fig. 4A). On plates lacking 5-FOA, all transformants grew because of the presence of the wild-type TIF4631 gene on the plasmid 
ycp50-TIF4631; URA3> (Fig. 4A, left plate). On plates containing 5-FOA, transformants carrying plasmids with deletions in the TIF4631 5'-UTR from position -530 (full length) to -112 (relative to the translation initiation codon) were able to grow, whereas transformants carrying plasmids with longer deletions (+22 to +230) were not able to grow (Fig. 4A, right plate). Transformants that grew on plates containing 5-FOA expressed full-length eIF4G1 as shown by Western blotting (Fig. 4B): the TIF4631 gene with only 112 nt upstream of the start AUG codon codes for the synthesis of eIF4G1 of the same size as the one encoded by the TIF4631 gene with 530 nt upstream of the AUG codon or by wild-type chromosomal TIF4631. These data demonstrate the presence of a promoter in the region -1 to -112 of the TIF4631 gene.
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, DNA controls). TIF4631 mRNA derived from wild-type cells as well as from cells carrying the shortest functional TIF4631 gene (deletion to position -112) gave rise to PCR products ending in the region of nucleotide positions -59 to -85. We conclude from these data that the longest form of mRNA produced by the promoter present in the TIF4631 5'-UTR (full length or deletion -112) carries an untranslated region of ~70–90 nt.
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).
We visualized the products of primer extension reactions by performing reverse transcriptase reactions using a 5'-(32P)-labeled oligonucleotide that hybridized close to the start AUG of TIF4631 mRNA. Two main signals were obtained when using total RNA from strain CWO4 that corresponds to transcription start sites at positions -38 and -2 (Fig. 5C, lane 1). Also, two longer products were detected in this reaction at positions around -390 and -580 (data not shown). We don’t know if those products were obtained because of DNA traces in our RNA preparations or if they correspond to real transcription start sites. The first explanation seems unlikely as control experiments with total RNA prepared from a yeast strain carrying a TIF4631 deletion (strain CBY1.1; Table 1) did not give any signal in our reverse transcription reactions (Fig. 5C, lane 2). In any case, our primer extension experiments confirm the existence of a main transcription start site located around position -38, which we also detected in our RACE experiments.
Because a stretch corresponding to nucleotides -580 to -3 of the 5'-UTR was found to be inhibitory for translation (Fig. 1), we next determined the translational activity of Photinus luciferase mRNAs with shorter 5'-UTR sequences derived from the TIF4631 gene. We produced capped Photinus luciferase mRNAs with the 5'-UTRs extending to positions -75 and -36 and tested them for in vitro translation in yeast extracts. The activities of these mRNAs were on the order of 1000-fold higher than for mRNA containing a 5'-UTR extending to position -508 (Fig. 6). These results indicate that TIF4631 mRNAs with 5'-UTRs of 36 or 75 nt are highly efficient for translation.
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DISCUSSION |
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. These data show that a DNA construct retaining sequences up to -112 is still functional, whereas a DNA construct starting at position +22 is not functional in vivo. DNA constructs with truncated TIF4631 genes starting at nucleotide positions +22, +130, and +230 cannot complement, although they encode mRNAs with an AUG codon at position 256 (amino acid 86). Initiation of translation at this AUG codon leads to the synthesis of N-terminally truncated eIF4G, which was shown to be functional in vivo (Tarun et al. 1997). This shows that the promoter driving the expression of the TIF4631 gene on the plasmid used in our complementation experiments is not plasmid-borne because such a promoter should lead to the synthesis of mRNAs encoding truncated but functional eIF4G1 protein.
By using Northern analysis, we previously identified a single TIF4631 mRNA of ~3800 nt (Goyer et al. 1993). In the course of this work, we decided to perform RT-PCR and RACE to identify the 5'-end of TIF4631 mRNAs because small differences in mRNA length cannot be determined as accurately by Northern analysis. Because the majority of TIF4631 mRNAs that we identified in wild-type yeast cells using RACE have their 5'-end at position -36 (Fig. 5B), primer extension experiments show two main mRNA bands at position -38 and -2 (Fig. 5C), and a truncated TIF4631 gene extending to position -112 encodes full-length eIF4G1 (Fig. 4B), we conclude that the promoter elements must be located in the nucleotide sequence between -36 and -112. This region contains two binding sites for transcription factor Bas2 (from -109 to -104 and from -45 to -39) and one binding site for transcription factors Gcr1 and Gcn4 (located in the region from -88 to -103; Fig. 5B; http://cgsigma.cshl.org/jian/). Perhaps, the Bas2 element located at position -104 to -109 is sufficient to drive transcription without further enhancer elements. Further experiments will be required to precisely map the DNA elements required for efficient transcription of TIF4631 mRNA.
Previously, longer 5'-UTRs for TIF4631 mRNAs detected by primer extension experiments have been reported (Goyer et al. 1993). We have also detected potentially longer transcripts in our primer extension experiments, but, as mentioned above, we do not know if they correspond to real mRNA 5'-ends or if they are artifacts. One possible error source is the presence of residual DNA in mRNA preparations. We observed longer DNA bands in our RT-PCR experiments when DNase treatment was insufficient (data not shown) and therefore always ran an RT-PCR control experiment without reverse transcriptase to make sure that all DNA bands detected originated from RNA (as shown in Fig. 3B). From our experiments, we cannot exclude that additional longer forms of TIF4631 mRNA are produced and perhaps translated by internal initiation. However, we state that longer forms of TIF4631 mRNA are not essential for yeast growth, although they may have a function under certain physiological conditions. From our in vitro data, we also conclude that there is no strong IRES sequence in the 5'-UTR of TIF4631.
We have not found any specific phenotype for yeast strains carrying TIF4631 genes with truncated 5'-UTRs. The generation time at 30°C in YPD was similar (100 min) for the strain CBY19 transformed with pRS313–112.4G, pRS313–170.4G, pRS313–320.4G, pRS313–370.4G, pRS313– 470.4G, pRS313–520.4G, or pRS313–530.4G after selection on 5-FOA. Furthermore, we did not detect significant differences between these strains in recovery after starvation, sporulation (diploid strains), or growth at lower (22°C) or higher (37°C) temperatures. We therefore conclude that the promoter in the region -112 to -36 is sufficient for growth under all these conditions by promoting the synthesis of mRNA with short 5'-UTR that is translated in a cap-dependent fashion.
Our findings are reminiscent of the situation in higher eukaryotes, where a strong promoter was found in the putative IRES of the gene encoding eIF4G1. This promoter also lies in a nucleotide sequence earlier believed to harbor an IRES (Han and Zhang 2001). As in our case, no evidence for an IRES could be found when a dicistronic mRNA containing this eIF4G1 sequence was translated in vitro or in vivo.
Finally, these results should remind us that second ORF translation from dicistronic mRNAs is insufficient to claim internal initiation (Kozak 2001) and that several additional control experiments have to be carried out, among them the determination of potential promoter activity in the putative IRES. Along this line, the presence of cryptic promoters in the genes TFIID, HAP4, and YAP1 encoding yeast transcription factors may be responsible for the expression of these proteins from dicistronic DNA constructs when encoded by the second ORF (Hecht et al. 2002).
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MATERIALS AND METHODS |
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.
Plasmids
The plasmids used in this study are shown in Table 2.
SP6R.P
The Photinus luciferase ORF was amplified by PCR on luciferase T7 DNA from Promega (plasmid provided with the kit TNT T7 Coupled Reticulocyte Lysate System) with a forward primer introducing a BamHI restriction site followed by an NcoI site (at the ATG start codon of the luciferase ORF) and the reverse primer introducing a SacI site. The resulting DNA fragment was inserted in BamHI/SacI-cut SP64 Poly(A) plasmid (Promega), producing plasmid SP6P. The plasmid SP6R.P was created introducing the Renilla luciferase ORF as an HindIII/BamH1 fragment into a HindIII/BamHI-cut plasmid SP6P.
SP6R.4G(-508/-3).P
The 5'-untranslated region of the TIF4631 gene from position -508 to -3 was amplified by PCR on genomic DNA of S. cerevisiae. The forward primer inserted a BamHI site at the 5'-end and the reverse primer an NcoI site (at the ATG start codon of the TIF4631 ORF) at the 3'-end. This sequence was then inserted into a BamH1/NcoI-cut plasmid SP6R.P.
pGal-R.P, pGal-R.4G(-508/-3).P, pGal-R.4G(-250/-3).P
These constructs were kindly provided by W. Zhou, who referred to them as pMyr-RP (pGal-R.P), pMyr-p150/RP (pGal-R.4G(-508/-3).P), and pMyr-p150/RP (250–508) (pGal-R.4G(-250/-3).P) (Zhou et al. 2001).
Deletion of nucleotide sequences in the 5'-region of the TIF4631 gene
The double-stranded nested deletion kit (Pharmacia) was used to produce deletions in the 5'-region of the TIF4631 gene on the plasmid pRS313.4G (vector pSR313 with inserted 3.5-kb BamHI/EcoRI fragment carrying the 5'-region [-530/-1] and the TIF4631 ORF with a His6x tag). This vector was digested with BamHI and SacI and the linerarized vector was incubated with exonuclease III at 28°C for different times to digest DNA from the 3'-end. S1 nuclease was then used to digest DNA single strands. Finally, the Klenow fragment of Pol I was used to produce blunt ends, and DNA was recircularized by ligation and transformed into Escherichia coli XL2blue. Plasmids from transformants were analyzed for the length of the 5'-region of the TIF4631 gene by restriction enzyme digestion and sequencing. Plasmids containing inserts starting at positions -520, -470, -370, -320, -170, -112, +22, +130, and +230 relative to the ATG start codon (called pRS313–530.4G, pRS313–520.4G, pRS313–470.4G, pRS313–370.4G, pRS313–320.4G, pRS313–170.4G, pRS313–112.4G, pRS313+22.4G, pRS313+130.4G, and pRS313+230.4G) were further analyzed.
Primers
The oligonucleotide primers used in this study are shown in Table 3.
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) was obtained by PCR on plasmid SP6 R.4G(-508/-3).P using the reverse primer SP6pA/rev and the forward primers T7–75/Nco1, T7–36, T7–36/Nco1, and T7–508/Nco1, which introduced a T7 promoter. DNA was purified by phenol/chloroform extraction and ethanol precipitation (2.5 volumes) in the presence of 2.5 M ammonium acetate. In vitro transcription reactions contained 1 mM ATP, UTP, and CTP; 0.2 mM GTP; 1 mM m7G(5')ppp(5')G (New England Biolabs); and (per microliter of reaction mixture) 40–50 ng of DNA, 0.8 U of RNasin, 1 U of RNA polymerase (SP6 or T7), and 6000 cpm of 35S-UTP (to quantify the RNA by measurement of UTP incorporation). After 2 h of incubation at 37°C, the RNA was phenol/chloroform-extracted and precipitated with ethanol (2.5 volumes) in the presence of 2.5 M ammonium acetate. Pellets were resuspended in DEPC-treated water.
In vitro translation
Yeast extracts were prepared as described (Altmann and Trachsel 1997). Briefly, cells were collected in the logarithmic growth phase and treated with ß-mercaptoethanol, followed by treatment with zymolyase and regeneration in rich medium containing 1 M Sorbitol. Spheroblasts were homogenized with a dounce homogenizer. After isolation of the S100 fraction by centrifugation, the extract was passed through a Sephadex G25 column. The OD260 peak of the void fraction was collected and stored in liquid nitrogen.
Extracts were treated for 10 min at room temperature with micrococcal nuclease (Amersham Pharmacia Biotech). Translation reactions (15 µL) contained 28 mM HEPES/KOH (pH 7.4), 180 mM KAc, 3 mM MgAc2, 1 mM ATP, 0.4 mM GTP, 12 mM creatine phosphate, 50 µg/mL creatine phosphokinase, 1.2 mM DTT, and 50 µM amino acids. The RNA concentrations used for translation are indicated in the figure legends. Translation mixes were incubated for 1 h at 20°C.
Luciferase assay
Photinus luciferase assays (50 µL) were done in Eppendorf tubes (saturated with BSA) in 20 mM Tris-phosphate (pH 7.8), 1 mM MgAc2, 2.7 mM MgSO4, 0.1 mM EDTA, 0.5 mM ATP, 34 mM DTT, 0.47 mM D-Luciferine (Sigma), and 0.27 mM Coenzyme A (Sigma). Measurements were done in a luminometer TD20/20 (Turner Design) by integrating the signal for 10 sec.
Dual luciferase assays were done using the Promega kit (dual luciferase reporter assay system) as recommended by the manufacturer using the luminometer TD20/20.
In vivo expression of dicistronic constructs
Wild-type CWO4, strain 334, and EGY48 (Invitrogen) were transformed with the plasmids pGal-R.P, pGal-R.4G(-508/-3).P, and pGal-R.4G(-250/-3).P using the lithium acetate method (Ito et al. 1983). Transformed cells were grown in minimal medium with 2% glucose at 30°C to exponential phase (OD600 ~ 0.6), washed with minimal medium, divided into two equal parts, and either incubated with 2% galactose and 1% raffinose (induction) or with 2% glucose (control). After 3 h of induction at 30°C, the cells were collected and resuspended in two volumes of 1x passive lysis buffer (Promega) containing 1 mg/mL porcine gelatine (PLB-GP). Cells were lysed by adding one volume of glass beads (45–65 µm) and vortexing four times for 30 sec. Extracts were centrifuged at 16,000g for 3 min at 4°C. Lysates (diluted 1/100 in PLB-GP buffer) were measured using the dual luciferase reporter assay system (Promega). Protein concentrations were determined with the Bio-Rad protein assay.
Complementation experiments
Plasmids pRS313 (control), pRS313+230.4G, pRS313+130.4G, pRS313+22.4G, pRS313–112.4G, pRS313–170.4G, pRS313–320.4G, pRS313–370.4G, pRS313–470.4G, pRS313–520.4G, and pRS313–530.4G were transformed into yeast strain CBY19 (Berset et al. 1998) using the lithium acetate method (Ito et al. 1983). The haploid CBY19 strain has both chromosomal gene copies (TIF4631 and TIF4632) disrupted and carries the wild-type TIF4631 gene under its own promoter on a URA3 plasmid (ycp50-TIF4631, URA3). The cells were analyzed for their ability to survive on 0.7% 5-FOA plates (loss of ycp50-TIF4631).
SDS polyacrylamide gel electrophoresis and Western blot analysis
Yeast cells were grown to exponential phase and harvested by centrifugation. The cell pellet from 1 mL of culture was resuspended in 250 mM Tris-HCl (pH 6.8), 10 mM DTT, 10% SDS, 0.1% ß-mercaptoethanol, 50% glycerol, and 0.5% bromophenol blue. Cells were lysed by heating to 100°C in this buffer for 1 min. Equal amounts of protein were loaded on 10% SDS polyacrylamide gels (Anderson et al. 1973; Berset et al. 1998) and blotted onto nitrocellulose for 45 min at 60 V in a Mini Trans Blot Cell (Bio-Rad). Blots were saturated with 2.5% BSA in TBS (10 mM Tris-HCl at pH 7.5, 150 mM NaCl) for 1 h at room temperature and incubated overnight with rat polyclonal antibodies against eIF4G1 (amino acids 542–883; 1:500 dilutions in TBS containing 0.5% BSA). After washing with TBS, blots were decorated for 1 h with peroxidase-conjugated rabbit anti-rat Ig (Dako) and stained with 0.018% chloronaphthol and 0.006% H2O2 in TBS. Equal protein loading was verified by Coomassie blue staining.
Isolation of total RNA from yeast cells
Yeast cells were harvested and resuspended in 10 mM Tris-HCl (pH 7.5), 2 mM EDTA, 150 mM LiCl (6 mL of buffer per gram of cells). Glass beads (45–60 µm, 10 g/g of cells), LiDS to 1% final concentration, and phenol/chloroform (10 mL/g of cells) were added. The mixture was vortexed three times for 30 sec and centrifuged at 5000g for 5 min at 4°C. The supernatant was re-extracted twice with phenol/chloroform and precipitated with 2.5 volumes ethanol containing 100 mM potassium acetate. RNA was resuspended in DEPC-treated water.
Isolation poly(A)+ RNA
Oligo(dT)25 dynabeads (Dynal) were used as recommended by the manufacturer: 300 µg of total RNA was incubated with 200 µL of dynabeads and poly(A)+ RNA eluted in 8 µL of DEPC-treated water. Poly(A)+ RNA was treated with RNase-free DNase (1 U/µL in 10 mM Tris-HCl at pH 8, 10 mM MgSO4, 1 mM CaCl2). DNase was inactivated by addition of 2 mM EGTA.
RT-PCR
Reverse transcription and PCR were carried out in a one-tube reaction in 20 mM Tris-HCl (pH 8.5), 50 mM KAc, 2.5 mM MgAc2, 10 mM DTT, 0.4 mM dNTPs, and 1 mM primers. Reverse transcription reactions (15 µL) contained 1.2 µL of poly(A)+ RNA and 3 U of AMV reverse transcriptase and were incubated for 45 min at 48°C.
For the PCR reaction, 0.1 U/µL of Taq DNA polymerase was added. Some cDNA preparations were diluted before PCR (see figure legends). Controls (minus AMV reverse transcriptase) never gave any signal.
RACE
The 5' RACE kit (Invitrogen) was used with poly(A)+ RNA as recommended by the manufacturer, except that AMV reverse transcriptase was used. The reverse transcription primer was MA150. PCR amplification of dC-tailed cDNA was done with primers MV07 and Abridge Anchor Primer. For the final amplification, MV07 and AUAP primers were used.
PCR product were purified through QIAGEN qiaquick columns as recommended and inserted into pGEM-T vector (Promega). Clones were analyzed by PCR, restriction digestion, and DNA sequencing.
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ACKNOWLEDGMENTS |
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Footnotes |
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Received May 21, 2003; accepted October 28, 2003.
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REFERENCES |
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Altmann, M. and Trachsel, H. 1997. Translation initiation factor-dependent extracts from yeast Saccharomyces cerevisiae. Methods 11: 343–352.[CrossRef][Medline]
Altmann, M., Sonenberg, N., and Trachsel, H. 1989. Translation in Saccharomyces cerevisiae: Initiation factor 4E-dependent cell-free system. Mol. Cell. Biol. 9: 4467–4472.
Altmann, M., Blum, S., Pelletier, J., Sonenberg, N., Wilson, T.M., and Trachsel, H. 1990. Translation initiation factor-dependent extracts from Saccharomyces cerevisiae. Biochim. Biophys. Acta 1050: 155–159.[Medline]
Anderson, C.W., Baum, P.R., and Gesteland, R.F. 1973. Processing of adenovirus 2-induced proteins. J. Virol. 12: 241–252.
Banroques, J., Delahodde, A., and Jacq, C. 1986. A mitochondrial RNA maturase gene transferred to the yeast nucleus can control mitochondrial mRNA splicing. Cell 46: 837–844.[CrossRef][Medline]
Belsham, G.J. and Jackson, R.J. 2000. Translation initiation on picornovirus RNA. In Translational control of gene expression (eds. N. Sonenberg et al.), pp. 869–900. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Belsham, G.J. and Sonenberg, N. 1996. RNA–protein interactions in regulation of picornavirus RNA translation. Microbiol. Rev. 60: 499–511.
Berset, C., Trachsel, H., and Altmann, M. 1998. The TOR (target of rapamycin) signal transduction pathway regulates the stability of translation initiation factor eIF4G in the yeast Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. 95: 4264–4269.
Borman, A.M. and Kean, K.M. 1997. Intact eukaryotic initiation factor 4G is required for hepatitis A virus internal initiation of translation. Virology 237: 129–136.[CrossRef][Medline]
Carter, M.S., Kuhn, K.M., and Sarnow, P. 2000. Cellular internal ribosome entry site elements and the use of cDNA microarrays in their investigation. In Translational control of gene expression (eds. N. Sonenberg et al.), pp. 615–635. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Cigan, A.M., Feng, L., and Donahue, T.F. 1988. tRNAimet functions in directing the scanning ribosome to the start site of translation. Science 242: 93–97.
Dever, T.E. 2002. Gene-specific regulation by general translation factors. Cell 108: 545–556.[CrossRef][Medline]
Dorokhov, Y.L., Skulachev, M.V., Ivanov, P.A., Zvereva, S.D., Tjulkina, L.G., Merits, A., Gleba, Y.Y., Hohn, T., and Atabekov, J.G. 2002. Polypurine (A)-rich sequences promote cross-kingdom conservation of internal ribosome entry. Proc. Natl. Acad. Sci. 99: 5301–5306.
Gingras, A.C., Raught, B., and Sonenberg, N. 1999. eIF4 initiation factors: Effectors of mRNA recruitment to ribosomes and regulators of translation. Annu. Rev. Biochem. 68: 913–963.[CrossRef][Medline]
Goyer, C., Altmann, M., Lee, H.S., Blanc, A., Deshmukh, M., Woolford, J.L., Trachsel, H., and Sonenberg, N. 1993. TIF4631 and TIF4632—Two yeast genes encoding the high-molecular-weight subunits of the cap-binding protein complex (eukaryotic initiation factor-4F) contain an RNA recognition motif-like sequence and carry out an essential function. Mol. Cell. Biol. 13: 4860– 4874.
Han, B. and Zhang, J.T. 2001. Regulation of gene expression by internal ribosome entry sites or cryptic promoters: The eIF4G story. Mol. Cell. Biol. 22: 7372–7384.
Hecht, K., Bailey, J.E., and Minas, W. 2002. Polycistronic gene expression in yeast versus cryptic promoter elements. FEMS Yeast Res. 2: 215–224.[Medline]
Hellen, C.U. and Sarnow, P. 2001. Internal ribosome entry sites in eukaryotic mRNA molecules. Genes & Dev. 15: 1593–1612.
Hershey, J.W.B. and Merrick, W.C. 2000. Pathway and mechanism of initiation of protein synthesis. In Translational control of gene expression (eds. N. Sonenberg et al.), pp. 33–88. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Iizuka, N., Najit, L., Franzusoff, A., and Sarnow, P. 1994. Cap-dependent and cap-independent translation by internal initiation of mRNAs in cell extracts prepared from Saccharomyces cerevisiae. Mol. Cell. Biol. 11: 7322–7330.
Ito, H., Fukuda, Y., Murata, K., and Kimura, A. 1983. Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 53: 163–168.
Jackson, R.J. 2000. Comparative view of initiation site selection mechanisms. In Translational control of gene expression (eds. N. Sonenberg et al.), pp. 127–183. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Jackson, R.J. and Kaminski, A. 1995. Internal initiation of translation in eukaryotes: The picornavirus paradigm and beyond. RNA 1: 985–1000.[Medline]
Komar, A.A., Lesnik, T., Cullin, C., Merrick, W.C., Trachsel, H., and Altmann, M. 2003. Internal initiation drives the synthesis of Ure2 protein lacking the prion domain and affects [URE3] propagation in yeast cells. EMBO J. 22: 1199–1209.[CrossRef][Medline]
Kozak, M. 1989. The scanning model for translation: An update. J. Cell Biol. 108: 229–241.
———. 2001. New ways of initiating translation in eukaryotes? Mol. Cell. Biol. 21: 1899–1907.
Pain, V.M. 1996. Initiation of protein synthesis in eukaryotic cells. Eur. J. Biochem. 236: 747–771.[Medline]
Paz, I., Abramovitz, L., and Choder, M. 1999. Starved Saccharomyces cerevisiae cells have the capacity to support internal initiation of translation. J. Biol. Chem. 274: 21741–21745.
Pestova, T.V., Shatsky, I.N., Fletcher, S.P., Jackson, R.J., and Hellen, C.U. 1998. A prokaryotic-like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initiation of hepatitis C and classical swine fever virus RNAs. Genes & Dev. 12: 67–83.
Sachs, A.B., Sarnow, P., and Hentze, M.W. 1997. Starting at the beginning, middle, and end: Translation initiation in eukaryotes. Cell 89: 831–838.[CrossRef][Medline]
Sikorski, R.S. and Hieter, P. 1989. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122: 19–27.
Tarun Jr., S.Z., Wells, S.E, Deardorff, J.A, and Sachs, A.B. 1997. Translation initiation factor eIF4G mediates in vitro poly(A) tail-dependent translation. Proc. Natl. Acad. Sci. 94: 9046–9051.
Thompson, S.R., Gulyas, K.D., and Sarnow, P. 2001. Internal initiation in Saccharomyces cerevisiae mediated by an initiator tRNA/eIF2-independent internal ribosome entry site element. Proc. Natl. Acad. Sci. 98: 12972–12977.
Wilson, J.E., Pestova, T.V., Hellen, C.U., and Sarnow, P. 2000. Initiation of protein synthesis from the A site of the ribosome. Cell 102: 511–520.[CrossRef][Medline]
Zhou, W., Edelman, G.M., and Mauro, V.P. 2001. Transcript leader regions of two Saccharomyces cerevisiae mRNAs contain internal ribosome entry sites that function in living cells. Proc. Natl. Acad. Sci. 98: 1531–1536.
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