In Vitro Translation of the Upstream Open Reading Frame in the Mammalian mRNA EncodingS-Adenosylmethionine Decarboxylase
2000; Elsevier BV; Volume: 275; Issue: 32 Linguagem: Inglês
10.1074/jbc.m003364200
ISSN1083-351X
AutoresAlexa Raney, Anita C. Baron, Gregory J. Mize, G. Lynn Law, David R. Morris,
Tópico(s)Cancer-related gene regulation
ResumoThe upstream open reading frame (uORF) in the mRNA encoding S-adenosylmethionine decarboxylase is a polyamine-responsive element that suppresses translation of the associated downstream cistron in vivo. In this paper, we provide the first direct evidence of peptide synthesis from theS-adenosylmethionine decarboxylase uORF using an in vitro translation system. We examine both the influence of cation concentration on peptide synthesis and the effect of altering the uORF sequence on peptide synthesis. Synthesis of wild type and altered peptides was similar at all concentrations of magnesium tested. In contrast, synthesis of the wild type peptide was more sensitive than that of altered peptides to elevated concentrations of the naturally occurring polyamines, spermidine and spermine, as well as several polyamine analogs. The sensitivity of in vitro synthesis to spermidine was influenced by both the amino acid sequence and the length of the peptide product of the uORF. Findings from the present study correlate with the effects of the uORF and polyamines on translation of a downstream cistron in vivo and support the hypothesis that polyamines and the structure of the nascent peptide create a rate-limiting step in uORF translation, perhaps through a ribosome stalling mechanism. The upstream open reading frame (uORF) in the mRNA encoding S-adenosylmethionine decarboxylase is a polyamine-responsive element that suppresses translation of the associated downstream cistron in vivo. In this paper, we provide the first direct evidence of peptide synthesis from theS-adenosylmethionine decarboxylase uORF using an in vitro translation system. We examine both the influence of cation concentration on peptide synthesis and the effect of altering the uORF sequence on peptide synthesis. Synthesis of wild type and altered peptides was similar at all concentrations of magnesium tested. In contrast, synthesis of the wild type peptide was more sensitive than that of altered peptides to elevated concentrations of the naturally occurring polyamines, spermidine and spermine, as well as several polyamine analogs. The sensitivity of in vitro synthesis to spermidine was influenced by both the amino acid sequence and the length of the peptide product of the uORF. Findings from the present study correlate with the effects of the uORF and polyamines on translation of a downstream cistron in vivo and support the hypothesis that polyamines and the structure of the nascent peptide create a rate-limiting step in uORF translation, perhaps through a ribosome stalling mechanism. S-adenosylmethionine decarboxylase upstream open reading frame high performance liquid chromatography N-(3-aminopropyl)-1,3-propanediamine N-(3-aminopropyl)-1,6-hexanediamine N1 N12-bis(ethyl)spermine The naturally occurring polyamines putrescine, spermidine, and spermine are small positively charged molecules that occur ubiquitously in living cells and are required for cell growth (1Marton L.J. Morris D.R. McCann P.P. Pegg A.E. Sjoerdsma A. Inhibition of Polyamine Biosynthesis: Biological Significance and Basis for New Therapies. Academic Press, Inc., New York1987: 79-105Google Scholar, 2Morris D.R. J. Cell. Biochem. 1991; 46: 102-105Crossref PubMed Scopus (56) Google Scholar). They have been implicated in many cellular processes, including DNA replication, transcription, and translation (1Marton L.J. Morris D.R. McCann P.P. Pegg A.E. Sjoerdsma A. Inhibition of Polyamine Biosynthesis: Biological Significance and Basis for New Therapies. Academic Press, Inc., New York1987: 79-105Google Scholar, 2Morris D.R. J. Cell. Biochem. 1991; 46: 102-105Crossref PubMed Scopus (56) Google Scholar). Control of intracellular polyamine levels is important, because depletion of polyamines leads to decreased cell growth and alterations in cellular differentiation (3Pegg A.E. McCann P.P. Am. J. Physiol. 1982; 243: C212-C221Crossref PubMed Google Scholar, 4Pegg A.E. Cancer Res. 1988; 48: 759-774PubMed Google Scholar, 5Marton L.J. Pegg A.E. Annu. Rev. Pharmocol. Toxicol. 1995; 35: 55-91Crossref PubMed Scopus (711) Google Scholar), whereas elevation of polyamines can be associated with cell toxicity and with oncogenic transformation (2Morris D.R. J. Cell. Biochem. 1991; 46: 102-105Crossref PubMed Scopus (56) Google Scholar, 6Heby O. Persson L. Trends Biochem. Sci. 1990; 15: 153-158Abstract Full Text PDF PubMed Scopus (496) Google Scholar). Thus, polyamine levels are tightly regulated by a variety of mechanisms, including feedback regulation of the expression, activity, and stability of key enzymes involved in polyamine biosynthesis (2Morris D.R. J. Cell. Biochem. 1991; 46: 102-105Crossref PubMed Scopus (56) Google Scholar, 6Heby O. Persson L. Trends Biochem. Sci. 1990; 15: 153-158Abstract Full Text PDF PubMed Scopus (496) Google Scholar, 7Large P.J. FEMS Microbiol. Rev. 1992; 8: 249-262Crossref PubMed Google Scholar, 8Grillo M.A. Colombatto S. Biochem. Soc. Trans. 1994; 22: 894-898Crossref PubMed Scopus (27) Google Scholar). S-Adenosylmethionine decarboxylase (AdoMetDC)1is a key regulated enzyme in the pathway of polyamine biosynthesis. AdoMetDC catalyzes the decarboxylation ofS-adenosylmethionine, thus generating then-propylamine donor for the conversion of putrescine to spermidine and of spermidine to spermine. Expression of AdoMetDC is regulated by exogenous signals and also by the endogenous level of polyamines, which influence its expression at multiple levels including transcription, translation, and enzyme stability (9Shirahata A. Pegg A.E. J. Biol. Chem. 1985; 260: 9583-9588Abstract Full Text PDF PubMed Google Scholar, 10Shirahata A. Pegg A.E. J. Biol. Chem. 1986; 261: 13833-13837Abstract Full Text PDF PubMed Google Scholar, 11Pajunen A. Crozat A. Janne O.A. Ihalainen R. Laitinen P.H. Stanley B. Madhubala R. Pegg A.E. J. Biol. Chem. 1988; 263: 17040-17049Abstract Full Text PDF PubMed Google Scholar, 12White M.W. Degnin C. Hill J. Morris D.R. Biochem. J. 1990; 268: 657-660Crossref PubMed Scopus (28) Google Scholar). Translational regulation of AdoMetDC may serve as a fast response mechanism (13Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Press, Cold Spring Harbor, NY1996Google Scholar), turning off polyamine synthesis quickly when intracellular levels reach a critical level. The mammalian AdoMetDC transcript contains an upstream open reading frame (uORF) specifying a peptide of six amino acids with the sequence MAGDIS (14Hill J.R. Morris D.R. J. Biol. Chem. 1992; 267: 21886-21893Abstract Full Text PDF PubMed Google Scholar). Studies in mammalian cells suggest that translation of this uORF is necessary for polyamine regulation of AdoMetDC (15Ruan H. Shantz L.M. Pegg A.E. Morris D.R. J. Biol. Chem. 1996; 271: 29576-29582Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). A point mutation in the AUG start codon abolishes regulation, and insertion of the uORF into the 5′ leader of a heterologous reporter gene confers polyamine regulation similar to that seen with the endogenous AdoMetDC transcript (15Ruan H. Shantz L.M. Pegg A.E. Morris D.R. J. Biol. Chem. 1996; 271: 29576-29582Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Inhibition of downstream translation by the AdoMetDC uORF requires its amino acid coding sequence but not its precise nucleotide sequence, because mis-sense codon substitutions but not synonymous substitutions abolish the suppressive influence of the uORF (15Ruan H. Shantz L.M. Pegg A.E. Morris D.R. J. Biol. Chem. 1996; 271: 29576-29582Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Saturation mutagenesis of the last three codons of the uORF in yeast demonstrate that only aspartic acid is tolerated in the fourth position, and only the homolog of isoleucine, valine, can substitute in the fifth position (16Mize G.J. Ruan H. Low J.J. Morris D.R. J. Biol. Chem. 1998; 273: 32500-32505Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The precise positioning of these two amino acids relative to the stop codon may also be important, because carboxyl-terminal extensions and truncations of the peptide product of the uORF abolish its suppressive influence (16Mize G.J. Ruan H. Low J.J. Morris D.R. J. Biol. Chem. 1998; 273: 32500-32505Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 17Hill J.R. Morris D.R. J. Biol. Chem. 1993; 268: 726-731Abstract Full Text PDF PubMed Google Scholar). These studies imply a rather precise interaction between the MAGDIS peptide and its regulatory target. According to the ribosome stalling model of polyamine regulation of AdoMetDC translation (17Hill J.R. Morris D.R. J. Biol. Chem. 1993; 268: 726-731Abstract Full Text PDF PubMed Google Scholar, 18Geballe A.P. Morris D.R. Trends Biochem. Sci. 1994; 19: 159-164Abstract Full Text PDF PubMed Scopus (195) Google Scholar), polyamines act to modulate an interaction of the fourth and fifth amino acids of the peptide product of the AdoMetDC uORF with some component in the translational apparatus, such as the peptidyl transferase center, the peptide channel of the ribosome, or a termination factor. The role of the sixth residue may be to position these residues in the correct orientation for this interaction to occur. This model predicts that a step in translation, such as chain termination or peptide release, would be inhibited through this interaction, causing the ribosome to stall over the termination codon. Ribosome stalling would block further scanning of ribosomes and suppress translation of both the uORF and the downstream cistron. This model implies that low polyamine levels would lead to relief of the blockade and allow translation of both the uORF and the downstream cistron. The ribosome stalling model of AdoMetDC translation is similar to that proposed for another sequence-dependent regulatory uORF found in theNeurospora crassa arg-2 transcript, with the exception that in this transcript the intracellular level of arginine regulates the stalling (19Wang Z. Sachs M.S. J. Biol. Chem. 1997; 272: 255-261Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 20Wang Z. Sachs M.S. Mol. Cell. Biol. 1997; 17: 4904-4913Crossref PubMed Scopus (108) Google Scholar). The ribosome stalling model makes certain predictions that can be tested in an in vitro translation system. First and foremost, the AdoMetDC uORF must be translated, and its translation should be influenced by the concentration of polyamines. The effect of polyamines on translation of the uORF should be influenced by specific changes in the amino acid sequence of the encoded peptide or in its length but not by changes in the ribonucleotide sequence that retain the amino acid coding content. Furthermore, for regulation of AdoMetDC to be a fine-tuned biological mechanism, we predict that uORF translation should be influenced by polyamines in a unique fashion not shared by other cellular cations such as magnesium. In the present study, these predictions were tested using an in vitro wheat germ translation system and a newly developed analytical procedure to measure synthesis of the six-residue peptide product of the uORF. Constructs were designed to produce capped transcripts driven by the T7 promoter that contain the AdoMetDC 5′ leader with wild type or altered uORFs (14Hill J.R. Morris D.R. J. Biol. Chem. 1992; 267: 21886-21893Abstract Full Text PDF PubMed Google Scholar) but not any of the open reading frame encoding AdoMetDC. The full 5′ leader is intact in these constructs, with the exception that the 14 nucleotides from the 5′ cap to the uORF in the mammalian transcript is replaced with 80 nucleotides from the pBluescript SK+ polylinker region. Furthermore, HindIII and BglII restriction enzyme sites were engineered for insertion of synthetic oligonucleotides encoding the uORFs. All constructs were verified by sequencing. Constructs were linearized withXbaI, purified, and used as templates for in vitro transcription reactions using a T7 transcription kit (Ambion, Austin, TX). Capped transcripts were made using trace amounts of [32P]UTP (3000 Ci/mmol; NEN Life Science Products) to quantitate RNA. RNA concentration was determined in triplicate by trichloroacetic acid precipitation and liquid scintillation counting, using the specific activity of the [32P]UTP. The integrity of the RNA and also the relative concentration was verified by resolving duplicate samples on an 8% denaturing acrylamide gel, followed by quantitation using the STORM PhosphorImager system and ImageQuant software (Molecular Dynamics, Sunnyvale, CA). RNA was stored in aliquots at −80 °C with RNasin (Promega, Madison, WI). Wheat germ extract was prepared essentially as described previously (21Anderson C.W. Straus J.W. Dudock B.S. Methods Enzymol. 1983; 101: 635-644Crossref PubMed Scopus (129) Google Scholar, 22Erickson A.H. Blobel G. Methods Enzymol. 1983; 96: 38-50Crossref PubMed Scopus (209) Google Scholar, 23Morch M.D. Drugeon G. Zagorski W. Haenni A.L. Methods Enzymol. 1986; 118: 154-164Crossref Scopus (34) Google Scholar). In vitro translation was performed in a total volume of 50 μl using a modified procedure (21Anderson C.W. Straus J.W. Dudock B.S. Methods Enzymol. 1983; 101: 635-644Crossref PubMed Scopus (129) Google Scholar, 22Erickson A.H. Blobel G. Methods Enzymol. 1983; 96: 38-50Crossref PubMed Scopus (209) Google Scholar, 23Morch M.D. Drugeon G. Zagorski W. Haenni A.L. Methods Enzymol. 1986; 118: 154-164Crossref Scopus (34) Google Scholar). The final concentration of the components in the translation reactions were: 1.2 mm ATP, 80 μm GTP, 0.4 mm each amino acid (excluding methionine), 9.6 mm creatine phosphate, 2.0 mm potassium hydroxide, 64 μg/ml creatine phosphokinase, 20 mm HEPES, 2 mmdithiothreitol, 1.65 mm magnesium (unless otherwise stated), 53 mm potassium acetate, 2 μCi/μl [35S]methionine (specific activity, 1175 Ci/mmol; NEN Life Science Products), RNA (8 ng/μl), RNasin (0.4 units/μl), and 2 mm bestatin aminopeptidase inhibitor (Sigma). To each reaction, 20 μl of wheat germ lysate was added. The concentration of endogenous polyamines in three independent lysate preparations was determined (24Seiler N. Methods Enzymol. 1983; 94: 10-20Crossref PubMed Scopus (73) Google Scholar), and the lysates were found to contribute 40–70 μm spermidine, 15–25 μm spermine, and no detectable putrescine, to the final reaction volume. Additional spermidine (Sigma) or spermine (Sigma) was added to the reactions as indicated under “Results.” Reactions were incubated 20 min (unless otherwise stated) at room temperature and were terminated by placing tubes on ice. Unlabeled carrier peptides (30 nmol) with the sequence MAGDIS (Alpha Diagnostic, San Antonio, TX) or MAGEIS, MAGDVS, MAGDLS, or MAGDI (United Biochemical Research, Inc., Seattle, WA) were added to samples as appropriate. Peptides were extracted from the translation mixtures by vortexing the samples with methanol (60 μl) andn-butanol (120 μl), incubating the samples at −80 °C (1 h), centrifuging the samples at 20,000 × g (10 min), and evaporating the resulting supernatant solutions to dryness (25Hackett P.B. Petersen R.B. Hensel C.H. Albericio F. Gunderson S.I. Palmenberg A.C. Barany G. J. Mol. Biol. 1986; 190: 45-57Crossref PubMed Scopus (36) Google Scholar). Extracted translation samples were resuspended in 30 μl of water (HPLC grade), followed by 130 μl water plus 0.1% trifluoroacetic acid (HPLC grade; Pierce). Samples were loaded onto a C18 reverse phase column (inner diameter, 4.6 mm × 75 mm; Altex, Berkeley, CA), linked to a BioCAD Sprint Perfusion Chromatography System (PerSeptive Biosystems, Inc., Framingham, MA). Prior to loading, the column was pre-equilibrated with water plus 0.1% trifluoroacetic acid. Peptides were eluted from the column using a gradient of acetonitrile (HPLC grade) plus 0.09% trifluoroacetic acid and a flow rate of 0.75 ml/min. The elution profile was monitored by absorbance at 210 nm, and the peptides were eluted from the column at 21.5 ± 1.5 min, depending on the sequence of the peptide. This was determined by the HPLC absorbance profiles of peptide standards (30 nmol of unlabeled carrier peptides; data not shown). Fractions containing the peptide (4 fractions, 250 μl each) were collected, pooled, and evaporated. Samples were resuspended in 20 μl of 50% acetonitrile and spotted on silica gel 60 plates (EM Separations, Gibbstown, NJ). Plates were developed 5 h inn-butanol:glacial acetic acid:water (4:1:1) and dried. Carrier peptides were visualized by spraying the plates with 0.5% ninhydrin in acetone. Plates were wrapped in plastic wrap, and in vitro translation products were quantified using the STORM PhosphorImager system and ImageQuant software. HPLC/HPLC analyses were performed as described above, except evaporated samples from the first HPLC run were derivatized (26Knapp D. Handbook of Analytical Derivatization Reactions. John Wiley and Sons, New York1979: 253Google Scholar) with acetic anhydride (Sigma) and anhydrous methyl alchohol (Sigma) and then reanalyzed by HPLC. Fractions of the acetylated peptide peak were collected and analyzed by liquid scintillation counting to quantitate translation products. The identity of the acetylated peptide peak was confirmed by mass spectroscopy. Control translation reactions containing brome mosaic virus RNA (Promega, Madison, WI) were performed as described above, except the final reaction volume was 25 μl, and reactions contained final concentrations of 0, 1, or 2 mm bestatin, 2.5 mm magnesium acetate, 140 mm potassium acetate, and 70 μm spermine. Reactions were analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. Protein synthesis was quantified by densitometry of the autoradiographs (Bio-Rad model GS-700 Imaging Densitometer). Synthetic peptides (30 nmol) were added to wheat germ translation mixtures containing 2 mm bestatin and 700 μm spermidine. Samples were incubated at room temperature or on ice (10 min), extracted, and analyzed by HPLC. The stability of the peptide was determined by comparing the HPLC peak heights of peptides incubated at room temperature versus those incubated on ice. Transcripts were added to wheat germ translation mixtures containing 700 μmspermidine and incubated at room temperature or on ice for 15 min. Transcripts were recovered by phenol/chloroform extraction and ethanol precipitation and were resolved on an 8% denaturing acrylamide gel. Transcript stability was determined in duplicate by comparing the intensity of samples incubated at room temperature versussamples incubated on ice, using the STORM PhosphorImager system. Capped transcripts of the AdoMetDC 5′ leader, containing either the wild type uORF or an altered version (Fig. 1), were synthesized and incubated in wheat germ translation mixtures with [35S]methionine. The stabilities of the transcripts in the wheat germ lysate were determined, and uniform recovery of all RNAs was obtained after incubation in translation mixtures containing 700 μm spermidine (data not shown). The peptide products encoded by the uORFs have half-lives of <1 min in wheat germ extracts (data not shown), and, thus, to stabilize the synthesized peptides, 2 mm of an aminopeptidase inhibitor (bestatin), was added to translation reactions. Using this concentration of bestatin, the recovery of wild type and altered peptides was found to be 75 ± 9% after incubation (10 min) in a wheat germ translation mixture containing 700 μm spermidine (data not shown). A similar situation was reported for the stability of other small peptides in rabbit reticulocyte lysates (25Hackett P.B. Petersen R.B. Hensel C.H. Albericio F. Gunderson S.I. Palmenberg A.C. Barany G. J. Mol. Biol. 1986; 190: 45-57Crossref PubMed Scopus (36) Google Scholar). To ensure that the presence of bestatin did not interfere with the translational machinery, control reactions using brome mosaic virus RNA were performed. The addition of a final concentration of 2 mm bestatin had no effect on brome mosaic virus RNA translation (data not shown). Peptide synthesis from the AdoMetDC uORF was analyzed according to the scheme in Fig. 2. Briefly, after incubation of the translation mixtures, unlabeled synthetic peptide was added to act as a carrier and to monitor recovery during extraction and chromatographic separations. Unlabeled and35S-labeled peptides were then extracted from the translation mixtures with a combination of methanol andn-butanol and purified by sequential HPLC and TLC. As shown by ninhydrin staining of the TLC plate in Fig. 2, uniform recovery of the carrier peptides was achieved through the chromatographic separations. A PhosphorImager scan of the same TLC plate showed co-migration of the 35S-labeled products with the ninhydrin-stained carrier peptides. 35S-Labeled products were detected from reaction mixtures containing either the wild type uORF (MAGDIS) or the altered uORF (MAGEIS) after incubation for 20 min at room temperature (lanes 3 and5). In contrast, no signal was observed in reaction mixtures either lacking added RNA (lane 1) or incubated on ice (lanes 2 and 4). To confirm peptide production from the uORF, translation reactions were analyzed by an alternate procedure (Fig.3). After initial extraction and HPLC as in Fig. 2, the recovered peptide peak from HPLC was acetylated. Acetylation lengthens the elution time of the peptides from the HPLC column by 2 min, as determined by mass spectroscopy of HPLC fractions (data not shown). As shown in Fig. 3, the acetylated radioactive products of a translation reaction containing the wild type uORF co-eluted from the HPLC column with the absorbance peak of the acetylated MAGDIS carrier peptide. A similar observation was made for translation mixtures containing the MAGEIS uORF, but no radioactive products were detected in control reactions lacking added RNA or incubated on ice (data not shown). This result, together with the co-migration of radioactive products and the corresponding carrier peptides on TLC, establishes that uORF-encoded peptides are synthesized in the wheat germ translation reactions. Protein synthesis is strongly influenced by multivalent cations, which probably act at multiple steps ranging from aminoacyl tRNA synthesis to ribosome stability (1Marton L.J. Morris D.R. McCann P.P. Pegg A.E. Sjoerdsma A. Inhibition of Polyamine Biosynthesis: Biological Significance and Basis for New Therapies. 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As shown in Fig. 4 (A and B), synthesis of both wild type (MAGDIS) and altered (MAGEIS) peptides responded to varying concentrations of magnesium and spermidine with the expected bell-shaped curves (28Igarashi K. Sugawara K. Izumi I. Nagayama C. Hirose S. Eur. J. Biochem. 1974; 48: 495-502Crossref PubMed Scopus (122) Google Scholar, 30Hunter A.R. Farrell P.J. Jackson R.J. Hunt T. Eur. J. Biochem. 1977; 75: 149-157Crossref PubMed Scopus (103) Google Scholar). The concentrations of magnesium and spermidine for optimal synthesis of the peptides were not far from physiological levels (30Hunter A.R. Farrell P.J. Jackson R.J. Hunt T. Eur. J. Biochem. 1977; 75: 149-157Crossref PubMed Scopus (103) Google Scholar, 34Tabor C.W. Tabor H. Pharmacol. Rev. 1964; 16: 245-300PubMed Google Scholar). Panels A and C of Fig. 4 show the final concentrations of magnesium in the translation reactions, whereas panels B andD show the final concentrations of addedspermidine. The wheat germ lysate contributes an additional 40–70 μm spermidine, 15–25 μm spermine, and no detectable putrescine to the final reaction volume of the reactions, as described under “Experimental Procedures”). At all concentrations of magnesium, synthesis of wild type and altered peptides was comparable, and, if anything, the wild type peptide was synthesized at a slightly greater rate (Fig. 4 A). Likewise, at concentrations of spermidine less than 300 μm, synthesis of the two peptides was comparable, but at concentrations of 500 μm and higher, synthesis of the wild type peptide was inhibited more strongly than that of the altered (Fig. 4 B). The differences in peptide synthesis in response to magnesium and spermidine concentration are observed clearly in the ratios of altered to wild type peptide synthesis plotted in Fig. 4 (C andD). This spermidine dose-response experiment was repeated three times, with different preparations of both wheat germ extract and RNA, with reproducible results. To investigate further the differences in peptide synthesis in response to cation concentration, time courses using low and high concentrations of magnesium and spermidine were carried out (Fig.5). As shown in Fig. 5, translation time courses of both wild type and altered uORFs exhibited the expected lag period (5 min), followed by linear translation (28Igarashi K. Sugawara K. Izumi I. Nagayama C. Hirose S. Eur. J. Biochem. 1974; 48: 495-502Crossref PubMed Scopus (122) Google Scholar, 30Hunter A.R. Farrell P.J. Jackson R.J. Hunt T. Eur. J. Biochem. 1977; 75: 149-157Crossref PubMed Scopus (103) Google Scholar). In translation mixtures lacking added spermidine and including low (1.65 mm) or high (4.5 mm) magnesium concentrations, the rate of translation of the wild type uORF was somewhat greater than that of the altered (Fig. 5, A and B). Specifically, the translation rate of the altered uORF is 0.64 of the wild type uORF at low magnesium (Fig. 5 A), and 0.88 at high magnesium (Fig. 5 B), as calculated from the 5–20 min portion of the curve. In contrast, in translation mixtures containing 700 μm spermidine, the effect was reversed and the translation rate of the altered uORF was 2.4-fold higher than the wild type (Fig. 5 C). This last experiment (Fig. 5 C) was repeated three times, with different preparations of wheat germ extract and RNA, and a 2–3-fold difference in translation rates was consistently observed. This difference was even more pronounced in translation mixtures containing 900 μm spermidine (4.5-fold; data not shown). The stabilities of the wild type and altered peptides and also the corresponding transcripts were shown to be identical after incubation in a wheat germ lysate containing an increased concentration of spermidine (700 μm; data not shown). Taken together, these results clearly demonstrate that translation of the wild type uORF is more sensitive to increased concentrations of spermidine than an altered uORF, and furthermore, this differential effect was not observed with magnesium. It was of interest to investigate whether the sensitivity of wild type uORF translation to spermidine resulted from the ribonucleotide sequence of the uORF, the sequence of the resulting peptide, and/or the length of the peptide product. To this end, translation of the wild type uORF and various altered uORFs (Fig. 1) were compared in wheat germ translation reactions with and without added spermidine (750 μm). As shown in TableI, translation of the wild type uORF, the MAGDVS uORF, and a uORF encoding the wild type peptide but with synomynous codon substitutions (WOBBLE), were highly sensitive to the addition of 750 μm spermidine. The influence of spermidine on translation of the wild type and WOBBLE uORFs was comparable, whereas translation of the MAGDVS uORF may have been slightly more sensitive. In contrast, translation of the MAGDLS and MAGDI uORFs were much less sensitive to spermidine than were either the MAGEIS or wild type uORFs. In fact, translation of MAGDLS and MAGDI uORFs may have been slightly stimulated by 750 μm spermidine (Table I). In a similar experiment, alteration of the termination codon from wild type (UAG) to UAA yielded a transcript equally as sensitive to spermidine as wild type, but an alteration to the UGA termination codon resulted in 1.6-fold less sensitivity, as determined by comparing the ratios (no added spermidine/750 μm spermidine; data not shown).Table ISequence dependence of the sensitivity of uORF translation to spermidineuORFNo added spermidine750 μm spermidineNo Added/750 μm spermidineMAGDIS42.8 ± 0.19.6 ± 0.34.5 ± 0.1MAGD VS61 ± 311 ± 15.8 ± 0.4WOBBLE30 ± 67.3 ± 0.74.2 ± 0.5MAG EIS39 ± 219 ± 32.2 ± 0.2MAGD LS79 ± 6100 ± 30.80 ± 0.03MAGDI77 ± 483 ± 20.94 ± 0.02Wheat germ translation reactions containing the appropriate uORF and either no added spermidine or 750 μm spermidine were carried out for 20 min and analyzed using the HPLC/TLC method (Fig. 2). The data represent the means ± S.D. of the mean of triplicate translation samples. The values shown are the intensities of the35S-labeled peptides from the PhosphorImager scan. These values were normalized to the translation sample with the highest intensity (MAGDLS, 750 μm spermidine), which was set at 100. Open table in a new tab Wheat germ translation reactions containing the appropriate uORF and either no added spermidine or 750 μm spermidine were carried out for 20 min and analyzed using the HPLC/TLC method (Fig. 2). The data represent the means ± S.D. of the mean of t
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