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• W2022344159 abstract "Spermidine-spermine N1-acetyltransferase (SSAT) is induced in response to an elevation in intracellular polyamine pools. The increased enzyme activity is the result of an increase in gene transcription, mRNA translation, and protein stability. Induction of SSAT by polyamine analogues can lead to intracellular polyamine depletion and apoptosis. The mechanism by which polyamines alter the translational efficiency of SSAT mRNA is not well understood. In this study, we investigated the regulation of SSAT translation by the polyamine analogue N1,N11-diethylnorspermine (DENSPM). DENSPM induced expression of both FLAG-tagged SSAT and SSAT fused to Renilla luciferase in a time- and concentration-dependent manner. This effect was not inhibited by actinomycin D indicating that changes in gene transcription did not explain the enhanced expression in the presence of DENSPM. Furthermore, because FLAG-SSAT did not contain the 5′- or 3′-untranslated regions of SSAT, translational regulation involved the coding sequence only. By contrast, cycloheximide completely inhibited induction by DENSPM, indicating a requirement for new protein synthesis. Deletion constructs identified two regions of the SSAT protein-coding RNA sequence that conferred polyamine responsiveness. Using these regions as probes in RNA electrophoretic mobility shift assays, we observed specific binding of a cytoplasmic protein. In addition, we found that the interaction between the RNA probes and the binding protein could be inhibited by DENSPM in a concentration-dependent manner. These results suggest that polyamines regulate SSAT mRNA translational efficiency by inhibiting a repressor protein from binding to regions of the coding sequence of the SSAT transcript. Spermidine-spermine N1-acetyltransferase (SSAT) is induced in response to an elevation in intracellular polyamine pools. The increased enzyme activity is the result of an increase in gene transcription, mRNA translation, and protein stability. Induction of SSAT by polyamine analogues can lead to intracellular polyamine depletion and apoptosis. The mechanism by which polyamines alter the translational efficiency of SSAT mRNA is not well understood. In this study, we investigated the regulation of SSAT translation by the polyamine analogue N1,N11-diethylnorspermine (DENSPM). DENSPM induced expression of both FLAG-tagged SSAT and SSAT fused to Renilla luciferase in a time- and concentration-dependent manner. This effect was not inhibited by actinomycin D indicating that changes in gene transcription did not explain the enhanced expression in the presence of DENSPM. Furthermore, because FLAG-SSAT did not contain the 5′- or 3′-untranslated regions of SSAT, translational regulation involved the coding sequence only. By contrast, cycloheximide completely inhibited induction by DENSPM, indicating a requirement for new protein synthesis. Deletion constructs identified two regions of the SSAT protein-coding RNA sequence that conferred polyamine responsiveness. Using these regions as probes in RNA electrophoretic mobility shift assays, we observed specific binding of a cytoplasmic protein. In addition, we found that the interaction between the RNA probes and the binding protein could be inhibited by DENSPM in a concentration-dependent manner. These results suggest that polyamines regulate SSAT mRNA translational efficiency by inhibiting a repressor protein from binding to regions of the coding sequence of the SSAT transcript. Spermidine-spermine N1-acetyltransferase (SSAT) 2The abbreviations used are: SSATspermidine-spermine N1-acetyltransferaseDENSPMN1,N11-diethylnorspermineREMSARNA electrophoretic mobility shift assayUTRuntranslated regionCMVcytomegalovirusHRPhorseradish peroxidaseORFopen reading frame. is a key enzyme in the degradation of the essential polyamines spermidine and spermine (1Casero R.A. Pegg Jr., A.E. FASEB J. 1993; 7: 653-661Crossref PubMed Scopus (393) Google Scholar, 2Wallace H.M. Fraser A.V. Hughes A. Biochem. J. 2003; 376: 1-14Crossref PubMed Scopus (778) Google Scholar). Normally, the intracellular level of SSAT is low, but it can be rapidly induced by elevating intracellular polyamine concentrations (3Fogel-Petrovic M. Vujcic S. Brown P.J. Haddox M.K. Porter C.W. Biochemistry. 1996; 35: 14436-14444Crossref PubMed Scopus (64) Google Scholar) or by treating cells with polyamine mimetics such as N1,N11-diethylnorspermine (DENSPM) or N1,N12-bis(ethyl)spermine (4Kramer D.L. Vujcic S. Diegelman P. White C. Black J.D. Porter C.W. Biochem. Soc. Trans. 1998; 26: 609-614Crossref PubMed Scopus (13) Google Scholar, 5Parry L. Balana Fouce R. Pegg A.E. Biochem. J. 1995; 305: 451-458Crossref PubMed Scopus (52) Google Scholar). SSAT expression is regulated at several different levels (5Parry L. Balana Fouce R. Pegg A.E. Biochem. J. 1995; 305: 451-458Crossref PubMed Scopus (52) Google Scholar, 6Fogel-Petrovic M. Shappell N.W. Bergeron R.J. Porter C.W. J. Biol. Chem. 1993; 268: 19118-19125Abstract Full Text PDF PubMed Google Scholar). Gene transcription is increased by polyamines via an Nrf-2-dependent pathway, leading to an increase in SSAT mRNA (7Wang Y. Xiao L. Thiagalingam A. Nelkin B.D. Casero Jr., R.A. J. Biol. Chem. 1998; 273: 34623-34630Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Moreover, polyamines stabilize SSAT mRNA (6Fogel-Petrovic M. Shappell N.W. Bergeron R.J. Porter C.W. J. Biol. Chem. 1993; 268: 19118-19125Abstract Full Text PDF PubMed Google Scholar) and increase translational efficiency (5Parry L. Balana Fouce R. Pegg A.E. Biochem. J. 1995; 305: 451-458Crossref PubMed Scopus (52) Google Scholar). Finally, elevated polyamine levels can lead to a stabilization of the SSAT protein by inhibiting its polyubiquitination and targeting to the proteasome (8Coleman C.S. Pegg A.E. Biochem. J. 2001; 358: 137-145Crossref PubMed Scopus (50) Google Scholar). N-terminal substituted polyamine analogues are not substrates for SSAT but appear to mimic the endogenous polyamine and cause an increase in intracellular SSAT activity that can be >1000-fold higher than that in untreated cells. The elevation in SSAT levels leads to a depletion of intracellular polyamines and induction of apoptosis (4Kramer D.L. Vujcic S. Diegelman P. White C. Black J.D. Porter C.W. Biochem. Soc. Trans. 1998; 26: 609-614Crossref PubMed Scopus (13) Google Scholar). The candidate drug DENSPM is currently under development as an anti-cancer agent (9Creaven P.J. Perez R. Pendyala L. Meropol N.J. Loewen G. Levine E. Berghorn E. Raghavan D. Invest. New Drugs. 1997; 15: 227-234Crossref PubMed Scopus (44) Google Scholar, 10Streiff R.R. Bender J.F. Invest. New Drugs. 2001; 19: 29-39Crossref PubMed Scopus (40) Google Scholar). spermidine-spermine N1-acetyltransferase N1,N11-diethylnorspermine RNA electrophoretic mobility shift assay untranslated region cytomegalovirus horseradish peroxidase open reading frame. The regulation of SSAT mRNA translation by polyamines is not fully understood. Fogel-Petrovic and coworkers (11Fogel-Petrovic M. Vujcic S. Miller J. Porter C.W. FEBS Lett. 1996; 391: 89-94Crossref PubMed Scopus (51) Google Scholar) showed that cycloheximide can increase SSAT mRNA in Malme-3 M cells, but removal of the protein synthesis inhibitor was not accompanied by an increase in enzyme activity. However, addition of polyamines led to an increased rate of SSAT translation, suggesting that protein synthesis required polyamines. Similarly, Parry and coworkers (5Parry L. Balana Fouce R. Pegg A.E. Biochem. J. 1995; 305: 451-458Crossref PubMed Scopus (52) Google Scholar) reported that N1,N12-bis(ethyl)spermine increased the amount of SSAT mRNA associated with the protein-synthesizing 80 S monosomes. They also concluded from deletion studies of an SSAT expression construct that the region of the mRNA responsive to N1,N12-bis(ethyl)spermine was located within the protein coding sequence. Thus, SSAT translation appears to be inhibited at low polyamine concentration, and this inhibition is released when polyamine levels are increased. SSAT activity is induced by a number of physiological stimuli, including oxidative stress (12Chopra S. Wallace H.M. Biochem. Pharmacol. 1998; 55: 1119-1123Crossref PubMed Scopus (75) Google Scholar), x-ray irradiation (13Mita K. Fukuchi K. Hamana K. Ichimura S. Nenoi M. Int. J. Radiat. Biol. 2004; 80: 369-375Crossref PubMed Scopus (10) Google Scholar), insulin-like growth factor-I (14Green M.L. Chung T.E. Reed K.L. Modric T. Badinga L. Yang J. Simmen F.A. Simmen R.C. Biol. Reprod. 1998; 59: 1251-1258Crossref PubMed Scopus (15) Google Scholar), cytotoxins (15Marverti G. Bettuzzi S. Astancolle S. Pinna C. Monti M.G. Moruzzi M.S. Eur. J. Cancer. 2001; 37: 281-289Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar), and heat shock (16Gerner E.W. Kurtts T.A. Fuller D.J. Casero Jr., R.A. Biochem. J. 1993; 294: 491-495Crossref PubMed Scopus (16) Google Scholar). Some of these stimuli do not appear to alter mRNA levels, suggesting that co- and/or post-translational mechanisms predominate. In the present study, we have investigated the translational regulation of the SSAT mRNA by DENSPM using two models, FLAG-tagged SSAT and SSAT fused to luciferase. Sequences located within the protein-coding region, and in close proximity to both the start and stop codons, are essential for the translational induction of SSAT transcript. We provide evidence for the binding of a cytosolic protein to the SSAT mRNA that can be displaced by DENSPM. Cloning of SSAT 5′-UTR and Coding Region—The SSAT 5′-UTR and various lengths of the coding sequence were cloned upstream and in-frame with the Renilla luciferase gene located in the XhoI/XbaI sites of pcDNA3.1 (rLuc). Initially, the SSAT 5′-UTR and the entire coding region except the terminal lysine and stop codon were amplified from a human breast cancer cDNA library (Prof. Peter Leedman, Western Australian Institute for Medical Research) using primers F1 and R1 (see supplemental Table S1 for oligonucleotides used in this study) and cloned into the NheI and BamHI sites of the vector pGL3 (Promega, Madison, WI). It was then removed by digestion with KpnI/BamHI and cloned into the same sites in rLuc such that the SSAT and luciferase sequences were in-frame to generate rLucSSAT510. Deletions of the coding region were achieved by amplifying SSAT using a common forward primer F2 and different reverse primers (R2, R3, R4, and R5), which were designed to generate products that contained the entire 5′-UTR only or together with 333, 166, or 68 bases of the coding region, respectively. The resulting constructs were named rLucSSATUTR, rLucSSAT333, rLucSSAT166, and rLucSSAT68 (shown in Fig. 1B). To make FLAG-tagged constructs, the coding region of SSAT was amplified using rLucSSAT510 as template and primers F3 and R6. Reverse primer R6 added the terminal lysine and stop codon missing from rLucSSAT510. The entire SSAT coding region was cloned into the HindIII and BamHI sites of p3XFLAG-CMV-7.1 expression vector (Sigma) yielding FLAG-SSAT513. A series of 3′ deletion constructs was made using the common forward primer F3 and various reverse primers (R7, R8, R9, and R10), which produced products containing the first 504, 498, 482, or 486 bases of the SSAT coding region, respectively. These constructs were named FLAG-SSAT504, FLAG-SSAT498, FLAG-SSAT492, and FLAG-SSAT486. A series of 5′ deletion constructs was made using various forward primers (F4, F5, and F6) with the common reverse primer R6, which produced products missing the first 15, 30, or 45 bases of the coding region, respectively. These constructs were named FLAG-SSAT15–513, FLAG-SSAT30–513, and FLAG-SSAT45–513. In addition, FLAG-tagged SSAT constructs were made that contained premature stop codons or base mutations. The primers used to make these constructs are shown in supplemental Table S1, and the location of premature stop codons and base mutations are shown in Fig. 5B. Constructs were verified by DNA sequencing. Cell Culture and Transient Transfections—HeLa (human cervical adenocarcinoma) cells were obtained from ATCC (Manassas, VA) and cultured in RPMI 1640 supplemented with 10% fetal bovine serum and incubated at 37 °C in an atmosphere of 5% CO2 in air. For luciferase studies, cells were seeded at a density of 2.5 × 105 cells/well (24-well plate) and incubated overnight at 37 °C. Cells were cotransfected in the absence of fetal bovine serum with 1 μg of rLucSSAT plasmid and 0.1 μg of the internal control plasmid pGL3-basic (Promega) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. After 24 h, cells were treated with up to 100 μm DENSPM (a kind gift from Dr. Carl Porter, Roswell Park Cancer Institute, Buffalo) for a further 24 h. Cells were then washed and assayed for luciferase activity using a Dual Luciferase Kit (Promega). For FLAG studies, cells were seeded and transfected as above with 1 μg of FLAG-SSAT plasmids, unless stated otherwise. Following transfection, cells were incubated overnight at 37 °C, and then treated with 10 μm DENSPM or vehicle for 4 h. In some experiments, cells were treated with actinomycin D (5 μg/ml) or cycloheximide (10 μg/ml). Cells were washed with phosphate-buffered saline and harvested by scraping into 0.4 ml of 20 mm Tris/1 mm EDTA buffer (pH 7.4) containing 1 mm dithiothreitol. Cells were then disrupted on ice by sonication, and the supernatants were cleared by centrifugation for 10 min at 16,000 × g (4 °C). Protein concentrations were determined by the method of Bradford (Bio-Rad), and equal amounts (10 μg) of each sample were separated on 12% SDS-polyacrylamide gels, transferred to nitrocellulose, and immunoblotted using anti-FLAG-M2 HRP monoclonal antibody (Sigma). MG132 Treatment and Immunoprecipitation—HeLa cells were seeded at a density of 1 × 106 cells/well (6-well plate) and co-transfected with 1 μg of pcDNA3-HA-ubiquitin and 2 μg of either FLAG-SSAT513 or FLAG-SSAT504 plasmid. After an overnight incubation, cells were co-treated with 10 μm DENSPM and 5 μm MG132 (Calbiochem) or vehicle (Me2SO) and incubated for a further 16 h. Cells were washed twice with phosphate-buffered saline and harvested by scraping into 0.8 ml of 20 mm Tris/1 mm EDTA buffer (pH 7.4) containing 1 mm dithiothreitol. Cells were then disrupted on ice by sonication, and the supernatants were cleared by centrifugation for 10 min at 16,000 × g (4 °C). Anti-FLAG-M2 monoclonal antibody (10 μg, F 3165, Sigma) was added, and the supernatants were rotated for 2 h at 4 °C. Protein G-Sepharose 4B (P 3296, Sigma) was then added, and the lysates were rotated for a further 1 h at 4 °C. Immunoprecipitates were collected by centrifugation, and the beads were washed three times with phosphate-buffered saline. The recovered proteins were separated on 12% SDS-polyacrylamide gels, transferred to nitrocellulose, and immunoblotted using anti-HA antibody (H 6908, Sigma) according to the manufacturer's instructions. RNA Extraction, Reverse Transcription, and Real-time PCR—For RNA studies, HeLa cells were seeded at 1 × 106 cells/well (6-well plate) and transfected with 2.5 μg of FLAG-SSAT513 plasmid. Cells were then incubated for 4 h in the absence or presence of 10 μm DENSPM, and total RNA was extracted using TRIzol reagent (Invitrogen) as outlined in the manufacturer's instructions. First strand cDNA was synthesized using SuperScript II reverse transcriptase (Invitrogen) as per the manufacturer's protocol. Reactions lacking reverse transcriptase also were performed to ensure no plasmid DNA contamination. Expression levels of FLAG-SSAT mRNA were quantified using the iCycler iQ Real-time PCR Detection System (Bio-Rad). First strand cDNA was amplified using specific primers for FLAG-SSAT (F10 and R7) or β-actin (F11 and R21). Reactions contained iQ Supermix (Bio-Rad), 6 pmol of each primer, and 1 μl of cDNA in a final volume of 25 μl. Samples were amplified using the following conditions: initial denaturation at 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 50 °C for 15 s, and extension at 72 °C for 30 s. A melting curve was obtained to verify specificity of the PCR. Samples were analyzed by the comparative CT method. In Vitro Transcription/Translation—Reaction mixtures contained 1 μg of rLucSSAT510 DNA and 40 μl of rabbit reticulocyte lysate (TnT Quick Coupled Transcription/Translation System, Promega), in a total volume of 50 μl. Initially, reactions were incubated at 30 °C for 30 min in the absence of methionine to synthesize transcript. Then, actinomycin D was added to a final concentration of 5 μg/ml to stop the transcription. Reactions were divided into two aliquots containing equal amounts of mRNA, and DENSPM (50 μm) or an equal volume of vehicle was added to each aliquot. The paired reactions were prewarmed, and protein synthesis was initiated by the addition of 20 μm methionine. Aliquots (5 μl) were removed at 0, 5, 10, 15, and 30 min and boiled immediately in Laemmli buffer. Samples were then separated on 12% SDS-polyacrylamide gels, transferred to nitrocellulose, immunoblotted using anti-Renilla luciferase monoclonal antibody (MAB4410, Chemicon), and quantified by densitometry (Quantity One software, Bio-Rad). Preparation of Cytoplasmic Extracts—HeLa cells were washed with cold phosphate-buffered saline and scraped into cytoplasmic extraction buffer (10 mm HEPES (pH 7.2), 3 mm MgCl2, 40 mm KCl, 5% glycerol, 0.2% Nonidet P-40, 1 mm dithiothreitol) containing protease inhibitor mixture (Sigma). Cell extracts were incubated on ice for 20 min and then centrifuged at 16,000 × g for 10 min at 4 °C, and the supernatant was retained. Extract was used immediately or snap-frozen in liquid nitrogen and stored at -80 °C until required. Protein concentrations were determined by the Bradford method (Bio-Rad). Preparation of RNA Transcripts—Fragments of the SSAT 5′ coding region were amplified by PCR using rlucSSAT510 as template and the common forward primer F7 and the reverse primers R16, R17, and R18. PCR products were digested with BamHI and HindIII and cloned into the same sites of pBluescript II KS+ (Stratagene) in the T7 sense orientation, giving the constructs P1–42, P1–77, and P1–165, where the number indicates the number of bases of the SSAT insert (beginning at the start codon). Fragments of the SSAT 3′ coding region were also amplified using the common reverse primer R19 and the forward primers F8 and F9, giving the constructs P433–513 and P333–513. P333–492 was made using forward primer F9 and reverse primer R20. All constructs were then linearized with HindIII and used as templates in in vitro transcription reactions. Linearized templates were transcribed using T7 RNA polymerase (Invitrogen) in reactions containing [α-32P]UTP (3000 Ci/mmol, GE Healthcare) as described elsewhere (17Thomson A.M. Rogers J.T. Walker C.E. Staton J.M. Leedman P.J. BioTechniques. 1999; 27: 1032-1042Crossref PubMed Scopus (47) Google Scholar). Full-length transcripts were isolated on 6% urea/acrylamide gels, eluted for 3 h at 22 °C in 0.5 m ammonium acetate/1 mm EDTA, and ethanol-precipitated to recover the RNA. Unlabeled RNA transcripts were synthesized as above but with 2.5 mm UTP. REMSAs—Binding reactions (10 μl) contained 10 μg of HeLa cytoplasmic extract and 100,000 cpm of 32P-Riboprobe (P1–42, P1–77, P1–165, or P333–513) and were performed as described elsewhere (17Thomson A.M. Rogers J.T. Walker C.E. Staton J.M. Leedman P.J. BioTechniques. 1999; 27: 1032-1042Crossref PubMed Scopus (47) Google Scholar). Briefly, reactions were incubated for 30 min at 22 °C, 0.3 unit of RNase T1 (Roche Applied Science) was added for 10 min, and the followed by heparin (Sigma, final concentration of 5 μg/μl unless stated otherwise) for 10 min. Samples were then electrophoresed on 5% native acrylamide gels, dried, and analyzed with a Personal Molecular Imager FX (Bio-Rad). In some assays, cell extracts were preincubated for 10 min at 22 °C with nonspecific or specific competitor unlabeled RNA (∼100× molar excess) or with DENSPM (0–50 μm). SSAT-luciferase Fusion Protein Is Induced by DENSPM—To determine whether SSAT fused to a reporter protein was inducible by DENSPM treatment, human cervical HeLa cells were transfected with rLucSSAT510 and then treated with increasing concentrations of drug. The SSAT-luciferase fusion protein showed almost a 10-fold increase in expression, with an EC50 of 2.7 ± 0.4 μm (Fig. 1A). A series of SSAT ORF deletion reporter constructs was generated (Fig. 1B) in an attempt to locate the region of SSAT that was responsive to DENSPM. The basal expression levels of the longer fusion proteins rLucSSAT333 and rLucSSAT166 were not different from rLucSSAT510, whereas those of rLucSSAT68 and rLucSSATUTR were significantly greater than rLucSSAT510 (Fig. 1C). The observed differences in basal expression levels were a result of differing fusion protein half-lives (Table 1). None of the C-terminal rLucSSAT deletion proteins were induced by DENSPM, including the control rLuc vector (Fig. 1C). Similar results were obtained using the human melanoma cell line MM2058 (data not shown). These results suggest that the induction of SSAT is dependent on the presence of the 3′ third of the coding region. To investigate the effect of DENSPM on the stability of SSAT-luciferase proteins, the inducible rLucSSAT510 and the non-inducible rLucSSAT333 proteins were treated with cycloheximide, and their half-lives were determined in the absence or presence of DENSPM. DENSPM had no significant effect on the half-life of either fusion protein (Table 1).TABLE 1Stability of SSAT-luciferase fusion proteins in HeLa cellsConstructsFusion protein half-life-DENSPM+DENSPMh (mean ± S.E.)rLuc-SSAT5101.17 ± 0.041.29 ± 0.14rLuc-SSAT3331.29 ± 0.111.31 ± 0.12rLuc-SSAT1661.12 ± 0.10NDaND, not determinedrLuc-SSAT681.91 ± 0.10NDrLuc-SSATUTR3.46 ± 0.11NDrLuc2.34 ± 0.19NDa ND, not determined Open table in a new tab DENSPM Enhances the Translation of SSAT-luciferase Fusion Protein in Vitro—The effect of DENSPM on the translation of rLucSSAT510 was investigated using an in vitro transcription/translation system. First, mRNA was synthesized from rLucSSAT510 plasmid in a reaction containing rabbit reticulocyte lysate, but lacking methionine. Actinomycin D (5 μg/ml) was added to stop the transcription, and the reaction was then divided into two equal aliquots, one treated with 50 μm DENSPM and the other with an equal volume of vehicle. Protein synthesis was initiated by the addition of methionine and the rate of protein synthesis measured over 30 min. Aliquots taken from each paired reaction at 0, 5, 10, 15, and 30 min were boiled immediately in Laemmli buffer and subjected to Western blot using an anti-Renilla luciferase antibody. The blots were quantified by densitometry, and the slope of the linear part of the curves was used to measure the rate of rLucSSAT protein synthesis (Fig. 2). The rate of protein synthesis in the presence of DENSPM was significantly greater than that in its absence (74.4 ± 3.4 and 19.8 ± 2.1 density units/min, respectively). Induction of FLAG-tagged SSAT by DENSPM Is via Translational Regulation—An inherent problem with the luciferase fusion proteins used in the first part of this study is the possibility of direct interference with the luciferase enzyme activity by the attached SSAT fragments. For that reason, FLAG-tagged SSAT was used as a model for the remainder of the study, and protein levels rather than enzyme activities were measured. Full-length SSAT was cloned into a FLAG vector (FLAG-SSAT513) and expressed in HeLa cells to investigate the mechanism by which DENSPM induces SSAT expression. In the absence of DENSPM treatment, no FLAG-SSAT protein was detectable by Western blot using an anti-FLAG HRP antibody (Fig. 3A). Upon DENSPM (10 μm) treatment there was a time-dependent increase in FLAG-SSAT protein production, with near maximal levels occurring by 8 h (Fig. 3A). To determine whether the induction of FLAG-SSAT required new mRNA synthesis, cells were treated with actinomycin D (5 μg/ml) for 10 min, and then with 10 μm DENSPM for 4 h. Induction of FLAG-SSAT by DENSPM was not affected by the presence of actinomycin D (Fig. 3B). By contrast, when cells were treated with the protein synthesis inhibitor cycloheximide (10 μg/ml) and DENSPM, induction was completely abolished (Fig. 3B). To determine if FLAG-SSAT mRNA was present in the absence of DENSPM, total RNA was extracted from control cells and cells that had been treated with 10 μm DENSPM for 4 h. Following reverse transcription, specific primers were used to quantify FLAG-SSAT transcript levels by real-time PCR. Transcript levels were not increased in the presence of DENSPM (Fig. 3C). In fact, transcript levels were reduced in the presence of DENSPM, which is consistent with increased translation and translation-dependent mRNA degradation (18Wilson T. Treisman R. Nature. 1988; 336: 396-399Crossref PubMed Scopus (506) Google Scholar). A previous study by Parry and coworkers (5Parry L. Balana Fouce R. Pegg A.E. Biochem. J. 1995; 305: 451-458Crossref PubMed Scopus (52) Google Scholar) also observed a decrease in SSAT mRNA levels following polyamine analogue treatment. This indicates that expression of FLAG-SSAT mRNA has little effect on SSAT protein levels. Combined, these results indicate that translation of FLAG-SSAT mRNA is blocked in the absence of DENSPM. In addition, because the SSAT 5′- and 3′-UTRs were not present in the plasmid, the translational control of SSAT by DENSPM is mediated by the protein coding region, which is in agreement with a previous study (5Parry L. Balana Fouce R. Pegg A.E. Biochem. J. 1995; 305: 451-458Crossref PubMed Scopus (52) Google Scholar). Induction of FLAG-SSAT by DENSPM Is Not Due to Protein Stabilization—SSAT is degraded via the ubiquitin/26 S proteasome pathway (19Coleman C.S. Pegg A.E. J. Biol. Chem. 1997; 272: 12164-12169Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), and it has been demonstrated that the C-terminal MATEE amino acid motif plays a critical role in polyamine-mediated stabilization of the protein (20Coleman C.S. Huang H. Pegg A.E. Biochemistry. 1995; 34: 13423-13430Crossref PubMed Scopus (54) Google Scholar). As a result, the lack of FLAG-SSAT protein detected in cells not treated with DENSPM may be due to rapid turnover of the protein. To address this possibility, HeLa cells were transiently transfected with plasmid containing either full-length SSAT (FLAG-SSAT513) or a truncated construct (FLAG-SSAT504), which produces a protein that is reported to be stabilized in the absence of polyamine analogue (19Coleman C.S. Pegg A.E. J. Biol. Chem. 1997; 272: 12164-12169Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Cells were then treated with 10 μm DENSPM in the absence or presence of the proteasomal inhibitor MG132 (20 μm). In cells not treated with DENSPM or MG132, no expression of either FLAG-SSAT513 or FLAG-SSAT504 was observed (Fig. 4A). When MG132 was added, a small amount of FLAG513 was observed in the absence of DENSPM. This low level of translation may be due to the presence of endogenous polyamines. In addition, no polyubiquitinated products were observed in lysates from cells treated with MG132 in the absence of DENSPM (Fig. 4B). These data indicate that the lack of FLAG-SSAT protein observed in the absence of DENSPM was not because of rapid protein degradation. Sequences Essential for DENSPM Induction of SSAT Are Located Both at the 5′- and 3′-Ends of the mRNA—To identify the region of the SSAT mRNA involved in SSAT translational induction, we constructed a series of FLAG-SSAT deletion mutants and determined the effect of DENSPM in transiently transfected HeLa cells (Fig. 5A). Because SSAT-luciferase fusion protein studies indicated that the 3′ third of the ORF was important, initial deletions were of this region. The FLAG-SSAT504 deletion had the same inducibility as full-length FLAG-SSAT513 (Fig. 5A). The next shortest construct, FLAG-SSAT498, was DENSPM-responsive but showed some leakiness with a small amount of FLAG-SSAT protein being produced in the absence of DENSPM. The FLAG-SSAT492 and FLAG-SSAT486 constructs were not inducible by DENSPM with similar amounts of FLAG-SSAT protein translated in the absence and presence of DENSPM (Fig. 5A). These results suggest that the coding sequence located between bases 492 and 504, relative to the SSAT start codon, serves to repress translation in the absence of DENSPM. Deletions also were performed on the 5′-end of the SSAT ORF. FLAG-SSAT15–513 and FLAG-SSAT30–513 constructs were inducible by DENSPM, but showed some leakiness with a small amount of FLAG-SSAT protein being translated in the absence of DENSPM (Fig. 5A). The FLAG-SSAT45–513 construct was not responsive to DENSPM, and there were similar levels of protein translated in the absence and presence of DENSPM (Fig. 5A). These results show that the first 45 bases of the SSAT coding region as well as the bases between 492 and 504 are required for DENSPM responsiveness. Functional mRNA instability elements have been detected within the coding regions of several mRNAs (21Tierney M.J. Medcalf R.L. J. Biol. Chem. 2001; 276: 13675-13684Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The bases located between 492 and 504 of the SSAT coding region bear resemblance to an mRNA instability element identified in plasminogen activator inhibitor type 2 as well as in five other mRNAs (21Tierney M.J. Medcalf R.L. J. Biol. Chem. 2001; 276: 13675-13684Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). To investigate the possibility that the involvement of this sequence in DENSPM responsiveness is due to inducible stability of the SSAT tran" @default.
• W2022344159 created "2016-06-24" @default.
• W2022344159 creator A5055028859 @default.
• W2022344159 creator A5058666484 @default.
• W2022344159 creator A5088709602 @default.
• W2022344159 date "2007-09-01" @default.
• W2022344159 modified "2023-10-16" @default.
• W2022344159 title "Polyamine-dependent Regulation of Spermidine-Spermine N1-Acetyltransferase mRNA Translation" @default.
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