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• W2018473684 abstract "GATA-4 is a key member of the GATA family of transcription factors involved in cardiac development and growth as well as in cardiac hypertrophy and heart failure. Our previous studies suggest that GATA-4 protein synthesis may be translationally regulated. We report here that the 518-nt long 5′-untranslated region (5′-UTR) of the GATA-4 mRNA, which is predicted to form stable secondary structures (–65 kcal/mol) such as to be inhibitory to cap-dependent initiation, confers efficient translation to monocistronic reporter mRNAs in cell-free extracts. Moreover, uncapped GATA-4 5′-UTR containing monocistronic reporter mRNAs continue to be well translated while capped reporters are insensitive to the inhibition of initiation by cap-analog, suggesting a cap-independent mechanism of initiation. Utilizing a dicistronic luciferase mRNA reporter containing the GATA-4 5′-UTR within the intercistronic region, we demonstrate that this leader sequence confers functional internal ribosome entry site (IRES) activity. The activity of the GATA-4 IRES is unaffected in trans-differentiating P19CL6 cells, however, is strongly stimulated immediately following arginine-vasopressin exposure of H9c2 ventricular myocytes. IRES activity is then maintained at submaximal levels during hypertrophic growth of these cells. Supraphysiological Ca2+ levels diminished stimulation of IRES activity immediately following exposure to vasopressin and inhibition of protein kinase C activity utilizing a pseudosubstrate peptide sequence blocked IRES activity during hypertrophy. Thus, our data suggest a mechanism for GATA-4 protein synthesis under conditions of reduced global cap-dependent translation, which is maintained at a submaximal level during hypertrophic growth and point to the regulation of GATA-4 IRES activity by sarco(ER)-reticular Ca2+ stores and PKC. GATA-4 is a key member of the GATA family of transcription factors involved in cardiac development and growth as well as in cardiac hypertrophy and heart failure. Our previous studies suggest that GATA-4 protein synthesis may be translationally regulated. We report here that the 518-nt long 5′-untranslated region (5′-UTR) of the GATA-4 mRNA, which is predicted to form stable secondary structures (–65 kcal/mol) such as to be inhibitory to cap-dependent initiation, confers efficient translation to monocistronic reporter mRNAs in cell-free extracts. Moreover, uncapped GATA-4 5′-UTR containing monocistronic reporter mRNAs continue to be well translated while capped reporters are insensitive to the inhibition of initiation by cap-analog, suggesting a cap-independent mechanism of initiation. Utilizing a dicistronic luciferase mRNA reporter containing the GATA-4 5′-UTR within the intercistronic region, we demonstrate that this leader sequence confers functional internal ribosome entry site (IRES) activity. The activity of the GATA-4 IRES is unaffected in trans-differentiating P19CL6 cells, however, is strongly stimulated immediately following arginine-vasopressin exposure of H9c2 ventricular myocytes. IRES activity is then maintained at submaximal levels during hypertrophic growth of these cells. Supraphysiological Ca2+ levels diminished stimulation of IRES activity immediately following exposure to vasopressin and inhibition of protein kinase C activity utilizing a pseudosubstrate peptide sequence blocked IRES activity during hypertrophy. Thus, our data suggest a mechanism for GATA-4 protein synthesis under conditions of reduced global cap-dependent translation, which is maintained at a submaximal level during hypertrophic growth and point to the regulation of GATA-4 IRES activity by sarco(ER)-reticular Ca2+ stores and PKC. The family of GATA transcription factors regulates differentiation, growth, and survival of a wide range of cell types (1Molkentin J.D. J. Biol. Chem. 2000; 275: 38949-38952Abstract Full Text Full Text PDF PubMed Scopus (727) Google Scholar). GATA-4 is a zinc finger-containing transcription factor, which is expressed, in various mesoderm-derived tissues such as the lung, liver, gonad, and gut where it regulates tissue-specific gene expression (2Peterkin T. Gibson A. Loose M. Patient R. Sem. Cell Dev. Biol. 2005; 16: 83-94Crossref PubMed Scopus (150) Google Scholar, 3Pikkarainen S. Tokola H. Kerkela R. Ruskoaho H. Cardiovasc. Res. 2004; 63: 196-207Crossref PubMed Scopus (307) Google Scholar). Consistent with the observed expression patterns, targeted disruption of GATA-4 in mice has elucidated important functions in each of these tissues. GATA-4 is also essential for proper cardiac morphogenesis and also plays an important role as one of the transcriptional factors, which regulates the hypertrophic response in cardiac myocytes (4Liang Q. De Windt L.J. Witt S.A. Kimball T.R. Markham B.E. Molkentin J.D. J. Biol. Chem. 2001; 276: 30245-30253Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 5Grepin C. Nemer G. Nemer M. Development. 1997; 124: 2387-2395Crossref PubMed Google Scholar, 6Aries A. Paradis P. Lefebvre C. Schwartz R.J. Nemer M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 6975-6980Crossref PubMed Scopus (227) Google Scholar). Several studies suggest that GATA-4 can be regulated at both the transcriptional level and by phosphomodulation during hypertrophy (1Molkentin J.D. J. Biol. Chem. 2000; 275: 38949-38952Abstract Full Text Full Text PDF PubMed Scopus (727) Google Scholar, 7Hasegawa K. Lee S.J. Jobe S.M. Markham B.E. Kitsis R.N. Circulation. 1997; 96: 3943-3953Crossref PubMed Scopus (154) Google Scholar, 8Herzig T.C. Jobe S.M. Aoki H. Molkentin J.D. Cowley Jr., A.W. Izumo S. Markham B.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7543-7548Crossref PubMed Scopus (179) Google Scholar). In cultured neonatal cardiomyocytes, electrical pacing-induced hypertrophy is associated with a significant increase in GATA-4 mRNA, suggesting a mechanism whereby total GATA-4 content is up-regulated during hypertrophy (9Xia Y. McMillin J.B. Lewis A. Moore M. Zhu W.G. Williams R.S. Kellems R.E. J. Biol. Chem. 2000; 275: 1855-1863Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Increases in GATA-4 phosphorylation mediated by Raf/Mek/Erk signaling subsequent to hypertrophic agonist administration have also been demonstrated (10Morimoto T. Hasegawa K. Kaburagi S. Kakita T. Wada H. Yanazume T. Sasayama S. J. Biol. Chem. 2000; 275: 13721-13726Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Our previous data (11Gera J.F. Mellinghoff I.K. Shi Y. Rettig M.B. Tran C. Hsu J.-H. Sawyers C.L. Lichtenstein A.K. J. Biol. Chem. 2004; 279: 2737-2746Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar) suggested that the GATA-4 mRNA is also regulated at the translational level and we undertook this study to evaluate the possibility that such control may play a role in augmenting GATA-4 potency during differentiation or hypertrophy in cardiac myocytes. Most mRNAs contain 5′-UTRs 3The abbreviations used are: UTR, untranslated region; IRES, internal ribosome entry site; ORF, open reading frame; uORF, upstream open reading frame; LUC, luciferase; mTOR, mammalian target of rapamycin; ITAF, IRES trans-acting factor; eIF-2α, eukaryotic translation initiation factor 2α; AVP, arginine vasopressin; ER, endoplasmic reticulum; BiP, immunoglobin heavy chain binding protein; PKC, protein kinase C; Cdk1, cyclin-dependent kinase 1; α-MHC, α-myosin heavy chain; RACE, rapid amplification of cDNA ends; nt, nucleotide. 3The abbreviations used are: UTR, untranslated region; IRES, internal ribosome entry site; ORF, open reading frame; uORF, upstream open reading frame; LUC, luciferase; mTOR, mammalian target of rapamycin; ITAF, IRES trans-acting factor; eIF-2α, eukaryotic translation initiation factor 2α; AVP, arginine vasopressin; ER, endoplasmic reticulum; BiP, immunoglobin heavy chain binding protein; PKC, protein kinase C; Cdk1, cyclin-dependent kinase 1; α-MHC, α-myosin heavy chain; RACE, rapid amplification of cDNA ends; nt, nucleotide. that are relatively unstructured and typically less than 100 nucleotides in length, which allows efficient cap-dependent translational initiation (12Hershey J.W. Merrick W.C. The Pathway and Mechanism of Initiation of Protein Synthesis. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000Google Scholar). However, the leaders of some cellular mRNAs are long, highly structured, and can contain multiple upstream AUG or CUG codons such that scanning ribosomes are unlikely to efficiently initiate translation. In a number of these mRNAs, translation initiation is mediated by cap-independent mechanisms via an internal ribosome entry site (13Hellen C.U.T. Sarnow P. Genes Dev. 2001; 15: 1593-1612Crossref PubMed Scopus (804) Google Scholar). IRES-mediated translation initiation can occur during a variety of physiological conditions and has been reported to promote initiation for several mRNAs during cell-cycle progression, differentiation, apoptosis, and during stress responses (14Spriggs K.A. Bushell M. Mitchell S.A. Willis A.E. Cell Death Differ. 2005; 12: 585-591Crossref PubMed Scopus (135) Google Scholar, 15Komar A.A. Hatzoglou M. J. Biol. Chem. 2005; 280: 23425-23428Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 16Stoneley M. Willis A.E. Oncogene. 2004; 23: 3200-3207Crossref PubMed Scopus (291) Google Scholar, 17Vagner S. Galy B. Pyronnet S. EMBO Rep. 2001; 2: 893-898Crossref PubMed Scopus (236) Google Scholar, 18Holcik M. Sonenberg N. Nat. Rev. Mol. Cell Biol. 2005; 6: 318-327Crossref PubMed Scopus (1029) Google Scholar). Previously, we identified several mRNAs which either increased or continued to be well translated during conditions when cap-dependent translation initiation was inhibited (11Gera J.F. Mellinghoff I.K. Shi Y. Rettig M.B. Tran C. Hsu J.-H. Sawyers C.L. Lichtenstein A.K. J. Biol. Chem. 2004; 279: 2737-2746Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 19Shi Y. Sharma A. Wu H. Lichtenstein A. Gera J. J. Biol. Chem. 2005; 280: 10964-10973Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). One of these mRNAs, GATA-4, increased its translational efficiency markedly following exposure of cells to the mTOR inhibitor rapamycin. Treatment with rapamycin results in the global inhibition of cap-dependent translation via a blockade to the formation of productive eIF4F initiation complexes (20Gingras A.-C. Raught B. Sonenberg N. Genes Dev. 2001; 15: 807-826Crossref PubMed Scopus (1182) Google Scholar). The human GATA-4 transcript has a 518 nucleotide 5′-UTR and contains 18 upstream initiation codons (21Huang W.-Y. Cukerman E. Liew C.-C. Gene (Amst.). 1995; 155: 219-223Crossref PubMed Scopus (43) Google Scholar). This prompted us to investigate the possibility of whether this mRNA could initiate translation in a cap-independent fashion and contain an IRES within its leader. Because GATA-4 has a prominent role in the regulation of muscle cell-specific differentiation and IRES-mediated translation may be enhanced during differentiation (22Gerlitz G. Jagus R. Elroy-Stein O. FEBS J. 2002; 269: 2810-2819Google Scholar, 23Krichevsky A.M. Metzer E. Rosen H. J. Biol. Chem. 1999; 274: 14295-14305Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), we analyzed the ability of this leader to mediate internal initiation during this process. We utilized the P19CL6 cell line model to investigate the possibility that the GATA-4 mRNA could initiate IRES-mediated translation during differentiation. The P19CL6 cell line is a clonal derivative of the P19 embryonal carcinoma cell line which efficiently differentiates into beating cardiomyocytes and produces characteristic muscle proteins (24Habara-Ohkubo A. Cell Struct. Funct. 1996; 21: 101-110Crossref PubMed Scopus (119) Google Scholar). GATA-4 is expressed in these cells, is induced upon differentiation, and is required to trans-activate muscle-specific genes (25Monzen K. Shiojima I. Hiroi Y. Kudoh S. Oka T. Takimoto E. Hayashi D. Hosoda T. Habara-Ohkubo A. Nakaoka T. Fujita T. Yazaki Y. Komuro I. Mol. Cell. Biol. 1999; 19: 7096-7105Crossref PubMed Scopus (214) Google Scholar). Additionally, because GATA-4 is known to be required for cardiac hypertrophy we determined whether IRES-mediated translation initiation of GATA-4 mRNA could occur following vasopressin-induced hypertrophy of H9c2 cardiomyocytes. We report here that the GATA-4 5′-UTR contains IRES activity, which is stimulated during hypertrophy but not during differentiation. Our data also suggest that GATA-4 IRES activity is enhanced coincident with elevated eIF-2α phosphorylation during hypertrophy. We propose that IRES-mediated translational initiation of GATA-4 mRNA allows continued protein synthesis under conditions of reduced cap-dependent initiation potential during the initial response to vasopressin and continues during hypertrophic growth. Moreover, our data suggest that sarco (endo)plasmic Ca2+ flux may play a role in the stimulation of GATA-4 IRES activity through a protein kinase C-dependent pathway during hypertrophy. Cell Lines and DNA Constructs—All cell lines were obtained from ATCC (American Type Culture Collection) and maintained in medium supplemented with 10% fetal bovine serum. P19CL6 cell lines expressing the dicistronic reporter mRNAs were generated by co-transfection with pcDNA3.1 and pRGATA-4F followed by G418 selection and subsequent screening for stable expression by Northern analysis. Stable clones were induced to differentiate under adherent conditions by plating at a density of 3.7 × 105 cells in a 60-mm culture dish with 1% dimethyl sulfoxide. The medium was changed every 2 days. Days of differentiation were numbered consecutively, with the first day of Me2SO treatment as day 0. The protein kinase C cell permeable myristoylated pseudosubstrate peptide 20–28 (12 μm) and nifedipine (1 μm) was obtained from Calbiochem, and Arg-vasopressin (1 μm) was from Sigma. 5′-RACE analysis of the GATA-4 mRNA was performed on total RNA from H9c2 or HeLa cells using the 5′/3′-RACE kit, 2nd Generation, as described by the manufacturer (Roche Applied Science). To construct the dicistronic reporters, the entire human GATA-4 5′-UTR (accession number NM_002052) was amplified from IMAGE clone 5744870 and inserted into the NcoI site of pRF (kindly provided by A. Willis, University of Nottingham, UK). The construction of pRmycF has been described previously (19Shi Y. Sharma A. Wu H. Lichtenstein A. Gera J. J. Biol. Chem. 2005; 280: 10964-10973Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). The GATA-4 5′-UTR was also inserted into a promoterless version of pRF (26Han B. Zhang J.-T. Mol. Cell. Biol. 2002; 22: 7372-7384Crossref PubMed Scopus (99) Google Scholar), (–)pRF (kindly provided by J.-T. Zhang, Indiana University) to generate –(p)RGATA-4F. A 46-bp HindIII-BamHI fragment containing a hairpin structure containing an 18-bp stem (ΔG =–61 kcal/mol) was liberated from pSP64-hp7 (27Belgrader P. Cheng J. Maquat L.E. Proc. Natl. Acad. Sci. 1993; 90: 482-486Crossref PubMed Scopus (154) Google Scholar) (kindly provided by L. Maquat, Roswell Park Cancer Institute) and inserted immediately upstream of the Renilla ORF in pRGATA-4F to generate phpRGATA-4F. To generate the constructs used in the monocistronic analysis of RNAs in vitro, the firefly luciferase ORF or the GATA-4 5′-UTR-firefly luciferase ORF sequences were amplified from pRGATA-4F and subcloned into the pSP64 poly(A) vector (Promega) to generate pSPpA and pSPGATA-4pA, respectively. Similarly, the dicistronic constructs used in the in vitro analyses were generated by amplifying a fragment spanning the Renilla ORF, the intercistronic region and the firefly ORF from pRF and subcloning this into pSP64 poly(A) to create cRFpoly(A)30. cRGATA-4Fpoly(A)30 and cRp27Fpoly(A)30 were created by amplification of the dicistronic regions of pRGATA-4F and pRp27F (19Shi Y. Sharma A. Wu H. Lichtenstein A. Gera J. J. Biol. Chem. 2005; 280: 10964-10973Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar), respectively, and subcloning into pSP64 poly(A) as before. The encephalomyocarditis virus (EMCV) IRES sequences were amplified from pIREShyg (Clontech) and inserted into the NcoI site of cRFpoly(A)30. All constructs were sequenced to confirm their integrity and primer information is available upon request. In Vitro Translation of Mono- and Dicistronic mRNA Reporters—The moncistronic and dicistronic plasmids were linearized and used as templates to in vitro transcribe the indicated RNAs using SP6 polymerase (19Shi Y. Sharma A. Wu H. Lichtenstein A. Gera J. J. Biol. Chem. 2005; 280: 10964-10973Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). These mRNAs were capped and subsequently used to program extracts of the indicated cell lines as described previously (19Shi Y. Sharma A. Wu H. Lichtenstein A. Gera J. J. Biol. Chem. 2005; 280: 10964-10973Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). The translation reactions were performed with the cap analog m7GpppG (Ambion) when indicated. Transient DNA and Dicistronic mRNA Transfections—The indicated reporter constructs were transfected into cells using Lipofectamine Plus (Invitrogen) and normalized for transfection efficiency by co-transfection with pSVβGal (Promega). Cells were harvested 18 h following transfection and Renilla, firefly, and β-galactosidase activities were determined (Dual-Glo luciferase and β-galactosidase assay systems, Promega). RNA transfection was performed as previously described (26Han B. Zhang J.-T. Mol. Cell. Biol. 2002; 22: 7372-7384Crossref PubMed Scopus (99) Google Scholar). Cells were harvested 12 h following RNA transfection, and luciferase activities determined. Relative luciferase values are expressed as activity per μg of protein. RNAi Analysis—Knockdown experiments targeting the pRF transcript were accomplished based on the strategy of Van Eden et al. (28Van Eden M.E. Byrd M.P. Sherrill K.W. Lloyd R.E. RNA. 2004; 10: 720-730Crossref PubMed Scopus (121) Google Scholar). A pool of double-stranded RNAs were synthesized (Dharmacon) targeting the Renilla luciferase ORF were annealed and co-transfected with the indicated mono or dicistronic plasmids into HeLa cells. The targeting sequences within the Renilla ORF used were 5′-AAAGTTTATGATCCAGAACAA-3′and 5′-AACAAAGGAAACGGATGATAA-3′. Metabolic Labeling, Immunoprecipitation, and in Vitro PKC Activity Assays—P19CL6 or H9c2 cells were pulse-labeled with [35S]methionine/cystine at a final concentration of 100 μCi/ml. Cells were harvested following the indicated treatments and lysates prepared in ice-cold radioimmune precipitation assay buffer (1× phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) with protease inhibitors added (Halt™ Protease Inhibitor Mixture EDTA-Free, Pierce). GATA-4 protein was immunoprecipitated overnight at 4 °C with 1 μg of anti-GATA-4 antibody and then collected with protein G-Sepharose (Amersham Biosciences, Piscataway, NJ). The immunoprecipitate was washed four times in radioimmune precipitation assay buffer and fresh protease inhibitors and the complex pelleted for resuspension in SDS sample buffer. The samples were separated by SDS-PAGE, the gels dried and visualized using a phosphorimager (Molecular Dynamics, Sunnyvale, CA). Phosphor-densitometry was performed using ImageQuant (Molecular Dynamics) software. Collectively, immunoprecipitation controls included equal numbers of cells plated per flask and equal amounts of quantitated total protein lysate per sample with equivalent amounts of antibody. PKC activity was determined using the SignaTECT® Protein Kinase C assay system (Promega, Madison, WI) as described by the manufacturer. Briefly, cell lysates were incubated with [γ-32P]ATP and PKC-biotinylated peptide substrate in substrate buffer at 30 °C for 5 min and subsequently spotted onto SAM biotin capture membrane (Promega). Membranes were washed and incorporated label measured by a scintillation counter. RNA and Protein Analysis—Northern analysis was performed as previously described (19Shi Y. Sharma A. Wu H. Lichtenstein A. Gera J. J. Biol. Chem. 2005; 280: 10964-10973Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) with RNA purified from the indicated cell lines or in vitro translation reactions. Following extraction with TRIzol, 3 μg of total RNA was loaded per lane in 1.4% formaldehyde-containing agarose gels, separated by electrophoresis and transferred to nylon membranes. Hybridization to riboprobes specific for the indicated transcripts was performed in ULTRAhyb hybridization buffer (Ambion). Blots were visualized by exposure to film or by a phosphorimager. RT-PCR was performed using the ImProm-II Reverse Transcription System (Promega) according to the instructions of the manufacturer. Polysome analysis was performed as previously described (11Gera J.F. Mellinghoff I.K. Shi Y. Rettig M.B. Tran C. Hsu J.-H. Sawyers C.L. Lichtenstein A.K. J. Biol. Chem. 2004; 279: 2737-2746Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). Briefly, cells were lysed in buffer containing 100 μg/ml cycloheximide at 4 °C. Following removal of mitochondria and nuclei, supernatants were layered onto 15–50% sucrose gradients and spun at 38,000 rpm for 2 h at 4 °C in a SW 40 rotor (Beckman Instruments). Gradients were fractionated into eleven 1-ml fractions using an ISCO Density Gradient Fractionator at a flow rate of 3 ml/min. The profiles of the gradients were monitored via UV absorbance at 260 nm. RNAs from the individual fractions were pooled into a non-ribosomal and monosomal containing pool and a polysomal pool. These RNAs (100 ng) were subsequently used in real-time quantitative RT-PCR analyses of the indicated transcripts using the QuantiTect SYBR Green RT-PCR kit (Qiagen) in an Eppendorf Mastercycler equipped with a realplex2 optical module (Eppendorf AG, Germany). Transcript-specific oligonucleotides for the indicated mRNAs were designed to amplify 150-bp fragments. Immunoblots were performed using standard procedures. Briefly, cells were lysed in 50 mm Tris, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 1 mm Na3VO4, 10 mm NaF, 2 mm phenylmethylsulfonyl fluoride, 0.5 mm EDTA, 10 μg/ml leupeptin, and 10 μg/ml aprotinin. Samples were resolved on 4–20% SDS-PAGE gels, transferred to polyvinylidene difluoride (0.2 μm) membranes and probed with antibodies to the following proteins: phospho-eIF-2α and eIF-2α (Cell Signaling), Cdk1 (Abcam), BiP (Imgenex), actin (Sigma), GATA-4, MLC-2, and α-MHC antibodies were all from Santa Cruz Biotechnology. The GATA-4 Leader Confers the Efficient Translation of Uncapped mRNA Reporters in Translationally Competent Cell Extracts—To begin to study the translational control of the human GATA-4 mRNA the transcriptional start site had to be determined and validated. We used 5′-RACE to isolate and characterize potential leaders. A single 5′-RACE product was amplified from total RNA extracted from either U87-MG or HeLa cells. The sequence was 518 nucleotides in length and was consistent with the previously published human 5′-UTR (21Huang W.-Y. Cukerman E. Liew C.-C. Gene (Amst.). 1995; 155: 219-223Crossref PubMed Scopus (43) Google Scholar). The GATA-4 leader is 67% G-C rich and contains 18 upstream initiation codons, several of which are in appropriate sequence context to support initiation by a scanning mechanism. Secondary structure prediction using the MFOLD algorithm of Zuker (29Zuker M. Nucleic Acids Res. 2003; 31: 3406-3415Crossref PubMed Scopus (10253) Google Scholar), predicted a highly stable structure with free energy values greater than –65 kcal/mol. We thus expected that the GATA-4 5′-UTR would be inhibitory to cap-dependent ribosomal scanning and may be translated in a cap-independent fashion. To determine if this was the case, we fused the GATA-4 5′-UTR immediately upstream of the firefly luciferase open reading frame in a SP6 promoter-based construct to generate pSPGATA-4pA as shown in Fig. 1A. In vitro transcribed mRNAs from the pSPGATA-4pA construct were either capped or not and used to program translation-competent cell-free extracts (30Carroll R. Lucaslenard J. Anal. Biochem. 1993; 212: 17-23Crossref PubMed Scopus (15) Google Scholar). As shown in Fig. 1B, uncapped pSPpA in vitro transcribed mRNA was poorly translated as compared with capped pSPpA mRNAs. However, when in vitro transcribed pSPGATA-4pA mRNAs were tested in an identical manner, uncapped transcripts containing the GATA-4 5′-UTR were translated efficiently at nearly the same levels as capped pSPGATA-4pA mRNAs or capped pSPpA mRNAs (Fig. 1B). Additionally we tested whether or not the cap-analog m7GpppG would effectively inhibit initiation of capped pSPpA or pSPGATA-4pA mRNAs in extracts. As can be seen in Fig. 1C, pSPpA RNA translation was inhibited by ∼80% in the presence of cap-analog, while pSPGATA-4pA was translated well at a comparable concentration of the analog. Northern analysis showed that similar amounts of pSPpA and pSPGATA-4pA mRNAs of the expected size could be detected in the in vitro translation reactions (Fig. 1D). These data suggested that the GATA-4 5′-UTR sequences present in the pSP-GATA-4pA construct do not affect steady-state mRNA levels, or are likely to contain splice sequences. Thus, the 5′-UTR of GATA-4 can confer efficient translation of uncapped reporter mRNAs. The Leader of the GATA-4 mRNA Enhances Translation in Vivo When Cap-dependent Translation Is Inhibited in cis—Because we had previously observed that the GATA-4 mRNA was well translated under conditions of reduced eIF-4F complex formation (11Gera J.F. Mellinghoff I.K. Shi Y. Rettig M.B. Tran C. Hsu J.-H. Sawyers C.L. Lichtenstein A.K. J. Biol. Chem. 2004; 279: 2737-2746Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar), we sought to determine whether a similar affect could be seen if its translation were inhibited in cis. We introduced a stable hairpin structure (ΔG =–61 kcal/mol) (27Belgrader P. Cheng J. Maquat L.E. Proc. Natl. Acad. Sci. 1993; 90: 482-486Crossref PubMed Scopus (154) Google Scholar) immediately distal to the SV40 promoter sequences in pGL to generate phpGL (Fig. 2A). This cis-acting structural element reduced the translation of the parental construct by 85% when transfected into HeLa cells (Fig. 2B). To assess whether the GATA-4 5′-UTR could mediate translation if cap-dependent translation was inhibited in this manner, we inserted the GATA-4 5′-UTR between the hairpin and the translation start site generating phpGATA-4L. This construct expressed 5-fold more luciferase activity in HeLa cell transfectants then the parental construct phpGL suggesting that the GATA-4 5′-UTR can promote translation initiation when cap-dependent scanning is reduced (Fig. 2B). To address whether cryptic promoters were present within the hairpin or GATA-4 5′-UTR sequences we removed the SV40 promoter sequences from phpGATA-4L to generate –(p)hpGATA-4L and tested HeLa transfectants for luciferase activity. As can be seen (Fig. 2B), no significant luciferase activity was detected suggesting that these sequences, in the context of the monocistronic reporter plasmid, do not support promoter activity. Dicistronic Reporter mRNAs Containing the GATA-4 5′-UTR within the Intercistronic Region Demonstrate IRES Activity—To determine whether the GATA-4 5′-UTR could function as an IRES in vivo, we introduced it into the intercistronic region within the dicistronic mRNA reporter plasmid pRF to generate pRGATA-4F (Fig. 3A). This plasmid contains the Renilla luciferase ORF as the first cistron and the firefly luciferase ORF as the second downstream cistron. In all the cell lines transfected with the pRGATA-4F construct downstream firefly luciferase activity was 6–10-fold higher as compared with values obtained for the control plasmid pRF (Fig. 3B). As a positive control, transfection with a construct baring the c-myc IRES in these lines also yielded firefly luciferase activities which were comparable to those obtained with pRGATA-4F. Renilla luciferase levels were comparable in all the cell lines tested, suggesting that the steady-state mRNA levels and the relative levels of cap-dependent translation of the dicistronic constructs were similar. Furthermore, Northern analysis revealed a single stable luciferase mRNA species of the expected size for each construct (Fig. 3C). In an effort to more carefully examine the possibility of monocistronic RNAs containing the firefly luciferase ORF being generated in vivo from these dicistronic mRNAs via aberrant splicing events we attempted to detect splice variants by RT-PCR. The integrity of the mRNAs was determined using primers spanning the Renilla and firefly cistrons (shown in Fig. 3A). As shown in Fig. 3D, a single RNA species of the expected molecular mass was amplified from HeLa cells transfected with pRGATA-4F. No other RNAs were observed suggesting that no aberrant splicing was taking place. These results support the notion that the GATA-4 5′-UTR can initiate translation internally and that the firefly luciferase activity observed is not likely because of cap-dependent initiation of shorter monocistronic reporter transcripts. Ribosomal Readthrough Does Not Occur in Dicistronic Constructs Containing the GATA-4 5′-UTR—To determine if the GATA-4 5′-UTR could function in a dicistronic construct by facilitating termination codon readthrough of the Renilla ORF through the intercistronic region we inserted the hairpin structure (Fig. 2A) upstream of the Renilla ORF within the dicistronic construct pRGATA-4F to generate phpRGATA-4F (Fig. 4A). If the GATA-4 5′-UTR sequences were mediating ribosomal readthrough of the Renilla termination codon, inhibiting translation of the upstream ORF should also result in a p" @default.
• W2018473684 created "2016-06-24" @default.
• W2018473684 creator A5039593366 @default.
• W2018473684 creator A5045364918 @default.
• W2018473684 creator A5054959208 @default.
• W2018473684 creator A5071456971 @default.
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• W2018473684 creator A5087531097 @default.
• W2018473684 creator A5089378437 @default.
• W2018473684 date "2007-03-01" @default.
• W2018473684 modified "2023-09-30" @default.
• W2018473684 title "Protein Kinase C Regulates Internal Initiation of Translation of the GATA-4 mRNA following Vasopressin-induced Hypertrophy of Cardiac Myocytes" @default.
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