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Transcriptional and Translational Regulation of the Léri-Weill and Turner Syndrome Homeobox Gene SHOX

2003; Elsevier BV; Volume: 278; Issue: 48 Linguagem: Inglês

10.1074/jbc.m306685200

1083-351X

Rüdiger J. Blaschke, Christine Töpfer, Antonio Marchini, Herbert Steinbeißer, Johannes W.G. Janssen, Gudrun Rappold,

RNA Research and Splicing

Regulation of gene expression is particularly important for gene dosage-dependent diseases and the phenomenon of clinical heterogeneity frequently associated with these phenotypes. We here report on the combined transcriptional and translational regulatory mechanisms controlling the expression of the Léri-Weill and Turner syndrome gene SHOX. We define an alternative promotor within exon 2 of the SHOX gene by transient transfections of mono- and bicistronic reporter constructs and demonstrate substantial differences in the translation efficiency of the mRNAs transcribed from these alternative promotors by in vitro translation assays and direct mRNA transfections into different cell lines. Although transcripts generated from the intragenic promotor (P2) are translated with high efficiencies, mRNA originating from the upstream promotor (P1) exhibit significant translation inhibitory effects due to seven AUG codons upstream of the main open reading frame (uAUGs). Site-directed mutagenesis of these uAUGs confers full translation efficiency to reporter mRNAs in different cell lines and after injection of Xenopus embryos. In conclusion, our data support a model where functional SHOX protein levels are regulated by a combination of transcriptional and translational control mechanisms. Regulation of gene expression is particularly important for gene dosage-dependent diseases and the phenomenon of clinical heterogeneity frequently associated with these phenotypes. We here report on the combined transcriptional and translational regulatory mechanisms controlling the expression of the Léri-Weill and Turner syndrome gene SHOX. We define an alternative promotor within exon 2 of the SHOX gene by transient transfections of mono- and bicistronic reporter constructs and demonstrate substantial differences in the translation efficiency of the mRNAs transcribed from these alternative promotors by in vitro translation assays and direct mRNA transfections into different cell lines. Although transcripts generated from the intragenic promotor (P2) are translated with high efficiencies, mRNA originating from the upstream promotor (P1) exhibit significant translation inhibitory effects due to seven AUG codons upstream of the main open reading frame (uAUGs). Site-directed mutagenesis of these uAUGs confers full translation efficiency to reporter mRNAs in different cell lines and after injection of Xenopus embryos. In conclusion, our data support a model where functional SHOX protein levels are regulated by a combination of transcriptional and translational control mechanisms. The human pseudoautosomal homeobox gene SHOX has recently been shown to encode a cell type-specific transcription factor involved in cell cycle and growth regulation (1Rao E. Blaschke R.J. Marchini A. Niesler B. Burnett M. Rappold G.A. Hum. Mol. Genet. 2001; 10: 3083-3091Crossref PubMed Scopus (87) Google Scholar). 1A. Marchini, S. Cladeira, R. J. Blaschke, T. Marttila, I. Melanchi, B. Häcker, E. Rao, M. Karperien, J. M. Wit, M. Tommasino, and G. A. Rappold, submitted for publication.1A. Marchini, S. Cladeira, R. J. Blaschke, T. Marttila, I. Melanchi, B. Häcker, E. Rao, M. Karperien, J. M. Wit, M. Tommasino, and G. A. Rappold, submitted for publication. Haploinsufficient loss of the SHOX gene causes short stature and has been correlated with variable skeletal phenotypes frequently observed in Léri-Weill and Turner syndrome patients (2Rao E. Weiss B. Fukami M. Rump A. Niesler B. Mertz A. Muroya K. Binder G. Kirsch S. Winkelmann M. Nordsiek G. Heinrich U. Breuning M.H. Ranke M.B. Rosenthal A. Ogata T. Rappold G.A. Nat. Genet. 1997; 16: 54-63Crossref PubMed Scopus (795) Google Scholar, 3Ellison J.W. Wardak Z. Young M.F. Gehron Robey P. Laig-Webster M. Chiong W. Hum. Mol. Genet. 1997; 6: 1341-1347Crossref PubMed Scopus (254) Google Scholar, 4Belin V. Cusin V. Viot G. Girlich D. Toutain A. Moncla A. Vekemans M. Le Merrer M. Munnich A. Cormier-Daire V. Nat. Genet. 1998; 19: 67-69Crossref PubMed Scopus (312) Google Scholar, 5Shears D.J. Vassal H.J. Goodman F.R. Palmer R.W. Reardon W. Superti-Furga A. Scambler P.J. Winter R.M. Nat. Genet. 1998; 19: 70-73Crossref PubMed Scopus (278) Google Scholar, 6Clement-Jones M. Schiller S. Rao E. Blaschke R.J. Zuniga A. Zeller R. Robson S.C. Binder G. Glass I. Strachan T. Lindsay S. Rappold G.A. Hum. Mol. Genet. 2000; 9: 695-702Crossref PubMed Scopus (340) Google Scholar). SHOX-deficient individuals exhibit a considerable phenotypic heterogeneity ranging from mild, barely detectable skeletal malformations to severe dysplasia adversely affecting the life of these patients (7Blaschke R.J. Rappold G.A. Trends Endocrinol. Metab. 2000; 11: 227-230Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 8Schiller S. Spranger S. Schechinger B. Fukami M. Merker S. Drop S.L. Troger J. Knoblauch H. Kunze J. Seidel J. Rappold G.A. Eur. J. Hum. Genet. 2000; 8: 54-62Crossref PubMed Scopus (130) Google Scholar). This phenotypic heterogeneity is clinically well documented but not understood on a molecular level. Although dominant negative effects of mutated gene products have been discussed (1Rao E. Blaschke R.J. Marchini A. Niesler B. Burnett M. Rappold G.A. Hum. Mol. Genet. 2001; 10: 3083-3091Crossref PubMed Scopus (87) Google Scholar), phenotypes caused by complete gene deletions argue for a quantitative threshold of functional protein to be necessary for normal development. Along these lines, the understanding of mechanisms regulating the level of functional protein will provide important clues to explain the SHOX-related phenotypes and their inherent phenotypic heterogeneity.Although initially transcriptional regulation has been correlated with a variety of human diseases, it has become increasingly clear that post-transcriptional processing and differential translation represent equally important checkpoints in the tissue-specific and disease-related control of gene expression (9Kozak M. Mamm. Genome. 2002; 13: 401-410Crossref PubMed Scopus (90) Google Scholar, 10Calkhoven C.F. Muller C. Leutz A. Trends Mol. Med. 2002; 8: 577-583Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 11Preiss T. Hentze M.W. Curr. Opin. Genet. Dev. 1999; 9: 515-521Crossref PubMed Scopus (114) Google Scholar). Among the various elements of mature mRNAs that are known to regulate translational efficiency, several recent investigations have emphasized the role of the 5′-untranslated region (UTR) 2The abbreviations used are: UTRuntranslated regionIRESinternal ribosomal entry sites.2The abbreviations used are: UTRuntranslated regionIRESinternal ribosomal entry sites. as major determinant controlling the initial steps of protein expression (12Mendell J.T. Dietz H.C. Cell. 2001; 107: 411-414Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 13Meijer H.A. Thomas A.A. Biochem. J. 2002; 367: 1-11Crossref PubMed Scopus (249) Google Scholar). Most eukaryotic mRNAs that exhibit high translational competence present themselves with short, 5′-m7GpppN (7-methylguanosine) cap containing UTRs that lack highly organized secondary structures. In contrast, several tightly regulated proteins including proto-oncogene products, growth factors, and their receptors as well as homeodomain proteins are encoded by mRNAs with long, complex 5′-UTRs with considerable secondary structures and multiple AUG codons upstream of the main open reading frame. Because these features interfere with the rate-limiting initial steps of the canonical cap-dependent translation mechanism and subsequent ribosomal scanning of the 5′-mRNA leader, they represent key indicators of mRNAs regulated on a translational level (14Davuluri R.V. Suzuki Y. Sugano S. Zhang M.Q. Genome Res. 2000; 10: 1807-1816Crossref PubMed Scopus (135) Google Scholar). In addition, these structural hallmarks have evoked and fueled a lively discussion about the capability of several cellular mRNAs to promote cap-independent translation by direct recruitment of ribosomes to internal ribosomal entry sites (IRES) (15Vagner S. Galy B. Pyronnet S. EMBO Rep. 2001; 2: 893-898Crossref PubMed Scopus (235) Google Scholar, 16Kozak M. Mol. Cell. Biol. 2001; 21: 1899-1907Crossref PubMed Scopus (141) Google Scholar, 17Schneider R. Agol V.I. Andino R. Bayard F. Cavener D.R. Chappell S.A. Chen J.J. Darlix J.L. Dasgupta A. Donze O. Duncan R. Elroy-Stein O. Farabaugh P.J. Filipowicz W. Gale M. Jr. Gehrke L. Goldman E. Groner Y. Harford J. B. Hatzglou M. He B. Hellen C. U. Hentze M. W.J. Hershey et al.Mol. Cell. Biol. 2001; 21: 8238-8246Crossref PubMed Scopus (44) Google Scholar, 18Han B. Zhang J.T. Mol. Cell. Biol. 2002; 22: 7372-7384Crossref PubMed Scopus (99) Google Scholar).We investigated the structural and functional properties of the 5′-UTR of the SHOX mRNA by computational, in vitro, cell culture based and in vivo analyses. In vitro and cell culture studies demonstrated an alternative intragenic promotor within exon 2 of the SHOX gene. Because this dual promotor structure leads to alternative mRNAs with different 5′ leaders, we analyzed and compared the translational efficiency of the two UTRs by direct RNA transfection assays. Here we provide evidence that the different 5′-UTRs exhibit differential translation efficiencies due to cell type-dependent inhibitory elements within the long SHOX mRNA variant. We finally integrate this data to propose a combinatorial model that takes into account transcriptional and translational mechanisms regulating the amount of functional SHOX protein.EXPERIMENTAL PROCEDURESPlasmid Constructs—The complete 5′-UTR of the SHOX mRNA was assembled from two genomic PCR fragments representing exon 1 and the SHOX non-coding portion of exon 2. PCRs were carried out with the HotProof polymerase system (Qiagen) using the following primers: Ex1for(SpeI), 5′-GGACTAGTCGCCTGCTTTTGCCCGGGTCCTGAG AA-3′; Ex1rev, 5′-CAAGCTCTGAGCGCAGGCCCCGAG-3′; Ex2for, 5′-GAAAACTGGAG TTGCTTTTCCTCCGG-3′; and Ex2rev(EcoRI), 5′-CGGAATTCCATGGCTGGGGCCGGGGC-3′. Both PCR fragments were digested with SpeI and EcoRI, respectively, cloned into a SpeI/EcoRIrestricted pBluescript plasmid (Stratagene), and sequence verified. All UTR-containing clones were derived from this plasmid (pBSK/SHOX-UTR). The expression constructs pHPL/UTR and pRF/UTR were generated by subcloning the complete SHOX 5′-UTR fragment from pBSK/SHOX-UTR via SpeI/NcoI into the respective parental vectors (19Stoneley M. Paulin F.E. Le Quesne J.P. Chappell S.A. Willis A.E. Oncogene. 1998; 16: 423-428Crossref PubMed Scopus (283) Google Scholar).Nested deletions of the SHOX 5′-UTR were obtained from pBSK/SHOX-UTR by PCR using the following forward primers: Ex2for(SpeI), 5′-GGACTAGTGAAAA CTGGAGTTTGCTTTTCCTCC-3′; Ex2Δ1for(SpeI), 5′-GGACTAGTAGGTGTACGGACGC CAAACAGT-3′; Ex2Δ2for-(SpeI), 5′-GGACTAGTCGGAGACCAGTAATTGCACCAGA-3′; and Ex2Δ3for(SpeI), 5′-GGACTACAGCGCTGGTGATCCACCCGCGCG-3′ in combination with the Ex2rev(EcoRI) oligo. Resulting PCR fragments were digested with SpeI/EcoRI, and cloned into pBluescript. The plasmid constructs pUTR-Luc, pEx2-Luc, pEx2Δ1-Luc, pEx2Δ2-Luc, and pEx2Δ3-Luc were generated by introducing an NcoI/SalI fragment containing the firefly luciferase coding cassette from the commercially available pGL3 vector (Promega) into the appropriately digested pBSK/UTR containing vectors.The plasmids pBSK/UTR-Xnoggin, pBSK/Ex2Δ3-Xnoggin, and pBSK/UTR-AUGm1–7-Xnoggin were generated by substituting the firefly coding sequence of the constructs pBSK/UTR-Luc, pBSK/Ex2Δ3-Luc and pBSK/UTR-AUGm1–7 by a noggin PCR fragment generated with the following primers: Xnoggin-for, 5′-GGCCATGGATCATTCCCAGTG CCTTGT-3′ and Xnoggin-rev, 5′-GGGTCTAGATCAATGATGATGATGATGATGGCATG AGCATTGCACTCGGAAATGACA-3′. The PCR fragment was cloned via NcoI/XbaI.Site-directed Mutagenesis—Upstream AUGs within the SHOX 5′-UTR were mutated using the QuikChange® Multi site-directed mutagenesis kit according to the procedure recommended by the supplier (Stratagene). Individual mutations were introduced into pBSK/SHOX-UTR with the following primers: uAUG1-mut, 5′-CTACTGCAAACAGA ATTGGGAGGGTGGACAGGCG-3′; uAUG2-mut, 5′-GACGCCAGGACGCGATTGAACCT CCGGGGCG-3′; uAUG3-mut, 5′-CCCTTCCAAAATTGGGATCTTTCCCC-3′; uAUG4/5-mut, 5′-GGACGCCAAACAGTGTTGAATTGAGAAGAAA GCCAATTGCCGG-3′; uAUG6-mut, 5′-CAGACAGGCAGCGCTTGGGGGGCTGGGC-3′; and uAUG7-mut, 5′-TAGTGAGATTTCC ATTGGAAAGGCGTAAAT-3′ resulting in the plasmids pBSK/UTR-AUGm1, pBSK/UTR-AUGm2, pBSK/UTR-AUGm3, pBSK/UTR-AUGm4/5, pBSK/UTR-AUGm6, and pBSK/UTR-AUGm7. The construct pBSK/UTR-AUGm1–7 combining mutations in all seven uAUGs was generated by mutating uAUG2 and uAUGs4/5, AUG6, and AUG7 of pBSK/UTR-AUGm1 and pBSK/UTR-AUGm3, respectively. The 5′ portion of pBSK/UTR-AUGm3–7 was subsequently exchanged for an SpeI/NarI fragment of pBSK/UTR-AUGm1–2 harboring mutations of uAUG1 and uAUG2. All mutations were verified by bidirectional sequence analysis and subcloned into pBSK/UTR-Luc via SpeI/NcoI.Cell Culture, DNA Transfections, and Luciferase Assays—U2Os, HEK293, and COS7 cells were maintained in Dulbecco's modified Eagle's medium/2 mm glutamine (PAA Laboratories) supplemented with 10% fetal calf serum (PAA Laboratories), 100 units/ml penicillin, and 100 μg/ml streptomycin. On the day of transfection, cells were splitted into 24-well plates (for luciferase assays) or 10-cm dishes (for RNA isolation) to a confluency of 50–60%. All cells were transfected with a 1:6 DNA/FuGENE6 (Roche Applied Science) ratio using 100 ng of plasmid DNA per 500 μl of cell culture medium. For luciferase assays the cells were lysed 36 h after transfection with 100 μl/well of Passive Lysis Buffer (Promega) for 15 min at room temperature and frozen at –80 °C for one hour. 10 μl of each cell lysate were assayed for Renilla and firefly luciferase activity with a dual injector 96-well plate luminometer (anthos) using the Dual-Luciferase® reporter assay system (Promega) as recommended by the manufacturer. All assays were performed at least three times and in triplicate.In Vitro RNA Synthesis and in Vitro Translation—Synthetic mRNAs were generated from various SHOX-UTR-Luciferase fusion constructs using the mMESSAGE mMACHINE™ or MEGAscript™ reaction systems (Ambion) according to the supplier's recommendations. Briefly, 1 μg of plasmid DNA linearized with XhoI were in vitro-transcribed with T3 polymerase in the presence of 6 mm CAP analog (m7G(5′)ppp(5′)G or A(5′)ppp(5′)G). The resulting mRNAs were digested with DNase I for 15 min at 37 °C, purified by column chromatography (Ambion), and analyzed on a formaldehyd containing 1% agarose gel. Purified mRNAs were photometrically analyzed and quantitated according to standard procedures. These synthetic mRNAs were used to prime in vitro translation reactions carried out with the Rabbit Reticulo Lysate System (Promega) according to the protocol provided. All reactions contained 1 μg of mRNA and 35 μl of nuclease-treated reticulo lysate in a final volume of 50 μl. 5 μl were withdrawn from this reaction in 10 min intervals, combined with an equal volume of 2× Passive Lysis Buffer (Promega), and firefly luciferase activity was determined as described above. Alternatively, pHPL- and pRF-derived plasmids were linearized with XhoI, column-purified (Qiagen), and directly used to prime coupled in vitro transcription/translation reactions (TNT) according to the manufacturer's instructions (Promega).RNA Transfection and Quantitation of Translation Efficiency—Synthetic mRNAs generated with the mMESSAGE mMACHINE™ or MEGAscript™ reaction systems (Ambion) were directly transfected into different cell lines using 2 μg RNA and 8 μl of TransMessenger™ Transfection Reagent (Qiagen) per well of a 6-well plate. Transfection procedure was carried out following the recommendations of the supplier. Transfected cells were trypsinized, washed once with phosphate-buffered saline, and the cell pellet was resuspended in 200 μl phosphate-buffered saline. 40 μl of this cell suspension were mixed with 10 μl of 5× Passive Lysis Buffer (Promega) and directly used for Luciferase assays. The remaining 160 μl of the cell suspension were used for isolation of total RNA by standard procedures (Qiagen). 1 μg aliquots of total RNA were reverse transcribed and analyzed by quantitative real time RT-PCR amplification with the FastStart reaction system (Roche Applied Science) in a LightCycler® instrument (Roche Applied Science) using the following primers: Luc1for, 5′-GGAGAGCAACT GCATAAGGC-3′, and Luc1rev, 5′-CATCGACTGAAATCCCTGGT-3′. The reactions contained 60 nmol MgSO4 in a final volume of 20 μl and were carried out under the following conditions: 95 °C/10 min; (95 °C/15 s, 60 °C/5 s, 72 °C/15 s)35X. Luciferase activities were corrected for RNA transfection efficiencies with the results from real time PCRs.Injection of Xenopus Embryos and Phenotype Evaluation—Eggs were obtained from Xenopus females injected with 300–400 units of human chorionic gonadotropin (Serva) and fertilized in vitro. The jelly coat was removed using a 2% cysteine solution (pH 8.0), and the embryos were microinjected in 1× MBS-H (88 mm NaCl, 1 mm KCl, 2.4 mm NaHCO3, 0.82 mm MgSO4, 0.41 mm CaCl2, 0.33 mm Ca(NO3)2, 10 mm HEPES (pH 7.4), 10 μg/ml streptomycinsulfate, and 10 μg/ml penicillin) and cultured in 0.1× MBS-H. The embryos were staged according to Nieuwkoop and Faber (20Nieuwkoop P.D. Faber J. Normal Table of Xenopus laevis (Daudin). North Holland Publishing Company, Amsterdam1967Google Scholar). Embryos were injected with mRNAs at the 4–8 cell stage into two ventral blastomeres with 5–10 nl of mRNA solutions. Embryos were then transferred to 0.1× MBS-H for cultivation up to tailbud stages (NF 33–35).RESULTSStructural Features of the SHOX 5′-UTR—The SHOXa and SHOXb mRNAs (accession numbers NM_000451 and NM_006883) share a complex 5′-untranslated region encoded by two exons joined via canonical donor-acceptor splice sites. This 5′-UTR comprises 694 nucleotides and exhibits an inconspicuous GC content of 63%. Beyond its over average length, however, it exhibits several unusual features. First, it contains a terminal oligopyrimidine tract of 12 nucleotides (TOP) and 7 AUG codons upstream of the major open reading frame (uAUGs). The environment of these uAUGs ranges between 44 and 67% identity to Kozak's consensus sequence (A/G)CC(A/G)CCAUGG (21Kozak M. Cell. 1986; 44: 283-292Abstract Full Text PDF PubMed Scopus (3565) Google Scholar, 22Peri S. Pandey A. Trends Genet. 2001; 17: 685-687Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) with uAUG1 and uAUG3 complying with the –3(A/G)/+4G rule for favorable translation initiation (23Kozak M. EMBO J. 1997; 16: 2482-2492Crossref PubMed Scopus (410) Google Scholar). All uAUGs are associated with open reading frames (uORFs) putatively encoding peptides with sizes between 2 and 29 amino acids, none of which exhibits any significant homologies to known peptides (Fig. 1). Second, predictions of its secondary structure using the mfold 3.1 algorithm (24Mathews D.H. Sabina J. Zuker M. Turner D.H. J. Mol. Biol. 1999; 288: 911-940Crossref PubMed Scopus (3198) Google Scholar) reveals an exceptionally strong folding of this 5′-UTR with a Gibbs free energy of ΔG = –282 kcal/mol for the most stable configuration. The individual stem loops, potentially interfering with ribosomal scanning (25Kozak M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8301-8305Crossref PubMed Scopus (395) Google Scholar), exhibit free energies in the range of ΔG = –11.2 to –24.2 kcal/mol. The capacity to form extraordinary stable secondary structures, the terminal oligopyrimidine tract, and the presence of multiple uAUGs strongly suggest regulatory properties of this 5′-UTR related to the control of SHOX gene expression on a translational level.Exon 2 of the SHOX Gene Harbors Alternative Promotor Activities—The calculated high degree of folding is indicative for IRES (15Vagner S. Galy B. Pyronnet S. EMBO Rep. 2001; 2: 893-898Crossref PubMed Scopus (235) Google Scholar) and has prompted us to first analyze the SHOX 5′-UTR translational activity by transfection assays using the reporter constructs pHPL/UTR and pRF/UTR (Fig. 2A). In pHPL a stable hairpin at the 5′-end of the SV40-driven transcription unit represses translation of a firefly luciferase encoding mRNA generated from the parental vector (19Stoneley M. Paulin F.E. Le Quesne J.P. Chappell S.A. Willis A.E. Oncogene. 1998; 16: 423-428Crossref PubMed Scopus (283) Google Scholar). As shown in Fig. 2B, the SHOX 5′-UTR is able to rescue this reporter gene expression if inserted between the 5′ hairpin structure and the firefly luciferase encoding sequence. Within different cell lines, the observed up-regulation varies between 25-fold (HEK293), 72-fold (COS7), and 95-fold in U2Os cells over wild type pHPL activity (data not shown). Also, the SHOX 5′-UTR up-regulates protein expression from the downstream reporter when inserted between the sea pansy (Renilla reniformis) and firefly encoding sequences of the bicistronic reporter vector pRF (Fig. 2, A and B).Fig. 2Exon 2 of theSHOXgene contains alternative promotor elements.A, constructs used for transient cell transfection assays. B, luciferase activities measured after transient transfections of pHPL, pHPL/UTR, pRF, and pRF/UTR into U2Os cells. The SHOX 5′-UTR is able to rescue reporter expression of pHPL and promote expression of the downstream (Firefly) reporter unit of the bicistronic vector pRF. C, Northern-blot analysis of total RNA from U2Os cells transfected with the plasmids constructs indicated on top. The strong signal in pHPL/UTR-transfected cells and the monocistronic transcript detected in RNA from cells transfected with pRF/UTR disclose a transcriptional start event within the SHOX 5′-UTR. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. D, luciferase activities of plasmid constructs indicated on the left. The strongest promotor activity is observed with a fragment that contains 298 bp preceding the SHOX AUG start codon. Negative regulatory elements reside between –498 and –298. Numbers indicate the length of the genomic fragments relative to the SHOX AUG start codon.View Large Image Figure ViewerDownload (PPT)In contrast to this remarkable up-regulation in cell culture-based assays, we did not detect any expression enhancing effects of the SHOX 5′-UTR in vitro, using pRF/UTR primed coupled TNT assays (data not shown). This prima facie discrepancy between cell culture-based and in vitro-derived results can be explained by the absence of cell-specific factors necessary for IRES utilization or the presence of intrinsic promotor elements that remain undetectable in T3 polymerase-driven in vitro TNT reactions. To distinguish between these two possibilities, we analyzed total RNA preparations from cells transfected with pHPL, pRF, and the corresponding 5′-UTR containing constructs. As shown by Northern blot analysis, all transfections yield RNAs complying in size with the expected SV40-driven transcription (Fig. 2C). However, we observed a significantly higher steady state level of firefly luciferase RNA in pHPL/UTR-transfected cells as compared with cells transfected with the parental plasmid (Fig. 2C, pHPL). The size of this transcript furthermore suggests a transcriptional start point about 600 bp downstream of the SV40 promotor within the SHOX 5′-UTR. In agreement with this interpretation, we detected a second firefly luciferase encoding RNA in pRF/UTR, but not in pRF-transfected cells corresponding in size to a monocistronic mRNA that originates from a transcription initiation event within the intercistronic SHOX 5′-UTR fragment (Fig. 2C, pRF). Both results clearly disfavor internal ribosomal entry underlying the observed up-regulation of reporter activity, but rather point to transcriptional initiation within the SHOX 5′-UTR. To corroborate the hypothesis of an alternative promotor, we generated the plasmid constructs pEX2-Luc, pEX2Δ1-Luc, pEX2Δ2-Luc, and pEX2Δ3-Luc by fusing different portions of genomic DNA residing upstream of the SHOX AUG start codon to a firefly luciferase reporter unit (Fig. 2D). Luciferase assays using extracts from cells transiently transfected with these plasmids confirmed transcription-promoting functions with the highest activity observed within a fragment of 298 bp directly preceding the SHOX AUG start codon (Fig. 2D).In conclusion, these results demonstrate that the SHOX gene is transcribed from at least two alternative promotors (P1 and P2) generating distinct mRNAs that encode identical proteins, but vary in their 5′-UTR sequences. These transcripts are hereafter referred to as type 1 and type 2 transcripts, respectively.The SHOX 5′-UTRs Differentially Regulate Translation Efficiency—As the promotor activity within exon 2 of the SHOX gene interferes with DNA transfection experiments, all subsequent analyses were directly based on in vitro generated mRNAs. To address the mechanism by which the two SHOX mRNA variants are translated, we generated in vitro transcripts from pBSK/UTR-Luc (type 1 mRNA) and pBSK/EX2Δ3-Luc (type 2 mRNA) in the presence of either a generic CAP analog (m7G(5′)ppp(5′)G; G-CAP) or an unmethylated variant (A(5′)ppp(5′)G; A-CAP) interfering with CAP-dependent translation initiation. The results from in vitro translation assays primed with these transcripts are shown in Fig. 3B. Although type 1 transcripts are translated at very low levels independently of the CAP structure, the unmethylated A-CAP substantially interferes with the high translation efficiency of type 2 variant. These results suggest that both type 1 and 2 transcripts do not support internal ribosomal entry, but require a CAP-dependent mechanism of translation initiation. Furthermore, they uncover considerable differences in translation competence between the 5′-UTRs of type 1 and type 2 transcripts. We next analyzed nested deletions of the type 1 SHOX 5′-UTR (Fig. 3A) by in vitro translation assays. As shown in Fig. 3C, translation efficiencies are indeed inversely related to the length of the 5′-UTR with an 85-fold difference between type 1 and type 2 variants. To confirm this in vitro-derived data in living cells, we transfected these mRNAs and quantitated their translational efficiency. As expected, we observed the same inverse correlation between length and translation activities in the osteogenic cell line U2Os (Fig. 3D). We next transfected the same in vitro generated mRNAs into different cell lines and determined their translational competence (Fig. 3E). Interestingly, this analysis not only confirms the translation-attenuating properties of the long 5′-UTR variant but also suggests some cell line-dependent responsiveness to the translation inhibitory elements presented by the different forms of the SHOX 5′-UTR (Fig. 3E). Taken together, these results provide compelling evidence that type 1 and type 2 transcripts generated from the alternative promotors P1 and P2 exhibit differential translation efficiencies due to inhibitory elements within the long SHOX 5′-UTR variant.Fig. 3The SHOX 5′-UTRs are differentially translated.A, schematic representation and gelelectrophoretic analysis of mRNAs transcribed in vitro from the plasmid constructs pUTR-Luc, pEX2-Luc, pEX2Δ1-Luc, pEX2Δ2-Luc, and pEX2Δ3-Luc. The structures of these constructs are diagrammatically shown in Fig. 2D. The free energies for the individual 5′-UTRs were predicted with the mfold 3.1 algorithm (bioinfo.math.rpi.edu). B, in vitro translation activity of type 1 and 2 5′-UTRs containing either a m7G(5′)ppp(5′)G (G-CAP) or an A(5′)ppp(5′)G (A-CAP). Although type 1 UTRs are translated with very low efficiencies, the A-CAP dramatically reduces the activity of the type 2 5′-UTR variant. C, in vitro translation activity of the nestedly deleted 5′-UTRs depicted in A. Luciferase activities are given in a logarithmic scale. D, translation efficiency of the same mRNAs in U2Os cells. Luciferase activity was measured in extracts from cells transfected with the in vitro transcribed mRNAs and corrected for RNA transfection efficiency by quantitative RT-PCR. Translational activity is inversely correlated with the length of the SHOX 5′-UTR. E, comparison of the translation efficiency in different cell lines. The activity of the short UTR form (EX2Δ3) was set to 100%.View Large Image Figure ViewerDownload (PPT)Upstream AUG Codons within the SHOX 5′-UTR Inhibit Translation in Vitro and in Vivo—Because the use of nested 5′ deletions does not allow to discriminate structural effects from the influence of uAUGs within the SHOX 5′-UTR, we generated constructs harboring mutations of individual uAUGs or different combinations thereof. In vitro transcription of these constructs yield mRNAs identical in size and with similar overall structural features of their 5′-UTRs (Fig. 4, A and B). These in vitro transcripts were directly transfected into U2Os cells and their translation efficiency determined. As shown in Fig. 4B, mutation of all seven uAUGs within type 1 transcripts yields translational activities comparable with the high efficiency of type 2 mRNAs. Therefore, we can exclude length and overall structure, but rather define the uAUGs as critical determinants for the observed translational down-regulation. We next investigated if this effect can be attributed to individual uAUGs by transfection of reporter RNAs harboring individual uAUG mutations. We can show that none of the individual mutations is able to confer substantial activity increase, whereas combined mutations of uAUG3–7 yield high translation efficiencies. These results argue for a concerted function of the SHOX uAUGs with a major contribution of uAUGs3–7. To confirm these results in vivo, we fused the wild type