It is by now well established that the estrogen receptor α (ERα) is transcribed from multiple promoters. One direct consequence of multiple promoters is the generation of mRNA variants with different 5′-untranslated regions (5′-UTRs). However, the potential roles of these individual mRNA variants are not known. All 5′-UTRs of ERα contain between one and six upstream open reading frames. In this study the effect of the 5′-UTRs of major human and mouse ERα mRNA variants on translation was evaluated. Some of the 5′-UTRs were found to strongly inhibit translation of the downstream open reading frame. Mutation of the upstream AUG codons partially or completely restored translation efficiency. A toeprinting analysis and assessment of the contribution of each AUG codon to the inhibitory effect on translation showed that leaky scanning and reinitiation occurs with these mRNAs. In conclusion, the upstream open reading frames in the 5′-UTRs of ERα mRNAs have the potential to regulate estrogen receptor α expression. It is by now well established that the estrogen receptor α (ERα) is transcribed from multiple promoters. One direct consequence of multiple promoters is the generation of mRNA variants with different 5′-untranslated regions (5′-UTRs). However, the potential roles of these individual mRNA variants are not known. All 5′-UTRs of ERα contain between one and six upstream open reading frames. In this study the effect of the 5′-UTRs of major human and mouse ERα mRNA variants on translation was evaluated. Some of the 5′-UTRs were found to strongly inhibit translation of the downstream open reading frame. Mutation of the upstream AUG codons partially or completely restored translation efficiency. A toeprinting analysis and assessment of the contribution of each AUG codon to the inhibitory effect on translation showed that leaky scanning and reinitiation occurs with these mRNAs. In conclusion, the upstream open reading frames in the 5′-UTRs of ERα mRNAs have the potential to regulate estrogen receptor α expression. estrogen receptor α untranslated region open reading frame upstream ORF upstream AUG cytomegalovirus Although estrogens are primarily recognized as female sex hormones that control the female secondary sexual characteristics, reproductive cycle, and pregnancy they are also involved in the development and maintenance of male reproductive organs (1Hess R.A. Bunick D. Lee K.H. Bahr J. Taylor J.A. Korach S.K. Lubahn D.B. Nature. 1997; 390: 509-512Crossref PubMed Scopus (772) Google Scholar) and in other physiological processes such as liver, fat, and bone metabolism and in cardiovascular and neuronal activity (2Norman A.W. Litwack G. Litwack G. Hormones. Academic Press, London1987: 550-560Google Scholar, 3George F.W. Wilson J.D. Knobil E. Neil J.D. Ewing L.L. Green-Wals G.S. Market C.L. Pfaff D.W. The Physiology of Reproduction. Raven Press, New York1988: 3-26Google Scholar, 4Auchus R.J. Fuqua S.A.W. Baillière's Clinical Endocrinology and Metabolism: Hormones, Enzymes and Receptors. Baillière Tindall, London1994: 433-449Google Scholar). The role of estrogens is also well established in several pathological processes such as osteoporosis (5Horowitz M.C. Science. 1993; 260: 626-627Crossref PubMed Scopus (428) Google Scholar), breast and endometrial cancers (6Henderson B.E. Ross R. Bernstein L. Cancer Res. 1988; 48: 246-253PubMed Google Scholar), and arteriosclerosis and Alzheimer's disease (4Auchus R.J. Fuqua S.A.W. Baillière's Clinical Endocrinology and Metabolism: Hormones, Enzymes and Receptors. Baillière Tindall, London1994: 433-449Google Scholar). The effects of estrogens are mediated by their intracellular receptors. To date, two estrogen receptors (members of the nuclear hormone receptors superfamily) have been described: estrogen receptor α (NR3A1) (7Green S. Walter P. Kumar V. Krust A. Bornert J.M. Argos P. Chambon P. Nature. 1986; 320: 134-139Crossref PubMed Scopus (2010) Google Scholar) and estrogen receptor β (NR3A2, Nuclear Receptors Nomenclature Committee, 1999; Refs. 8Nuclear Receptors Nomenclature Committee Cell. 1999; 97: 161-163Abstract Full Text Full Text PDF PubMed Scopus (964) Google Scholar and 9Kuiper G.G. Enmark E. Pelto-Huikko M. Nilsson S. Gustafsson J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5925-5930Crossref PubMed Scopus (4268) Google Scholar). The estrogen receptor α (ERα)1 gene is transcribed from multiple promoters that give rise to mRNA variants with distinct 5′-untranslated regions (Ref. 10Kos M. Reid G. Denger S. Gannon F. Mol. Endocrinol. 2001; 15: 2057-2063Crossref PubMed Scopus (232) Google Scholar and references therein). In total, seven (A, B, C, D, E, F, and T) and six (A, B, C, E, F, and H) promoters are known for human and mouse ERα gene, respectively, and presumably these lists are not exhaustive. These promoters are utilized in a tissue-specific manner resulting in different levels of expression of mRNA variants in individual tissues (11Grandien K. Backdahl M. Ljunggren O. Gustafsson J.A. Berkenstam A. Endocrinology. 1995; 136: 2223-2229Crossref PubMed Google Scholar, 12Denger S. Reid G. Kos M. Flouriot G. Parsch D. Brand H. Korach K.S. Sonntag-Buck V. Gannon F. Mol. Endocrinol. 2001; 15: 2064-2077Crossref PubMed Scopus (142) Google Scholar, 13Flouriot G. Griffin C. Kenealy M.-R. Sonntag-Buck V. Gannon F. Mol. Endocrinol. 1998; 12: 1939-1954Crossref PubMed Google Scholar, 14Donaghue C. Westley B.R. May F.E. Mol. Endocrinol. 1999; 13: 1934-1950PubMed Google Scholar, 15Kos M. O'Brien S. Flouriot G. Gannon F. FEBS Lett. 2000; 477: 15-20Crossref PubMed Scopus (53) Google Scholar, 16Osterlund M.K. Grandien K. Keller E. Hurd Y.L. J. Neurochem. 2000; 75: 1390-1397Crossref PubMed Scopus (85) Google Scholar). The different expression of human A, B, and C variants in normal and in cancerous breast tissue and tumor-derived cell lines has also been observed (11Grandien K. Backdahl M. Ljunggren O. Gustafsson J.A. Berkenstam A. Endocrinology. 1995; 136: 2223-2229Crossref PubMed Google Scholar,17Weigel R.J. Crooks D.L. Iglehart J.D. deConinck E.C. Cell Growth Differ. 1995; 6: 707-711PubMed Google Scholar). The alternative utilization of multiple promoters might serve to fine tune ERα levels within the cell. This could occur at the level of transcript production, transcript stability, or, as each variant has a different 5′-UTR, efficiency of translation of the transcript. It is known that in general the secondary structure and/or the presence of AUGs and associated open reading frames (ORFs) in the 5′-UTRs can regulate the translation of the main ORF (for review, see Refs.18Kozak M. J. Cell Biol. 1991; 115: 887-903Crossref PubMed Scopus (1475) Google Scholar, 19van der Velden A.W. Thomas A.A. Int. J. Biochem. Cell Biol. 1999; 31: 87-106Crossref PubMed Scopus (321) Google Scholar, 20Morris D.R. Geballe A.P. Mol. Cell. Biol. 2000; 20: 8635-8642Crossref PubMed Scopus (581) Google Scholar). There are two examples of such regulation in the nuclear hormone receptor family. Several short upstream ORFs (uORFs) in the mouse retinoic acid receptor β2 mRNA act to regulate the tissue-specific expression of the receptor at the translational level (21Zimmer A. Zimmer A.M. Reynolds K. J. Cell Biol. 1994; 127: 1111-1119Crossref PubMed Scopus (77) Google Scholar, 22Reynolds K. Zimmer A.M. Zimmer A. J. Cell Biol. 1996; 134: 827-835Crossref PubMed Scopus (65) Google Scholar). Recently the importance of a uORF for expression of the glucocorticoid receptor has also been reported (23Diba F. Watson C.S. Gametchu B. J. Cell. Biochem. 2001; 81: 149-161Crossref PubMed Scopus (46) Google Scholar). The ERα genomic unit might be a good candidate for such regulation as it has a plethora of mRNAs with different 5′-UTRs that contain between one and six uORFs. In this study the effect of upstream AUGs (uAUGs) on the translation of major ERα mRNA variants in human and mouse was analyzed in transient transfection assays and by in vitrotoeprinting. The 5′-UTRs of the human ERα C, E, and F mRNAs showed a moderate negative effect on the translation of a reporter gene. However, human ERα T and mouse ERα C and F 5′-UTRs significantly suppressed the translation of a reporter gene. Mutation of the uAUGs partially or completely restored the translation efficiency of these mRNAs. The extent to which different uAUGs influence the translation of the downstream ORF with regard to their homology to a consensus Kozak sequence (24Kozak M. Nucleic Acids Res. 1987; 15: 8125-8132Crossref PubMed Scopus (4549) Google Scholar) and the location of the associated uORFs relative to the main ORF translation start site was analyzed for the human T and mouse F variants. Toeprinting analysis revealed that leaky scanning occurs with mRNAs bearing these 5′-UTRs and excluded the internal ribosomal entry site-mediated translation as a major mechanism for translation of these mRNAs. This study shows that 5′-UTRs might play an important role in the regulation of ERα expression in some target tissues. GenBankTMaccession numbers of sequences used in this study are listed below. Note that the mouse F 5′-UTRs result from splicing of the exon F1 to the exon F2, respectively (10Kos M. Reid G. Denger S. Gannon F. Mol. Endocrinol. 2001; 15: 2057-2063Crossref PubMed Scopus (232) Google Scholar). The human sequences used were: cDNA, NM_000125; A, NM_000125; C, X62462; E, X86816/AJ002561; F, U68068/AJ002562; T, AJ421639. The mouse sequences used were: cDNA, M38651; C, M38651; F1, AJ272164; F2, AJ272165. The coding sequence of firefly luciferase was amplified from pGL3 basic plasmid (Promega) using the following primers: sense, 5′-GCGGGATCCATGACCATGGAAGACGCCAAAAACATAAAGAAAGGC-3′; antisense 5′-ATAGGGCCCTTACAATTTGGACTTTCCGCCCTTCTTGGCC-3′. The sense primer introduced a BamHI site and initiator ATG sequence of ERα (underlined) that when joined to luciferase forms aBstXI restriction site. The antisense primer contained anApaI site at its 5′-end. The amplified product was directionally cloned into the BamHI and ApaI sites downstream of the CMV promoter in pcDNA3.1-Hygro(+) vector (Invitrogen). The obtained construct, pcDNA3.1-Luc, was used as a parental vector for subcloning various 5′-UTRs of ERα. The 5′-UTRs of human and mouse ERα were amplified by reverse transcription-PCR from human endometrium, liver, or testis and mouse liver RNA using a common antisense primer for human (5′-GGCGTCTTCCATGGTCATGGTAAGTGGCAGCCGGCG-3′) and mouse (5′-GGCGTCTTCCATGGTCATGGTAAGTGGCAGCCGGCG-3′) that introduced the beginning of the luciferase coding sequence behind the initiator ATG of ERα and formed the BstXI as described above. The 5′-UTRs of transcribed mRNAs contained 59 bp of vector sequence at their 5′-end followed by ERα 5′-UTRs fused to luciferase coding sequence. The sense primers, containing an NheI site at their 5′-end, were as follows: human A, 5′-CGCGCTAGCAGGAGCTGGCGGAGGGCG-3′; human C, 5′-CGCGCTAGCTTCACACACTGAGCCACTCGC-3′; human E, 5′-CGCGCTAGCAGTCAGAGAAATAATCGCAGAGCCTC-3′; human F, 5′-CGCGCTAGCACCAAAACTGAAAATGCAGGCTCCATGC-3′; human T, 5′-CGCGCTAGCCTCTTGCCTGCCGCCATGTAAGAAGC-3′; mouse C, 5′-CGCGCTAGCATCACACACCGCGCCACTCGATCATTCG-3′; and mouse F, 5′-CGCGCTAGCGAAAACACAAGGCTCCATGCTCAGC-3′. The resulting PCR products were cloned into the NheI and BstXI sites of pcDNA3.1-Luc. The human ERα expression vector plasmid HEO (pSG5-ERα) was kindly provided by P. Chambon (25Green S. Issemann I. Sheer E. Nucleic Acids Res. 1988; 16: 369Crossref PubMed Scopus (618) Google Scholar). The mouse ERα-expressing vector was constructed by subcloning the ERα coding sequence from pMT2-MOR (kindly provided by M. G. Parker) into the EcoRI site of the pSG5 vector (25Green S. Issemann I. Sheer E. Nucleic Acids Res. 1988; 16: 369Crossref PubMed Scopus (618) Google Scholar). The pSG5-Renilla vector was kindly provided by M. Hentze and contains the Renilla luciferase coding sequence cloned between the SmaI and BamHI sites of pSG5. Site-directed mutagenesis was performed according to the procedure in the QuikChange site-directed mutagenesis kit (Stratagene). All the constructs were sequenced. MDA-231, 293, MCF-7, HepG-2, HeLa, and NIH-3T3 cell lines were maintained in with and at in a The were into (for luciferase or (for RNA such that they were on the of were performed using transfection for NIH-3T3 or for other cell lines following the of the One of vector containing various 5′-UTRs and of pSG5-Renilla vector were used in each The were for In the effect of ERα on translation efficiency of vector was transfection were for with and assays (Promega) were performed following the of the of cell were into a and the was for with the of of luciferase or on a All were performed at in RNA from in the was using the RNA kit according to the from the the RNA was and to assays were performed as described J. D.W. J. D.W. Press, Scholar). The for firefly luciferase and Renilla luciferase were as A of firefly luciferase gene was amplified from pGL3 basic vector (Promega) using the following sense and antisense primers, which and EcoRI sites at the and of the amplified The PCR was into (Stratagene). sense and antisense were used to a of Renilla luciferase from plasmid (Promega) that was into BamHI and EcoRI sites of constructs were and transcribed with RNA according to the in the presence of using a of to of were to and on a The obtained were using The toeprinting analysis was performed as described M. Nucleic Acids Res. 1998; PubMed Scopus Google Scholar). the constructs containing various human and mouse ERα 5′-UTRs upstream of the firefly luciferase coding sequence were by with and transcribed using RNA according to the using of in the presence of and and a at was to a of and the was for RNA was with RNA by and in in a of One of RNA were with of primer in by to for followed by a at The was to the in containing of of and or as was and the internal of in the was between and The were at for and on A was analyzed by primer in a containing of and of (Invitrogen). The were for at the was by with of and of the were on a the were with the primer and using the kit according to the In the following ERα mRNA variants are by their and the uAUGs and uORFs are by their from the 5′-end of the The 5′-UTRs of major human and mouse ERα mRNA variants used in this study are in The of the variants are between and analysis revealed the presence of AUGs and associated ORFs upstream of the main ORF translation start site in variants. The numbers of uAUGs one in human A and and mouse C, two in human C, in human F, in mouse F, and six in human The mouse F has the Kozak for translation M. Nucleic Acids Res. 1987; 15: 8125-8132Crossref PubMed Scopus (4549) Google Scholar) an A at and at The other uAUGs are in a or a translation with the two AUGs of the main The of the associated uORFs from a codon E, and to codons The human and uORFs the AUG into the ERα The human A 5′-UTR, has the secondary as by J. M. J. Mol. Biol. 1999; PubMed Scopus Google Scholar), with a and the individual with a of The was in the human F with with are not to translation M. Proc. Natl. Acad. Sci. U. S. A. 1986; PubMed Scopus Google Scholar, M. Mol. Cell. Biol. PubMed Scopus Google Scholar, M. J. Biol. 1991; Full Text PDF PubMed Google Scholar, A. A. S. G. 2001; PubMed Scopus Google Scholar). it is that of the 5′-UTRs translation of the downstream ORF to their secondary To the effect of various 5′-UTRs on translation of the main the constructs containing different 5′-UTRs upstream of a luciferase reporter gene were with a Renilla plasmid into human breast cell or mouse cell NIH-3T3 The parental vector has a of uAUGs and as a control for translation efficiency. The firefly luciferase were for transfection efficiency using the Renilla luciferase activity and also with mRNA expression in The human A the translation of luciferase with the parental The human C, E, and F 5′-UTRs a moderate negative effect on translation efficiency. However, the human T and mouse C and F the translation of the luciferase to or of the Mutation of upstream AUGs to restored or the translation efficiency of human 5′-UTRs not of The of uAUGs an effect on the human T it the translation by were obtained using other cell lines such as HeLa, MCF-7, and not with an ERα-expressing vector or transfection into the cell was performed to ERα has an effect on translation of different mRNA variants. However, in translation efficiency has been observed in the presence or in the of not These that upstream AUG in 5′-UTRs of the multiple mRNA of ERα variants can regulate the expression on the level of The mouse F and human T which the translation from the downstream luciferase ORF contain six and The of individual uAUGs to the of translation were using constructs with uAUGs or their into the of the mouse F or not have a effect on the It was the of the that was for of the in translation efficiency of the of human T or not the of the However, the of the translation efficiency The of and the translation and restored the efficiency of translation of this to the level of the parental vector The of the which is in the frame with the not the efficiency of the uAUGs the translation an the level of the To the AUG codons in mRNAs containing various 5′-UTRs of human and mouse ERα the toeprinting analysis was performed one to uAUGs were observed in that these mRNAs are by leaky A of the presumably to a Kozak and on the AUG In the of human A and to the main ORF AUGs were also Several that were by of that not to AUG were also observed by in is The human T was analyzed by of the codons into codons at the 5′-end of the and the downstream uAUGs of codons to at downstream uAUGs and in a of the from the 5′-end of the It is that the the main ORF AUGs were observed the of The of the might the potential of the AUG to serve as an This analysis also the of the observed A not to AUG was observed in in which the uAUGs were in This might be by a of the secondary structure that is by the presence of the at the The ERα mRNAs are transcribed from multiple promoters that are of genomic region (10Kos M. Reid G. Denger S. Gannon F. Mol. Endocrinol. 2001; 15: 2057-2063Crossref PubMed Scopus (232) Google Scholar). have that the expression of the ERα in human and mouse is by 5′-UTRs on the level of the human A, C, E, and F 5′-UTRs not inhibit translation of downstream the mouse C and F and the human T 5′-UTRs inhibitory effects to various G. Griffin C. Kenealy M.-R. Sonntag-Buck V. Gannon F. Mol. Endocrinol. 1998; 12: 1939-1954Crossref PubMed Google Scholar, 14Donaghue C. Westley B.R. May F.E. Mol. Endocrinol. 1999; 13: 1934-1950PubMed Google Scholar, 15Kos M. O'Brien S. Flouriot G. Gannon F. FEBS Lett. 2000; 477: 15-20Crossref PubMed Scopus (53) Google Scholar, 16Osterlund M.K. Grandien K. Keller E. Hurd Y.L. J. Neurochem. 2000; 75: 1390-1397Crossref PubMed Scopus (85) Google Scholar). This to a of the by which the levels of the ERα are in target the observed tissue-specific expression of ERα mRNA variants (11Grandien K. Backdahl M. Ljunggren O. Gustafsson J.A. Berkenstam A. Endocrinology. 1995; 136: 2223-2229Crossref PubMed Google Scholar). the major mRNA variants in human and known estrogen target are A and C G. Griffin C. Kenealy M.-R. Sonntag-Buck V. Gannon F. Mol. Endocrinol. 1998; 12: 1939-1954Crossref PubMed Google Scholar). The that human C has inhibitory human A the translation of the main A and C ERα promoters might be utilized in these tissues in to levels of ERα the the human T 5′-UTR, which is at levels in testis H. Kos M. Denger S. Flouriot G. J. Gannon F. Reid G. Endocrinology. PubMed Scopus Google Scholar), translation It is known that estrogen receptors are important for testis development and for normal as to levels of estrogens or and development to male reproductive L. 2001; PubMed Scopus Google Scholar and references therein). regulation of ERα expression in testis is to be The translational efficiency of the human T mRNA might that levels of ERα are in the target cell. the of regulation of translation efficiency to be a uORF in the retinoic acid receptor β2 mRNA its translation in and not in other Refs. A. Zimmer A.M. Reynolds K. J. Cell Biol. 1994; 127: 1111-1119Crossref PubMed Scopus (77) Google Scholar and 22Reynolds K. Zimmer A.M. Zimmer A. J. Cell Biol. 1996; 134: 827-835Crossref PubMed Scopus (65) Google Scholar). The extent to which 5′-UTRs influence the translation might also of development or on The performed in a of different cell might not be to such It has also been reported in the that by short uORFs can regulate expression of the retinoic acid receptor β2 or the glucocorticoid receptor K. Zimmer A.M. Zimmer A. J. Cell Biol. 1996; 134: 827-835Crossref PubMed Scopus (65) Google Scholar, F. Watson C.S. Gametchu B. J. Cell. Biochem. 2001; 81: 149-161Crossref PubMed Scopus (46) Google Scholar). However, of the ERα uORFs is to of these the and of uORFs in different 5′-UTRs is well between mouse and of these human and mouse C are partially mouse C is significantly and it that by uORFs in various ERα 5′-UTRs influence the expression of ERα. The toeprinting analysis showed that on multiple upstream and the mRNAs containing various 5′-UTRs of human or mouse ERα are by a leaky scanning the obtained with mouse F and human T 5′-UTRs the scanning and reinitiation of translation M. Cell. 15: Full Text PDF PubMed Scopus Google Scholar) of these mRNAs P. Mol. Cell. Biol. 1986; 6: PubMed Scopus Google Scholar). In this the ribosomal to the and the mRNA it the AUG codon it the AUG is not recognized to Kozak or secondary the this AUG and at AUG resulting in leaky scanning (for review, see Refs. 20Morris D.R. Geballe A.P. Mol. Cell. Biol. 2000; 20: 8635-8642Crossref PubMed Scopus (581) Google Scholar M. 1999; PubMed Scopus Google Scholar). the the can at downstream from the or at the codon of the The efficiency of reinitiation on a downstream AUG also on the between the of upstream ORF and the AUG M. Mol. Cell. Biol. 1987; PubMed Scopus Google Scholar, Mol. Cell. Biol. 1994; PubMed Scopus Google Scholar, S. J. 1995; PubMed Google Scholar). this is short M. Mol. Cell. Biol. 1987; PubMed Scopus Google Scholar) the is to the it the and it to on the downstream This for the contribution of individual uAUGs of the human T to of translation efficiency This contains six uORFs The uORFs upstream of the main and the that they have a moderate effect on translation efficiency. This is by of individual or which not the translation of luciferase the translation of or are to each or be to strongly inhibit translation of the main ORF as they downstream of the main ORF initiator the translation of the on the to and or The of or have a effect in the presence of The this The of translation of luciferase and of and translation The of not have The of the uAUGs the translation a with the sequence. to the human T 5′-UTR, the mouse F contains a of uORFs. The and upstream of the main The translation be to be by the and The has a Kozak the the translation of the main ORF the The analysis this the toeprinting analysis showed that the translation occurs at the several uAUGs in 5′-UTRs the downstream at or the AUG of the main the as it might be that the human and F 5′-UTRs not inhibit the translation of luciferase significantly as their uAUGs are not in Kozak and uORFs upstream of the main ORF However, it was not that the human A not inhibit the translation to an extent to mouse C or F 5′-UTRs as the uORFs of 5′-UTRs in the from the main ORF and A the A that on this the secondary structure in human A the of the main AUG or mechanism such as internal entry is for this effect is not of uAUGs in human 5′-UTRs restored or the translation of the of mouse C or F 5′-UTRs uAUGs restored the translation efficiency to or of the parental It is that a negative in mouse in the common of the 5′-UTRs between the common site and main which is between mouse and can influence the translation from the main However, is to this In have in this study that multiple 5′-UTRs and influence the expression of ERα in human and The that human A and C are with a efficiency human T strongly the translation of the downstream ORF are of the observed effects are by upstream have that mRNAs containing human or mouse ERα 5′-UTRs are by leaky scanning and
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