Translation Initiation of the Insulin-like Growth Factor I Receptor mRNA Is Mediated by an Internal Ribosome Entry Site

The insulin-like growth factor I receptor (IGF-IR) is a heterotetrameric receptor mediating the effects of insulin-like growth I and other growth factors. This receptor is encoded by an mRNA containing an unusually long, G-C-rich, and highly structured 5′ untranslated region. Using bicistronic constructs, we demonstrated here that the 5′ untranslated region of the IGF-IR allows translation initiation by internal ribosome entry and therefore constitutes an internal ribosome entry site. In vitro cross-linking revealed that this internal ribosome entry site binds a protein of 57 kDa. Immunoprecipitation of UV cross-linked proteins proved that this protein was the polypyrimidine tract-binding protein, a well known regulator of picornavirus mRNA translation. The efficiency of translation of the endogenous IGF-IR mRNA is not affected by rapamycin, which is a potent inhibitor of cap-dependent translation. This result provides evidence that the endogenous IGF-IR mRNA is translated, at least in part, through a cap-independent mechanism. This is the first report of a growth factor receptor containing sequence elements that allow translation initiation to occur by internal initiation. Because the IGF-IR has a pivotal function in the cell cycle, this mechanism of translation regulation could play a crucial role in the control of cell proliferation and differentiation. The insulin-like growth factor I receptor (IGF-IR) is a heterotetrameric receptor mediating the effects of insulin-like growth I and other growth factors. This receptor is encoded by an mRNA containing an unusually long, G-C-rich, and highly structured 5′ untranslated region. Using bicistronic constructs, we demonstrated here that the 5′ untranslated region of the IGF-IR allows translation initiation by internal ribosome entry and therefore constitutes an internal ribosome entry site. In vitro cross-linking revealed that this internal ribosome entry site binds a protein of 57 kDa. Immunoprecipitation of UV cross-linked proteins proved that this protein was the polypyrimidine tract-binding protein, a well known regulator of picornavirus mRNA translation. The efficiency of translation of the endogenous IGF-IR mRNA is not affected by rapamycin, which is a potent inhibitor of cap-dependent translation. This result provides evidence that the endogenous IGF-IR mRNA is translated, at least in part, through a cap-independent mechanism. This is the first report of a growth factor receptor containing sequence elements that allow translation initiation to occur by internal initiation. Because the IGF-IR has a pivotal function in the cell cycle, this mechanism of translation regulation could play a crucial role in the control of cell proliferation and differentiation. insulin-like growth factor I insulin-like growth factor I receptor vascular smooth muscle cell untranslated region internal ribosome entry site encephalomyocarditis virus polypyrimidine tract-binding protein insulin-like growth factor II Renilla luciferase gene,FLuc, Firefly luciferase gene nucleotide(s) base pair(s) Dulbecco's modified Eagle's medium Insulin-like growth factor I (IGF-I)1 is a ubiquitous peptide that has a fundamental role in both prenatal and postnatal development (1Werner H. Adamo M. Roberts Jr., C.T. LeRoith D. Vitam. Horm. 1994; 48: 1-58Crossref PubMed Scopus (74) Google Scholar, 2Jones J.I. Clemmons D.R. Endocr. Rev. 1995; 16: 3-34Crossref PubMed Google Scholar). The effects of IGF-I are mediated through the IGF-I receptor (IGF-IR),which resembles the insulin receptor in primary and tertiary structure (3Ullrich A. Gray A. Tam A.W. Yang-Feng T. Tsubokawa M. Collins C. Henzel W. Le Bon T. Kathuria S. Chen E. Jacobs S. Francke U. Ramachandran J. Fujita-Yamaguchi Y. EMBO J. 1986; 5: 2503-2512Crossref PubMed Scopus (1511) Google Scholar). The IGF-IR plays a central role during the cell cycle, as demonstrated by the fact that overexpression of IGF-IR abrogates requirements for exogenous growth factors (4Pietrzkowski Z. Lammers R. Carpenter G. Soderquist A.M. Limardo M. Phillips P.D. Ullrich A. Baserga R. Cell Growth Differ. 1992; 3: 199-205PubMed Google Scholar). In vascular smooth muscle cells (VSMCs),the IGF-IR mediates the mitogenic effects of various growth factors such as platelet-derived growth factor, endothelial growth factor, fibroblast growth factor β, thrombin, and angiotensin II, consistent with an important role in cardiovascular growth responses (5Pfeifle B. Boeder H. Ditschuneit H. Endocrinology. 1987; 120: 2251-2258Crossref PubMed Scopus (85) Google Scholar, 6Coppola D. Ferber A. Miura M. Sell C. D'Ambrosio C. Rubin R. Baserga R. Mol. Cell. Biol. 1994; 14: 4588-4595Crossref PubMed Scopus (268) Google Scholar, 7Delafontaine P. Lou H. J. Biol. Chem. 1993; 268: 16866-16870Abstract Full Text PDF PubMed Google Scholar, 8Delafontaine P. Anwar A. Lou H. Ku L. J. Clin. Invest. 1996; 97: 139-145Crossref PubMed Scopus (74) Google Scholar). A variety of studies have shown that relatively small changes in IGF-IR expression have important proliferative or antiproliferative effects (9Delafontaine P. Meng X.P. Ku L. Du J. J. Biol. Chem. 1995; 270: 14383-14388Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 10Du J. Delafontaine P. Circ. Res. 1995; 76: 963-972Crossref PubMed Scopus (74) Google Scholar, 11Baserga R. Cancer Res. 1995; 55: 249-252PubMed Google Scholar). However, studies characterizing growth factor and tumor suppressor gene regulation of the IGF-IR have focused on transcriptional mechanisms (12Werner H. Karnieli E. Rauscher F.J. LeRoith D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8318-8323Crossref PubMed Scopus (337) Google Scholar, 13Ohlsson C. Kley N. Werner H. LeRoith D. Endocrinology. 1998; 139: 1101-1107Crossref PubMed Scopus (136) Google Scholar, 14Du J. Peng T. Scheidegger K.J. Delafontaine P. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 2119-2126Crossref PubMed Scopus (59) Google Scholar, 15Du J. Meng X.-P. Delafontaine P. Endocrinology. 1996; 137: 1378-1384Crossref PubMed Scopus (28) Google Scholar, 16Scheidegger K.J. Du J. Delafontaine P. J. Biol. Chem. 1999; 274: 3522-3530Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Some features of the IGF-IR gene suggest that it could be regulated at the posttranscriptional level. Like housekeeping genes, which appear to be transitionally regulated, the IGF-IR promoter gene is devoid of TATA and CAAT elements and is also very GC-rich. Moreover, the human and rat 5′ untranslated regions (UTRs) are unusually long, 1038 and 943 bp, respectively, and the rat 5′ UTR has been reported to possess two additional AUGs upstream in frame with the initiator AUG (17Werner H. Stannard B. Bach M.A. LeRoith D. Roberts Jr., C.T. Biochem. Biophys. Res. Commun. 1990; 169: 1021-1027Crossref PubMed Scopus (68) Google Scholar). In eukaryotic cells, different modes of translation initiation are used depending on the nature of the mRNA to be translated and on the physiological state of the cell. The most frequently used are the “scanning mechanism” and “internal initiation.” In the scanning mechanism, the initiation of translation requires the formation of a “43 S complex,” which binds to the 5′-m7G cap structure of the mRNA and scans along the 5′ UTR up to the initiator AUG. Then, the 60 S subunit attaches to this complex and translation is initiated (18Kozak M. J. Cell Biol. 1989; 108: 229-241Crossref PubMed Scopus (2810) Google Scholar). However, the presence of highly structured 5′ UTR will cause the repression of this cap-dependent mechanism, and many mRNAs with structured 5′ UTRs are poorly translated using this mechanism (19Pelletier J. Sonenberg N. Cell. 1985; 40: 515-526Abstract Full Text PDF PubMed Scopus (388) Google Scholar). A cap-independent mechanism called internal initiation was first demonstrated in picornaviruses, which lack a 5′-m7G cap and have long structured 5′ UTRs in their RNA. The presence of an internal ribosome entry site (IRES) has been shown in different picornaviruses, such as encephalomyocarditis virus (EMCV), human rhinoviruses, and hepatitis A virus (see Ref. 20Jackson R.J. Hunt S.L. Reynolds J.E. Kaminski A. Sarnow P. Cap-independent Translation. Springer-Verlag, Berlin1995Google Scholar for review). This mechanism requires secondary structures that allow ribosomes to bind directly next to the initiator AUG and permit translation to start without previous scanning. Regulation of IRESs sometimes also involves cellular factors such as the polypyrimidine tract-binding protein (PTB), a member of the heterogeneous nuclear ribonucleoprotein family involved in nuclear splicing regulation (21Hellen C.U. Witherell G.W. Schmid M. Shin S.H. Pestova T.V. Gil A. Wimmer E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7642-7646Crossref PubMed Scopus (293) Google Scholar). Recently, IRES elements have been found in several cellular mRNAs. These include growth factors such as vascular endothelial growth factor (22Huez I. Creancier L. Audigier S. Gensac M.C. Prats A.C. Prats H. Mol. Cell. Biol. 1998; 18: 6178-6190Crossref PubMed Scopus (245) Google Scholar), fibroblast growth factor (23Vagner S. Gensac M.C. Maret A. Bayard F. Amalric F. Prats H. Prats A.C. Mol. Cell. Biol. 1995; 15: 35-44Crossref PubMed Scopus (289) Google Scholar), and IGF-II (24Teerink H. Voorma H.O. Thomas A.A. Biochim. Biophys. Acta. 1995; 1264: 403-408Crossref PubMed Scopus (54) Google Scholar). IRESs are also found in a variety of other mammalian genes: proto-oncogenes (c-myc, (25Nanbru C. Lafon I. Audigier S. Gensac M.C. Vagner S. Huez G. Prats A.C. J. Biol. Chem. 1997; 272: 32061-32066Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar) and c-sis (26Bernstein J. Sella O. Le S.Y. Elroy-Stein O. J. Biol. Chem. 1997; 272: 9356-9362Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar)), transcription factors (NF-κB repressing factor) (27Oumard A. Hennecke M. Hauser H. Nourbakhsh M. Mol. Cell. Biol. 2000; 20: 2755-2759Crossref PubMed Scopus (55) Google Scholar), hematopoiesis transcription factors (28Pozner A. Goldenberg D. Negreanu V. Le S.Y. Elroy-Stein O. Levanon D. Groner Y. Mol. Cell. Biol. 2000; 20: 2297-2307Crossref PubMed Scopus (119) Google Scholar), cardiac voltage-gated potassium channel (Kv1.4 (29Negulescu D. Leong L.E. Chandy K.G. Semler B.L. Gutman G.A. J. Biol. Chem. 1998; 273: 20109-20113Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar)), and the β subunit of mitochondrial H+-ATP synthase (30Izquierdo J.M. Cuezva J.M. Biochem. J. 2000; 346: 849-855Crossref PubMed Scopus (44) Google Scholar). In this study, we investigated whether IGF-IR mRNA translation could be driven by an internal initiation mechanism. We provide evidence for the first time that the mRNA encoding a growth factor receptor (IGF-IR) is translated by an internal initiation mechanism. Moreover, we show that the 5′ UTR of the IGF-IR mRNA can be considered to possess an IRES that binds These have important for mechanisms of control of IGF-IR expression and mechanisms of cellular growth smooth muscle cells rat as S. Jr., M.A. J. Cell Biol. PubMed Scopus Google Scholar). in with and cells in without with and in with and cells in with and The rat IGF-I receptor sequence and 5′ UTR was in J. Delafontaine P. Circ. Res. 1995; 76: 963-972Crossref PubMed Scopus (74) Google Scholar). The to the other of the 5′ UTR was by H. Werner The was by rat IGF-IR by J. the of The was by of J. Delafontaine P. Circ. Res. 1995; 76: 963-972Crossref PubMed Scopus (74) Google Scholar) the site of the was and the of the sequence for the 5′ UTR of the was by the of the site of the 5′ UTR This the sequence for the 5′ UTR and of the of the bicistronic The gene was by and the site of the Firefly gene was the the site of In the called the transcription of the first to is the control of the The sequence for the 5′ UTR of the IGF-IR mRNA was by using as and and which site and a The was in the the and containing the sequence for the 5′ UTR was in the of the A by using was by of a and in site of upstream of luciferase The was called containing of the sequence for the 5′ was by of of and in the site of cells in and with of bicistronic well with cells and to the by using the in a cells for and with containing in the presence or of various of UV cross-linking to Huez (22Huez I. Creancier L. Audigier S. Gensac M.C. Prats A.C. Prats H. Mol. Cell. Biol. 1998; 18: 6178-6190Crossref PubMed Scopus (245) Google Scholar). and rat as cells in and by The cell was in of and The was at for and the was to and at A was and by at the site. and and by at the and was and by at the site. with of with of for at in containing and in a of to and with UV at the and with at for The with a of of and of A at for with of at for was and the for at and on a The was in for and to UV cross-linking as the to with and and with of by E. or a at the on protein The with the proteins by and by cells for and with containing in the presence or of of of ribosomes was on as A. D. L. T. J. 1994; PubMed Scopus Google Scholar). in in the presence of the and in cells in containing and by of to a of and by two at and The was a in the containing and in a at for at of the of the was with and for and of the RNA. was in of The of the various mRNAs the different was by The IGF-IR and the of and the of D. J. Mol. Biol. PubMed Scopus Google Scholar), The and by The for at and was using a The features of the IGF-IR mRNA 5′ UTR to whether this region the to a highly structured secondary of the rat and human IGF-IR 5′ UTR secondary structures using and the structures with the F. P. P. C. B. Res. 1997; PubMed Scopus Google Scholar). base is along this 5′ which has a of and several a long polypyrimidine upstream of the initiator AUG and was of the rat and human 5′ UTR the at the of this sequence revealed a site for Moreover, the sequence is in a structure which is an important of picornavirus IRES S.Y. Jr., Res. 1997; PubMed Scopus Google Scholar). These features to whether the rat IGF-IR mRNA can be translated by internal ribosome the presence of an IRES in the 5′ UTR of the IGF-IR we used a bicistronic in which the first is translated by a cap-dependent scanning mechanism and the requires translation by internal entry Sarnow P. PubMed Scopus Google Scholar). The bicistronic was by the gene as the first the control of the and the gene as the The sequence for the or the 5′ UTR of the IGF-IR mRNA or as well as that for the encephalomyocarditis virus IRES as a control and These cells and for the two luciferase The the two was and the using the as the was in cells with in which the was the control of the in cells with the control In cells with the was that of the control We also whether the 5′ UTR of the rat IGF-IR mRNA can internal initiation in human cells and not In cells with which a of the sequence for the 5′ UTR of the IGF-IR translation of the gene was the was that with the We also the which the of the 5′ and of the sequence in the This was used to that the IRES was for the 5′ UTR this was of translation of with the that the IRES was not to translation initiation the first or by a mechanism of we a upstream of the The for are in B. of encoded by the first of was that of by the In a was upstream of the of the was as that of the and was These that translation of the was of that of the first of the was initiated the IRES in the rat 5′ UTR that the IGF-IR 5′ UTR an we investigated to translation in the presence of which was known to cap-dependent translation both in and in vitro L. A.C. Sonenberg N. EMBO J. 1996; 15: PubMed Scopus Google Scholar). this bicistronic mRNAs containing the IRES or the IGF-IR 5′ UTR in cells In the the translation of was the translation of the control of the IRES was cap-independent a a of at of the cap-dependent translation of the cap-independent translation was not with the in which the translation of was the control of the IGF-IR 5′ UTR A was with a of at of the IGF-IR 5′ UTR the to translation in a cap-independent as the not proved that the IGF-IR 5′ UTR could translation through a cap-independent mechanism. The of on the efficiency of translation of the endogenous IGF-IR mRNA was by the of IGF-IR mRNA and a fibroblast cell was used it IGF-IR mRNA for by mRNA was used as a control mRNA initiated by cap-dependent translation. cells for with or without rapamycin, and the of IGF-IR and mRNAs was of IGF-IR mRNA control and cells the different was very and of IGF-IR mRNA was with containing at least two ribosomes in control and not a in the IGF-IR mRNA In was mRNA with in cells with in control cells in the of mRNA in containing two ribosomes was mRNA can bind the in mRNA containing to to to a of the efficiency of translation. In not the efficiency of translation of the endogenous IGF-IR proteins are known to with the IRES of the and mammalian mRNAs. whether the IGF-IR 5′ UTR was to bind we used rat and UV cross-linking we whether the of the UTR containing the site (see was to bind proteins in to the of the 5′ containing the and the first of the was with and of proteins cross-linked to the was by cross-linked of 60 and and both the nature of The of the was demonstrated by In the presence of a or of both and We investigated the that the protein could to We used a to the IRES as a control for The protein to the with a factor to the IRES and Moreover, of the protein was and a or of IRES was used as an protein with an of was not by this was an was used and the 5′ the site of with the protein, we using the different in A. the not the In the was The protein was to bind to both and and However, this protein not at with the This that the presence of the is for of the These are consistent with the that the protein could be and with the secondary structure of the region of the by this protein the is also in the 5′ UTR of the human IGF-IR UV cross-linking with of human two proteins of 60 and revealed with which to the rat IGF-IR 5′ These two with and that at was found for These revealed that the proteins be in rat and human and that to bind to the rat IGF-IR 5′ the of the protein, UV cross-linked with and and using a The complex was with both the and the containing the of the IGF-IR 5′ UTR mRNA The protein was also of UV cross-linked human with was in The of this protein result of this protein with or Immunoprecipitation with protein and protein and not to that the and was the complex was by using the This revealed a in both cell which at 60 kDa. This result demonstrated that the was in the UV cross-linked IGF-I receptor gene expression is by a of physiological and cell In this study, we that initiation of translation of this growth factor receptor mRNA can occur by internal ribosome a additional of control of this is the first IRES to be in the mRNA of a growth factor Because the rat IGF-IR 5′ UTR is long and a the the of ribosomes to along the IGF-IR 5′ UTR mRNA to the scanning was ribosome entry was first for picornavirus J. Sonenberg N. PubMed Scopus Google different elements involved in the internal ribosome entry are well The rat 5′ UTR mRNA not the of picornavirus such as a or Wimmer E. Sarnow P. Cap-independent Translation. Scholar). of the secondary structure revealed that this 5′ UTR mRNA a structure of two different The first two to be the site for T.V. C.U. Wimmer E. J. PubMed Google Scholar) and a of the a sequence upstream of the as is the of a of picornavirus IRESs Cell. 1992; Full Text PDF PubMed Scopus Google Scholar). Moreover, this structure is in a for other cellular such as vascular endothelial growth factor and (22Huez I. Creancier L. Audigier S. Gensac M.C. Prats A.C. Prats H. Mol. Cell. Biol. 1998; 18: 6178-6190Crossref PubMed Scopus (245) Google Scholar, M. Le 1998; 16: PubMed Scopus Google Scholar). The secondary structure of the 5′ UTR of the human IGF-IR mRNA revealed rat and human 5′ the human 5′ UTR is the rat and a upstream of the initiator AUG. Moreover, the two upstream AUGs for the rat IGF-IR mRNA are not found in the human IGF-IR We investigated the of the rat IGF-IR 5′ UTR to internal translation initiation. this we used the bicistronic Sarnow P. PubMed Scopus Google Scholar). The translation efficiency of the was the IGF-IR 5′ UTR mRNA was in the and the efficiency of the IGF-IR IRES was that of the control we whether this IRES was also of cellular cap-dependent translation by These first in vitro and demonstrated that the IGF-IR 5′ UTR the to translation the cap-dependent translation is This result was also in as the endogenous IGF-IR mRNA was The of the IGF-IR IRES to be an important is in the first of the 5′ and it will be of primary to whether this IRES is of as for the protein mRNA Proc. Natl. Acad. Sci. U. S. A. 2000; 97: PubMed Scopus Google Scholar). UV cross-linking with rat vascular smooth muscle cell and human cells revealed that at least two proteins can bind to the rat IGF-IR 5′ UTR mRNA and that the using rat and human cell very Moreover, proteins both with the IRES and the rat IGF-IR 5′ UTR The protein could to the protein known to with IRES T. 1990; PubMed Scopus (33) Google Scholar). We demonstrated by that the protein to the splicing on the secondary the site be the structure This was by the fact that the of this structure the of the in human and rat cellular also known as is a nuclear protein, in in the of many cells, which binds to elements A. S. C. G. Res. 1992; 20: PubMed Scopus Google Scholar). The role of this factor both in the splicing mechanism and in translation initiation it has been shown that this protein the of different IRESs R. R. M. Mol. Cell. Biol. 2000; 20: PubMed Scopus Google Scholar). The of IRESs has been is evidence that posttranscriptional regulation translation plays a role in expression of several proto-oncogenes and protein The is a regulator of cell and and it was demonstrated that the IRES was during M. M. M. Mol. Cell. Biol. 2000; 20: PubMed Scopus Google Scholar). Moreover, IRESs found in mRNA that the translation of important mammalian such as fibroblast growth and platelet-derived growth A mechanism mediated by an IRES to the mRNA such an The vascular endothelial growth factor IRES is during protein is I. A. P. R. Z. E. Mol. Cell. Biol. 1998; 18: PubMed Scopus Google Scholar), an important role in The platelet-derived growth factor IRES is during cell in which protein are also (26Bernstein J. Sella O. Le S.Y. Elroy-Stein O. J. Biol. Chem. 1997; 272: 9356-9362Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). regulation allows translation that are not for cap-dependent such as or evidence has shown that the IGF-IR is at a in the control of cell growth and that a is for the mitogenic effects of a variety of growth platelet-derived growth factor (5Pfeifle B. Boeder H. Ditschuneit H. Endocrinology. 1987; 120: 2251-2258Crossref PubMed Scopus (85) Google Scholar), endothelial growth factor D. Ferber A. Miura M. Sell C. D'Ambrosio C. Rubin R. Baserga R. Mol. Cell. Biol. 1994; 14: 4588-4595Crossref PubMed Scopus (268) Google Scholar), angiotensin II, and P. Anwar A. Lou H. Ku L. J. Clin. Invest. 1996; 97: 139-145Crossref PubMed Scopus (74) Google Scholar, P. M. Du J. 1996; PubMed Scopus Google Scholar). of the IGF-IR is and overexpression of the receptor has IGF-IR gene expression be regulated to protein at the time and In cells, cap-dependent translation is and proteins of the cap-dependent are poorly We can that the internal ribosome entry allow the translation of proteins such as growth factor to permit the cell to in In this it is to that a reported that in human cells, the of IGF-IR 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). This is consistent with the mechanism of cap-independent translation initiation of the IGF-IR as has been for the insulin receptor on G. Sarnow P. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar). we have shown that IGF-IR on without IGF-IR mRNA that control mechanisms could be important K.J. Delafontaine P. J. Biol. Chem. 2000; Full Text Full Text PDF Scopus Google Scholar). In we have shown that the 5′ UTR of the IGF-IR mRNA an IRES that binds and allows cap-independent translation. Because of the role by the IGF-IR in cell control and have important for mechanisms of and cell proliferation and mechanisms for control of gene We E. Wimmer for the of the