The Two Upstream Open Reading Frames of Oncogene mdm2 Have Different Translational Regulatory Properties

Few details are known of the mechanisms through which multiple upstream open reading frames (uORFs) interact to regulate translation in higher eukaryotes. The predominant transcript of oncogene mdm2 in normal human cells (L-mdm2) contains two upstream open reading frames in its 5′ leader. Elimination of these two uORFs raises the translational efficiency of the transcript by over 10-fold in HeLa cells. The 5′-most uORF (uORF1) alone suppresses downstream translational activity by over 5-fold, whereas uORF2 contributes <2-fold to the inhibition by the intact leader. The different activities of the two uORFs do not depend on the nucleotide sequence surrounding the uORFs in the 5′ leader, the order of the two uORFs in the 5′ leader, or the occurrence of secondary structure or rare codons within the uORFs. Specific features of the amino acid sequence encoded by uORF1 contribute to its stronger suppressive activity, suggesting that it belongs to the class of “sequence-specific” uORFs. The weaker inhibitory activity inherent in uORF2 is potentiated by a sub-optimal nucleotide context surrounding its initiator AUG. The occurrence of two uORFs with differing activities in both the human gene and the mouse orthologue suggests that this pair of elements may play a fundamental role in regulating expression of the mdm2 gene. Few details are known of the mechanisms through which multiple upstream open reading frames (uORFs) interact to regulate translation in higher eukaryotes. The predominant transcript of oncogene mdm2 in normal human cells (L-mdm2) contains two upstream open reading frames in its 5′ leader. Elimination of these two uORFs raises the translational efficiency of the transcript by over 10-fold in HeLa cells. The 5′-most uORF (uORF1) alone suppresses downstream translational activity by over 5-fold, whereas uORF2 contributes <2-fold to the inhibition by the intact leader. The different activities of the two uORFs do not depend on the nucleotide sequence surrounding the uORFs in the 5′ leader, the order of the two uORFs in the 5′ leader, or the occurrence of secondary structure or rare codons within the uORFs. Specific features of the amino acid sequence encoded by uORF1 contribute to its stronger suppressive activity, suggesting that it belongs to the class of “sequence-specific” uORFs. The weaker inhibitory activity inherent in uORF2 is potentiated by a sub-optimal nucleotide context surrounding its initiator AUG. The occurrence of two uORFs with differing activities in both the human gene and the mouse orthologue suggests that this pair of elements may play a fundamental role in regulating expression of the mdm2 gene. Open reading frames in the 5′ leaders of messenger RNAs, which are termed upstream open reading frames (uORFs), 1The abbreviations used are: uORF, upstream open reading frame; hORF, human ORF; mORF, mouse ORF; hGH, human growth hormone; S-mdm2, short form of mdm2 RNA; L-mdm2, long form of mdm2 RNA; AdoMetDC, S-adenosylmethionine decarboxylase; IRES, internal ribosome entry. are emerging as important mediators of transcript-specific translational control (1Geballe A.P. Sachs M.S. Sonenberg N. Hershey J.W.B. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Press, Cold Spring Harbor, NY2000: 595-614Google Scholar, 2Morris D.R. Geballe A.P. Mol. Cell. Biol. 2000; 20: 8635-8642Crossref PubMed Scopus (568) Google Scholar). uORFs can occur singly and multiply, depending on the particular transcript, and there are regulatory implications associated with both of these situations. In order for a single uORF to regulate ribosome access to downstream coding sequences, it seems that two conditions must be met. 1) Translation of the uORF must block reinitiation that could lead to translation further downstream on the mRNA; and 2) there must be a regulated mechanism for attenuating or bypassing the block. “Sequence-dependent uORFs” block movement of ribosomes on the 5′ leader as a result of specific interactions of uORF-encoded nascent peptides with targets that are thought to be components of the translation machinery (2Morris D.R. Geballe A.P. Mol. Cell. Biol. 2000; 20: 8635-8642Crossref PubMed Scopus (568) Google Scholar). Ribosomes have been found arrested on sequence-dependent uORFs either during the elongation phase of translation or at termination, depending on the sequence of the particular peptide and, therefore, the identity of its target. Another situation leading to inhibition is where a uORF is out of frame with and overlapping the initiator AUG of the major open reading frame. One way of regulating both overlapping uORFs (3Harding H.P. Novoa I. Zhang Y.H. Zeng H.Q. Wek R. Schapira M. Ron D. Mol. Cell. 2000; 6: 1099-1108Abstract Full Text Full Text PDF PubMed Scopus (2415) Google Scholar) and sequence-specific uORFs is through modulated recognition of the initiation codon of the uORF, which leads to “leaky scanning.” Another mechanism of regulation is mediated through elimination of inhibitory uORFs in alternative forms of transcripts (4Waterhouse P. Khokha R. Denhardt D.T. J. Biol. Chem. 1990; 265: 5585-5589Abstract Full Text PDF PubMed Google Scholar, 5Brown C.Y. Mize G.J. Pineda M. George D.L. Morris D.R. Oncogene. 1999; 18: 5631-5637Crossref PubMed Scopus (93) Google Scholar). Specific regulation of sequence-dependent uORFs occurs when interaction of the nascent peptide with its target is modulated (2Morris D.R. Geballe A.P. Mol. Cell. Biol. 2000; 20: 8635-8642Crossref PubMed Scopus (568) Google Scholar). In addition to single uORFs, multiple uORFs are found in mammalian genes, particularly those encoding oncoproteins, growth factors, and growth factor receptors (2Morris D.R. Geballe A.P. Mol. Cell. Biol. 2000; 20: 8635-8642Crossref PubMed Scopus (568) Google Scholar, 6Kozak M. Nucleic Acids Res. 1987; 15: 8125-8148Crossref PubMed Scopus (4168) Google Scholar, 7Geballe A.P. Morris D.R. Trends Biochem. Sci. 1994; 19: 159-164Abstract Full Text PDF PubMed Scopus (195) Google Scholar). A fungal model for regulation through multiple uORFs is the yeast GCN4 gene, the translation of which is controlled through interactions of four uORFs (8Hinnebusch A.G. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Press, Cold Spring Harbor, NY1996: 199-244Google Scholar). There is no similarly detailed model for regulation through multiple uORFs in higher eukaryotes. One of the human oncogene mdm2 transcripts contains two uORFs, and these two elements cooperate to inhibit translation of the oncoprotein product (5Brown C.Y. Mize G.J. Pineda M. George D.L. Morris D.R. Oncogene. 1999; 18: 5631-5637Crossref PubMed Scopus (93) Google Scholar). Inhibition by the two uORFs seems to be of biological significance, because a class of tumors arises from overexpression of a shortened mdm2 mRNA with both uORFs eliminated (5Brown C.Y. Mize G.J. Pineda M. George D.L. Morris D.R. Oncogene. 1999; 18: 5631-5637Crossref PubMed Scopus (93) Google Scholar, 9Landers J.E. Cassel S.L. George D.L. Cancer Res. 1997; 57: 3562-3568PubMed Google Scholar, 10Landers J.E. Haines D.S. Strauss J.F. George D.L. Oncogene. 1994; 9: 2745-2750PubMed Google Scholar, 11Capoulade C. Bressacdepaillerets B. Lefrere I. Ronsin M. Feunteun J. Tursz T. Wiels J. Oncogene. 1998; 16: 1603-1610Crossref PubMed Scopus (116) Google Scholar). This “short form” of the mdm2 mRNA (S-mdm2) is generally present at low levels in normal cells, and its 5′ leader contains no obvious regulatory elements. mRNAs containing the S-mdm2 leader are very efficiently loaded with ribosomes (5Brown C.Y. Mize G.J. Pineda M. George D.L. Morris D.R. Oncogene. 1999; 18: 5631-5637Crossref PubMed Scopus (93) Google Scholar). The “long form” transcript (L-mdm2 mRNA) is constitutively expressed and has a relatively long 5′ leader of 297 nucleotides. mRNAs containing the L-mdm2 5′ leader are >10 times less efficiently translated than S-mdm2 mRNAs in the cell types tested. This inhibitory activity of the L-mdm2 leader is due to the presence of the two uORFs, which seem to cooperate in inhibiting translation (5Brown C.Y. Mize G.J. Pineda M. George D.L. Morris D.R. Oncogene. 1999; 18: 5631-5637Crossref PubMed Scopus (93) Google Scholar). The structures of the human and mouse L-mdm2 5′ leaders are diagramed in Fig. 1. Both uORFs in human mdm2 are 15 codons in length (including the termination codons) and share four codons in common (excluding the initiator methionine). Both uORFs are required for maximal inhibition of translation by the L-mdm2 leader, but uORF1 seems to contribute more to inhibition than uORF2 (5Brown C.Y. Mize G.J. Pineda M. George D.L. Morris D.R. Oncogene. 1999; 18: 5631-5637Crossref PubMed Scopus (93) Google Scholar). Two uORFs are also found in the mouse L-mdm2 leader (Fig. 1), suggesting that this conserved arrangement could have functional significance. The hORF2 and mORF2 sequences (hORF2 and mORF2 refer to the human and mouse versions, respectively) have diverged widely between the two species, whereas hORF1 and mORF1 are conserved in both overall length and coding at four of thirteen positions. Three of the four amino acids conserved in the peptide products of mORF1 and hORF1 are also conserved in hORF2 (alanine 5, proline 11, and arginine 12). Glycine is present in position 9 of uORF1 from both species, but not in the uORF2 of either species. The goal of the studies reported in this paper was to define those aspects of the structure of the L-mdm2 5′ leader that contribute to the distinct properties of mdm2 uORF1 and uORF2. Reporter Constructs—Chimeric expression constructs between the 5′ leader of the mdm2 gene and human growth hormone (hGH) as a reporter gene were detailed previously (5Brown C.Y. Mize G.J. Pineda M. George D.L. Morris D.R. Oncogene. 1999; 18: 5631-5637Crossref PubMed Scopus (93) Google Scholar). Building on our previously described construct, pGH-E1, by means of “back-to-back” PCR (12Hemsley A. Arnheim N. Toney M.D. Cortopassi G. Galas D.J. Nucleic Acids Res. 1989; 17: 6545-6551Crossref PubMed Scopus (416) Google Scholar), uORFs 1 and 2 were deleted from the 5′ leader and replaced with restriction sites MluI and StyI, The used sites in for the MluI and and, for the and The the of in of the uORFs. uORF1 was the MluI and uORF2 was the the 5′ mdm2 leader with the of the restriction through of the sites in initiation and expression constructs between S-adenosylmethionine and were described previously G.J. Morris D.R. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). the and sites the uORF, encoding the uORF were the 5′ leader by constructs were to to and sequence of the cells were in with and The for of and for expression were described previously G.J. Morris D.R. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), with a as expression cells were in in the of the was G.J. Morris D.R. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), and of the were and for the cells were for of of growth hormone and for mRNA were as of in the was with by were to within the of the for the was by PCR of a by R. used were and The PCR product was and for the was internal was from the and to with the with A was out as described C.Y. Morris D.R. of mRNA and Press, Scholar). A was used for and was used for the was out in and the mRNA levels were to the levels of of of the L-mdm2 5′ on Translational of the L-mdm2 5′ leader previously that both hORF1 and hORF2 were to of translation of associated reporter gene (5Brown C.Y. Mize G.J. Pineda M. George D.L. Morris D.R. Oncogene. 1999; 18: 5631-5637Crossref PubMed Scopus (93) Google Scholar). further restriction sites were the 5′ leader in the uORFs which to be the constructs in of the uORFs. was used as the reporter gene Morris D.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar) in these and the are as translational which is as of to mRNA G.J. Morris D.R. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). In the were with a control in which the initiator AUG codons of both human uORFs been to (Fig. in Fig. the L-mdm2 leader with both human uORFs intact (Fig. the translational efficiency of the to of the The activities of the constructs with hORF1 (Fig. or hORF2 (Fig. were and of the activities with the constructs by in constructs from which to and translational to the uORFs of and not of restriction sites the uORFs not regulatory The that which contains has the activity of the with hORF2 this in inhibitory activity was due to position within the 5′ leader, the of the two human uORFs were constructs and as in Fig. The the hORF1 is more inhibitory than hORF2 of position in the leader and, therefore, the surrounding sequence (Fig. with and with in the position in the 5′ leader, hORF1 is more inhibitory than of sequence from the human 5′ leader between the two human uORFs in the L-mdm2 leader no on its inhibitory activity not the that the surrounding sequence not the inhibitory activities of the two human uORFs. There is a of position within the to the 5′ seems to the inhibitory activity of both uORFs (Fig. with and with This may from the known in recognition of initiator AUG with to the M. Gene Google Scholar). in the the mouse L-mdm2 mRNA also contains two uORFs. The human and mouse uORF1 sequences have in whereas the two are between the two (Fig. the two mouse uORFs were the human L-mdm2 leader (Fig. of downstream translation was to that with the human uORFs. The of the mouse and human uORFs to constructs (Fig. constructs and that the mORF1 is less inhibitory than whereas human and mouse have of the of uORF1 to the functional properties of mdm2 hORF1 in it was the 5′ leader in of the The regulatory properties of the leader and its uORF have been G.J. Morris D.R. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, A. C. Morris D.R. J. Biol. Chem. Full Text Full Text PDF PubMed Google Scholar, A. Mize G.J. Morris D.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Morris D.R. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, Morris D.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The activities of the mdm2 uORFs were in with uORF sequence in which the initiator AUG codon been by to a (Fig. for a in which the regulatory activities of the uORF been through a acid codon to arginine (Fig. for the activity of a uORF with no suppressive properties in the 5′ leader G.J. Morris D.R. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). There was translation present in the construct, which from translation of the uORF, by reinitiation at the downstream reporter gene (2Morris D.R. Geballe A.P. Mol. Cell. Biol. 2000; 20: 8635-8642Crossref PubMed Scopus (568) Google Scholar). The human mdm2 when the (Fig. downstream translation by whereas the mouse uORF1 (Fig. was less with the from the constructs (Fig. Translational by human uORF2 (Fig. was over times less than by The activities of the uORFs in this context were very to those found in the context of the 5′ leader (Fig. the that the surrounding sequence not contribute to the suppressive activities of these uORFs. The inhibitory activity of human uORF2 is very to the uORF that was to its regulatory properties constructs hORF2 and in Fig. it seems that the human and mouse must have properties that the inhibition of downstream seems that uORF1 must be translated in order to these because of the human initiator AUG a that is in activity from the uORF2 and uORF constructs with initiation codons (Fig. the activities of constructs and a secondary structure for human uORF1 with a of it that this structure not contribute to the inhibitory activity of the with this of in the codons of the secondary structure and coding no on the inhibitory activity of this uORF not There are two relatively rare codons within those encoding proline and arginine which have in of and these two codons were to the suppressive activity of the uORF, were to and which the amino acids and have of and In not this no in expression with the hORF1 construct, which contains the human the suppressive activity of hORF1 be due to the presence of these two relatively rare One between human uORF1 and uORF2 is the context surrounding the initiation uORF1 in both human and mouse has a context M. Cell. Full Text PDF PubMed Scopus Google Scholar) with at position human uORF2 has at that the of hORF2 with (Fig. the sequence as in inhibition of downstream translation (Fig. Two and at position 2 of also the suppressive activity of the uORF by the (Fig. constructs and The that these codon have in common is the initiation context by a at position hORF2 with the initiation context is less suppressive than hORF1 (Fig. these that at factor the suppressive activity of uORF2 is initiation be that the hORF2 constructs with the initiation are more suppressive that the uORF, Fig. with Fig. suggesting that this uORF2 may have inhibitory properties of its of the context surrounding the initiation there is inherent in inhibitory activity between hORF1 and hORF2 (Fig. the for this in activity, the human uORFs were and were in which these were between the uORFs, and the were in the leader (Fig. constructs and are hORF1 and and these the activities Fig. with Fig. The of initiation context is important in these for the between and the constructs (hORF2 and respectively) can be for by in initiation context the activities in Fig. with those in Fig. constructs with the A must be in order to between the two uORFs. In this was as a of the in hORF1 and hORF2 of constructs with and with that from hORF1 has suppressive activity (Fig. The can be with to by with and with A of leads to the that this of hORF1 is more inhibitory than the from hORF2 constructs with with with and with the in activity between the is than that found between the and, in is it is found the four the stronger translational activity of hORF1 to hORF2 seems to be the result of The is the stronger initiation context present in The is the amino acid sequence of the peptide product of in which contributes to its there seems to be a of the amino acid sequence of C. mdm2 hORF1 seems to be a of the class of sequence-dependent uORFs, the activities of which are mediated by the encoded peptides (2Morris D.R. Geballe A.P. Mol. Cell. Biol. 2000; 20: 8635-8642Crossref PubMed Scopus (568) Google Scholar). of on uORF1 of of the two human mdm2 uORFs that the inhibitory activity of uORF1 in from the amino acid sequence of the peptide this hORF1 and the activities of the uORFs the reporter gene containing the 5′ leader, as The activity of and that the of the peptide product was to play a role in the inhibitory properties of this 9 is conserved between the human and mouse but occurs in this position in human uORF2 (Fig. the conserved to in of the inhibitory activity of human uORF1 (Fig. the of the 9 on inhibition by hORF1 was by of a at position (Fig. activities of constructs and were with cell and the are those in Fig. The and are conserved between human uORF1 and as is in mouse uORF1 (Fig. A of at and the inhibitory activity of human uORF1 (Fig. suggesting a role for this of the peptide in This was not in the of Fig. because of the in this between human uORFs 1 and The activities of constructs and (Fig. that the of the peptide could play a role in the of proline and arginine both to (Fig. no on inhibition by This result suggests that these two conserved (Fig. 1) do not play a role in the inhibitory activity of the the peptide by the no on the activity of the peptide by (Fig. constructs and mdm2 is because of the important role that the in and in cell growth J. Trends 17: Full Text Full Text PDF PubMed Scopus Google Scholar, D. M. PubMed Scopus Google Scholar, J. G. 2000; PubMed Scopus Google Scholar). The of of mdm2 expression are as a result of and in the of control of mdm2 expression T. M. Mol. 1999; PubMed Google Scholar), translation a target for regulation of this gene. class of tumors from of translation of mdm2 (5Brown C.Y. Mize G.J. Pineda M. George D.L. Morris D.R. Oncogene. 1999; 18: 5631-5637Crossref PubMed Scopus (93) Google Scholar, 9Landers J.E. Cassel S.L. George D.L. Cancer Res. 1997; 57: 3562-3568PubMed Google Scholar, 10Landers J.E. Haines D.S. Strauss J.F. George D.L. Oncogene. 1994; 9: 2745-2750PubMed Google Scholar, 11Capoulade C. Bressacdepaillerets B. Lefrere I. Ronsin M. Feunteun J. Tursz T. Wiels J. Oncogene. 1998; 16: 1603-1610Crossref PubMed Scopus (116) Google Scholar), which the of regulation at this Two uORFs occur in the long form of mdm2 mRNA in both human and the two for which have these uORFs have a major on translation of this form of the mRNA in HeLa cells (5Brown C.Y. Mize G.J. Pineda M. George D.L. Morris D.R. Oncogene. 1999; 18: 5631-5637Crossref PubMed Scopus (93) Google uORFs 1 and 2 contribute to the on features of the uORF, it to have the on translation in both species. Both human and mouse have for translation with at position in human and at the position in In as in this features of the coding sequence of human uORF2 its on downstream translation to the of uORF2 in downstream of uORF1 George D.L. J. PubMed Scopus Google Scholar), contribute to recognition by a reinitiation mechanism (2Morris D.R. Geballe A.P. Mol. Cell. Biol. 2000; 20: 8635-8642Crossref PubMed Scopus (568) Google Scholar), in to where the between the two uORFs is J.E. Cassel S.L. George D.L. Cancer Res. 1997; 57: 3562-3568PubMed Google Scholar). features of the of both mouse and human to on downstream In to the structure of uORF1 in both seems to be for recognition and inhibition of downstream The initiation context in both human and mouse is with in position and a at the coding sequence of uORF1 contributes to its inhibitory This of the coding sequence is not due to secondary structure or the occurrence of rare the of uORF1 on translation of downstream seems to be mediated by the structure of the encoded The of the encoded peptide and, in the at position seems important in the inhibitory activity of the uORF1 mdm2 uORF1 seems to the of sequence-dependent uORFs (2Morris D.R. Geballe A.P. Mol. Cell. Biol. 2000; 20: 8635-8642Crossref PubMed Scopus (568) Google Scholar) in that within the coding the inhibitory activity, in the codons have no the of mdm2 uORF1 seems more than that of sequence-dependent uORFs that have been two of the uORFs, those from the mammalian gene and the gene, activities can be by G.J. Morris D.R. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, Geballe A.P. J. PubMed Google Scholar), which are thought to interactions between the uORF-encoded peptides and In the in hORF1 that has been to its inhibitory activity is the of the block of four amino acids in the in either the structure of the hORF1 peptide or its with its target is also by the functional interaction between and are conserved as and in human and 9 to as found at this position in human the inhibitory activity of but the of this was by at position The known sequence-dependent uORFs seem to by inhibiting movement of ribosomes the on the 5′ leader. This occurs as a result of the interaction between the nascent peptides and targets (2Morris D.R. Geballe A.P. Mol. Cell. Biol. 2000; 20: 8635-8642Crossref PubMed Scopus (568) Google Scholar). the and the ribosomes seem to at the termination translation of the uORF A. Mize G.J. Morris D.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Geballe A.P. Mol. Cell. Biol. 16: PubMed Scopus Google Scholar). with these two uORFs, the between the in the of the peptide and the is in the peptide with its target to the addition or of a single codon activity in 2Morris D.R. Geballe A.P. Mol. Cell. Biol. 2000; 20: 8635-8642Crossref PubMed Scopus (568) Google Scholar). In sequence-dependent uORF, that from the gene, ribosomes at the elongation P. Sachs M.S. Mol. Cell. Biol. 1998; 18: PubMed Scopus Google Scholar, A. Sachs M.S. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) and seems to be less in the position of the inhibitory peptide sequence to the termination In this human mdm2 uORF1 seems to be to in that or a codon from the of the uORF no on its inhibitory is that the of the 5′ leaders of the human and mouse mdm2 mRNAs both two uORFs with In both uORF1 is inhibitory to downstream translation and, in sequence between the two species, whereas uORF2 no inhibitory properties and is There are in both yeast (8Hinnebusch A.G. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Press, Cold Spring Harbor, NY1996: 199-244Google Scholar) and (3Harding H.P. Novoa I. Zhang Y.H. Zeng H.Q. Wek R. Schapira M. Ron D. Mol. 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Gene 6: PubMed Scopus Google Scholar), to block ribosome upstream of the The presence of at the of the L-mdm2 leader could a regulated mechanism for bypassing the inhibitory of the two uORFs. A regulated also for the of translation initiation at internal AUG of the L-mdm2 transcript, which a with biological properties PubMed Scopus Google Scholar).