Translational Regulation

When ODC is induced, the total increase in ODC activity is usually much larger than that found in ODC mRNA level, suggesting also the involvement of posttranscrip-tional mechanisms. The increase in ODC activity is often explained by a stabilization of the enzyme against degradation. However, in some experimental systems, the rise in ODC activity cannot be explained only by an increase in ODC mRNA and a stabilization of the enzyme protein, also indicating an increase in the efficiency by which the ODC mRNA is translated (14,49,50).

The polyamines exert a strong feedback control of ODC (3,9,51). An excess of polyamines rapidly downregulates ODC activity, whereas a cellular polyamine deficiency induces a compensatory upregulation of ODC activity. Part of this feedback regulation of ODC is caused by changes in the turnover of the enzyme (10). The

Fig. 1. Myc binding elements (E-boxes) and single A/G nucleotide polymorphism in intron 1 of the human ODC gene.

Fig. 1. Myc binding elements (E-boxes) and single A/G nucleotide polymorphism in intron 1 of the human ODC gene.

mechanisms involved in the polyamine-mediated control of ODC degradation have been shown to be unique to this enzyme (see subheading 4.). Besides changes in ODC turnover rate, the feedback control of ODC appears to involve changes in the ODC synthesis rate (3,9,51). However, the polyamine-mediated changes in enzyme synthesis are not explained by any changes in the amount of ODC mRNA, suggesting that the effects of the polyamines are on the translational level rather than on the transcription or stability of the ODC mRNA (3,51).

The expression of ODC is also strongly affected by the osmolarity of the growth medium (52-54). Exposure of cells to a hypertonic medium rapidly results in a marked reduction of ODC activity, whereas exposure to a hypotonic growth medium results in a dramatic increase in ODC activity within a very short time after the onset of the osmotic shock. The mechanisms involved are still not fully understood. In some systems, the changes in ODC activity are correlated with changes in the ODC mRNA content, whereas in other systems the phenomenon appears to involve mainly, if not exclusively, translational and posttranslational mechanisms (52,54).

Mammalian ODC mRNA, as with many of the other mRNAs encoding growth-related proteins, belongs to the rare group of mRNAs with a very long 5' untranslated region (UTR) (55,56). The 5' UTR of mammalian ODC mRNA is about 300 nucleotides long and has a very high GC content. The high ratio of GC to AU increases the possibility of strong secondary structures being formed in this part of the mRNA. These structures may negatively affect the translation of the message. Furthermore, the 5' UTR of ODC mRNA also contains an upstream open reading frame, which may suppress the translation. Using various expression systems, it has been shown that the 5' UTR of ODC mRNA strongly inhibits the translation of subsequent reporter genes (57-59). Most of this suppressive effect on translation is mapped to the first part of the 5' UTR. This part of the mRNA is particularly G/C-rich and is expected to form a stable stem loop, which could negatively affect translation. Interestingly, it has been demonstrated that tissue extracts contain a 58-kDa protein that appear to bind specifically to this part of the ODC mRNA (60). However, the biological function of this protein is still unknown. Most of the ODC mRNA is found associated with fractions containing ribosomal subunits and monosomes in polysome profiles, which indicates the mRNA is indeed poorly translated in vivo (61,62).

The 3' UTR of ODC mRNA is also relatively long (>300 nucleotides). However, the frequency of G and C in the 3' UTR is much less compared with that of the 5' UTR, which suggest that this part of the ODC mRNA contains less stable secondary structures than the 5' UTR (63). Nevertheless, it has been demonstrated that the 3' UTR

Fig. 2. The cap-dependent initiation complex. The initiation factor eIF4F consists of eIF3, eIF4A, and eIF4G.

may interact with the 5' UTR of ODC mRNA in such a way that the suppressive effect of the 5' UTR on translation is reduced (57,59). The underlying mechanism is not yet known.

One of the limiting factors for the initiation of translation has been suggested to be the initiation factor, eIF4E (64). In particular mRNAs with long and structured 5' UTRs is affected by low levels of eIF4E (65). eIF4E is important in the early process of initiation by bringing the 5' methylated guanosine cap structure of the mRNA and the 40S ribosomal subunit closer together (Fig. 2). The eIF4E has two binding sites: one for the cap structure of the mRNA and one for another initiation factor, eIF4G, which in turn is joined to the 40S ribosomal subunit by the initiation factor eIF3. Thus the initiation factors eIF4E, eIF4G, and eIF3 form a bridge between the cap structure and the 40S ribosomal subunit. Besides binding to eIF4G, eIF3 may bind directly to the mRNA. The eukaryotic initiation factor 4G is a large protein and has binding sites for eIF4A, eIF4E, and eIF3. The initiation factor eIF4A contains a RNA helicase activity, which is important for the melting of secondary structures of the 5' UTR. Thus the initiation factors eIF4E, eIF4G, and eIF4A form a large complex, which sometimes is referred to as eIF4F. When all the components needed for initiation are brought together the ribosomal subunit "scans" the mRNA for the correct initiation codon, starting from the cap end of the mRNA (66). This scanning procedure may be suppressed by strong secondary structures in the 5' UTR (67). However, the presence of helicase activity in eIF4A facilitates a continued scanning (68).

Most mRNAs coding for proto-oncogenes or other growth-related proteins have long G/C-rich 5' UTRs. These mRNAs are especially dependent on the initiation factor eIF4E for their translation. Interestingly, a number of growth factors are known to induce the transcription of eIF4E mRNA and the promoter of the eIF4E gene contains c-Myc-responsive motifs, indicating that c-Myc may also transactivate this gene (69). Thus eIF4E is an important factor in the control of cell growth and proliferation, which is supported by the fact that increased expression of eIF4E stimulates DNA synthesis and cell-cycle progression, but inhibits apoptosis (70,71). Furthermore, forced overexpression of eIF4E has been demonstrated to induce transformation of various rodent cell lines and thus eIF4E may play a role in tumorigenesis (72,73).

As with other mRNAs coding for growth factors, the ODC mRNA has a long and highly structured 5' UTR. Overexpression of ODC has been demonstrated to induce transformation, and may thus be considered as a proto-oncogene (22). The expression of ODC has been shown to be strongly affected by eIF4E. Cells having increased levels of eIF4E exhibit increased ODC activity, which appears to be caused by reduced translational suppression exerted by the secondary structures of the ODC mRNA 5' UTR (74,75). Antisense eIF4E RNA, on the other hand, has been shown to increase the suppression of ODC mRNA translation, resulting in a decrease in cellular ODC activity (76). Interestingly, the eIF4E-induced transformation appears to be related to ODC. The transformation has been demonstrated to be reversed by treatment with the ODC inhibitor DFMO or by expression of an ODC dominant-negative mutant, suggesting a role for ODC in the transformation mechanism (77,78).

Most mRNAs in the cell are being translated in a cap-dependent manner. However, a minor fraction of the mRNAs appears to be translated without preceding cap binding and scanning (79,80). Thus the mechanism seems to involve an "internal initiation," in which the ribosomal complex binds directly to an internal site close to the initiation codon on the 5' UTR. This site is called an "internal ribosomal entry site" (IRES), and was first demonstrated to exist in picornavirus mRNA (81,82). These mRNAs are uncapped mRNAs with a high degree of secondary structures (as well as upstream AUGs) in their 5' UTR. Cellular infection with picornavirus results in an inhibition of the host cap-dependent translation resulting from cleavage of eIF4G by a viral protease (82). However, because the IRES-dependent initiation of the picornavirus mRNA is not affected cellular protein synthesis is mainly directed toward picorna protein production. Internal initiation is not dependent on eIF4E or the amino terminal part of eIF4G (which is cleaved off by the picorna protease), but is otherwise dependent on virtually the same initiation factors as cap-dependent initiation.

An increasing number of mammalian IRES-containing mRNAs have been identified during the last decade. A large fraction of these mRNAs code for proteins involved in the regulation of cell proliferation or embryonic development (e.g., various growth factors and proto-oncogenes) (83). Characteristically, their 5' UTRs are very long and highly structured, often with one or several AUGs upstream to the main start codon. ODC mRNA also has been suggested to contain an IRES, which functions exclusively during the G2/M phase of the cell cycle (84). During the cell cycle, there are two peaks of ODC activity; one during the G1/S boundary and one during the G2/M transition (50,85). The first peak in ODC activity is corresponding to a general rise in protein synthesis and is probably the result of normal cap-dependent translation. However, the timing of the second peak coincides with a period of the cell cycle when the cap-dependent translation is markedly inhibited. Results from a study by Pyronnet et al. (84) indicated that the rise in ODC synthesis during the G2/M phase was a result of internal initiation at an IRES. This IRES was located close to the initiation AUG and demonstrated to contain a pyrimidine-rich sequence similar to that of the picornavirus 5' UTR. During the G2/M phase of the cell cycle, the polyamines are believed to be essential for the formation of the mitotic spindle and the chromatin condensation. In addition, Pyronnet et al. (84) demonstrated that c-Myc mRNA, which also has been suggested to contain an IRES, was translated during the G2/M phase of the cell cycle. Thus, IRES-dependent initiation may be an important mechanism in the synthesis of some proteins during specific phases of the cell cycle (e.g., mitosis) when cap-dependent protein synthesis is inhibited. However, in spite of the vast experimental data supporting the existence of IRES in mammalian mRNAs, this notion is not without controversy (86-90).

ODC expression is highly regulated by the polyamines (3,9). A major part of this control is carried out at a posttranslational level. However, part of the feedback regulation of ODC exerted by the polyamines appears to be at the translational level (61,91). ODC synthesis is downregulated in the presence of an excess of polyamines and upregulated in situations of polyamine deficiency. The cellular ODC mRNA level, on the other hand, is not affected by changes in cellular polyamine content, indicating a translational mechanism.

As described earlier, ODC mRNA has a long G/C rich 5' UTR, which may be involved in the translational control of the enzyme. However, results from experiments in which the 5' UTR was added to the mRNAs of various reporter genes, as with P-galactosidase, CAT, and luciferase, indicated that the 5' UTR was not important for the polyamine-mediated regulation of ODC synthesis (57,62). Moreover, no difference in feedback regulation of ODC synthesis existed between Chinese hamster ovary cells expressing the full-length ODC mRNA and those expressing an ODC mRNA lacking the major part of the 5' UTR, demonstrating that the polyamine-mediated translational control of ODC is not dependent on the 5' UTR of the mRNA (92).

Exposure of cells to hypotonic stress has been shown to strongly induce ODC activity (52-54). The increase, which is very fast, occurs independent of a suppression of general protein synthesis. Results from various studies indicate that the increase in ODC activity is caused by both a decrease in degradation and an increase in synthesis. The level of ODC mRNA does not usually change, indicating that the change in ODC synthesis is mainly an increase in translation, rather than a change in transcription. Using a series of stable transfectants of Chinese hamster ovary cells expressing ODC mRNAs with various truncations in the 5' and 3' UTRs it was demonstrated that the hypotonic induction of ODC mRNA translation was highly dependent on the presence of the 3' UTR, but not of the 5' UTR (93). Cells expressing ODC mRNAs with various deletions in the 5' UTR still induced ODC, whereas cells expressing ODC mRNAs without the 3' UTR did not, or only slightly, induce ODC after exposure to hypotonic stress. Thus the 3' UTR of ODC mRNA appears to affect the translation of the message in some way. Interestingly, the 3' UTR of ODC mRNA has been shown to partially neutralize the inhibition exerted by the 5' UTR, indicating that this part of the ODC mRNA may play a role in the translational control of the enzyme by interacting with the 5' UTR (57,59). However, it appears that such an interaction is not a prerequisite for the hypotonic induction of ODC expression since the induction occurred also in cells expressing ODC mRNA without the 5' UTR (93).

That the 3' UTR of an mRNA also may be participating in the regulation of translation has attracted increased attention during recent years. A number of protein factors and sequence elements associated with the effects on translation mediated by the 3' UTR have been described (94-96). However, the exact mechanism by which the 3' UTR of ODC mRNA affects the translation of the message during hypotonic stress remains to be established.

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