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Ornithine decarboxylase (ODC) is the ratelimiting enzyme in the biosynthesis of polyamines, which are required for optimal cell growth and proliferation. ODC is overexpressed in many tumors and, conversely, its overexpression induces transformation. We have previously reported that ODC mRNA alternative splicing relieves the translation repression normally imposed by a long and structured 5' untranslated region (UTR), and that the ODC 5' UTR contains an internal ribosome entry site (IRES). Here we show that ODC IRES activity is enhanced following inclusion of alternative sequences generated by splicing at cryptic acceptor sites. Furthermore, the alternative ODC IRES is more sensitive to cell-cycledependent changes in the rate of translation. These findings uncover a new biological property of differentially spliced transcripts. This is the first example of alternative splicing that modulates mRNA translation through the cell cycle in a cap-independent manner.

The eukaryotic mRNA 5' cap structure facilitates translation. However, cap-dependent translation is impaired at mitosis, suggesting a cap-independent mechanism for mRNAs translated during mitosis. Translation of ornithine decarboxylase (ODC), the rate-limiting enzyme in the biosynthesis of polyamines, peaks twice during the cell cycle, at the G1/S transition and at G2/M. Here, we describe a cap-independent internal ribosome entry site (IRES) in the ODC mRNA that functions exclusively at G2/M. This ensures elevated levels of polyamines, which are implicated in mitotic spindle formation and chromatin condensation. c-myc mRNA also contains an IRES that functions during mitosis. Thus, IRES-dependent translation is likely to be a general mechanism to synthesize short-lived proteins even at mitosis, when cap-dependent translation is interdicted.

Gastrin via its G-protein coupled specific receptor induces transcription of c-fos and c-jun genes through a ras-MAPK pathway. Ornithine Decarboxylase (ODC), a growth regulated proto-oncogene, was chosen to investigate gastrin effects on translation initiation of mRNAs exhibiting a 5'UnTranslated Region (5'UTR) responsible for translation repression in quiescent cells. In AR4-2J tumoral cells, we first demonstrated that gastrin increases ODC mRNA translation. Transient transfections with various CAT chimeric constructs suggested a direct involvement of the 5'UTR in this observation. Translation of this group of mRNAs is enhanced by the availability of the cap-binding protein (eIF4E) that is increased after phosphorylation of its specific binding protein eIF4E-BP1. We found that AR4-2J cells over-expressed eIF4E protein which was not modulated by gastrin treatment. Rapamycin which inhibits 4E-BP1 phosphorylation, completely prevents gastrin-mediated increase of ODC translation indicating that 4E-BP1 could be involved in regulating ODC translation. Implication of 4E-BP1 in mediating gastrin effects is corroborated by the capacity of the ligand to affect 4E-BP1 phosphorylation. These results indicate that gastrin enhances ornithine decarboxylase mRNA translation through a rapamycin sensitive pathway and provide the first evidence in the control of 4E-BP1 phosphorylation after occupancy of a G protein-coupled receptor.

The ornithine decarboxylase enzyme (ODC) is the key regulator of polyamine synthesis and is a member of the cellular proto-oncogene family. Its expression becomes constitutively activated by carcinogens, viruses, and oncogenes. ODC mRNA has a long 5' untranslated region that could be important in the regulation of enzyme levels by affecting translation. To test this hypothesis, we have determined the role of this region on the constitutive ODC hyperexpression measured in AR4-2J cells, an azaserine-induced, tumor-derived pancreatic acinar cell line. Construction of expression vectors in which ODC 5' leader sequence was placed flanking the chloramphenicol acetyltransferase reporter gene allowed us to identify three AR4-2J specific, different alternatively spliced ODC 5' leaders. The 5' ends of exons 2 and 3 were lengthened by 17 and 13 bases, respectively. Translation performed in a cell-free system as well as in COS7 transient transfection experiments demonstrated that AR4-2J isoforms induce a strong increase in the rate of translation. These results provide evidence that alternative splicing observed in tumoral cells, coupled with translation regulation, relieves the translation repression mediated by the long and structured 5' untranslated region of the ODC proto-oncogene.