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Polyamines regulate both transcription and translation of the gene encoding ornithine decarboxylase antizyme in mouse
J Nilsson1, S Koskiniemi, K Persson
1Department of Cellular and Developmental Biology, Umeå University, Sweden.
Abstract:
The degradation of ornithine decarboxylase (ODC) is mediated by antizyme, a protein regulated by the end-products of ODC activity, the polyamines. High levels of polyamines induce a +1 ribosomal frameshift in the translation of the rat antizyme message leading to the expression of a full-length protein. We have studied whether the regulation of antizyme expression occurs only at the level of translation or whether polyamine levels also affect the transcription of the antizyme gene. Thus, we have cloned and sequenced the mouse homologues of the rat ODC-antizyme gene and cDNA. Northern blot analysis shows that although high concentrations of polyamines do not affect the steady-state levels of antizyme message in L1210 leukemia cells, polyamine depletion using 2-(difluoromethyl)ornithine [Orn(F2Me)] leads to a marked decrease in mRNA levels. Results of transient transfections of luciferase-reporter-gene constructs driven by antizyme promoter fragments in untreated and Orn(F2Me)-treated Balb/C 3T3 cells indicate that the transcription of the antizyme gene is altered upon polyamine depletion. The amount of antizyme protein on Western blots was also altered by polyamine depletion and addition, and the polysomal distribution of antizyme message suggests a general translational increase of the message when polyamine concentrations are high. These results indicate a role for polyamines in the transcriptional and translational regulation of ornithine decarboxylase antizyme.
Insights
Polyamines regulate ornithine decarboxylase (ODC) antizyme expression at both transcriptional and translational levels. Polyamine depletion decreases antizyme gene transcription and mRNA levels, impacting ODC activity.
Area of Science:
- Molecular Biology
- Biochemistry
- Gene Regulation
Background:
- Ornithine decarboxylase (ODC) activity is regulated by polyamines, its end-products.
- Antizyme protein mediates ODC degradation and its expression is linked to polyamine levels.
- Previous studies indicated translational regulation via ribosomal frameshifting.
Purpose of the Study:
- To investigate if polyamine levels affect antizyme gene transcription in addition to translation.
- To clone and characterize mouse homologues of the ODC-antizyme gene and cDNA.
- To elucidate the role of polyamines in both transcriptional and translational control of antizyme.
Main Methods:
- Cloning and sequencing of mouse ODC-antizyme gene and cDNA.
- Northern blot analysis to assess mRNA levels under varying polyamine conditions.
- Transient transfection assays with luciferase-reporter constructs to study promoter activity.
- Western blot analysis to quantify protein levels.
- Polysomal distribution analysis of antizyme mRNA.
Main Results:
- High polyamine concentrations did not alter steady-state antizyme mRNA levels in L1210 cells.
- Polyamine depletion using 2-(difluoromethyl)ornithine [Orn(F2Me)] significantly decreased antizyme mRNA levels.
- Antizyme gene transcription was altered by polyamine depletion, as shown by reporter gene assays.
- Polyamine depletion and addition affected antizyme protein levels.
- Polysomal distribution suggested increased translational efficiency of antizyme mRNA at high polyamine concentrations.
Conclusions:
- Polyamines play a role in the transcriptional regulation of the antizyme gene.
- Polyamines also influence the translational regulation of antizyme.
- These findings reveal a dual role for polyamines in controlling ODC antizyme expression at both transcription and translation.