A mammalian sequence-dependent upstream open reading frame mediates polyamine-regulated translation in yeast

In mammals, control of S-adenosylmethionine decarboxylase (AdoMetDC) translation is one component of a feedback network that regulates intracellular levels of the polyamines, spermidine, and spermine. AdoMetDC mRNA from mammals contains a highly conserved upstream open reading frame (uORF) within its leader sequence that confers polyamine-regulated suppression of translation on the associated downstream cistron. This regulation is mediated through an interaction that depends on the amino acid sequence of the uORF-encoded hexapeptide. It remains to be shown whether polyamines participate directly in this interaction or indirectly through a specialized signal transduction pathway. We show that Saccharomyces cerevisiae does not have a uORF associated with its AdoMetDC gene (SPE2) and that ribosome loading on the SPE2 mRNA is not positively influenced by polyamine depletion, as it is in mammalian cells. Nevertheless, the mammalian AdoMetDC uORF, when introduced into a polyamine auxotroph of yeast, conferred polyamine regulation of both translational efficiency and ribosome loading on the associated mRNA. This regulatory activity depended on the amino acid sequence encoded by the fourth and fifth codons of the uORF, as in mammalian cells. The fact that the regulatory properties of this mammalian translational control element are quite similar in both mammalian and yeast cells suggests that a specialized signal transduction pathway is not required. Rather, it seems likely that polyamines may be directly participating in an interaction between the uORF-encoded peptide and a constitutive component of the translation machinery, which leads to inhibition of ribosome activity.     

On the translational control of ornithine decarboxylase expression by polyamines

Ornithine decarboxylase (ODC, EC 4.1.1.17) expression is subject to negative feedback regulation by the polyamines. The results of previous studies favor either translational or post-translational regulation. To facilitate further analysis of the mechanism by which polyamines affect ODC expression we have used a cell line (L1210-DFMOr) that overproduces ODC. This cell line was isolated by selection for resistance to the antiproliferative effect of the ODC inhibitor alpha-difluoromethylornithine (DFMO). These cells respond similarly to polyamine depletion and repletion as do their wild-type counterparts. When L1210-DFMOr cells were grown in the presence of 20 mM DFMO (i.e., when their polyamine content was reduced to an extent that still permitted a normal growth rate) ODC represented 4-5% of the soluble protein synthesized. After transfer of the cells to a medium lacking DFMO (i.e., when their polyamine pools were repleted), the rate of incorporation of [35S]methionine into ODC was one order of magnitude lower. Since this difference in incorporation of radioactivity into ODC remained the same irrespective of the pulse-label time used (between 2 and 20 min) it is likely to represent a true difference in ODC synthesis rate. Consequently, the pulse-label experiments cannot be explained by rapid degradation of the enzyme during the labeling period. The difference in ODC synthesis rate was not accompanied by a corresponding difference in the steady-state level of ODC mRNA. Analyses of the distribution of ODC mRNA in polysome profiles did not demonstrate any major difference between cells grown in the absence or presence of DFMO, even though the ODC synthesis rate differed by as much as 10-fold. However, the distribution of the ODC mRNA in the polysome profiles indicated that the message was poorly translated. Thus, most of the ODC mRNA was present in fractions containing ribosomal subunits or monosomes. Inhibition of elongation by cycloheximide treatment resulted in a shift of the ODC mRNA from the region of the gradient containing ribosomal subunits to that containing mono- and polysomes, indicating that most of the ODC mRNA was accessible to translation. Taken together these data lend support to a translational control mechanism which involves both initiation and elongation.     

Regulation of ornithine decarboxylase mRNA translation by polyamines. Studies using a cell-free system and a cell line with an amplified ornithine decarboxylase gene

The translational control of ornithine decarboxylase (ODCase) by polyamines has been studied using a cellular as well as a cell-free system. A mutant L1210 cell line, in which ODCase represents 4-5% of all soluble protein synthesized, was isolated by stepwise selection for resistance to the ODCase inhibitor 2-difluoromethylornithine (DFMO). The exceptionally high expression of ODCase in these cells was due to amplification of the ODCase gene. When the cells were grown in the absence of DFMO, dramatic increases in cellular putrescine and spermidine levels occurred. These increases were accompanied by a rapid decrease in ODCase synthesis. The change in ODCase synthesis was not associated with an alteration in the amount of ODCase mRNA, demonstrating a translational control in these cells. The effects of polyamines on ODCase mRNA translation were also studied in rabbit reticulocyte lysates using mRNA isolated from the DFMO-resistant cells. Low concentrations of spermidine stimulated synthesis of ODCase and that of total protein, when added to gel-filtered lysates. Notably, optimal stimulation of ODCase synthesis was achieved at a spermidine concentration lower than that required for an optimal rate of total protein synthesis. Higher concentrations of spermidine were inhibitory, and their effects of ODCase synthesis were stronger than on protein synthesis in general, resulting in a decrease in the fraction of protein synthesis accounted for by ODCase. The present results demonstrate that at least part of the feedback regulation of ODCase exerted by the polyamines is due to direct inhibition of ODCase mRNA translation.     

Increased efficiency of translation of ornithine decarboxylase mRNA in mitogen-activated lymphocytes

Ornithine decarboxylase (ODC) mRNA was elevated ninefold by 6 h following concanavalin A (ConA) stimulation of bovine lymphocytes. Comparison of the increases in ODC mRNA and ODC activity revealed a fivefold discrepancy, which is consistent with a change in efficiency of translation of ODC mRNA. In resting cells, 45% of the total ODC mRNA was associated with particles sedimenting at about 40 S, and therefore was not translated. The untranslated ODC mRNA in resting cells could be completely shifted into polysomes by a 15-min treatment of the cells with appropriate concentrations of cycloheximide. In activated cells, the proportion of ODC mRNA in untranslated material was reduced to 18%. This shift in distribution of ODC mRNA occurred between 6 h and 12 h following mitogen stimulation with no increase in the cellular level of this message. The rate of synthesis of ODC protein was found in increase twofold between 6 h and 12 h, paralleling the increase in the amount of ODC mRNA associated with polysomes. Thus, in this time frame, a decrease in the amount of untranslated ODC mRNA with a corresponding increase in the amount associated with polysomes leads to an increase in the biosynthesis of ODC with no change in the cellular level of the message. These changes in translational efficiency were not observed with actin mRNA.     

Translational control mechanism of ornithine decarboxylase by asparagine and putrescine in primary cultured hepatocytes

Asparagine stimulated the translation of ornithine decarboxylase (ODC) mRNA more than 10-fold in cultured hepatocytes which had been pretreated with glucagon in simple salt/glucose medium. Putrescine suppressed the increase in the rate of ODC synthesis caused by asparagine without significant change in the amount of ODC mRNA, suggesting that putrescine inhibited the effect of asparagine at least in part at the level of translation. Polysomal distribution of ODC mRNA was analyzed to examine the site of translational regulation by these effectors. In uninduced hepatocytes, most of the ODC mRNA was sedimented slightly after the 40 S ribosomal subunit. This ODC mRNA was sequestered from translational machinery since it was not shifted to the polysome fraction when peptide elongation was specifically inhibited by a low concentration of cycloheximide. In asparagine-treated cells, 40% of total ODC mRNA was in the polysomal fraction and formed heavier polysomes, indicating that asparagine stimulated both recruitment of ODC mRNA from the untranslatable pool and the initiation steps of translation. Putrescine did not change the distribution pattern of ODC mRNA on polysomes significantly. Thus, 30% of ODC mRNA remained on polysomes even when ODC synthesis was completely inhibited by putrescine. Paradoxically more than 70% of ODC mRNA was shifted into polysomes by putrescine in the presence of low concentrations of cycloheximide. These results, together with changes in the polysome profile, suggested that putrescine nonspecifically stimulated the recruitment of ODC mRNA from the untranslatable pool, whereas it specifically inhibited its translation at both the initiation and the elongation steps.     

Shut-off of actin biosynthesis in adenovirus serotype-2-infected cells

Adenovirus produces a dramatic shut-off of host protein synthesis after infection of HeLa cells. The level of actin messenger RNAs remained relatively unchanged after viral infection, when assayed by in vitro translation and two-dimensional gel electrophoresis analysis of the proteins or hybridization of the total cytoplasmic RNAs to the human actin gene. The distribution of actin mRNA in the polyribosomes is altered after adenovirus infection, with small polyribosomes and monoribosomes of the infected cells occupied by actin messages untranslatable in a rabbit reticulocyte lysate. The large polyribosomes still retain enough functional mRNAs to provide significant levels of actin protein in a rabbit reticulocyte in vitro translation system. In contrast, in homologous infected cell lysates, the translation of exogenous actin mRNA is greatly reduced when compared to uninfected HeLa cell lysates. In nuclease-treated uninfected or infected HeLa cell-free extracts, translation of viral mRNA is equally efficient and higher than that of actin mRNA. Thus, translational regulatory mechanisms which include inactivation of a part of the actin mRNA population accompanied by displacement to small polysomes and/or virus-induced modification of the cellular translational machinery to discriminate against cellular actin mRNA seem to account for the sharp reduction in actin protein synthesis of adenovirus-infected cells.     

Effect of inhibitors of S-adenosylmethionine decarboxylase on the contents of ornithine decarboxylase and S-adenosylmethionine decarboxylase in L1210 cells.

Treatment of L1210 cells with either of two inhibitors of S-adenosylmethionine decarboxylase (AdoMetDC), namely 5'-deoxy-5'-[N-methyl-N-[2-(amino-oxy)ethyl])aminoadenosine or 5'-deoxy-5'-[N-methyl-N-(3-hydrazinopropyl)]aminoadenosine, produced a large increase in the amount of ornithine decarboxylase (ODC) protein. The increased enzyme content was due to a decreased rate of degradation of the protein and to an increased rate of synthesis, but there was no change in its mRNA content. The inhibitors led to a substantial decline in the amounts of intracellular spermidine and spermine, but to a big increase in the amount of putrescine. These results indicate that the content of ODC is negatively regulated by spermidine and spermine at the levels of protein translation and turnover, but that putrescine is much less effective in bringing about this repression. Addition of either spermidine or spermine to the cells treated with the AdoMetDC inhibitors led to a decrease in ODC activity, indicating that either polyamine can bring about this effect, but spermidine produced effects at concentrations similar to those found in the control cells and appears to be the physiologically important regulator. The content of AdoMetDC protein (measured by radioimmunoassay) was also increased by these inhibitors, and a small increase in its mRNA content was observed, but this was insufficient to account for the increase in protein. A substantial stabilization of AdoMetDC occurred in these cells, contributing to the increased enzyme content, but an increase in the rate of translation cannot be ruled out.     

Uptake and utilization of mRNA by myogenic cells in culture

Primary chick myoblast cultures demonstrate the ability to take up exogenously supplied polyadenylated RNA and express the encoded information in a specific manner. This expression is shown to exhibit tissue specificity. Analysis of creatine kinase activity monitored at various times of incubation in the presence of either polyadenylated or nonpolyadenylated RNA indicates that only the poly(A)+ mRNA is capable of being actively translated. Radioactively labled poly(A)+ mRNA is taken up by the cell cultures in a time-dependent manner and subsequently shown to be associated with polysomes. This association with polysomes does not occur in the presence of puromycin and is unaffected by actinomycin D. Thus, nonspecific interaction with polysomes and induction of new RNA synthesis are ruled out and the association of the exogenously supplied poly(A)+ mRNA with polysomes is indicative of its translation in the recipient cells. When heterologous mRNA (globin) is supplied to the myoblasts, it is also taken up and properly translated. In addition, exogenously supplied myosin heavy chain mRNA is found associated with polysomes consisting of 4-10 ribosomes in myoblast cell cultures while in myotubes it is associated with very large polysomes, thus reflecting the different translational efficiencies that this message exhibits at two very different stages of myogenesis. The results indicate that muscle cell cultures can serve as an in vitro system to study translational controls and their roles in development.     

Feedback control of ornithine decarboxylase expression by polyamines. Analysis of ornithine decarboxylase mRNA distribution in polysome profiles and of translation of this mRNA in vitro.

Cell growth and differentiation require the presence of optimal concentrations of polyamines. Ornithine decarboxylase (ODC) catalyses the first and rate-controlling step in polyamine synthesis. In studies using cultures of Ehrlich ascites-tumour cells, we have shown that the expression of ODC is subject to feedback regulation by the polyamines. A decrease in the cellular polyamine concentration results in a compensatory increase in the synthesis of ODC, whereas an increase in polyamine concentration results in suppression of ODC synthesis. These changes in ODC synthesis were attributed to changes in the efficiency of ODC mRNA translation, because the steady-state amount of ODC mRNA remained constant. We now show that the number of ribosomes associated with ODC mRNA is low, and that the increase in ODC mRNA translation takes place without a shift in the distribution of ODC mRNA towards larger polysomes. This finding indicates that the polyamines regulate the efficiency of ODC mRNA translation by co-ordinately affecting the rates of initiation and elongation. By analysing ODC mRNA translation in vitro, using a rabbit reticulocyte lysate, polyadenylated RNA from a cell line with an amplified ODC gene, and a monospecific anti-ODC antibody, we also show that spermidine, but not putrescine, exerts a direct regulatory effect on ODC synthesis.     

Effects of polyamine depletion on serum stimulation of quiescent 3T3 murine fibroblast cells

Numerous reports have shown that polyamines are required for cell proliferation. A current model for regulating commitment to DNA replication in cultured fibroblasts stimulated from quiescence by serum addition postulates sequential action by specific growth factors. To temporally localize polyamine-dependent steps within this defined sequence, mouse Balb/c-3T3 fibroblasts were partially depleted of polyamines by treatment with DL-alpha-difluoromethylornithine (DFMO), next rendered quiescent by serum deprivation, then stimulated by 10% serum with or without exogenous putrescine (Pu). Depletion of polyamines was verified by HPLC, and entry of cells into S phase was monitored by autoradiography. After 24 h of incubation with [3H]-thymidine, polyamine-depleted cells had labeling indices similar to quiescent cells if they were serum-stimulated without Pu, but progressed to S phase to the same degree as control cultures if polyamines were restored by adding Pu at the time of serum stimulation. These observations suggested that commitment of quiescent cells to DNA replication may require polyamines. To determine if polyamine-dependent steps occur during the pre-commitment period (up to 12 h after serum stimulation) or only in traverse of G1 (12 h to 24 h, post-commitment), polyamine-depleted quiescent cells were serum-stimulated for 12 h without Pu, then returned to low serum with Pu. Labeling indices of these cultures remained nearly as low as those of unstimulated cells. Reducing serum concentration from 10% to 0.5% at 12 h after stimulation did not effect labeling indices of control cells not depleted of polyamines by DFMO. These results supported the postulated requirement for polyamines during pre-commitment events. However, polyamine-deficient quiescent cells serum-stimulated without Pu for periods longer than 24 h had labeling indices at 36 and 48 h significantly greater than at 24 h. This suggested that polyamine depletion may decrease the rate at which quiescent cells commit to DNA replication, rather than producing an absolute blockade during the pre-commitment period.     

Translational regulation of ornithine decarboxylase and other enzymes of the polyamine pathway

It has long been known that polyamines play an essential role in the proliferation of mammalian cells, and the polyamine biosynthetic pathway may provide an important target for the development of agents that inhibit carcinogenesis and tumor growth. The rate-limiting enzymes of the polyamine pathway, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), are highly regulated in the cell, and much of this regulation occurs at the level of translation. Although the 5' leader sequences of ODC and AdoMetDC are both highly structured and contain small internal open reading frames (ORFs), the regulation of their translation appears to be quite different. The translational regulation of ODC is more dependent on secondary structure, and therefore responds to the intracellular availability of active eIF-4E, the cap-binding subunit of the eIF-4F complex, which mediates translation initiations. Cell-specific translation of AdoMetDC appears to be regulated exclusively through the internal ORF, which causes ribosome stalling that is independent of eIF-4E levels and decreases the efficiency with which the downstream ORF encoding AdoMetDC protein is translated. The translation of both ODC and AdoMetDC is negatively regulated by intracellular changes in the polyamines spermidine and spermine. Thus, when polyamine levels are low, the synthesis of both ODC and AdoMetDC is increased, and an increase in polyamine content causes a corresponding decrease in protein synthesis. However, an increase in active eIF-4E may allow for the synthesis of ODC even in the presence of polyamine levels that repress ODC translation in cells with lower levels of the initiation factor. In contrast, the amino acid sequence that is encoded by the upstream ORF is critical for polyamine regulation of AdoMetDC synthesis and polyamines may affect synthesis by interaction with the putative peptide, MAGDIS.     

Translational regulation of human beta interferon mRNA: association of the 3'' AU-rich sequence with the poly(A) tail reduces translation efficiency in vitro.

The 3' AU-rich region of human beta-1 interferon (hu-IFN beta) mRNA was found to act as a translational inhibitory element. The translational regulation of this 3' AU-rich sequence and the effect of its association with the poly(A) tail were studied in cell-free rabbit reticulocyte lysate. A poly(A)-rich hu-IFN beta mRNA (110 A residues) served as an inefficient template for protein synthesis. However, translational efficiency was considerably improved when the poly(A) tract was shortened (11 A residues) or when the 3' AU-rich sequence was deleted, indicating that interaction between these two regions was responsible for the reduced translation of the poly(A)-rich hu-IFN beta mRNA. Differences in translational efficiency of the various hu-IFN beta mRNAs correlated well with their polysomal distribution. The poly(A)-rich hu-IFN beta mRNA failed to form large polysomes, while its counterpart bearing a short poly(A) tail was recruited more efficiently into large polysomes. The AU-rich sequence-binding activity was reduced when the RNA probe contained both the 3' AU-rich sequence and long poly(A) tail, supporting a physical association between these two regions. Further evidence for this interaction was achieved by RNase H protection assay. We suggest that the 3' AU-rich sequence may regulate the translation of hu-IFN beta mRNA by interacting with the poly(A) tail.     

Polyamines control human chorionic gonadotropin production in the JEG-3 choriocarcinoma cell

The effect of inhibition of ornithine decarboxylase with difluoromethylornithine (DFMO) and the resultant lowering of polyamine levels upon human chorionic gonadotropin (hCG) production in JEG-3 choriocarcinoma cells was investigated. DFMO (10 mM) totally inhibited ornithine decarboxylase activity. In DFMO-treated cells, cellular spermidine concentrations fell to nondetectable levels (less than 1% of control values) within 24 h and spermine concentrations were reduced to 41.9% of controls over 6 days. DFMO caused a 70-80% inhibition of hCG production. Levels of mRNA for both the alpha and beta subunits of hCG were also inhibited relative to mRNA for tubulin. Exogenous putrescine normalized hCG production in a dose-dependent manner. Other diamines, including cadaverine, 1,3-diaminopropane, 1,6-diaminohexane, and 1,7-diaminoheptane, were ineffective in reestablishing hCG production in DFMO-treated cells. Dibutyryl cAMP (1 mM) stimulated hCG production and increased levels of mRNA for the alpha and beta subunit 5-40-fold in both DFMO-treated and control cells. Polyamines appear to have a fundamental role in hCG production in JEG-3 choriocarcinoma cells. However, dibutyryl cAMP can partially overcome or circumvent the requirement for polyamines in hCG biosynthesis.     

Effect of polyamines on the activity of malarial alpha-like DNA polymerase

DNA polymerase from the malarial parasite Plasmodium falciparum required Mg2+ for activity, Putrescine (1 mM) caused a twofold increase in enzyme activity in the presence of a suboptimal concentration of MgCl2 (2 mM). Spermidine (1.5-2.0 mM) or spermine (0.1-0.3 mM) increased the activity of malarial DNA polymerase, in the presence of 2 mM MgCl2, by factors of 6 and 3-5, respectively. The activity of DNA polymerase from calf thymus or from NIH 3T3 cells transformed by the ras oncogene were not stimulated by these polyamines to the same extent. These findings suggest that in malaria-infected erythrocytes, polyamines, at physiological concentrations, serve as a cofactor for the parasitic alpha-like DNA polymerase. Malarial parasites grown in cultured human erythrocytes did not synthesize DNA after treatment with alpha-difluoromethylornithine, which caused polyamine depletion in the infected cells. DNA synthesis was resumed after adding putrescine to the polyamine-depleted cultures. DNA synthesis was also initiated when actinomycin D was added along with putrescine to polyamine-depleted cells. It thus appears that polyamines are essential for the translation of the DNA polymerase mRNA and that polyamines play an important role in regulating the cell cycle of the malarial parasite.     

Modulation of tumor cell proliferation and apoptosis by polyamine depletion in cells of head and neck squamous cell carcinomas

These studies were carried out to examine the capacity of alpha-difluoromethylornithine (DFMO) to modulate cell proliferation and apoptosis in cells of squamous cell carcinomas (SCCs) of the head and neck. Exposure of cells to DFMO (5 mM for 48 h) depleted intracellular putrescine and spermidine levels (greater than 5-fold) and inhibited proliferation of the cells without manifestation of cytotoxicity as measured by a clonogenic assay. Exposure of the cells to DFMO did not influence the survival response after exposure to single-dose radiation between 0 and 10 Gy. Treatment of polyamine-depleted cells with 200 nM staurosporine amplified apoptosis 65% (1.65-fold) over that in controls, as determined by flow cytometry. The increased apoptosis after DFMO treatment was effectively inhibited by the addition of 1 mM putrescine or spermidine. Cleavage of poly(ADP-ribose) polymerase (PARP) illustrated that the staurosporine treatment induced apoptosis in the cells within 6 h. Analysis of PARP cleavage indicated that treatment with DFMO accelerated the kinetics of progression of apoptosis but did not influence the sensitivity of cells to 10 nM-1 microM staurosporine. These data suggest an involvement of endogenous polyamines in modulation of proliferation kinetics and apoptosis in human SCCs and suggest opportunities to explore new therapeutic strategies in head and neck cancer patients to be treated with radiation therapy.