The role of protein synthesis in the decay of phosphoenolpyruvate carboxykinase messenger RNA.

The role of protein synthesis in the control of phosphoenolpyruvate carboxykinase (PEPCK; 4.1.1.32) mRNA turnover was studied in FTO-2B rat hepatoma cells. A previous study demonstrated that incubation of these cells with cAMP prolongs the half-life of the otherwise short-lived PEPCK mRNA. The decay rate of PEPCK mRNA was also slowed in cells incubated with cycloheximide, but not in cells incubated with other translation inhibitors, such as puromycin or pactamycin, even though protein synthesis was inhibited 85-95% by these agents. No correlation was noted between the rate of L-[3H]valine incorporation into cellular proteins and PEPCK mRNA half-life, suggesting that protein synthesis per se is not required for breakdown of the mRNA. Exposure of cells to the translation initiation inhibitor pactamycin together with cycloheximide abolished the "slowing" effect of cycloheximide, and PEPCK mRNA decayed at the same rate as in cells incubated in the presence of pactamycin alone. In contrast, pactamycin did not reverse the effect of cAMP, and the mRNA decayed at the same slow rate in cells incubated in the presence of either (Bu)2cAMP alone or (Bu)2cAMP together with pactamycin. Since pactamycin promotes polysomes dissociation, these results suggest that cAMP enhances the stability of a polysome-free PEPCK mRNA. Furthermore, these results strongly indicate that neither the rapid decay of PEPCK mRNA nor the cAMP-mediated stabilization of the mRNA requires on-going protein synthesis.     

The estrogen-regulated destabilization of Xenopus albumin mRNA is independent of translation

Protein synthesis inhibitors have been shown to increase the stability of a number of labile mRNAs. In Xenopus laevis serum albumin mRNA is destabilized in the liver cell cytoplasm following estrogen administration. The present study examined the effect of translation inhibitors on this process. The initiation inhibitor 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide causes accumulation of albumin mRNA in 20-80S mRNP particles whereas the elongation inhibitor cycloheximide causes albumin mRNA to accumulate in polysomes. Neither inhibitor blocked the disappearance of albumin mRNA from liver cell cytoplasm when added with estradiol to the medium of liver explant cultures. We conclude that unlike a number of labile mRNAs the instability of Xenopus albumin mRNA following estradiol is independent of translation.     

Autoregulation of tubulin expression is achieved through specific degradation of polysomal tubulin mRNAs

We have utilized protein synthesis inhibitors to investigate the autoregulatory mechanism that uses the concentration of unpolymerized tubulin subunits to specify tubulin mRNA content in animal cells. Puromycin and pactamycin, both of which remove RNAs from polysomes, completely unlink tubulin RNA content from the level of free subunits, whereas pretreatment of cells with cycloheximide, which traps mRNAs onto stalled polyribosomes, enhances the specific degradation of tubulin RNAs in response to increases in the subunit content. Moreover, in the absence of protein synthesis inhibitors, the tubulin RNAs that are lost from cells with elevated free tubulin subunit levels are those that are associated with polyribosomes. Further, beta-tubulin mRNAs encoding a truncated translation product of only 26 amino acids (and that cannot be polyribosomal) are not substrates for autoregulation. We conclude that autoregulation of tubulin synthesis is achieved by specifically altering the stability of tubulin RNAs that are bound to polyribosomes.     

Accelerated poly(A) loss and mRNA stabilization are independent effects of protein synthesis inhibition on alpha-tubulin mRNA in Chlamydomonas.

In Chlamydomonas, the usual rapid degradation of tubulin mRNAs induced by flagellar amputation is prevented by inhibition of protein synthesis with cycloheximide. Evidence is presented that the ability of cycloheximide to stabilize alpha-tubulin mRNA depends on the time of addition. Addition of cycloheximide to cells before induction strongly stabilizes the induced mRNAs, while addition after their synthesis stabilizes them only transiently. Moreover, cycloheximide inhibition does not stabilize the same alpha-tubulin mRNA species in uninduced cells. These results suggest that cycloheximide is not acting to stabilize the induced alpha-tubulin mRNAs simply by preventing ribosome translocation. The stabilized state of tubulin mRNA was found to correlate with its occurrence on smaller polysomes but larger EDTA-released mRNP particles than the unstable state. A second effect of cycloheximide on the metabolism of induced tubulin mRNAs is to accelerate complete poly(A) removal. This effect of cycloheximide inhibition, unlike stabilization, occurs whenever cycloheximide is added to cells, and appears unrelated to stabilization. The effect is shown to be mRNA-specific; poly(A)-shortening on the rbcS2 mRNA is not altered in the presence of cycloheximide, nor do completely deadenylated molecules accumulate. Experiments in which cells were released from cycloheximide inhibition suggest that deadenylated alpha-tubulin mRNAs may be less stable than their polyadenylated counterparts during active translation.     

Translational alterations in maize leaves responding to pathogen infection, paraquat treatment, or heat shock : polysome dissociation and accumulation of a 57 kilodalton protein

Translational alterations occur in maize (Zea mays L.) leaves stressed by pathogen infection or herbicide paraquat treatment. These translational changes include: (a) dissociation of large polysomes to small polysomes, monosomes, and subunits; (b) a decreased rate of total protein synthesis; and (c) a reduced synthesis of several proteins by polysomes in vitro. The polysome dissociation was neither due to an extraction artifact nor to degradation of RNA by RNase. The protein patterns of polysomes isolated from leaves inoculated with Bipolaris maydis at 6 to 48 hours showed an increase in the intensity of a 57 kilodalton protein. When inoculated with less virulent pathogens, such as B. zeicola, Exserohilum turcicum, or Colletotrichum graminicola, the protein was accumulated in polysomes of leaves at 24 to 48 hours after inoculation. The 57 kilodalton protein was also accumulated in polysomes of maize leaves responding to heat shock or herbicide paraquat treatments. The purified 57 kilodalton protein reassociated with polysomes isolated from healthy leaves and inhibited polysomal translation in vitro. Since the 57 kilodalton protein is rapidly accumulated in maize polysomes in response to various biological and environmental stresses and may affect protein synthesis, it may be involved in translational regulation of maize leaves during stress response.     

Cyclin B Translation Depends on mTOR Activity after Fertilization in Sea Urchin Embryos

The cyclin B/CDK1 complex is a key regulator of mitotic entry. Using PP242, a specific ATP-competitive inhibitor of mTOR kinase, we provide evidence that the mTOR signalling pathway controls cyclin B mRNA translation following fertilization in Sphaerechinus granularis and Paracentrotus lividus. We show that PP242 inhibits the degradation of the cap-dependent translation repressor 4E-BP (eukaryotic initiation factor 4E-Binding Protein). PP242 inhibits global protein synthesis, delays cyclin B accumulation, cyclin B/CDK1 complex activation and consequently entry into the mitotic phase of the cell cycle triggered by fertilization. PP242 inhibits cyclin B mRNA recruitment into active polysomes triggered by fertilization. An amount of cyclin B mRNA present in active polysomes appears to be insensitive to PP242 treatment. Taken together, our results suggest that, following sea urchin egg fertilization, cyclin B mRNA translation is controlled by two independent mechanisms: a PP242-sensitive and an additional PP242-insentitive mechanism.     

Translational control and the cytoskeleton in Physarum polycephalum

Translationally active plasmodia of the syncytial slime mold Physarum polycephalum develop into translationally dormant sclerotia during starvation. Although functional mRNA and ribosomes exist in sclerotia, protein synthesis is suppressed at the level of initiation. To test the possibility that alterations in the cytoskeleton may limit protein synthesis, we have examined the distribution of polysomes and actin mRNA in the cytoskeletal (CSK) and soluble (SOL) fractions of Triton X-100-extracted plasmodia and sclerotia. Most of the polysomes and actin mRNA were located in the CSK of plasmodia, while most of the ribosomes and actin mRNA were located in the SOL of sclerotia. The results suggest that ribosomes and mRNA shift from the CSK to the SOL as protein synthesis is suppressed during starvation. Plasmodia and sclerotia can be induced to accumulate excess polysomes by treatment with low levels of the elongation inhibitor cycloheximide. Treatment of plasmodia with cycloheximide caused excess polysomes to accumulate in the SOL, suggesting that the CSK contains a limited capacity for binding translational components and that the association of polysomes with the cytoskeleton is not required for protein synthesis. Treatment of sclerotia with cycloheximide, however, caused polysomes and actin mRNA to accumulate in the CSK, suggesting that the sclerotial cytoskeleton, although depleted in ribosomes and mRNA, is capable of binding translational components. It is concluded that alterations in the sclerotial cytoskeleton are not involved in translational control.     

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.     

Role of protein synthesis in decay and accumulation of mRNA during spore germination in the cellular slime mold Dictyostelium discoideum.

Spore germination in Dictyostelium discoideum is a particularly suitable model for studying the regulation of gene expression, since developmentally regulated changes in both protein and mRNA synthesis occur during the transition from dormant spore to amoeba. The previous isolation of three cDNA clones specific for mRNA developmentally regulated during spore germination allowed for the quantitation of the specific mRNAs during this process. The three mRNAs specific to clones pLK109, pLK229, and pRK270 have half-lives much shorter (minutes) than those of constitutive mRNAs (hours). Using spore germination as a model, we studied the roles of ribosome-mRNA interactions and protein synthesis in mRNA degradation by using antibiotics that inhibit specific reactions in protein biosynthesis. Cycloheximide inhibits the elongation step of protein synthesis. Polysomes accumulate in inhibited cells because ribosomes do not terminate normally and new ribosomes enter the polysome, eventually saturating the mRNA. Pactamycin inhibits initiation, and consequently polysomes break down in the presence of this drug. Under this condition, the mRNA is essentially free of ribosomes. pLK109, pLK229, and pRK270 mRNAs were stabilized in the presence of cycloheximide, but pactamycin had no effect on their normal decay. Since it seems likely that stability of mRNA reflects the availability of sites for inactivation by nucleases, it follows that in the presence of cycloheximide, these sites are protected, presumably by occupancy by ribosomes. No ribosomes are bound to mRNA in the presence of pactamycin, and therefore mRNA degrades at about the normal rate. The data further indicate that a labile protein is probably not involved in mRNA decay or stabilization, since protein synthesis is inhibited equally by both antibiotics. We conclude that it may be important to use more than one type of protein synthesis inhibitor to evaluate whether protein synthesis is required for mRNA decay. The effect of protein synthesis inhibition on mRNA synthesis and accumulation was also studied. mRNA synthesis continues in the presence of inhibitors, albeit at a diminished rate relative to that of the uninhibited control.