Translation of brain messenger ribonucleic acids in homologous,heterologous and mixed cell free systems

Optimum conditions were determined for translation of rat brain messenger RNA in vitro using three heterologous systems (wheat germ, Krebs ascites cell and reticulocyte) and a homologous system containing ribosomal subunits and factors from brain. The four systems showed similarities, as well as differences, in regard to their requirements. Although spermine partially replaced magnesium ions in all the four, it stimulated protein synthesis in the extracts of reticulocyte and wheat germ, but not in those of ascites cell or brain. When potassium ions were added as acetate instead of chloride, amino acid incorporation was enhanced and the optimum was shifted to much higher concentrations of potassium (110–120 mM) than was observed with KCl (80 mM). These differences were probably due to inhibition by high concentrations of chloride when KCl was used as the sole source of potassium.Under optimum conditions for each system, translation of brain messenger RNA in the brain system was inferior to the other three extracts, when based on equivalent amounts of ribosomes present in the reaction mixture. However, the homologous system was able to sustain linear incorporation of amino acid for a much longer period than the others, indicating that homologous factors may play a role in the translation of brain messenger RNA.     

Inhibition of translation of mRNAs for ornithine decarboxylase and S-adenosylmethionine decarboxylase by polyamines

The effect of spermidine and spermine on the translation of the mRNAs for ornithine decarboxylase and S-adenosylmethionine decarboxylase was studied using a reticulocyte lysate system and specific antisera to precipitate these proteins. It was found that the synthesis of these key enzymes in the biosynthesis of polyamines was much more strongly inhibited by the addition of polyamines than was either total protein synthesis or the synthesis of albumin. Translation of the mRNA for S-adenosylmethionine decarboxylase was maximal in a lysate which had been substantially freed from polyamines by gel filtration. Addition of 80 microM spermine had no significant effect on total protein synthesis and stimulated albumin synthesis but reduced the production of S-adenosylmethionine decarboxylase by 76%. Similarly, addition of 0.8 mM spermidine reduced the synthesis of S-adenosylmethionine decarboxylase by 82% while albumin and total protein synthesis were similar to that found in the gel-filtered lysate. Translation of ornithine decarboxylase mRNA was greater in the gel-filtered lysate than in the control lysate but synthesis of ornithine decarboxylase was stimulated slightly by low concentrations of polyamines and was maximal at 0.2 mM spermidine or 20 microM spermine. Higher concentrations were strongly inhibitory with a 70% reduction occurring at 0.8 mM spermidine or 150 microM spermine. Further experiments in which both polyamines were added together confirmed that the synthesis of ornithine and S-adenosylmethionine decarboxylases were much more sensitive to inhibition by polyamines than protein synthesis as a whole. These results indicate that an important part of the regulation of polyamine biosynthesis by polyamines is due to a direct inhibitory effect of the polyamines on the translation of mRNA for these biosynthetic enzymes.     

Transport of ornithine carbamoyltransferase precursor into mitochondria. Stimulation by potassium ion, magnesium ion, and a reticulocyte cytosolic protein(s)

The precursor of rat liver ornithine carbamoyltransferase (EC 2.1.3.3) synthesized in vitro was taken up and processed to the mature enzyme by isolated rat liver mitochondria. Potassium ion, magnesium ion, and a reticulocyte cytosolic protein(s), in addition to the precursor and the mitochondria, were required for maximal transport and processing of the precursor. The concentrations of potassium and magnesium ions required for maximal transport and processing were about 120 and 0.8-1.6 mM, respectively. Dialyzed postribosomal supernatant of rabbit reticulocyte lysate (36 mg of protein/ml), in combination with potassium and magnesium ions, stimulated the transport and processing severalfold. The stimulatory activity of the dialyzed lysate was inactivated by trypsin treatment or heating at 100 degrees C for 2 min. No significant amount of the precursor was associated with the mitochondria when incubation was performed in the absence of these components. These results suggest that potassium ion, magnesium ion, and a reticulocyte cytosolic protein(s) stimulate the binding and transport of the ornithine carbamoyltransferase precursor to the mitochondria. Dialyzed supernatant of rabbit erythrocyte lysate was equally effective in stimulating the precursor transport and processing, and a dialyzed cytosol fraction of Ehrlich ascites cells was partly stimulatory. On the other hand, dialyzed cytosol fractions of rat liver and rat kidney, and dialyzed supernatant of wheat germ extracts did not stimulate the precursor transport and processing but rather inhibited it.     

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.     

The role of mRNA competition in regulating translation. III. Comparison of in vitro and in vivo results

Competition of encephalomyocarditis virus, reovirus, and L-cell mRNAs for a message-discriminatory component was studied in vitro. The data were analyzed qualitatively to determine the relative initiation efficiencies among the various mRNAs. The effects of potassium chloride concentration, magnesium acetate concentration, and m7G methylation on mRNA competition in vitro were also studied. These results were correlated with translation rates in vivo for the same mRNAs, to determine if the sites of competition in vitro and in vivo are the same. It was found that under a particular set of magnesium acetate and potassium chloride concentrations, the order of mRNA initiation efficiencies was the same both in vivo and in vitro, suggesting that the same limiting message-discriminatory factor is regulating initiation rates in both cases. This can only be accomplished in a competitive situation when RNA is in molar excess relative to the discriminatory component.     

Distribution of mRNAS of fibrinogen polypeptides and albumin in free and membrane-bound polyribosomes and induction of alpha-fetoprotein mRNA synthesis during liver regeneration after partial hepatectomy

To study the effect of regenerative response of the liver following partial hepatectomy on the synthesis of major plasma proteins (secretory proteins), we have determined the sequence contents and the distribution of albumin and fibrinogen polypeptide mRNAs in rat liver at intervals after partial hepatectomy and sham operation. Using a quantitative technique for the isolation of polyribosomes, we demonstrated that the distribution of RNA between free and membrane-bound polyribosomal fraction was unchanged in these experiments. There was no shift in the polyribosomal population to favor free polyribosomes after partial hepatectomy. However, there was a dramatic increase (5-6-fold) of the fibrinogen polypeptide mRNA concentration during the first 24 h after resection. In contrast, the albumin mRNA concentration decreased (2-3-fold). There were no alpha-fetoprotein mRNA sequences detectable in any liver RNA fraction in these experimental animals. In sham-operated rats with intact livers, similar changes of fibrinogen polypeptide and albumin mRNA concentrations as described in regenerating liver after partial hepatectomy, were observed. These results suggest that albumin and fibrinogen synthesis after partial hepatectomy is reciprocally regulated at the mRNA level and represents a nonspecific acute phase response to surgical trauma.     

Changes in the sequence content of albumin mRNA and in its translational activity in the rat liver with age

To investigate the regulation of age-related changes in albumin synthesis in the rat liver, total postnuclear RNA and polyribosomes, both membrane-bound and free, were prepared from livers of rats of different ages. By the use of a specific complementary DNA probe, the albumin mRNA sequence content was quantitated in these RNA fractions. These studies showed a specific increase in albumin mRNA sequence content in total postnuclear RNA and membrane-bound polyribosomes at between 12 and 24 months of age. Between 24 and 36 months of age, the increase in the amount of albumin mRNA in these two fractions was due only to an increase in liver weight. The increase in albumin mRNA sequence content was not found in the poly(A)⁺ fraction but in the RNA extracted from the void of oligo(dT)-cellulose column chromatography. The isolated polyribosomes were translated in a cell-free system to assess age-related changes in total protein and albumin synthesis due to translational control. No changes with age were found in the translational capacity of membrane-bound and free polyribosomes per RNA unit. Immunoprecipitation of the synthesized albumin in the translation products revealed that albumin synthesis in the cell-free system is not increased proportionally with the elevated albumin mRNA level between 12 and 24 months of age. This indicates that albumin mRNAs present in the livers of old rats are biologically less active than those found in younger animals.     

Translation kinetics in cultured mouse hepatoma cells. Regulation of albumin synthesis by amino acids

The synthesis of albumin in the liver has been shown to correlate with the availability of essential amino acids in the diet. We have investigated this phenomenon in the highly differentiated mouse hepatoma cell line, Hepa. Cells were grown for three days in complete medium with daily changes. The cells were then incubated for 22 h in media containing varying concentrations of individual essential amino acids. The deficient media were then changed; 1.5 h later the cells were labeled for 0.5 h with [3H]leucine. Albumin was immunoprecipitated and total protein was acid-precipitated from postribosomal supernatants of detergents-solubilized cells. With the exception of isoleucine, the relative rates of albumin synthesis decreased as a function of amino acid concentration from 4.3% in complete medium to 2.5% in totally deficient media. This specific reduction in albumin synthesis was confirmed by analysis of labeled Hepa proteins displayed on sodium dodecyl sulfate/polyacrylamide gels. Essential amino acid limitation reduced total protein synthesis by 50%. This is the result of a decrease in the translation efficiency of total mRNA from 5 to 3 polypeptides/message min-1 and is consistent with a reduction in the initiation rate. In contrast, the 70% decrease in albumin synthesis was a result of a reduced number of functional albumin messages/cell. The translation efficiency of these albumin messages remained unchanged at 1.     

Translation of chicken interferon messenger in a cell-free protein synthesis system and in a cell culture

A highly effective cell-free system for protein synthesis was obtained from rabbit reticulocytes and for the first time used for synthesis of biologically active chicken interferon. The optimal conditions for translation of its mRNA were developed. The translation efficacy in the cell-free system was 10-50 times higher than that in the culture of heterologous cells. The higher the purity level of RNA, the higher the translation level. With respect to poly (A+) RNA sedimenting in the sucrose gradient 9S the efficacy reached 2560 units per 1 microgram of RNA. By the content of poly (A), sequences and rate of the sedimentation, mRNA of the chicken interferon was similar to that of the human fibroblast cell interferon. The possible translation of mRNA of the chicken interferon at low concentrations of exogenic potassium ions in the cell-free system is explained by production of interferon in infected cells where the concentration of the intracellular potassium significantly decreases which is indicative of the mRNA interferon similarity with virus templates. It was found that only albino New Zealand rabbits, but also chinchilla may be used for preparation of the cell-free protein synthesizing system. Various exogenic templates in the mRNA-dependent cell-free system prepared from reticulocyte nonfractionated lysate by treatment with micrococcal nuclease stimulated the protein synthesis by 7-15 times.     

Protein synthesis with membrane-bound polysomes and albumin messenger RNA from livers of mutant mice

Summary Investigation of deficiencies in serum protein synthesis resulting from deletion-mutations at the albino locus in mice was continued usingin vitro conditions. Previous work showed that although total protein synthesis was only slightly lower in livers from albinos, newly synthesized protein appearing in plasma was 22% of that in controls. It was thought that the disorganized endoplasmic reticulum and Golgi apparatus, characteristic for the liver (and kidney) of these mutants, might be responsible for the observed deficiencies. In the present study mebrane-bound polysomes isolated from the livers of newborn albinos were 55% (c^3H/c^3H strain) and 62% (c^14CoS/c^14CoS strain) as efficient as those from normal littermates in incorporating radioactive leucine into protein in a cell-free system. These differences could not be eliminated by the addition of excess liver mRNA, exogenous soluble factors or by the exchange of cell sap between albino and control polysomes. In an earlier study albino liver slices synthesized only 13% (or 17% per mg of total protein synthesized) as much albumin as controls. We have now found that the level of albumin poly(A)⁺-RNA isolated from albino livers and assayed with a reticulocyte lysate, was almost as high (85%) as in controls. It was concluded that the very low level of albumin synthesis in albino livers did not result from a deficiency of albumin mRNA. Whether the rate-limiting step in synthesis of albumin in mutant livers is at the level of translation or processing for secretion requires further investigation.     

Distribution of mRNAs of fibrinogen polypeptides and albumin in free and membrane-bound polyribosomes and induction of α-fetoprotein mRNA synthesis during liver regeneration after partial hepatectomy

To study the effect of regenerative response of the liver following partial hepatectomy on the synthesis of major plasma proteins (secretory proteins), we have determined the sequence contents and the distribution of albumin and fibrinogen polypeptide mRNAs in rat liver at intervals after partial hepatectomy and sham operation. Using a quantitative technique for the isolation of polyribosomes, we demonstrated that the distribution of RNA between free and membrane-bound polyribosomal fraction was unchanged in these experiments. There was no shift in the polyribosomal population to favor free polyribosomes after partial hepatectomy. However, there was a dramatic increase (5–6-fold) of the fibrinogen polypeptide mRNA concentration during the first 24 h after resection. In contrast, the albumin mRNA concentration decreased (2–3-fold). There were no α-fetoprotein mRNA sequences detectable in any liver RNA fraction in these experimental animals. In sham-operated rats with intact livers, similar changes of fibrinogen polypeptide and albumin mRNA concentrations as described in regenerating liver after partial hepatectomy, were observed. These results suggest that albumin and fibrinogen synthesis after partial hepatectomy is reciprocally regulated at the mRNA level and represents a nonspecific acute phase response to surgical trauma.     

Differential expression of albumin and alpha-fetoprotein genes in fetal tissues of mouse and rat

We have carried out a comparative analysis of the expression of the albumin and alpha-fetoprotein (AFP) genes in yolk sac and liver at different stages of fetal and postnatal life, in rat and mouse. Albumin and AFP mRNA levels were examined in these tissues by R0t analysis of RNA excess-cDNA hybridization data and/or by Dot blot hybridization. In addition, size analysis of the mRNA sequences were performed by electrophoretic fractionation on agarose gels containing methylmercury hydroxide and hybridization to radioactive cloned rat and mouse albumin and AFP cDNA probes. In the mouse, substantial amounts of albumin mRNA molecules were found in the yolk sac at different stages of development, while minimal levels of albumin mRNA sequences were detected in the rat yolk sac. The mouse yolk sac albumin mRNA molecules were found to be associated with the polysomes and to be functional in cell-free translation systems. In the rat, a reciprocal relationship appears to exist between the concentrations of the two mRNAs in yolk sac and embryonic liver. In contrast, in the mouse a parallel increase in both albumin and AFP mRNA levels was found in these tissues during fetal development. These results suggest that the expression of the albumin and AFP genes may be subjected to different regulatory events in these two members of the Muridae family.     

Isolation of rat liver albumin messenger RNA.

Rat liver albumin messenger RNA has been purified to apparent homogeneity by means of polysome immunoprecipitation and poly(U)-Sepharose affinity chromatography. Specific polysomes synthesizing albumin were separated from total liver polysomes through a double antibody technique which allowed isolation of a specific immunoprecipitate. The albumin-polysome immunoprecipitate was dissolved in detergent and the polysomal RNA was separated from protein by sucrose gradient centrifugation. Albumin mRNA was then separated from ribosomal RNA by affinity chromatography through the binding of poly(U)-Sepharose to the polyadenylate 3' terminus of the mRNA. Pure albumin mRNA migrated as an 18 S peak on 85% formamide-containing linear sucrose gradients and as a 22 S peak on 2.5% polyacrylamide gels in sodium dodecyl sulfate. It coded for the translation of authentic liver albumin when added to a heterologous protein-synthesizing cell-free system derived from either rabbit reticulocyte lysates or wheat germ extracts. Translation analysis in reticulocyte lysates indicated that albumin polysomes were purified approximately 9-fold from total liver polysomes, and that albumin mRNA was purified approximately 74-fold from albumin polysomal RNA. The total translation product in the mRNA-dependent wheat germ system, upon addition of the pure mRNA, was identified as authentic albumin by means of gel electrophoresis and tryptic peptide chromatography.     

Reversion by hypotonic medium of the shutoff of protein synthesis induced by encephalomyocarditis virus.

Infection of human HeLa cells by picornaviruses produces a drastic inhibition of host protein synthesis. Treatment of encephalomyocarditis virus-infected HeLa cells with hypotonic medium reversed this inhibition; no viral protein synthesis was detected. The blockade of viral translation by hypotonic conditions was observed for a wide range of multiplicities of infection. However, only with low virus-to-cell ratios did cellular protein synthesis resume. The ratio of cellular to viral mRNA translation was strongly influenced by the concentration of monovalent ions present in the culture medium: a high concentration of NaCl or KCl favored the translation of viral mRNA and strongly inhibited cellular protein synthesis, whereas the opposite was true when NaCl was omitted from the culture medium. Once viral protein synthesis had been blocked by hypotonic medium treatment, it resumed when the infected cells were placed in a normal or hypertonic medium, indicating that the viral components synthesized in the infected cells were not destroyed by this treatment. These observations reinforced the idea that ions play a role in discriminating between viral and cellular mRNA translation in virus-infected animal cells.     

Alterations in albumin secretion and total protein synthesis in livers of thyroidectomized rats.

Perfused rat livers and isolated rat hepatocytes exhibited a 50% decrease in the secretion of both albumin and total secretory proteins after thyroidectomy. In contrast, synthesis of non-secretory proteins was decreased by only 20% from the rates observed in liver preparations from euthyroid rats. These observations suggested a disproportionate effect of thyroidectomy on the synthesis of secretory proteins compared with non-secretory proteins. Disproportionate decreases in the synthesis of albumin in other endocrine-deficient states such as hypophysectomy and diabetes had previously been shown to be associated with decreases of similar magnitude in the relative abundance of albumin-mRNA sequences. In contrast, thyroidectomy did not affect the activity or amount of albumin mRNA in total liver poly(A)-containing RNA when assayed by cell-free translation and by hybridization with complementary DNA, respectively. Furthermore, labelling experiments in vivo demonstrated that albumin synthesis represented 12.9 +/- 0.5% and 12.4 +/- 0.4% of total protein synthesis in livers of thyroidectomized and euthyroid rats respectively. Therefore the fall in secretion of albumin and total secretory protein after thyroidectomy did not appear to be a reflection of disproportionate decreases in the synthesis of these proteins. Instead, defects in steps involved in the post-synthetic processing and secretion of albumin are suggested. A number of comparisons, including ribosome half-transit times, the size distributions of total and albumin-synthesizing polyribosomes, and the fraction of RNA present as inactive ribosomes, provided evidence that the overall decrease in protein synthesis after thyroidectomy was not due to generalized alterations in translational processes. Instead, the decrease in total protein synthesis appeared to reflect the RNA content of the liver, which fell in proportion to th decrease in protein synthesis.     

Triiodothyronine increases translatable albumin messenger RNA in Rana catesbeiana tadpole liver

The effects of both 3,5,3'-triiodo-L-thyronine and spontaneous metamorphosis on Rana catesbeiana liver mRNA were studied using in vitro translation of isolated liver poly(A)+ RNA in a rabbit reticulocyte lysate system. Conventional phenol extraction methods yielded degraded RNA due to high levels of endogenous ribonucleases released upon homogenization of Rana catesbeiana liver. Isolation of intact total RNA was achieved using the potent ribonuclease denaturant, guanidinium thiocyanate. Adult bullfrog serum albumin was purified to homogeneity and a monospecific antibody was elicited against it. A serum protein of 23,000 daltons that migrated near serum albumin on a 6% native gel was also purified to homogeneity. A monospecific antibody was also raised against this protein. Both antibodies were used to quantitatively immunoprecipitate the in vitro translation products of poly(A)+ RNA isolated at intervals following a single injection of triiodothyronine or during various stages of spontaneous amphibian metamorphosis. Triiodothyronine caused a sevenfold increase in translatable albumin mRNA and a threefold increase in translatable mRNA for the 23,000 dalton protein. These increases are consistent with a nuclear initiated mechanism for thyroid hormone action during amphibian metamorphosis.     

Changes in collagen and albumin mRNA in liver tissue of mice infected with Schistosoma mansoni as determined by in situ hybridization

We have employed in situ hybridization to evaluate the molecular mechanisms responsible for hypoalbuminemia and increased liver collagen content in murine schistosomiasis. Results were compared using a simplified method of hybridizing isolated hepatocytes from Schistosoma mansoni-infected and normal mouse liver with mouse albumin (pmalb-2) and chick pro-alpha 2(l) collagen (pCg45) probes. Whereas hepatocytes from infected mice showed significantly less albumin mRNA than hepatocytes from control, there were more grains of procollagen mRNA in hepatocytes from infected as compared with control liver. Hybridization of infected liver tissue sections with the collagen probe showed more grains per field in granulomas than in liver regions, whereas with the albumin probe there was more hybridization in liver tissue than in granulomas. These results suggest that in murine schistosomiasis a reduction in albumin mRNA sequence content may be associated with decreased albumin synthesis and ultimately leads to hypoalbuminemia. In addition, although the granuloma seems to be the primary source of type I collagen synthesis, hepatocytes are also capable of synthesizing collagen, especially under fibrogenic stimulation.     

N1-monoacetylation abolishes the inhibitory effect of spermine and spermidine in the reticulocyte lysate translation system

At optimum magnesium, the translation of rat heart mRNA in the nuclease treated rabbit reticulocyte lysate system was inhibited by low concentrations of spermidine or spermine but not of putrescine. Spermidine and spermine cause a general reduction in the translation of all the heart mRNAs since no differential effects were observed when the translation products were examined by gel electrophoresis. Spermine was a five times more potent inhibitor than spermidine but no inhibition was obtained with N1-acetylspermidine or N1-acetylspermine. Since analyses of endogenous polyamines demonstrate that the inhibitory concentrations of spermine could be obtained by converting a small fraction of the endogenous spermidine to spermine, these results indicate that interconversions of the polyamines might be a sensitive regulatory mechanism for protein synthesis.