Role of tissue specific factors in the translation of brain messenger ribonucleic acids in vitro

The activity of brain ribosomal subunits was examined by measuring (a) the saturation of ribosomes with nascent polypeptides and the rates of release of completed chains; (b) interaction of the subunits with brain messenger RNA to form polysomal aggregates; (c) formation of polyphenylalanine in the presence of polyuridylic acid at high magnesium concentration and (d) inhibition by aurine tricarboxylic acid. The results showed that a portion of the subunits were defective in forming initiation complexes with brain messenger RNA, but translated polyuridylate efficiently. The subunits that did form polysomes were more competent than the heterologous systems (derived from Krebs ascites cells, reticulocytes or wheat germ) in carrying out reinitiations of brain mRNA translation.Both the homologous and the heterologous systems translated brain mRNA and synthesized the two brain specific proteins S-100 and the neuron specific enolase, indicating that each of the systems had all the necessary factors. However, homologous initiation factors, aminoacyl-tRNA synthetases and transfer RNAs were more effective, particularly at suboptimal concentrations. Our results suggest that discriminative translation of brain messenger RNA may take place based on relative proportions of required components in the reaction milieu rather than by the presence or absence of one or more special messenger RNA specific factors.     

Newly Synthesized mRNA Is Translated during the Initial Imbibition Phase of Germinating Maize Embryo

RNA synthesis is activated in the cells of the plant embryo very soon after the start of seed imbibition. We previously reported that mainly heterogeneous nuclear RNA is synthesized in the radicle of Zea mays embryo during the first hours of germination. The present study was undertaken in order to detect the time of appearance of the newly synthesized messenger RNA in the polysomes of germinating maize axes.

Free polysomes were prepared from embryonic axes rehydrated for 2 hours in the presence of radioactively labeled uridine. These polysomes were shown to be labeled and to contain labeled particles sedimenting, after dissociation with EDTA, in the 10S to 40S region of a sucrose gradient. The labeled polysomal RNA migrates heterogeneously in a gel with a mean size corresponding to about 16S, and 60% of these molecules are polyadenylated.

The data indicate that the newly synthesized RNA associated with the polysomes after 2 h of germination consists of messenger RNA molecules. Analysis of the polysomes prepared 0.5 and 1 h after the start of imbibition suggests that translation of the newly synthesized messenger RNA probably occurs within the 1st hour of imbibition of the isolated axis, thus well before the completion of the initial water uptake.

     

Polysomal and non-polysomal messenger RNA in neuroblastoma cells: Lack of correlation between polyadenylation or initiation efficiency and messenger RNA location

Neuroblastoma cytoplasm was fractionated on sucrose gradients into polysomes (>90 S) and non-polysomal particles (<90 S). Purified RNA from these fractions was translated using a wheat germ lysate and translation products were compared by two-dimensional gel electrophoresis. Non-polysomal messenger RNA directed the synthesis of a specific subset of polysomal mRNA translation products. Careful comparison of individual translation products demonstrated that specific mRNAs were not randomly distributed between polysomes and the non-polysomal fraction.Fractionation of both RNA populations into polyadenylated (poly(A)⁺) and non-adenylated (poly(A)^?) species indicated that specific, abundant non-polysomal mRNAs were not less adenylated than their polysomal counterparts. Furthermore, comparison of translation products from assays of subsaturating and supersaturating RNA concentrations demonstrated that no simple correlation could be made between the relative initiation efficiency of a specific mRNA and its distribution between polysomes and non-polysomal particles.     

STABILITIES OF NUCLEAR AND MESSENGER RNA MOLECULES IN SEA URCHIN EMBRYOS

The kinetics of accumulation of radioactive adenosine in adenosine triphosphate and in RNA of nuclear, cytoplasmic, and polysomal fractions of sea urchin embryos have been analyzed. 85% of the RNA synthesized decays in the nucleus with an apparently uniform half-life of about 7 min. The remaining 15% goes to the cytoplasm, mostly entering polysomes, and decays with a quite uniform half-life of about 75 min. The nuclear RNA accounts for one-third and the cytoplasmic RNA accounts for two-thirds of the total unstable RNA which accumulates at steady state in the embryo. The size distribution of short-labeled nuclear RNA is very similar to that of long-labeled messenger RNA, when both are extracted directly from the cells without a previous cell fractionation.     

In Vitro Synthesis of Wheat (Triticum aestivum L.) Storage Proteins

Free and membrane-associated polysomes were isolated in approximately equal amounts from endosperm of wheat kernels harvested 20 days after anthesis. The presence of heparin in the homogenizing buffer minimized polysome degradation. Ribonucleic acid from the isolated polysomes, when translated in vitro in a wheat germ system, yielded products ranging in size from about 12,000 to about 80,000 daltons, including at least two polypeptides that co-migrated with seed extract proteins in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The nature of the translation products of free and membrane-associated RNA are distinctly different, with membrane-associated RNA yielding a higher proportion of polypeptides in the size range of 30,000 to 37,000 daltons. Analysis of membrane-associated 3′-terminal polyadenylyl-containing RNA in vitro translation products, by solubility in 70% ethanol and by immunoprecipitation, indicates that the 33,000- to 37,000-dalton polypeptides contain gliadins, and the analysis provides evidence that these proteins are synthesized in association with membranous cell organelles. Gliadin polypeptides synthesized in vitro are larger than authentic gliadins and probably are precursors which, in vivo, undergo modification to yield the smaller final products.     

Kinetic estimation of the base sequence complexity of poly(A)-containing mRNA in Ehrlich-ascites-tumor cells and its abundance in nuclear RNA.

Poly(A)-containing messenger RNA was isolated from polysomes of Ehrlich ascites tumor cells, and analyzed for sequence complexity by hybridization to its complementary DNA. The results indicate the presence of about 27,000 diverse mRNA species in mouse Ehrlich ascites tumor cells. Total nuclear RNA was also hybridized to cDNA transcribed from polysomal poly(A)-containing mRNA up to an rot of 3,000 M . s. It was found that all classes of the polysomal poly(A)-containing mRNA sequences were also present in the nucleus, although the distribution varied. About 2% of the total nuclear RNA sequences were expressed as total polysomal poly(A)-containing mRNA. We also report that the total percentage of the haploid mouse genome transcribed in Ehrlich cells is significantly higher than that found in other mouse cells previously examined for poly(A)-containing mRNA sequence complexity.     

Metabolism of Poly(A) in Plant Cells: Discrete Classes Associated with Free and Membrane-bound Polysomes

In the subapical region of dark-grown pea epicotyls about 40% of the total polysomes are associated with membranes. The presence of poly(A) in polysomal mRNA was detected by hybridization of unlabeled RNA with (3)H-poly(U). Both free mRNA and messenger ribonucleoprotein particles in polysomes hybridize with (3)H-poly(U) quantitatively. The binding of (3)H-poly(U) to polysomes is increased by treatment with the detergent sodium dodecyl sulfate. Since detergent influenced the (3)H-poly(U) binding more in membrane-bound polysomes than in free, there may be more protein(s) associated with the poly(A) portion of the mRNA in membrane-bound polysomes. Analysis of the poly(A) segments isolated from the mRNA of these two classes of polysomes indicates that there are discrete classes of poly(A) and they appear to be differentially associated with free and membrane-bound polysomes. Mean size distribution of poly(A) in free polysomes is larger than in membrane-bound polysomes.Following treatment (2 days) with the plant growth hormone indoleacetic acid, there is a gradual decrease in the mean length of total poly(A), which appears to correspond to a decrease in the size of the polysomes and their associated mRNA.     

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.     

Segregation of specific classes of messenger RNA into free and membrane-bound polysomes

Analysis of mRNA populations from rat liver rough microsomes and free polysomes by homologous and heterologous cDNA . mRNA hybridization shows that the two mRNA populations are distinct, demonstrating that specific mRNA classes are efficiently segregated for translation in association with endoplasmic reticulum membranes. We estimate that approximately 90% of the mRNA in membrane-bound polysomes contains a diverse set of messengers with a minimum of 500--2000 different species although approximately 5--8 messengers may constitute 25--30% of the mRNA mass. The complexity of the mRNA population of free polysomes appears to be comparable to that estimated for total liver poly(A) + mRNA by other investigators, and is likely to be substantially greater than that of the bulk of bound mRNA. In addition, mRNA in free polysomes lacks the high abundance class characteristic of mRNA-bound polysomes. The substantial complexity of the bound mRNA population suggests that the segregation of polysomes in rough microsomes is not limited to a small class specialized in manufacturing secretory proteins, but extends to polysomes engaged in the synthesis of proteins for intracellular distribution. The segregation of specific messengers into the free and membrane-bound classes was abolished when polysome disassembly was induced by administration of ethionine. Thus, messenger RNA molecules themselves lacked the capacity for segregation, although they contain information for segregation which is expressed during translation. These findings are consistent with the presence of signal sequences in nascent polypeptides which determine the attachment of ribosomes to endoplasmic reticulum membranes.     

PROTEIN SYNTHESIS BY CEREBRAL POLYSOMES PRETREATED WITH PUROMYCIN

Abstract— Polysomes prepared from rat cerebral microsomes, following preincubation with a high concentration of puromycin (2.5 mM) in the presence of rat liver soluble enzymes, were very similar to normal polysomes in yield, A 260_nm:A 280_nm ratio and in absorbance profile on sucrose density gradients. However, the capacity for amino acid incorporation was inhibited by more than 50 per cent by puromycin treatment. The extent of inhibition far exceeded what could be expected from the amount of residual puromycin bound to polysomes, suggesting that some essential step in polypeptide synthesis was damaged. An examination of the labelled polypeptides, using sucrose density gradient centrifugation, showed that most of the new chains synthesized by puromycin-polysomes were released into solution. However, small amounts of polypeptides of high specific radioactivity were distributed among the polysomal aggregates. In contrast to normal polysomes, the specific radioactivity of puromycin polysomes was the highest in aggregates of six or more ribosomes and declined sharply at the levels of trimers and dimers. It is suggested that cerebral polysomes pretreated with puromycin become defective in the termination mechanism with the consequence that even though they are capable of moving at least short distances on the messenger RNA and of releasing the polypeptide chains formed, a concomittant release of monomeric ribosomes is obstructed. This may result in the‘clogging’of the terminus of the mRNA, thus blocking further polypeptide synthesis.     

Isolation of Undegraded Free and Membrane-Bound Polysomal mRNA from Rat Brain

A new procedure is described for the isolation of undegraded free and membrane-bound polysomal mRNA from rat brain in which polysomes are simultaneously separated from soluble components of the cell and slowly sedimenting ribosome species and concentrated by rate-zonal centrifugation on short linear sucrose gradients. This avoids the problem encountered in conventional procedures of pelleting polysomes along with membranous materials that are not solubilized by detergents and that appear to contain traces of nucleases. It also permits a more thorough analysis of the mRNA populations actively engaged in protein synthesis, since both polyadenylated and nonpolyadenylated mRNAs are isolated together. Moreover, the likelihood of sedimenting nonpolysomal mRNP particles along with polysomes is reduced by using rate-zonal rather than pelleting centrifugation. The translational activity in vitro of free and membrane-bound polysomal RNA prepared in this way is high and is about 1.5 times that of RNA prepared by a conventional pelleting technique. The difference is attributable to better preservation of large mRNAs, as inferred from two-dimensional gel electrophoretic analysis of translation product abundance. The recovery of both classes of polysomal RNA is about 90%. The method is simple, efficient, and adapted for isolation of small amounts of polysomal RNA.     

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.     

Large heterogeneous nuclear ribonucleic acid has three times as many 5'' caps as polyadenylic acid segments, and most caps do not enter polyribosomes.

The rate of synthesis in Chinese hamster cells of 5' cap structures, m7 GpppNmp, in large (greater than 700 bases) heterogeneous nuclear ribonucleic acid (RNA) molecules is two to three times faster than the synthesis of 3'-terminal polyadenylic acid segments. As judged by presence of caps, newly synthesized polysomal messenger RNA, exclusive of messenger RNA the size of histone messenger RNA, is more than 90% in the polyadenylated category. It appears, therefore, that between half and two-thirds of the long capped heterogeneous nuclear RNA molecules do not contribute a capped polysomal derivative to the cytoplasm. There are capped, nonpolysomal, non-polyadenylated molecules with a rapid turnover rate that fractionate with the cytoplasm. These metabolically unstable molecules either could represent leakage into the cytoplasm during fractionation or could truly spend a brief time in the cytoplasm before decay.     

Synthesis of S100 Protein on Free and Membrane-Bound Polysomes of the Rabbit Brain

Abstract: Free and membrane-bound polysomes were isolated from the cerebral hemispheres and cerebellum of the young adult rabbit. The two polysomal populations were translated in an mRNA-dependent cell-free system derived from rabbit reticulocytes. Analysis of the [³⁵S]methionine-labeled translation products on two-dimensional polyacrylamide gels indicated an efficient separation of the two classes of brain polysomes. The relative synthesis of S100 protein by free and membrane- bound polysomes was determined by direct immuno-precipitation of the cell-free translation products in the presence of detergents to reduce nonspecific trapping. Synthesis of S100 protein was found to be twofold greater on membrane-bound polysomes compared with free polysomes isolated from either the cerebral hemispheres or the cerebellum. In addition, the proportion of poly- (A+)mRNA coding for SlOO protein was also twofold greater in membrane-bound polysomes compared with free polysomes isolated from the cerebral hemispheres. These results indicate that the cytoplasmic S100 protein is synthesized predominantly on membrane-bound polysomes in the rabbit brain. We suggest that the nascent S100 polypeptide chain translation complex is attached to the rough endoplasmic reticulum by an ionic interaction involving a sequence of 13 basic amino acids in S100 protein.     

Specific control of messenger translation in Drosophila oocytes and embryos

The distribution of messenger RNA between polysomes and mRNP in oocytes and embryos of Drosophila melanogaster has been studied by in vitro translational analysis. Poly(A)⁺ RNA was purified from polysomes or mRNA from mature oocytes and young embryos. The messenger populations were translated in vitro and the peptides synthesized were separated by two-dimensional electrophoresis. Analysis of the 2D gel patterns enabled the detection of three peptides coded by messengers present predominantly in the mRNA pools of mature oocytes. When DNA-binding peptides were selected from the in vitro translation products, they showed, after separation by two-dimensional electrophoresis, less than 100 spots. The analysis of the 2D gels indicated that three DNA-binding peptides are coded by messengers present only in the mRNP of the oocytes. These messengers are later found in the polysomal fraction of embryos.     

Glucocorticoid-mediated selective reduction of functioning collagen messenger ribonucleic acid

Polysomes were isolated from both lung and dermis of neonatal rats and the poly(A) RNA therein was isolated by oligo(dT)-cellulose chromatography. The RNA fractions were then translated in the nuclease-treated reticulocyte lysate in the presence of radioactive proline. Optimal collagen and noncollagen protein synthesis directed by lung poly(A) RNA occurred at 0.7 mm magnesium and 100 mm potassium. The RNA fraction directed the synthesis of both proα₁ and proα₂ chains as determined by polyacrylamide gel electrophoresis. The poly(A) RNA isolated from both lung and dermis polysomes of rats treated with triamcinolone diacetate synthesized significantly less collagen peptides as determined by collagenase digestion as did the RNA isolated from polysomes of nontreated animals. Noncollagen protein synthesis was decreased to a lesser extent than collagen synthesis. Glucocorticoid treatment did not affect the ability of either polysomes in wheat germ extract or polysomal poly(A) RNA in nuclease-treated reticulocyte lysate to synthesize prolyl hydroxylase as determined by immunoprecipitation. These data indicate the glucorticoid-mediated inhibition of collagen polypeptide synthesis does not result from nonspecific effect on total cellular protein synthesis of normal fibroblasts, but a selective reduction of poly some-associated messenger RNA. Furthermore these data provide a molecular basis for selective inhibitory effect of synthetic anti-inflammatory steroids on collagen synthesis.     

Biosynthesis of ribosomal proteins by poly(A)-containing mRNAs from rat liver in a wheat germ cell-free system and sizes of mRNAs coding ribosomal proteins

(1) Poly(A)-containing mRNAs from total polysomal RNA of regenerating rat liver were incubated with [3H]leucine in a wheat germ cell-free system. Ribosomal proteins were purified as described previously [1], and with two-dimensional gel electrophoresis. The proteins on the gel except for less basic protein had appreciable radioactivity, whereas the surrounding areas had very low radioactivity. Acetic acid-soluble proteins labeled in this system were subjected to three-dimensional gel electrophoresis [2]. Except for L1 and L2 proteins, each of the ribosomal proteins, including less basic ones, showed a major radioactive peak coinciding with the protein band on SDS gel. Thus, the wheat germ cell-free system completely translates almost all mRNAs for individual ribosomal proteins. Equimolar amounts of almost all ribosomal proteins were synthesized in the presence of the saturating concentration of mRNAs. (2) Free polysomes from regenerating rat liver were fractionated into three sizes. Each class of polysomes was incubated with [3H]leucine. Ribosomal proteins with molecular weights of 40 000 to 21 000 were mainly synthesized by Fraction B (5-14 monomeric ribosomes), L1 and L2 [2] with 60 000 and 54 000, by Fraction C (greater than 15 monomeric ribosomes) and B, and ribosomal proteins smaller than 20 000 by Fractions A (less than pentamer) and B. (3) mRNAs from rat liver total polysomes were fractionated into seven classes by size and each was translated in the wheat germ extract. Ribosomal proteins with molecular weights of 54 000 to 30 000 were mainly synthesized by mRNAs of 12 to 14.5 S, ribosomal proteins of 35 000 to 22 000 by those of 9.5 to 12 S, ribosomal proteins of 22 000 to 13 000 by those of 7 to 9.5 S, and smaller ribosomal proteins by those smaller than 7 S. These results indicate that individual ribosomal proteins are synthesized by monocistronic mRNAs, the lengths of which are proportional to the molecular weights of the corresponding ribosomal proteins.     

Changes in proteins and isoenzymes in leaves of wheat when infected by stem rust

Summary Biuret assay, gel electrophoresis and immunochemistry were used to study concentrations, forms and activities of proteins of uredospores of Puccinia graminis Pers. f. sp. tritici, in healthy wheat leaves, wheat leaves that had been inoculated with incompatible races of stem rust and leaves which had become rusted.The soluble proteins of primary leaves increased by 25–117% following infection by compatible races of stem rust. There was a corresponding decrease of proteins in uninfected younger leaves. Infection by an incompatible strain of rust led to a temporary 29% increase in soluble proteins.Immunoelectrophoresis and gel electrophoresis of infected leaves showed the presence in them of the forms of malate dehydrogenase, glucose-6-phosphate dehydrogenase, catalase and -amylase characteristic of the rust fungus. In the infected leaves, the activity of certain bands of host glucose-6-phosphate dehydrogenase and catalase changed with the development of the pathogen; the malate dehydrogenase and -amylase of the host were unaffected. In leaves inoculated with an incompatible race there were no obvious changes of any of these enzymes.     

Large-sized polysomes in Chironomus tentans salivary glands and their relation to Balbiani ring 75S RNA

Polysomes from the salivary glands of Chironomus tentans were investigated to determine whether Balbiani ring 75S RNA is incorporated into polysomal structures, and thus probably acts as messenger RNA. A new extraction technique for obtaining ribonucleoproteins was applied that gives a high yield of polysomes with only moderate degradation of the cytoplasmic, high molecular weight RNA. The polysomes sedimented in a broad region (200-2,000S) with a peak value of about 700S, which suggested that they were partly of very large sizes. This was confirmed by visualization of the polysomes in the electron microscope: 400S polysomes contained mainly 11-16 ribosomes, and 1,500S polysomes about 60 ribosomes per polysome. However, polysomes containing 100 or more ribosomes were also observed. It was further established that most of the cytoplasmic 75S RNA was located in polysomes, preferentially in the most rapidly sedimenting ones. From the available information on Balbiani ring RNA in cytoplasm and the present demonstration of 75S RNA molecules in polysomes, it was concluded that at least some Balbiani ring RNA, generated as 75S RNA within the Balbiani rings, eventually enters polysomes without being measurably changed in size. The present information on the potential amino acid coding sequences in 75S RNA is discussed in relation to the large size of the polysomes observed.     

In vivo and in vitro processing of seed reserve protein in the endoplasmic reticulum: evidence for two glycosylation steps

Cotyledons of the common bean (Phaseolus vulgaris L.) synthesize large amounts of the reserve protein phaseolin. The polypeptides are synthesized on membrane-bound polysomes, pass through the endoplasmic reticulum (ER) and accumulate in protein bodies. For a study of the biosynthesis and processing of phaseolin, developing cotyledons were labeled with radioactive amino acids, glucosamine and mannose, and isolated fractions (polysomal RNA, polysomes, and rough ER) were used for in vitro protein synthesis. Newly synthesized phaseolin present in the ER of developing cotyledons can be fractioned into four glycopolypeptides by SDS PAGE. In vitro synthesis with polysomal RNA results in the formation of two polypeptides by polysome run-off shows that glycosylation is a co-translational event. The two unglycosylated polypeptides formed by polysome run-off are slightly smaller than the two polypeptides formed by in vitro translation of isolated RNA, indicating that a signal peptide may be present on these polypeptides. Run-off synthesis with rough ER produces a pattern of four polypeptides similar to the one obtained by in vivo labeling. The two abundant glycopolypeptides formed by polysome run-off. This result indicates the existence of a second glycosylation event for the abundant polypeptides. Inhibition of glycosylation by Triton X-100 during chain-completion with rough ER was used to show that these two glycosylation steps normally occur sequentially. Both glycosylation steps are inhibited by tunicamycin. Analysis of carhohydrate to protein ratios of the different polypeptides and of trypsin digests of polypeptides labeled with [(3)H]glucosamine confirmed the conclusion that some glycosylated polypeptides contain two oligosaccharide chains, while others contain only one. An analysis of tryptic peptide maps shows that each of the unglycosylated polypeptides is the precursor for one glycosylated polypeptide with one oligosaccharide chain and one with two oligosaccharide chains.