Free and membrane-bound polyribosomes in normal and Rauscher-virus-infected mouse spleen cells

1. Free and membrane-bound polyribosomes and ribosomal monomers were isolated from normal and Rauscher-virus-infected mouse spleens by means of discontinuous sucrose density gradients. 2. The addition of ribonuclease inhibitor from rat liver was essential to protect these polyribosomes from degradation. To separate the smooth and rough membranes from ribosomal monomers an additional centrifugation step through a continuous sucrose density gradient was necessary. 3. After infection a marked increase in rRNA from both membrane-bound and free polyribosomes was observed. Treatment of the membrane-bound polyribosomes with sodium deoxycholate yielded only 80S particles even when ribonuclease inhibitor was added. 4. A striking feature of the infected spleen was the occurrence of large polyribosomes. Up to 40 monomers per polyribosome could be counted on electron micrographs.     

The effect of pressure on fetal and neonatal liver mitochondrial membranes

The effects of pressure on late fetal and neonatal rat liver mitochondria have been investigated. High hydrostatic pressure, as produced by isopycnic centrifugation in sucrose and glycogen gradients, altered the mitochondrial membranes of 1- and 7-day-old rats. Most of the mitochondrial enzymes, chosen for their known submitochondrial location, had a trimodal distribution in the sucrose gradients. In the glycogen gradients, a shift of the mitochondria to a lower density was noticed. Fetal liver mitochondria were resistant to the hydrostatic pressure exerted during isopycnic centrifugation experiments under different conditions such as sucrose and glycogen density gradients. The submitochondrial compartment tracer enzymes exhibited an unimodal distribution. Experimental temperatures set at 15 degrees C had a protective effect from hydrostatic pressure alterations in the neonatal liver mitochondria, whereas no effects were noticeable in the fetal mitochondria. Experiments in a hydraulic compression chamber showed that outer membranes of fetal mitochondria were more fragile and the inner membranes more resistant to compression than in the early stages after birth.     

Polyribosome size analysis. Measurement of number-average polyribosome sizes

The analysis of translational efficiencies of specific mRNAs requires a determination of the polyribosome size. The appropriate value to use in such calculations is the number-average size. A method is described for accurately measuring the number-average size of total and of specific protein synthesizing polyribosomes using isokinetic sucrose density gradients and 125I-labeled antibodies. By this method, we demonstrated that albumin synthesizing polyribosomes from a serum albumin secreting mouse hepatoma cell line exist over a broad range from trimers to 20-mers (mean 6-10). The specificity of antibody interaction with polyribosomes was demonstrated using cells not synthesizing mouse serum albumin, and by demonstrating that 125I-anti ovalbumin does not bind to mouse hepatoma polyribosomes. Treatment of the mouse hepatoma cells with 1 MUM cycloheximide shifted practically all of the monomers into polyribosomes resulting in an increase in the number-average size of the albumin synthesizing polyribosomes. Cycloheximide treatment, however, did not eliminate the size heterogeneity in the albumin synthesizing polyribosomes.     

Differential effects of iron and inflammation on ferritin synthesis on free and membrane-bound polyribosomes of rat liver.

We have examined the distribution of ferritin mRNA to free and endoplasmic reticulum (ER)-bound liver polyribosomes during inflammation and iron treatment of rats. Postnuclear tissue supernatants were fractionated on a discontinuous sucrose gradient developed to separate free and bound polyribosomes. Total RNA recovered averaged 3.2 mg/g tissue, 40% of which was with ER and 30% with the free polyribosomes, about 25% being with the postribosomal/RNP fraction. Slot-blot hybridization of equal portions of RNA revealed that 12 h after injection of turpentine to induce inflammation, ferritin mRNA was concentrated on the ER-bound polyribosomes, while it was concentrated on the free polyribosomes 2 h after injection of ferric ammonium citrate. Differences were highly significant, based on multiple determinations and densitometry. Profiles of ferritin mRNA distribution on linear sucrose gradients corroborated the differential findings. Concentrations of total ferritin mRNA per gram liver doubled with iron treatment but were not significantly different 12 h after turpentine treatment. At the same time point after turpentine, ferritin protein synthesis was increased twofold, as measured by the 1 h incorporation of [14C]leucine. We conclude that a significant portion of ferritin mRNA always associates with the ER-bound polyribosomes, and that inflammation and iron differentially alter the polysomal distribution of ferritin mRNA, suggesting that two different kinds of mRNA may be involved.     

Alterations in the protein synthetic apparatus of Friend erythroleukemia cells during their erythroid differentiation

The changes in rate of protein synthesis and cell division and the distribution of polyribosomes and globin mRNA on the polyribosomes of Friend erythroleukemia (FL) cells exposed to 2% DMSO and maintained at low cell density, were examined at different times after exposure to DMSO. The rate of protein synthesis and the capacity of cells to divide declined in concert to 50% of the level found in untreated cell cultures at 24 hours after exposure. Thereafter these rates recovered to 70% of the rate found in untreated control cultures until 96 hours post-exposure and then irreversibly declined as the cells lost the capacity to divide. The proportion of ribosomes present as polyribosomes in cells exposed to DMSO paralleled the capacity of these cells to synthesize protein. The distribution of polyribosomes analyzed by sedimentation in sucrose gradients demonstrated that a discrete, abundant class of polyribosomes composed of pentamers to heptamers appeared as early as 48 hours after exposure to DMSO. The appearance of an abundant class of polyribosomes was correlated with globin synthesis by demonstrating that a discrete class of polyribosomes arises in cells treated with the inducers hexamethylene bisacetamide and hemin.     

Polyribosomal structures inactive for protein synthesis present in erythroid cells during terminal stages of differentiation.

During the terminal stages of differentiation nucleated erythroid cells from the fetal mouse synthesize hemoglobin at a lower rate because after the last cycle of cell division about half of their polyribosomal structures are rendered inactive for protien synthesis though they maintain their aggregated shape. Partially inactive polyribosomes are tested in comparison with normal polyribosomes for the capacity to support polypeptide chain synthesis in cell-free conditions. The following observations are made: a) no difference is found for the profile on sucrose density gradients; b) partially inactive polyribosomes carry growing polypeptide chains in reduced amounts in comparison with active polyribosomes; c) partially inactive polyribosomes are not capable to release "run off" 80 S ribosomal monomers and to dissociate to active ribosomal subunits. These data are interpreted as the evidence for a block of chain termination producing inactivation of polyribosomes during the late maturation of nucleated erythroid cells.     

Characterization of Late Polyoma mRNA

Polyoma-infected mouse kidney cell cultures were labeled with [³H]uridine for 3 h late in the lytic cycle (26 to 29 h after infection) and RNA was extracted from cytoplasm and nuclei and from isolated polyribosomes. Sedimentation velocity analysis in sucrose gradients showed that polyoma-specific “giant” and 26S RNAs are present exclusively in the nucleus. RNA associated with cytoplasmic polyribosomes was analyzed by sedimentation in aqueous sucrose density gradients and dimethylsulfoxide sucrose gradients, as well as by polyacrylamide gel electrophoresis. Polyoma-specific RNA in polyribosomes consists of at least two classes, with sedimentation coefficients of 16 (major fraction) and 19S (minor fraction) in aqueous sucrose gradients and 15 and 17S, respectively, in dimethylsulfoxide gradients. Estimates based on dimethylsulfoxide gradient and analysis suggest a molecular weight of approximately 500,000 for 16S RNA and 700,000 for 19S RNA. These polyoma RNAs seem to undergo reversible conformational changes under the different conditions of analysis. We cannot exclude the possibility that they contain more than one molecular species.     

Isolation of ribonuclease-free intact polyribosomes from rat kidney.

High levels of RNAase present in rat kidney have prevented isolation of intact polyribosomes from this tissue. This problem has been circumvented by a thorough in situ arterial perfusion of rat kidney, coupled with homogenization of the perfused rat kidney in heparin and detergents-fortified high-speed supernatant prepared from rat liver. This procedure reduced RNAase activity in the homogenate by as much as 70%. Sedimentation of the polyribosomes from this homogenate through a layer of 2.0 M sucrose resulted in a 78--80% yield of polyribosomes from the rat kidney. The resulting polyribosomal pellet contained less than 8% of the RNAase activity present in polyribosomes from non-perfused rat kidney. The remaining RNAase activity was separated from the larger polyribomes by sucrose density gradient centrifugation. The majority of the polyribosomes were larger than tetramers. This procedure also incrased both the yield and size of polyribosomes from rat and mouse liver.     

Differences in sequence content of nuclear and cytoplasmic polyribosomal RNA from adenovirus-infected cells.

RNA molecules from nuclear and cytoplasmic polyribosomes of adenovirus-infected HeLa cells were compared by hybridization to analyse the sequence content. Nuclear polyribosomes were released by exposure of intact detergent-washed nuclei to poly(U) and purified. Cytoplasmic polyribosomes were also purified from the same cells. To show that nuclear polyribosomes contain ribosomes linked by mRNA, polyribosomes were labelled with methionine and uridine in the presence of actinomycin D in adenovirus-infected cells. Purified nuclear polyribosomes were treated with EDTA under conditions which dissociate polyribosomes into ribosomes and subunits with a simultaneous release of mRNA, and sedimented. The treatment dissociated these polyribosomes, releasing the mRNA from them. Radiolabelled total RNA from each polyribosome population was fractionated in sucrose gradients into several pools or hybridized to intact adenovirus DNA to select virus-specific RNA. Sucrose-gradient-fractionated pool-3 RNA (about 28S) and virus-specific RNA were then hybridized to fragments of adenovirus DNA cleaved by restriction endonucleases SmaI, HindIII and EcoRI by the Southern-blot technique and by filter hybridization. The results showed that nuclear RNA contained sequences, from about 0 to 18 map units, which were essentially absent from cytoplasmic RNA. Furthermore, the amount of virus-specific RNA for a particular sequence was also different in the two populations.     

Effect on lysosomes of invertase endocytosed by rat-liver

The intracellular localization of invertase endocytosed by rat liver was investigated by analytical centrifugation in sucrose and Percoll gradients of mitochondrial fractions originating from rats killed 15 h after injection. After isopycnic centrifugation in a sucrose gradient, invertase is located in higher density zones than acid hydrolases. The difference between the distribution of invertase and that of acid hydrolases increases with the amount of invertase injected. When the invertase dose is sufficiently high, a change of lysosomal enzyme distribution is clearly visible. It consists in the shift of a proportion of these enzymes to higher density regions where invertase is located. The proportion of hydrolase activity affected by invertase is different for each enzyme measured; it is the least pronounced for acid phosphatase, and most for acid deoxyribonuclease and arylsulfatase. A pretreatment of the rat with Triton WR 1339 considerably decreases the equilibrium density of structures bearing invertase. Nevertheless invertase distribution is quite distinct from that of the bulk of lysosomal enzymes that are recovered in lower density zones of the gradient; on the other hand the invertase injection to rats treated with Triton WR 1339 causes a spreading of the acid hydrolase distribution towards higher density zones. The distribution of acid hydrolases and invertase in a Percoll gradient depends on the sucrose concentration of the solvent. It is shifted towards higher densities when the sucrose concentration increases. The phenomenon is more important for invertase. These results are best explained by supposing that invertase accumulates in a distinct population of lysosomes that can be individualized as a result of the density increase they are subjected to by the invertase they accumulate. It is proposed that these lysosomes mainly originate from non-parenchymal cells of the liver.     

Polyribosome distribution in regenerating livers of irradiated adrenalectomized rats.

The effect of gamma-radiation (1800 rad) on polyribosome distribution in the regenerating livers of intact and adrenalectomized rats was studied both before and after partial hepatectomy. The animals were divided into four sub-groups: (1) control; (2) irradiated only; (3) partially hepatectomized; and (4) irradiated partially hepatectomized. The relative distribution of lighter oligosomes to heavier polyribosomes was analysed by sucrose density gradient centrifugation (10-40 per cent). Partial hepatectomy by itself increased the proportion of heavier polyribosomes in both intact and adrenalectomized rats. However, when gamma-rays were delivered 2 hours before or 2 hours after partial-hepatectomy, the formation of heavy polyribosomes was decreased for at least 24 hours after hepatectomy in the adrenal intact rats. This depression was not maintained until later times (48 and 72 hours after hepatectomy) when an increase in the proportion of heavy polyribosomes relative to light oligosomes was observed. In addition, irradiation did not measurably affect the distribution of polyribosomes in regenerating hepatocytes of adrenalectomized rats at any time after partial hepatectomy.     

Requirement of protein synthesis for the degradation of host mRNA in Friend erythroleukemia cells infected wtih herpes simplex virus type 1.

We describe experiments which demonstrate that shortly after infection of Friend erythroleukemia cells with herpes simplex virus (HSV), polyribosomes dissociate and cellular mRNA degrades. Analysis of infected cell extracts on sucrose density gradients demonstrates that the majority of the polyribosomes have dissociated to monoribosomes at 2 h postinfection. Physical measurements of infected-cell RNAs support this conclusion and demonstrate that the polyadenylated RNAs decrease in size. The degradation of mRNA is apparently a stochastic process as judged by the failure to detect a shift in the Crt1/2 when polyadenylated RNA extracted from infected cells at different times is hybridized to globin complementary DNA. In experiments designed to determine whether dissociation of polyribosomes is sufficient to cause degradation of globin mRNA, the amount of globin mRNA in uninfected cells did not change when cells were treated with NaF or pactamycin at concentrations sufficient to dissociate all polyribosomes. In cells infected with UV-irradiated virus polyribosomes dissociate but globin mRNA does not degrade, suggesting that it is possible to separate dissociation from degradation.     

Translational regulation of ferritin synthesis in rat spleen: effects of iron and inflammation

Translational control of ferritin synthesis was studied in rat spleen, and compared with that for liver, heart and brain, in response to iron and inflammation. Spleen concentrations of total RNA in the ribonucleoprotein (mRNP) fraction was comparable to that for liver, while polyribosomal RNA was less. Both fractions were ten-fold lower in heart and brain. In untreated animals, the mRNP fraction of all tissues had the largest portion of the ferritin mRNA, as determined by slot blot hybridization with 32P-labeled cDNA for the L subunit. Acute treatment with ferric ammonium citrate shifted the spleen ferritin mRNA to the polyribosome fraction. This was also so in liver but not in the heart and brain which took up much less iron. The findings were confirmed by hybridization studies of mRNPs and polyribosomes separated in sucrose gradients. Turpentine-induced inflammation also caused a shift in ferritin mRNA from the mRNP to the polyribosome fraction of spleen and liver, over 12 h. We conclude that as in liver, spleen ferritin synthesis is under translational control by iron, and that both tissues also respond to inflammation by shifting of ferritin mRNA to the polyribosomes.     

Proteins associated with the messenger ribonucleoprotein particle for the estrogen-regulated apolipoprotein II mRNA.

The stability of the mRNA for apolipoprotein (apo) II is regulated by estrogen [Gordon et al. (1988) J. Biol. Chem. 263, 2625-2631]. On the hypothesis tha estrogen regulation of apoII mRNA stability is mediated through mRNA-protein interaction, we have examined the messenger ribonucleoprotein particle (mRNP) for apoII mRNA following release from chicken liver polyribosomes. Polyribosomes containing undegraded apoII mRNA were obtained when tissue was homogenized without detergent, and polyribosomes were isolated following simultaneous addition of detergent and magnesium to a 20000g supernatant. ApoII mRNP released by EDTA sedimented at 12-18 S in sucrose gradients, and banded at rho = 1.4 g/mL in CsCl isopycnic centrifugation, indicative of a 3:1 ratio of protein to mRNA. A fraction in which apoII mRNP was enriched to 40-50% of total mRNP was prepared by successive size fractionation steps on sucrose gradients. Proteins associated with sucrose gradient enriched apoII mRNP were examined by iodination of UV-cross-linked proteins followed by SDS-polyacrylamide gel electrophoresis. Comparisons of proteins in highly enriched apoII mRNP to proteins in mRNP from non-estrogen-treated rooster liver did not reveal any differences. This result suggests that the major proteins associated with apoII mRNA are mRNP proteins also associated with the bulk of liver mRNAs.     

Eukaryotic Polyribosome Profile Analysis

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase or the elongation phase by a variety of cellular stresses such as amino acid starvation or growth factor withdrawal. Alternatively, translation of individual mRNAs can be regulated by mRNA localization or the presence of cognate microRNAs. Studies of protein synthesis frequently utilize polyribosome analysis to shed light on the mechanisms of translation regulation or defects in protein synthesis. In this assay, mRNA/ribosome complexes are isolated from eukaryotic cells. A sucrose density gradient separates mRNAs bound to multiple ribosomes known as polyribosomes from mRNAs bound to a single ribosome or monosome. Fractionation of the gradients allows isolation and quantification of the different ribosomal populations and their associated mRNAs or proteins. Differences in the ratio of polyribosomes to monosomes under defined conditions can be indicative of defects in either translation initiation or elongation/termination. Examination of the mRNAs present in the polyribosome fractions can reveal whether the cohort of individual mRNAs being translated changes with experimental conditions. In addition, ribosome assembly can be monitored by analysis of the small and large ribosomal subunit peaks which are also separated by the gradient. In this video, we present a method for the preparation of crude ribosomal extracts from yeast cells, separation of the extract by sucrose gradient and interpretation of the results. This procedure is readily adaptable to mammalian cells.     

Isolation and Characterization of Two Cell Types of Coxiella burneti Phase I

Two morphologically distinct cell types of Coxiella burneti phase I have been separated on the basis of unique buoyant densities. When centrifuged to equilibrium in cesium chloride or density gradients of sucrose or Renografin, the cells band in two zones. Electron micrographs of ultrathin sections of the two cesium chloride-separated cell types indicate a considerable number of morphological differences. The lower-density cells are small, compact, and rodshaped and have very dense nucleoids. The cell type of highest density is larger, rounded, and more pleomorphic, and the nucleoid filaments are more dispersed. The two cell types are nearly identical in sedimentation rates, and both infect chick yolk sac cells and are lethal to chick embryos. They convert to a mixture of cell types when cultured separately. Treatment with Formalin induces all cells to band at the same position when centrifuged to equilibrium in cesium chloride. The cell type variance was found to be independent of the antigenic phase phenomenon of C. burneti.     

Association of selenium with tissue membranes of ovine and rat tissues

Rats and lambs were injected with⁷⁵Se-selenite and the microsomal fraction obtained by differential centrifugation from homogenates of muscle, heart, liver, brain, kidney, and testes. These fractions were subsequently centrifuged on sucrose density gradients from 25 to 45% and the distribution of selenium-75 between the labeled membranes of different densities monitored by scintillation counting. The majority of the selenium-75 was associated with membranes of similar density in muscle, heart, liver, and testes from both rats and lambs. The selenium-75 was distributed between many different density membranes in brain from both lambs and rats. The selenium-75 was associated predominantly with one density membrane from ovine kidney but with several density membranes from rat kidney.     

Biosynthesis in vitro of tryptophanase by polyribosomes from induced cultures of Escherichia coli

1. Polyribosomes were isolated from Escherichia coli grown in media in which tryptophanase is induced and in which it is repressed. The polyribosomes from the induced bacteria had a small amount of tryptophanase activity associated with them. 2. A portion of the enzyme activity remained bound to polyribosomes during centrifuging in sucrose gradients. 3. Incubation of tryptophanase-containing polyribosomes with puromycin released enzyme activity. 4. The binding of the enzyme to the polyribosomes did not depend on the presence of DNA. 5. When the polyribosomes were incubated under conditions of protein synthesis with supernatant fraction obtained from repressed bacteria, a small but statistically significant increase in enzyme activity was produced. 6. When a radioactive amino acid was included in the incubation mixture for the tryptophanase system a radioactive protein was obtained whose chromatographic, electrophoretic and sedimentation properties were identical with those of tryptophanase. 7. The amount of incorporation was consistent with the amount of new enzyme synthesis predicted by the increase in enzyme activity. Both radioactive incorporation and increase in enzyme activity were shown to be energy-dependent and also negative controls were obtained by using zero-time incubations or polyribosomes isolated from either repressed cells or a mutant lacking the ability to produce tryptophanase. 8. The distribution of radioactive leucine in the carboxyl region of the newly labelled tryptophanase was examined by digesting the labelled protein with carboxypeptidases. It was shown that the radioactivity was more highly concentrated towards the carboxyl terminus when the incubation times for protein synthesis were shorter (implying that, with longer incubation times, longer lengths of polypeptide chain contained radioactive amino acid residues).     

Linking mRNA turnover and translation: assessing the polyribosomal association of mRNA decay factors and degradative intermediates

mRNA decay is a multistep process, often dependent on the active translation of an mRNA and on components of the translation apparatus. In Saccharomyces cerevisiae, several trans-acting factors required for mRNA decay associate with polyribosomes. We have explored the specificity of the interactions of these factors with polyribosomes, using sucrose gradient sedimentation analysis of the yeast UPF1 protein to test whether such interactions are altered when polyribosomes are disrupted by treatment with EDTA, digestion with micrococcal nuclease, or shifting of cells containing a temperature-sensitive eIF3 mutation to the nonpermissive temperature. These experiments, as well as others assaying the strength of factor association in high-salt sucrose gradients, lead us to conclude that Upf1p is tightly bound to the smallest polyribosomes, but not to the 40S or 60S ribosomal subunits. Similar experimental approaches were used to determine whether mRNA decay initiates prior to mRNA release from polyribosomes. Using sucrose gradient fractionation and Northern blotting, we can detect the polysomal association of a PGK1 mRNA decay intermediate and conclude that mRNA decay commences while an mRNA is still being translated.     

Association of D-RNA with Polyribosomes in the Soybean Root

Ribosomes from soybean root tips were shown to consist of about 75% polyribosomes based on size distribution on sucrose gradients. Electronmicrographs showed the presence of ribosome clusters in normal preparations and only monomers and dimers in ribonuclease-treated preparations. The polyribosome preparations, but not single ribosomes, showed good in vitro amino acid incorporating activity.