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PubMed1975

Preferential inhibition by homopolyribonucleotides of the methylation of ribosomal ribonucleic acid and disruption of the production of ribosomes in a rat tumor.

The literature indicates that some mechanism other than the interferon or host-mediated immune enhancement might also be responsible for an antitumor effect of polyinosinate-polycytidylate [poly(I)-poly(C)]. We have examined the effect of this drug on the synthesis of ribosomes and other macromolecules in a rat tumor, the Novikoff ascites hepatoma. The nucleolus was one of the primary targets affected by the administration of poly(I)-poly(C) in vivo. A progressive decline of the activity of nucleolar ribosomal RNA methylases began within 2 hr, followed by a decline of the nucleolar RNA content. The activity of nucleolar RNA polymerase was inhibited only at later time intervals. Labeling of tumor macromolecules in vivo revealed that the methylation of ribosomal RNA and the production of ribosomes, particularly in the small subunits, were immediately and progressively affected, followed by inhibition of the synthesis of DNA, RNA, and protein at later times. In addition, poly(I)-poly(C) also induced disaggregation of polyribosomes and restricted the movements of nuclear RNA to cytoplasm and of cytoplasmic protein to nucleus. These in vivo effects of poly(I)-poly(C) on tumor cells was observed neither on the host livers nor on livers of normal rats. Studies on isolated nucleoli showed that the in vitro addition of polyinosinate and several other compounds actively inhibited tumor ribosomal RNA methylases but were devoid of inhibitory effect against liver ribosomal RNA methylases; these results augment other studies in the literature in suggesting a selective effect of the polyinosinate moiety on tumor cells. We conclude from this study that initial impairment of the methylation of ribosomal precursor RNA, following exposure of tumor cells to poly(I)-poly(C), is responsible for the destruction of ribosomes, preferentially the small subunits, during the maturation processes. Failure to provide new ribosomes thus triggers the events limiting the growth of tumor cells.

Animals↗
PubMed1977

Control of ribosomal RNA synthesis in Escherichia coli. II. Ribosomal RNA synthesis in isolated nucleoids.

The effect of amino acid-starvation on the transcription in vitro of overall RNA and ribosomal RNA was investigated using nucleoids prepared from the exponentially growing and the amino acid-starved cells of rel+ and rel- strains of Escherichia coli. In this system, the synthesis of RNA is exclusively due to elongation of the chains which have been initiated in vivo. The amounts of overall and ribosomal RNA synthesized per unit of DNA in the nucleoids were analyzed for each preparation. The following observations have been made. (1) The total RNA synthesis per unit of DNA in the nucleoids from the amino acid-starved rel+ and rel- cells was not significantly different from each other. (2) The preferential ribosomal RNA synthesis occurred in the nucleoids from the growing cells; the ribosomal RNA synthesis was restricted in the nucleoids from the starved rel+ cells, while no restriction was observed in the nucleoids from the starved rel- cells. The results suggest that the ribosomal RNA synthesis is regulated at the initiation or less likely elongation level of the transcription. (3) A ribosomal RNA of a discrete size of about 30S was synthesized in the nucleoids. No mature ribosomal RNA species was produced in this system. The 30S RNA is probably a primary transcript of ribosomal RNA genes containing 23S, 16S and 5S mature ribosomal RNA sequences.

Cell-Free System↗
PubMed1976

Effect of starvation of the distribution of free and membrane-bound ribosomes in rat liver and on the content of phospholipid and glycogen in purified ribosomes.

The distribution of free and membrane-bound ribosomes in liver in response to starvation has not been clearly defined. An investigation has been made of the effects of starvation on the content of DNA, RNA, protein, phospholipid and glycogen in rat liver, on the distribution of free and membrane-bound ribosomes, and on the content of phospholipid and glycogen in free and bound ribosome fractions. The results indicate that starvation can produce up to a 50% reduction in hepatic ribosomes without altering either the fraction of rRNA relative to the total RNA or the distribution of free and membrane-bound ribosomes. In addition, the degree of contamination of isolated ribosomes with membranous material does not fluctuate with changes in the nutritional status of the animal. The results suggest that the relative capacities for protein synthesis among the two ribosome compartments are maintained during the early stages of starvation. Further, co-sedimentation with glycogen is not responsible for the presence of membranous materials in purified ribosomes.

Animals↗
PubMed1978

Ribosomal proteins in growing and starved Tetrahymena pyriformis. Starvation-induced phosphorylation of ribosomal proteins.

The complements of ribosomal proteins in growing and starved cells of Tetrahymena pyriformis strain GL were examined by two-dimensional gel electrophoresis. In growing cells, the 40-S ribosomal subunit contained 30 proteins, 4 of which migrated toward the anode at pH 8.6, while the 60-S ribosomal subunit contained 46 proteins, 9 of which migrated toward the anode at pH 8.6. When exponentially growing cells were transferred into a non-nutrient medium pronounced phosphorylation of a single 40-S ribosomal subunit protein, S6, was induced. The phosphorylation was very specific; more than 99.5% of the [32P]phosphate incorporated into ribosomal proteins was associated with S6. Phosphate was incorporated into S6 as O-phosphoserine and O-phosphothreonine. Two-dimensional gel electrophoresis indicated that the complement of proteins associated with the ribosomes isolated from starved cells differed from that of growing cells. Careful examination, however, suggested that except for the phosphorylation of certain ribosomal proteins in starved cells, the observed differences did not reflect starvation-induced changes in vivo, but most probably different levels of artifactual modifications (limited proteolysis) during the preparation of the ribosomes.

Animals↗
PubMed1975

Adenosine 3'5'-m onophosphate dependent phosphorylation of ribosomes and ribosomal subunits from bovine corpus luteum.

In a previous publication the purification and properties of two protein kinases (KI and KII) from a soluble fraction of bovine corpus luteum and the stimulation of the latter fol. Chem. 248,494-501). We have now studied the effects oc cyclic AMP and luteinizing hormone on ribosomal protein phosphorylation of corpus luteum by protein kinase II. Protein kinase II catalyzed the phosphorylation of ribosomes by transfer of terminal phosphate of ATP to ribosomal proteinsmextraction with hot trichloroacetic acid and non-aqueous solvent revealed that about 80% of total radioactivity incorporated remain associated with the protein residue. Radioactivity was identified in the phosphoserine and phosphothreonine residues of polypeptides by high voltage paper electrophoresis; The extent of phosphorylation was stimulated by cyclic AMP but not by luteinizing hormonemat least 9 proteins of 80-S ribosomes and 12 proteins of the 60-S ribosomal subunit were phosphorylated in the presence of cyclic AMP as resolved by urea polyacrylamide gel electrophoresis. However, only one major and four minor bands were phosphorylated in the ase of 40-S ribosomal subunit under the influence of cyclic AMP. The ribosomal protein phosphorylation catalyzed by protein kinase II is regulated by cyclic AMP wherease luteinizing hormone has no effect on ribosome phosphorylation.

Animals↗
PubMed1979

Reversible modification of Escherichia coli ribosomes with 2,3-dimethylmaleic anhydride. A new method to obtain protein-deficient ribosomal particles.

Treatment of Escherichia coli ribosomes with the protein reagent 2,3-dimethylmaleic anhydride is accompanied by inactivation of polypeptide polymerization and by dissociation of ribosomal proteins. Regeneration of the modified amino groups at pH 6.0 is followed by reactivation and reconstitution of the ribosomes. Prior to regeneration of the amino groups, ribosomal particles and split proteins can be separated by centrifugation, which allows the preparation of new protein-deficient particles. The ribosomal particles obtained by three successive treatments with 2,3-dimethyl-maleic anhydride at a molar ratio of reagent to ribosome equal to 16,000 lack proteins S1, S2, S3, S5, S10, S13, S14, L7, L8, L10, L11, L12, and L20 and have lost part of proteins S4, L1, L6, L16, and L25. This new procedure to obtain protein-deficient ribosomal particles is mild and might be useful to dissociate other protein-containing structures in addition to ribosomes.

Escherichia coli↗
PubMed1979

Binding of magnesium ions and ethidium bromide: comparison of ribosomes and free ribosomal RNA.

Comparative studies of free ribosomal RNA and ribosomes were made with two probes, Mg++ ions and ethidium bromide, which interact with RNA in different ways. Mg++. E. coli 16 S rRNA and 30 S ribosomes were equilibrated with four different buffers. Equilibration required several days at 4 degrees and several hours at 37 degrees. In all buffers ribosomes bound more Mg than free rRNA, the difference sometimes reaching 20--30%. Ribosomes were more resistant than free rRNA to heat denaturation and their denaturation was more highly cooperative. Ribosomes that bound more Mg++ had higher denaturation temperatures. Ethidium bromide. Fluorescence enhancement studies of ethidium intercalation showed the free 16 S rRNA to have 50--80 binding sites per molecule. A large fraction of these sites were present and accessible in the ribosome, but their ethidium-binding constants were reduced by an order of magnitude. In addition, free rRNA contained a small number of very strong binding sites that were virtually absent in the ribosomes.

Chemical Phenomena↗
PubMed1978

Characterization of the ribosomal binding site in rat liver rough microsomes: ribophorins I and II, two integral membrane proteins related to ribosome binding.

Rat liver rough endoplasmic reticulum membranes (ER) contain two characteristic transmembrane glycoproteins which have been designated ribophorins I and II and are absent from smooth ER membranes. These proteins (MW 65,000 and 63,000 respectively) are related to the binding sites for ribosomes, as suggested by the following findings: i) The ribophorin content of the rough ER membranes corresponds stoichiometrically to the number of bound ribosomes; ii) ribophorins are quantitatively recovered with the bound polysomes after most other ER membrane proteins are dissolved with the nonionic detergent Kyro EOB; iii) in intact rough microsomes ribophorins can be cross-linked chemically to the ribosomes and therefore are in close proximity to them. Treatment of rough microsomes with a low Triton-X-100 concentration leads to the lateral displacement of ribosomes on the microsomal surface and to the formation of aggregates of bound ribosomes in areas of membranes which frequently invaginate into the microsomal lumen. Subfractionation of Triton-treated microsomes containing invaginations led to the recovery of smooth and "rough-inverted" vesicles. Ribophorins were present only in the latter fraction, indicating that both proteins are displaced together with the ribosomes when these aggregate without detaching. Measurements of the ribosome-binding capacity of rough and smooth microsomal membranes reconstituted after solubilization with detergents suggest that ribophorins are necessary for in vitro ribosome binding. Ribophorin-like proteins were found in rough microsomes obtained from secretory tissues of several animal species. The two proteins present in rat lacrimal gland microsomes have the same mobility as hepatocyte ribophorins and cross-react with antisera against them.

Animals↗
PubMed1979

Kinetic studies on ribosomal proteins assembly in preribosomal particles and ribosomal subunits of mammalian cells.

Proteins were isolated from 80-S preribosomal particles and ribosomal subunits of murine L5178Y cells after short and longer periods of incubation with tritiated amino acids. The labeling patterns of ribosomal proteins were compared by two-dimensional polyacrylamide gel electrophoresis. The analysis of isotopic ratios in individual protein spots showed marked differences in the relative kinetics of protein appearance within nucleolar peribosomes and cytoplasmic subunits. Among the about 60 distinct proteins characterized in 80-S preribosomes, 9 ribosomal proteins appeared to incorporate radioactive amino acids more rapidly. These proteins become labeled gradually in the cytoplasmic ribosomal subunits. It was found that one non-ribosomal protein associated with 80-S preribosomes takes up label far more quickly than other preribosomal polypeptides. It is suggested that this set of proteins could associate early with newly transcribed pre-rRNA, more rapidly than others after their synthesis on polyribosomes, and could therefore play a role in the regulation of ribosome synthesis. In isolated 60-S and 40-S ribosomal subunits, we detected five proteins from the large subunit and four proteins from the small subunit which incorporate tritiated amino acids more quickly than the remainder. These proteins were shown to be absent or very faintly labeled in 80-S preribosomal particles, and would associate with ribosomal particles at later stages of the maturation process.

Animals↗
PubMed1977

Studies on native ribosomal subunits from rat liver. Purification and characterization of a ribosome dissociation factor.

A population of free, native ribosomal 40S subunits, that do not react with 60S subunits to form 80S ribosomes, has been identified in the postmicrosomal fraction of rat liver homogenates. A protein (IF-3) has been purified from high salt (0.88 M KCI) extracts of native 40S subunits by gradient centrifugation and by ammonium sulfate fractionation; it prevents the reassociation of subunits and to a limited extent dissociates ribosomes to subunits. The activity is measured by ultracentrifugation of the reaction products on linear sucrose gradients, or with an assay developed in this laboratory that couples dissociation with the 60S-specific peptidyltransferase reaction; the latter procedure measures the amount of 60S subunits released from ribosomes or remaining in incubations in the presence of IF-3. Dissociation factor activity is recovered from most of the particles that are resolved by zonal centrifugation of the total "native subunits" obtained from the postmicrosomal fraction; the highest concentration of IF-3, however, appears to be associated with native 40S subunits. The purified dissociation factor IF-3 is composed of about ten polypeptides and the molecular weight is estimated to be between 500 000 and 700 000, on the basis of glycerol and cesium chloride gradient centrifugation. When purified 40S subunits react with IF-3 or when 80S ribosomes are dissociated by IF-3, a product is formed which is dependent on the concentration of the protein factor and has the characteristics of a 40SIF-3 complex; centrifugation of the complex on sucrose and cesium chloride gradients suggests that the complex consists of 1 equiv of each of the two components. Although dissociation factor IF-3 appears to react in a specific manner with free or ribosome-associated 40S subunits, the reaction with subunits differs in several respects from that with ribosomes. The dissociation factor also appears to interact with 60S subunits but multiple complexes are formed, some with more than 1 IF-3 equiv per 60S particle. The IF-3 converts 40S dimers (55S particles) to the 40S-IF-3 complex and dissociates free, native 80S particles present in the postmicrosomal fraction, but it does not affect polysome-associated ribosomes engaged in protein synthesis.

Animals↗
PubMed2021

RP-REP Ribosomal Profiling Reports: an open-source cloud-enabled framework for reproducible ribosomal profiling data processing, analysis, and result reporting.

Ribosomal profiling is an emerging experimental technology to measure protein synthesis by sequencing short mRNA fragments undergoing translation in ribosomes. Applied on the genome wide scale, this is a powerful tool to profile global protein synthesis within cell populations of interest. Such information can be utilized for biomarker discovery and detection of treatment-responsive genes. However, analysis of ribosomal profiling data requires careful preprocessing to reduce the impact of artifacts and dedicated statistical methods for visualizing and modeling the high-dimensional discrete read count data. Here we present Ribosomal Profiling Reports (RP-REP), a new open-source cloud-enabled software that allows users to execute start-to-end gene-level ribosomal profiling and RNA-Seq analysis on a pre-configured Amazon Virtual Machine Image (AMI) hosted on AWS or on the user's own Ubuntu Linux server. The software works with FASTQ files stored locally, on AWS S3, or at the Sequence Read Archive (SRA). RP-REP automatically executes a series of customizable steps including filtering of contaminant RNA, enrichment of true ribosomal footprints, reference alignment and gene translation quantification, gene body coverage, CRAM compression, reference alignment QC, data normalization, multivariate data visualization, identification of differentially translated genes, and generation of heatmaps, co-translated gene clusters, enriched pathways, and other custom visualizations. RP-REP provides functionality to contrast RNA-SEQ and ribosomal profiling results, and calculates translational efficiency per gene. The software outputs a PDF report and publication-ready table and figure files. As a use case, we provide RP-REP results for a dengue virus study that tested cytosol and endoplasmic reticulum cellular fractions of human Huh7 cells pre-infection and at 6 h, 12 h, 24 h, and 40 h post-infection. Case study results, Ubuntu installation scripts, and the most recent RP-REP source code are accessible at GitHub. The cloud-ready AMI is available at AWS (AMI ID: RPREP RSEQREP (Ribosome Profiling and RNA-Seq Reports) v2.1 (ami-00b92f52d763145d3)).

AMI↗
PubMed1977

Ribosomal RNA genes of Saccharomyces cerevisiae. II. Physical map and nucleotide sequence of the 5 S ribosomal RNA gene and adjacent intergenic regions.

A DNA fragment containing the structural gene for the 5 S ribosomal RNA and intergenic regions before and after the 35 S ribosomal RNA precursor gene of Saccharomyces cerevisiae has been amplified in a bacterial plasmid and physically mapped by restriction endonuclease cleavage and hybridization to purified yeast 5 S ribosomal RNA. The nucleotide sequence of the DNA fragments carrying the 5 S ribosomal RNA gene and adjacent regions has been determined. The sequence unambiguously identifies the 5 S ribosomal RNA gene, determines its polarity within the ribosomal DNA repeating unit, and reveals the structure of its promoter and termination regions. Partial DNA sequence of the regions near the beginning and end of the 35 S ribosomal RNA gene has also been determined as a preliminary step in establishing the structure of promoter and termination regions for the 35 S ribosomal RNA gene.

Base Sequence↗
PubMed1977

The ribosomes of Drosophila. Normal and defective ribosome biosynthesis in Drosophila cell cultures.

The assembly of proteins and RNA into mature ribosomal subunits has been studied in Drosophila cell cultures by pulse-chase experiments. Pulse labeled rRNA has a transit time of 3 h, while the transfer of ribosomal protein occurs completely within 30 min. Inhibition of protein synthesis by cycloheximide results in an almost immediate cessation of ribosome assembly, a result which indicates that no large pool of free ribosomal proteins exists in the cell. Substituting pre-ribosomal RNA with the analogue 5-fluorouridine (5-FU) results in a cessation of ribosome muturation. Under these conditions at least three large subunit proteins continue to accumulate on pre-existing cytoplasmic subunits, indicating an exchange. A portion of ribosomal subunit proteins synthesized in the presence of 5-FU can be recovered in cytoplasmic subunits once the effect of 5-FU has been reversed. This is most easily interpreted in terms of their stabilization on substituted pre-rRNA within the nucleolus, and subsequent utilization on unsubstituted RNA.

Adenosine↗
PubMed1977

On the control of ribosomal protein biosynthesis in Escherichia coli. I. Studies on ribosomal protein biosynthesis in amino acid-starved cells.

The rate of individual ribosomal protein synthesis relative to total protein synthesis has been determined in Escherichia coli rel+ and rel- cells, under valyltRNA deprivation. These strains have a temperature-sensitive valyl-tRNA synthetase. Starvation was obtained following transfer to the cells to non-permissive temperature. Ribosomal proteins were obtained by treatment of either total lysates of freeze-thawed lysozyme spheroplasts or ammonium sulphate precipitate of ribosomes, with acetic acid. Differential labelling of the ribosomal proteins was observed in both strains: proteins from the rel+ strain appear more labelled than those from the rel- strain, the rate of labelling of individual proteins being about the same in both strains. Moreover ribosomal proteins were found as stable during starvation as total protein. It is thus concluded that in starving cells individual ribosomal proteins are not synthesized at equal rates. This indicates that the synthesis of ribosomal proteins is not only under the control of the rel gene.

Amino Acids↗
PubMed1979

Ribosomal proteins synthesis and exchange in the absence of 28-S and 18-S ribosomal RNA synthesis in L5178Y cells.

The effect of the adenosine analogue toyocamycin on ribosomal proteins synthesis and assembly within ribosomal particles was investigated in the murine cells, L5178Y. The analogue was used for periods not exceeding 5 h, at a concentration which permits the synthesis of ribosomal precursor RNA but inhibits the maturation process. The following observations were made: 1. Ribosomal proteins, synthesized de novo in the presence of the drug, were associated with toyocamycin-containing 45-S pre-rRNA in preribosomal-like 80-S ribonucleoproteins which accumulated in the nucleolus. Two-dimensional electrophoresis revealed a full protein complement of these particles, although minor discrepancies were observed in the relative proportions of a limited number of polypeptides. 2. In the absence of 28-S and 18-S rRNA formation, a surprisingly high proportion of newly synthesized ribosomal proteins were incorporated into high-salt washed ribosomal subunits. The extent of individual protein exchange as well as their apparent turnover rates were markedly heterogeneous. Most of these exchangeable proteins were shown to be labeled rapidly in ribosomal subunits of normal cells. Some alternative interpretations of these results are discussed.

Animals↗
PubMed1978

The ribosomes of Plasmodium berghei: isolation and ribosomal ribonucleic acid analysis.

Ribosomes and high molecular weight ribosomal ribonucleic acid (rRNA) from the blood stages of Plasmodium berghei parasites were studied in preparations free from host ribosome contamination. Purified malarial ribosomes were isolated in high yield from a population of ultrastructurally intact, viable parasites by hypertonic lysis with Triton X-100 and differential centrifugation. These ribosomes were shown to be derived from active polysomes and could be dissociated into subunits by puromycin-0.5 M KCl treatment. Malarial rRNA extracted from purified 40S and 60S ribosomal subunits was characterized by electrophoretic, sedimentation and base ratio analyses. Like certain other protozoa, the P. berghei 40S ribosomal subunit possessed an exceptionally large RNA species (mol. wt 0.9 X 10(6), while RNA isolated from the parasite's 60S subunit (mol. wt 1.5 X 10(6)) was specifically 'nicked' to produce one large component (mol.wt 1.2 X 10(6)) and one small component (mol.wt 0.3 X 10(6)) in equimolar quantities. These rRNA's migrate identically on polyacrylamide gels after heating to 63 degrees C for 5 min or under denaturing conditions in the presence of formamide, indicating an absence of aggregation and non-specific degradation of the rRNA species. Base composition studies showed P. berghei rRNA to be low in guanosine and cytosine content, as is the case for protozoa generally.

Animals↗