Transport of messenger RNA into different classes of membrane-associated polyribosomes in Ehrlich-ascites-tumor cells.

The membrane-bound polyribosomes in Ehrlich ascites tumor cells can be separated into a loosely bound and a tightly bound fraction by means of a high salt treatment. Both membrane fractions as well as the free polyribosomes in the supernatant synthesize about the same set of proteins, suggesting a close relationship between these polyribosome fractions in the Ehrlich cell. Relatively high concentrations of cycloheximide do not prevent newly synthesized poly(A)-containing mRNA from entering the tightly bound polyribosome fraction. Nor had treatment of the cells with puromycin in the presence of cycloheximide, which released about 70% of the nascent chains, any significant effect on the entrance of newly synthesized mRNA into tightly bound polyribosomes. These results suggest that in ehrlich ascites tumor cells nascent polypeptide chains are not involved in the binding of polyribosomes to membranes.     

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.     

Three-Dimensional Organization of Polyribosomes–A Modern Approach

Polyribosomes in cells usually have a certain structural organization whose significance has not yet been elucidated. The development of cryo electron tomography has provided a new approach to study polyribosome structure. New data confirm or correct observations made earlier by classical techniques of electron microscopy. The existence of circular and linear (zigzag) topology of polyribosomes was confirmed, and their relationship with the frequently observed tworow forms was clarified. Contacts between ribosomes have been identified in densely packed three-dimensional helical polyribosomes. At the same time, modern cell-free translation systems have opened the possibility of investigating polyribosomes on mRNA of a given structure to elucidate the mechanism of polyribosome structure formation, especially of circular polyribosomes. There is an increasing amount of data supporting the idea of interdependence between polyribosome structure and their translational activity. Moreover, participation of polyribosomes in mRNA transport and localization of protein synthesis in the cell has been shown. Improvement of the resolution and the development of the cryo electron tomography technique for the analysis of polyribosomes in situ will enable further progress in understanding the process of protein synthesis in cells.     

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.     

Changes in ribosome-polyribosome balances in chick muscle cells during tissue dissociation, development in culture, and exposure to simplified culture-medium

The populations of polyribosomes, monomeric ribosomes, and ribosomal subunits are described from the time of tissue explantation to the time of complete muscle differentiation in primary cultures of chick muscle cells. There is extensive degradation of polyribosomes, and a net loss of ribosomes recovered, as cells of embryonic muscle are dissociated with proteolytic enzymes. The cells rapidly restore a high polysome: monomeric ribosome ratio. This recovery of the polyribosome population occurs before there is any detectable net increase in ribosome number. Ribosome production begins after a lag of approximately 15 hours in culture. Number of ribosomes/cell triples by 60 hours, at which time cell fusion (myotube formation) is complete. Unlike developing muscle in vivo, cultured cells have a very reduced pool of monomeric ribosomes. Medium simplification experiments done with fully differentiated cultures show, however, that monomers accumulate during starvation. These monomers reassociate to form polyribosomes during medium replenishment. Subunit complements are maintained at a constant level regardless of nutritional conditions. These features of cultured muscle are discussed as possible tools for further study of muscle development.     

Indirect immunoprecipitation by rat liver polyribosomes using antibodies to tyrosine aminotransferase]

A fraction of rat liver polyribosomes is isolated, which in its immunochemical characteristics considerably enriched with polyribosomes capable to synthesize hydrocortisone-induced liver tyrosine aminotransferase isoenzyme. This specific polyribosome fraction was purified by immunochemical fractionation of total liver polyribosomes using indirect precipitation. The content of polyribosomes in immunoprecipitates comprise 0.4-0.8% of its initial amount (before immunochemical fractionation). The ratio of specific polyribosomes in immunoprecipitates varies from 20 to 45%, which corresponds to 25-100-fold purification. The data obtained suggest that the method of indirect precipitation can be an efficient step in the isolation procedure of individual mRNA.     

Changes in the population of polyribosomal containing red cells of peripheral blood of rainbow trout, Salmo gairdneri Richardson, following starvation and bleeding

Peripheral blood of trout contained two populations of red cells: those with polyribosomes located in the cytoplasm, and those without polyribosomes. Starvation of trout for 30 days was accompanied by a proportional decline of the polyribosomal-containing (PRC) red cells. One week after a 15% bleeding of both fed and starved animals fed individuals showed a proportional decline of PRC red cells whilst starved fish showed a proportional increase of the same cell population. In fed individuals the bleeding response was accompanied by the appearance of many red cells with senescence-related characteristics. PRC cells in both groups of animals were arbitrarily subdivided into three subgroups according to the density of polyribosomes present. No statistically demonstrable differences were evident between the means of the three PRC cell groups of control animals and those subjected to starvation and bleeding. However, there was an apparent rise in the proportion of red cells with the highest density of polyribosomes as a result of both treatments.     

The 5' end of the pea ferredoxin-1 mRNA mediates rapid and reversible light-directed changes in translation in tobacco

Ferredoxin-1 (Fed-1) mRNA contains an internal light response element (iLRE) that destabilizes mRNA when light-grown plants are placed in darkness. mRNAs containing this element dissociate from polyribosomes in the leaves of transgenic tobacco (Nicotiana tabacum) plants transferred to the dark for 2 d. Here, we report in vivo labeling experiments with a chloramphenicol acetyl transferase mRNA fused to the Fed-1 iLRE. Our data indicate that the Fed-1 iLRE mediates a rapid decline in translational efficiency and that iLRE-containing mRNAs dissociate from polyribosomes within 20 min after plants are transferred to darkness. Both events occur before the decline in mRNA abundance, and polyribosome association is rapidly reversible if plants are re-illuminated. These observations support a model in which Fed-1 mRNA in illuminated leaves is stabilized by its association with polyribosomes, and/or by translation. In darkness a large portion of the mRNA dissociates from polyribosomes and is subsequently degraded. We also show that a significant portion of total tobacco leaf mRNA is shifted from polyribosomal to non-polyribosomal fractions after 20 min in the dark, indicating that translation of other mRNAs is also rapidly down-regulated in response to darkness. This class includes some, but not all, cytoplasmic mRNAs encoding proteins involved in photosynthesis.     

Some effects of antibiotics on bacterial polyribosomes as studied by gel electrophoresis

Bacterial polyribosomes possess characteristic electrophoretic mobilities in agarose-acrylamide composite gels. In cells whose normal protein synthesis is inhibited by certain antibiotics, the resolution of the gel electrophoresis technique has permitted the detection of specific increases in the mobility of the polyribosomes. Antibiotics producing these changes in polyribosome mobility include inhibitors of the 30 S as well as the 50 S subunit.The in vivo action of streptomycin has been studied in some detail. Streptomycin alters the polyribosomes of sensitive strains, haploid as well as heterodiploid, but does not alter polyribosomes of strains resistant to or dependent upon streptomycin. Streptomycin-altered polyribosomes are stable in vivo for more than one hour and exhibit a considerably prolonged run-off time following rifampicin treatment. They are also significantly more resistant to the in vitro RNase degradation than control ribosomes. The subunit composition ($\text{50}\text{S}\text{30}\text{S}$) of the altered polyribosomes remains unchanged from the control (1:1).Since the electrophoretic mobility of monosomes remains unchanged during the antibiotic treatment, the evidence presented suggests that the alteration of polyribosome mobility involves a stacking of the ribosomes on mRNA.     

Protein synthesis during the transition from the resting to the growing state in suspension cultures of Paul's Scarlet rose cells

Cells derived from Paul's Scarlet rose ( Rosa sp. ) were grown in the chemically defined medium of Nesius. When a stationary phase culture was diluted with fresh medium, growth was initiated after a pronounced lag period. DNA replication, as revealed by thymidine labeling and autoradiography, did not begin until 36 h, and mitotic figures were not observed until 48 h after dilution. A 10–15 fold increase in the rate of protein synthesis occurred during the lag period. This was brought about by a 3.5 fold increase in the amount of ribosomal RNA per cell, plus a doubling of both the percentage of ribosomes that are present as polyribosomes and the average number of ribosomes per polyribosome. The spectrum of polypeptides synthesized by these cells during the lag and early log periods of growth was examined. Polyribosomes were extracted from the cells at intervals preceding and accompanying the initiation of proliferative growth. The polyribosomes were translated in a wheat germ cell-free protein synthesizing system and the ³⁵S-methionine-labeled translation products were separated on polyacrylamide slab gels and by 2-dimensional gel electrophoresis. Comparatively few differences were observed between stationary phase, lag phase and log phase cells in terms of the spectrum of polypeptides synthesized in vitro. However, these various phases of the growth cycle could be characterized by a relatively high rate of synthesis of a few specific polypeptides. That is, while most proteins are synthesized throughout the growth cycle and even in non-growing cells at approximately the same relative rates, there are a few variable proteins whose synthesis marks a particular phase of the growth cycle.     

Localization of Polyribosomes Containing Alkaline Phosphatase Nascent Polypeptides on Membranes of Escherichia coli

A procedure has been developed for extracting membranes from bacterial cells under conditions that keep a large fraction of bacterial polyribosomes intact. Freeze-thawing spheroplasts in the presence of deoxyribonuclease, followed by differential centrifugation, permits a separation of free and membrane-associated polyribosomes. The latter fraction contains as much as 40% of cell ribosomal ribonucleic acid (RNA) and 55% of cell messenger RNA (mRNA). Nascent polypeptides were divided almost equally between the two fractions, but 70 to 80% of alkaline phosphatase nascent chains, detected both chemically and immunologically, were derived from polyribosomes associated with the bacterial membrane. Analysis of the fractions for mRNA specific for the lac and trp operons by RNA-deoxyribonucleic acid hydridization showed somewhat larger amounts on membrane than on free polyribosomes, but enrichment for nascent alkaline phosphatase (a secreted protein) on membranes was consistently greater, suggesting that polyribosomes making secreted proteins are more tightly bound to membranes. Electron micrographs of the membrane preparations show relatively intact membranes with clusters of polyribosomes on their inner surfaces.     

Phytochrome-mediated assembly of polyribosomes in etiolated bean leaves. Evidence for post-transciptional regulation of development.

Light operating through phytochrome controls the proportion of total ribosomes present as polyribosomes in etiolated leaves of Phaseolus vulgaris. Similar responses but with slightly different time courses are elicited by brief red light treatment and by continuous far-red or white light. The increase in polyribosome proportions after red light treatment reaches a maximum within 2 h, after which the proportion steadily declines. Light treatment appears to lead to increased proportions of polyribosomes in higher size classes. This is most evident with continuous white light, but probably also occurs with red and far-red light. The increase in polyribosomes is due principally to cytoplasmic ribosomes although proportionately greater effects are observed in chloroplast ribosomes. Although cordycepin inhibits RNA synthesis by 85-90% within 3 h there is no depression of the light-mediated increase in polyribosome proportions when leaves are preincubated in the inhibitor for 3 h. The data therefore indicate that phytochrome is capable of controlling protein synthesis, and thus development, at a post-transciptional level.     

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.     

Polyribosomes associated with microfilaments in cultured lens cells

Epithelial hamster lens cells, transformed by SV40 can be grown in suspension culture. Triton X-100 extraction of these cells grown under conditions when ribosome run off is blocked releases about 40% of the total amount of polyribosomes, designated as free- and loosely-bound polyribosomes. The Triton ghosts retain the remaining polysomal population which can be released by a combined treatment with deoxycholate and Nonidet P 40. Electron microscopic examination of the ghosts reveals microfilament-associated ribosome clusters next to a fraction of polysomes still attached to membranes. Preincubation of the cells with cytochalasin D prior to polyribosome isolation enables us to discriminate between these two latter polysome populations. The experiments indicate that about 25% of the polyribosomes are attached to microfilaments, while the remaining 35% are tightly bound to the membranes of the endoplasmic reticulum. When the different polyribosome classes were translated in a reticulocyte lysate, no significant differences could be observed in the patterns of the newly synthesized polypeptides. In all cases actin was one of the major products synthesized de novo.     

Occurrence, isolation, and characterization of polyribosomes in yeast

This report details the procedural requirements for preparing cell-free extracts of yeast rich in polyribosomes. This enabled us to demonstrate the occurrence of polyribosomes in yeast, to show their role in protein synthesis, and to devise methods for their resolution and isolation. When certain precautions are met (the use of log phase cells, rapidly halting cell growth, gentle methods of disruption, sedimentation through exponential density gradients, etc.), individual polyribosome size classes ranging up to the heptosome can be fractionated and separated from their nearest neighbors. Larger size classes are resolved partially among themselves, free of smaller polyribosomes. This was confirmed by extensive electron micrographic studies of material from the various fractions obtained upon density gradient centrifugation of yeast extracts. Modifications of the gradients and procedure should allow fractionation and isolation of the larger polyribosomes, including those containing polycistronic messages. Yeast polyribosomes are disaggregated to single ribosomes by longer term grinding, cell disruption by the French pressure cell, the Hughes press, or by incubation with dilute RNAse. Yeast polyribosomes are active in the incorporation of amino acids into polypeptide; the single ribosomes exhibit only slight activity. The latter activity is probably due to the presence of a small fraction of monosomes still containing mRNA. Poly-U stimulates amino acid incorporation only in the single ribosomes.     

Autodegradation of rat liver polyribosomes in vitro]

In vihro decay of rat liver polyribosomes is studied under conditions of occuring phosphorolytic activity of polynucleotidephosphorylase (PNPase) and, in contrast, excluding PNPase activity (in the absence of orthophosphate). The rate of polyribosome degradation is found to be higher in the presence of orthophosphate, than in its absence. Sedimentation coefficient of ribosomes formed from degraded polyribosomes in the absence of orthophosphate is 70S, while that of ribosomes formed under phosphorolysis is 67S. It is suggested that polyribosome degradation in the presence of orthophosphate is carried out by the combined action of 5'-RNase and PNPase. 5'-RNase degrades mRNA and rRNA without disturbing the integrity of ribosomal complexes, while PNPase phosphorolyzes the fragments formed from the 3'-OH terminal, successively releasing ribosomes. The rate of polysome inactivation in the presence of orthophosphate (half-life period being approximately 17 min) is considerably higher than that in the absence of orthophosphate (half-life period being 37 min). The amount of active polysomes in samples incubated for 3 hours both in the presence and in the absence of orthophosphate is 16% and 24% of the total amount of active polysomes, respectively. It is suggested that there are a certain type of polyribosomes, which comprise about 8% of polyribosomes that can be inactivated only by PNPase.     

The selectivity and stoicheiometry of membrane binding sites for polyribosomes, ribosomes and ribosomal subunits in vitro.

Differences in the binding sites for polyribosomes, template-depleted ribosomes and large ribosomal subunits were found in microsomal derivatives of the rough endoplasmic reticulum. 1. The stoicheiometry of polyribosome and ribosome interaction in vitro with membranes was shown to be influenced by the relative concentration of interactants and the duration of their mixing. Large ribosomal subunits required a more prolonged mixing schedule to achieve saturation of membranes than did polyribosomes. 2. By using a procedure which minimized the effects on binidng by the stoicheiometric variables, competition between populations of polyribosomes, ribosomes and subunits for membrane sites showed that subunits, and to a lesser extent ribosomes, failed to block polyribosome attachment. 3. Polyribosomes isolated from liver, kidney and hepatoma 5123C entirely bound to a common membrane site, but some polyribosomes from myeloma MOPC-21 bound to other sites, perhaps influenced by their unique nascent proteins. 4. Subunit-binding sites appear on rough membranes only after endogenous polyribosomes have been removed, but no evidence that resulting changes in surface constituents are responsible was found. Large-subunit binding was largely abolished by lowering MgC12 concentration of 0.1 mM, whereas under the same conditions polyribosome binding was undiminished. 5. The large-subunit site appears to be distinct from the polyribosome site not only in the restriction of its affinity for particles but also spatially, to the extent that bound subunits do not hinder access of polyribosomes to their sites.     

The interaction between newly-formed mRNA and ribosomal particles during retarded translation]

The formation of polyribosomes in mouse liver cells at the reduced-rate translation was studied by treatment with cycloheximide (CHI) and aurintricarboxylic (ATA) acid. An increase of polypeptide synthesis time by 1.7-2.7 times (0.5 mg CHI per 25 g of weight or 15 mg ATA per 25 g) leads to a delay of the entrance of newly formed cytoplasmic D-RNA into polyribosomes. These results are in agreement with the model of polyribosome formation from ribonucleoprotein precursors containing cytoplasmic D-RNA. On the other hand, in the presence of a CHI dose (5 mg/25 g) causing a dramatic (240-fold) increase of polypeptide synthesis time, the kinetics of entrance of newly formed D-RNA into polyribosomes does not differ from the normal one, and amount of the incorporated mRNA is even somewhat higher than under normal conditions. It is suggested that in this situation ribosomes are moving along the newly formed mRNA, and their movement is not accompanied by the synthesis of completed polypeptide chain.     

Combined use of oligo(dt) and 28S cDNA probes for the quantitation of total mRNA in polyribosomes: Application to the castration-induced atrophy of the rat prostate

The castration-induced atrophy of the rat prostate was used as a model for the validation of a sensitive technique allowing the quantitation of total mRNA in polyribosomes. Electron micrographs of polyribosome samples showed a decrease in polyribosomes length 7 days after castration (GDX). Specificity of labeled oligo(dt) probe for poly(A) was demonstrated and the technique was successfully applied to demonstrate that GDX is associated with a decrease in poly(A) mRNA content of polyribosomes. Provided that normalization of the hybridization signal for mRNA is achieved with a rRNA cDNA probe, the assay therefore represents a suitable tool for further studies regarding the translational regulation of total and/or specific mRNAs.