In Vitro Synthesis of Proteins by Membrane-Bound Polyribosomes from Vesicular Stomatitis Virus-Infected HeLa Cells

Membrane-bound polysomes from vesicular stomatitis virus (VSV)-infected HeLa cells synthesize predominantly three proteins in an in vitro protein synthesizing system. These three proteins have different molecular weights than the viral structural proteins, i.e., 115,000, 88,000, and 72,000. Addition of preincubated L or HeLa cell S10 or HeLa cell crude initiation factors stimulates amino acid incorporation and, furthermore, alters the pattern of proteins synthesized. Stimulated membrane-bound polysomes synthesize predominantly viral protein G and lesser amounts of N, NS, and M. In vitro synthesized proteins G and N are very similar to virion proteins G and N based on analysis of tryptic methionine-labeled peptides. Most methionine-labeled tryptic peptides of virion G protein contain no carbohydrate moieties, since about 90% of sugar-labeled peptides co-chromatograph with only about 10% of methionine-labeled peptides. Sucrose gradient analysis of the labeled RNA present in VSV-infected membrane-bound polysomes reveals a relative enrichment in a class of viral RNA sedimenting slightly faster than the total population of the 13 to 15S mRNA, as compared to a VSV-infected crude cytoplasmic extract. A number of proteins, other than the viral structural proteins, are synthesized in the cytoplasm of five lines of VSV-infected cells. One of these proteins has the same molecular weight as the major in vitro synthesized protein, P(88). In vitro synthesized protein P(88) does not appear to be a precursor of viral structural proteins G, N, or M based on pulse-chase experiments and tryptic peptide mapping. Nonstimulated membrane-bound polysomes from uninfected HeLa cells synthesize the same size distribution of proteins as nonstimulated VSV-infected membrane-bound polysomes.     

Direct association of messenger RNA-containing ribonucleoprotein particles with membranes of the endoplasmic reticulum in ethionine-treated rat liver.

The administration of ethionine to female rats causes breakdown of hepatic polysomes. The fate of the mRNA molecules after polysome breakdown was investigated by measuring the amount of poly(A)-containing mRNA in membranous and non-membranous fractions obtained from the cytoplasm of ethionine-treated rat liver. The amount of poly(A)-containing mRNA in the membrane fraction of ethionine-treated liver was found to be the same as that of normal liver. When poly(A)-containing mRNAs from various fractions were translated in a wheat germ system and the products were isolated by immunoprecipitation, the albumin-specific mRNA was found exclusively in the membrane fraction of both normal and ethionine-treated livers. The membrane-bound mRNA in ethionine-treated liver, selectively labeled with [14C]orotate, was banded in CsCl gradient centrifugation at 1.42 g/ml which corresponds to the previously reported mRNA-containing ribonucleoprotein particles. From these results, we concluded that even after the polysome disaggregation by ethionine, most of the mRNA of membrane-bound polysomes remains attached to the endoplasmic reticulum membranes independently of ribosomes and the nascent polypeptide chains.     

Sub-cellular localization of vesicular stomatitis virus messenger RNAs.

Vesicular stomatitis virus (VSV) messenger RNAs (mRNAs) appear to be compartmentalized within the infected HeLa cells. Analysis by polyacrylamide gel electrophoresis in formamide of the RNA associated with the membrane bound polyribosomes from VSV-infected cytoplasmic extracts shows predominantly one size class of VSV mRNA, which is absent from the remaining cytoplasm. These results are consistent with the mRNA for the viral glycoprotein being exclusively associated with membrane bound polysomes since the latter have been shown to synthesize mainly the virion glycoprotein in an $\text{in vitro}$ translation system.     

In Vitro Protein-Synthesizing Activity of Vesicular Stomatitis Virus-Infected Cell Extracts

Crude cytoplasmic extracts from vesicular stomatitis virus (VSV)-infected HeLa cells incorporate radioactive amino acids into hot trichloroacetic acid-precipitable material linearly for 10 to 20 min. The material synthesized in vitro corresponds in molecular weight to four of the five VSV structural proteins. However, synthesis of the viral glycoprotein (G) is significantly reduced, whereas the relative amounts of viral structural proteins L and NS synthesized are increased compared with the ratio of the proteins found in the virion. Fractionation of a VSV-infected crude cytoplasmic extract into a cytoplasmic pellet (20,000 x g for 30 min) and a cytoplasmic supernatant results in a significant reduction in protein synthesizing activity of both fractions, although both contain polysomes. The products synthesized by a cytoplasmic supernatant-directed system included all the VSV structural proteins except the glycoprotein, whereas in an in vitro system directed by the cytoplasmic pellet there is a marked reduction in synthesis of the nucleoprotein (N) and also a small relative increase in synthesis of the glycoprotein. Addition of uninfected, preincubated HeLa or L-cell S10 or a HeLa ribosomal fraction to the VSV-infected cytoplasmic pellet results in a 30- to 60-fold stimulation of (35)S-methionine incorporation. However, these uninfected extracts do not stimulate (35)S-methionine incorporation by the infected crude cytoplasmic extract or the cytoplasmic supernatant. The products synthesized by the stimulated cytoplasmic pellet now include sizeable amounts of the glycoprotein in addition to the other VSV structural proteins.     

Translational control of protein synthesis after infection by vesicular stomatitis virus.

Four hours after infection of BHK cells by vesicular stomatitis virus (VSV), the rate of total protein synthesis was about 65% that of uninfected cells and synthesis of the 12 to 15 predominant cellular polypeptides was reduced to a level about 25% that of control cells. As determined by in vitro translation of isolated RNA and both one- and two-dimensional gel analyses of the products, all predominant cellular mRNA's remained intact and translatable after infection. The total amount of translatable mRNA per cell increased about threefold after infection; this additional mRNA directed synthesis of the five VSV structural proteins. To determine the subcellular localization of cellular and viral mRNA before and after infection, RNA from various sizes of polysomes and nonpolysomal ribonucleoproteins (RNPs) was isolated from infected and noninfected cells and translated in vitro. Over 80% of most predominant species of cellular mRNA was bound to polysomes in control cells, and over 60% was bound in infected cells. Only 2 of the 12 predominant species of translatable cellular mRNA's were localized to the RNP fraction, both in infected and in uninfected cells. The average size of polysomes translating individual cellular mRNA's was reduced about two- to threefold after infection. For example, in uninfected cells, actin (molecular weight 42,000) mRNA was found predominantly on polysomes with 12 ribosomes; after infection it was found on polysomes with five ribosomes, the same size of polysomes that were translating VSV N (molecular weight 52,000) and M (molecular weight 35,000) mRNA. We conclude that the inhibition of cellular protein synthesis after VSV infection is due, in large measure, to competition for ribosomes by a large excess of viral mRNA. The efficiency of initiation of translation on cellular and viral mRNA's is about the same in infected cells; cellular ribosomes are simply distributed among more mRNA's than are present in growing cells. About 20 to 30% of each of the predominant cellular and viral mRNA's were present in RNP particles in infected cells and were presumably inactive in protein synthesis. There was no preferential sequestration of cellular or viral mRNA's in RNPs after infection.     

Cell-free translation of simian virus 40 16S and 19S L-strand-specific mRNA classes to simian virus 40 major VP-1 and minor VP-2 and VP-3 capsid proteins.

Simian virus 40 capsid proteins VP-1, VP-2, and VP-3 have been synthesized in wheat germ and reticulocyte cell-free systems in response to either poly(A)-containing mRNA from the cytoplasm of infected cells or viral RNA purified by hybridization to simian virus 40 DNA linked to Sepharose. All three viral polypeptides synthesized in vitro are specifically immunoprecipitated with anti-simian virus 40 capsid serum. VP-2 and VP-3 are related by tryptic peptide mapping to each other but not to VP-1. The most abundant class of L-strand-specific viral mRNA, the 16S species, codes for the major capsid protein. The relatively minor 19S class directs the cell-free synthesis of VP-1, VP-2, and VP-3. Whether the 19S RNA represents more than one distinct species of mRNA is not yet clear. VP-1 mRNA can be isolated from the cytoplasm, detergent-washed nuclei, and the nuclear wash fraction. The mRNA from the nuclear wash fraction is enriched for VP-2 mRNA when compared to other viral or cellular polypeptides.     

Physical aspects and cytoplasmic distribution of messenger RNA in mouse kidney.

As a prerequisite to examining mRNA metabolism in compensatory renal hypertrophy, polyadenylated RNA has been purified from normal mouse kidney polysomal RNA by selection on oligo(dT)-cellulose. Poly(A)-containing RNA dissociated from polysomes by treatment with 10 mM EDTA and sedimented heterogeneously in dodecyl sulfate-containing sucrose density gradients with a mean sedimentation coefficient of 20 S. Poly(A) derived from this RNA migrated at the rate of 6-7 S RNA in dodecyl sulfate-containing 10% polyacrylamide gels. Coelectrophoresis of poly(A) labeled for 90 min with poly(A) labeled for 24 h indicated the long-term labeled poly(A) migrated faster than pulse-labeled material. Twenty percent of the cytoplasmic poly(A)-containing mRNA was not associated with the polysomes, but sedimented in the 40-80 S region (post-polysomal). Messenger RNA from the post-polysomal region had sedimentation properties similar to those of mRNA prepared from polysomes indicating post-polysomal mRNA was not degraded polysomal mRNA. Preliminary labeling experiments indicated a rapid equilibration of radioactivity between the polysomal and post-polysomal mRNA populations, suggesting the post-polysomal mRNA may consist of mRNA in transit to the polysomes.     

Cytoskeleton involvement in the distribution of mRNP complexes and small cytoplasmic RNAs

These studies were designed to determine whether small cytoplasmic RNAs and two different mRNAs (actin mRNA and histone H4 mRNA) were uniformly distributed among various subcellular compartments. The cytoplasm of HeLa S3 cells was fractionated into four RNA-containing compartments. The RNAs bound to the cytoskeleton were separated from those in the soluble cytoplasmic phase and each RNA fraction was further separated into those bound and those not bound to polyribosomes. The four cytoplasmic RNA fractions were analysed to determine which RNA species were present in each. The 7 S RNAs were found in all cytoplasmic fractions, as were the 5 S and 5.8 S ribosomal RNAs, while transfer RNA was found largely in the soluble fraction devoid of polysomes. On the other hand a group of prominent small cytoplasmic RNAs (scRNAs of 105-348 nucleotides) was isolated from the fraction devoid of polysomes but bound to the cytoskeleton. Actin mRNA was found only in polyribosomes bound to the cytoskeleton. This mRNA was released into the soluble phase by cytochalasin B treatment, suggesting a dependence upon actin filament integrity for cytoskeletal binding. A significant portion of several scRNAs was also released from the cytoskeleton by cytochalasin B treatment. Analysis of the spatial distribution of histone H4 mRNAs, however, revealed a more widely dispersed message. Although most (60%) of the H4 mRNA was associated with polyribosomes in the soluble phase, a significant amount was also recovered in both of the cytoskeleton bound fractions either associated or free of polyribosome interaction. Treatment with cytochalasin B suggested that only cytoskeleton bound, untranslated H4 mRNA was dependent upon the integrity of actin filaments for cytoskeletal binding.     

RNA metabolism and membrane-bound polysomes in relation to globulin biosynthesis in cotyledons of developing field beans (Vicia faba L.).

In cotyledon cells of developing field beans the RNA content per cell does not change in the second half of developmental period 2, whereas globulin biosynthesis continues. The constant RNA content per cell results from an equilibrium between RNA synthesis and degradation. All types of RNA are synthesized until the end of globulin biosynthesis, but poly(A)-containing RNA was preferentially labelled during maximum globulin formation. During stage 2 of seed development of poly(A)-containing RNA fraction represents a discrete peak in the 12--18-S region on agarose gels and corresponds to the peak of poly(A)-containing RNA isolated from polysomes. alpha-Amanitin inhibits selectively the labelling of poly(A)-containing RNA and concomitantly globulin formation. Translation of total poly(A)-containing RNA, free and membrane-bound polysomes in a cell-free wheat germs demonstrates that the globulins are preferentially produced on membrane-bound polysomes and that poly(A)-containing RNA includes the mRNA for both vicilin and legumin.     

Isolation of mRNA from membrane-bound polysomes synthesizing Sepia officinalis haemocyanin in Xenopus laevis oocytes

Isolation and translation in Xenopus laevis oocytes of the mRNAs from the nuclear pellet and the cytoplasmic supernatant of the branchial glands from Sepia officinalis, homogenized in the presence of vanadyl-ribonucleoside complexes, revealed that the membrane-bound polysomes for haemocyanin sedimented together with the nuclei. Centrifugation on a 15-50% sucrose gradient of these polysomes, released by treatment with Triton X-100, yielded a major peak of heavy ones. The messenger fraction, isolated from this heavy fraction, directed the synthesis of the large 390 000 Mr haemocyanin polypeptide chain in oocytes. A large mRNA on heavy membrane-bound polysomes thus synthesizes the whole haemocyanin chain of S. officinalis.     

Studies on the mechanism for entry of vesicular stomatitis virus glycoprotein g mRNA into membrane-bound polyribosome complexes

Glycoprotein mRNA (G mRNA) of vesicular stomatitis virus is synthesized in the cytosol fraction of infected HeLa cells. Shortly after synthesis, this mRNA associates with 40S ribosomal subunits and subsequently forms 80S monosomes in the cytosol fraction. The bulk of labeled G mRNA is then found in polysomes associated with the membrane, without first appearing in the subunit or monomer pool of the membrane-bound fraction. Inhibition of the initiation of protein synthesis by pactamycin or muconomycin A blocks entry of newly synthesized G m RNA into membrane-bound polysomes. Under these circumstances, labeled G mRNA accumulates into the cytosol. Inhibition of the elongation of protein synthesis by cucloheximide, however, allows entry of 60 percent of newly synthesized G mRNA into membrane-bound polysomes. Furthermore, prelabeled G mRNA associated with membrane-bound polysomes is released from the membrane fraction in vivo by pactamycin or mucomycon A and in vitro by 1mM puromycin - 0.5 M KCI. This release is not due to nonspecific effects of the drugs. These results demonstrate that association of G mRNA with membrane-bound polysomes is dependent upon polysome formation and initiation of protein synthesis. Therefore, direct association of the 3' end of G mRNA with the membrane does not appear to be the initial event in the formation of membrane-bound polysomes.     

Labelling kinetics of RNA containing poly(a) in liver subcellular fractions

Kinetics of incorporation of (3H) uridine into cytoplasmic RNA fractions of rat liver is investigated. The fractions include free and membrane bound polysomes, rough membranes sedimenting with mitochondria and free cytoplasmic RNA particles. (1) Poly(A) containing RNA, isolated by oligo-dT cellulose, amounts to 0.4% of the total RNA in the homogenate, 0.5% in bound polysomes, 3.4% in free polysomes and 16% in free cytoplasmic RNA particles. (2) The rate of (3H) uridine incorporation into RNA lacking poly(A) proceeds uniformly in all subcellular fractions except for free cytoplasmic RNA particles, which accumulate negligible amounts of radioactivity. (3) The initial labelling of RNA containing poly(A) is most active in free cytoplasmic RNA particles supporting their identity as mRNA en route to polysomes. The initial specific radioactivities decrease in the following order: homogenate, bound polysomes, rough membranes sedimenting with mitochondria, free polysomes. The data suggest that mRNA is supplied to free and membrane-bound polysomes via different routes. The kinetic analysis indicates that free cytoplasmic RNA particles may be a precursor of mRNA of free polysomes rather than that of bound polysomes. (4) The kinetic differences of free and membrane bound polysomes are also demonstrated by comparing the radioactivity of RNA containing poly(A) to the total radioactivity at various incorporation times. In bound polysomes this decreases from 31% at 1 h to 10% at 25 h, whereas in free polysomes the corresponding ratio increases from 10 to 13%. RNA containing poly(A) of free cytoplasmic RNA particles represents 64% of the total radioactivity throughout the experiment.     

Translation in vivo and in vitro of proteins resembling apoproteins of rat plasma very low density lipoprotein.

Antibodies raised against rat plasma apoVLDL and a purified fraction of arginine-rich peptides (ARP) were labeled with Na125I and were shown to bind to polyribosomes isolated from rat liver. Antibody fractions enriched by selective affinity chromatography exhibited increased levels of binding to polysomes. Anti-apoVLDL immunoreactivity was further resolved into anti-ARP and anti apoB components, each reactive with a distinct polysome population. Binding was specific for rat polysomes, and was directed toward nascent polypeptide chains. About 2% of normal rat liver polysomes were recovered by indirect immunoprecipitation with anti-apoVLDL. Ribonucleic acid (RNA) extracted from this immunoprecipitate contained species with polyadenylate (poly[A] sequences characteristic of eukaryotic messenger RNA (mRNA). These species, purified by affinity chromatography on poly(U)-Sepharose, stimulated the in vitro synthesis of immunoprecipitable apoVLDL-like proteins by about 17-fold when compared to unfractionated rat liver mRNA. Most of the in vitro translation products precipitated by purified anti-ARP migrated identically on polyacrylamide gel electrophoresis with unlabeled purified ARP. Some implications of these findings with respect to plasma VLDL biosynthesis are discussed.     

Synthesis of exported proteins by membrane-bound polysomes from Escherichia coli.

A membrane-bound fraction of polysomes of Escherichia coli has been isolated after lysis of cells without the use of lysozyme. Protein-synthesis studies in vitro show that membrane-bound and free polysomes are different in the following respects. 1. Membrane-bound polysomes synthesize proteins which are exported from the cell. The products include proteins of the outer membrane and a secreted periplasmic protein, the maltose-binding protein. 2. The major product synthesized by free polysomes is elongation factor Tu, a soluble cytoplasmic protein. 3. The activity of membrane-bound polysomes in vitro is more resistant to puromycin than is the activity of free polysomes. In addition, the mRNA associated with membrane-bound polysomes is more stable than the bulk of cellular mRNA as revealed by studies with rifampicin.     

Characterization and translation of methylated and unmethylated vesicular stomatitis virus mRNA synthesized in vitro by ribonucleoprotein particles from vesicular stomatitis virus-infected L cells.

Ribonucleoprotein particles isolated from extracts of vesicular stomatitis virus (VSV) -infected L cells synthesized in vitro four classes of polyadenylated RNA sedimenting at 29S, 19S, 17S, and 13S. When synthesized in vitro in the presence of the methyl donor S-adenosyl methionine, these RNA species contained the following 5'-terminal structures: (i) m7G5ppp5'AmpAp(70%) ; (ii) m7G5'ppp5'AmpAmpNp (20%) and (iii) pppAp (10%). In the presence of the methylation inhibitor S-adenosylhomocysteine, however, the mRNA contained the 5'-terminal structures G5'ppp5'Ap (80%) and pppAp (20%). The mRNA's synthesized in vitro were translated in the homologous ascites and the heterologous wheat embryo cell-free systems. In both, the products were shown by sodium dodecyl sulfate gel electrophoresis and by immunoprecipitation to contain all five viral proteins, L, G, N, NS, and M. The presumed precursor to the G protein (G*) was also identified by fingerprint analysis. Methylated VSV mRNA was more active in protein synthesis than unmethylated mRNA in both the ascites system and the wheat embryo systems. Addition of S-adenosylmethionine stimulated translation of unmethylated mRNA in the wheat embryo but not in the ascites extract. S-adenosylhomocysteine, however, by preventing mRNA methylation inhibited the translation of unmethylated VSV mRNA in both systems. The mRNA methylating activity present in wheat embryo S-30 extracts was recovered in the ribosome-free supernatant fraction (S-150) and was insensitive to the protein synthesis inhibitor pactamycin.     

Cytoplasmic distribution of pulse-labelled poly(A)-containing RNA,particularly 26 S RNA,during myoblast growth and differentiation

Total poly(A)-containing RNA in different polysomal and supernatant cytoplasmic fractions was analysed after pulse-labelling in dividing myoblasts and fused myotubes. In particular, the peak of 26 S RNA (putative messenger for the large subunit of myosin) is located in a light region of the gradient coinciding with the monosome-trisome fractions prior to fusion, and is found in the heavy polysomes only after fusion. These heavy polysomes are free (i.e. not membrane bound). Treatment of the light part of the polysome gradient with EDTA shows that the 26 S RNA found here does not exist as part of a polysomal complex, but is present as a ribonucleoprotein particle cosedimenting in this region. Previous experiments had indicated that in actively dividing myoblasts 26 S RNA has a relatively short half-life but that it becomes “stable” after the cessation of mitosis just prior to fusion. RNA chase experiments performed in the present study show that the “short-lived” 26 S RNA from dividing myoblasts, which is present as a ribonucleoprotein particle, does not enter the heavy polysomes. In contrast, the more stable 26 S RNA also initially present as a ribonucleoprotein, just prior to and in the early stages of fusion, can be shown by chase experiments to enter the heavy polysomes later in fusion. Hence accumulation of 26 S RNA seems to precede its activation as a messenger.     

Comparison of populations of mRNA coding fumarase in rat brain and liver

The populations of mRNA encoding mitochondrial and cytosolic fumarases in the poly A+ RNA fractions purified from polysomes of rat brain and liver were examined. When the in vitro translation products programmed by the poly A+ RNA fraction obtained from rat brain were purified by immunoprecipitation with anti-fumarase antibody and analyzed by SDS polyacrylamide gel electrophoresis and fluorography, only one polypeptide of 50 KD was detected as a precursor of fumarase. In contrast, by the same method, two polypeptides of 50 KD and 45 KD, which is the same size as mature fumarase, were detected as precursors of rat liver fumarase. These results suggest that rat brain polysomes contain only one population of mRNA coding a 50 KD precursor of mitochondrial fumarase with little or no mRNA of the cytosolic fumarase, whereas rat liver polysomes contain two types of mRNA for mitochondrial and cytosolic fumarases, respectively. These findings are consistent with the fact that the brain is the only organ in rats known not to contain cytosolic fumarase.