PROTEIN SYNTHESIS IN PANCREAS OF FASTED PIGEONS

The regulation of protein synthesis in the pigeon has been studied by comparing the capability of cell-free amino acid incorporating systems of membrane-bound and membrane-free polysomes prepared from fasted and fed birds. New methods were developed for isolating polysomes since techniques used for other tissues did not provide quantitative recovery of polysomal RNA. The sucrose gradient profile of polysomes from pigeon pancreas showed a predominance of trisome species. Although initiation factors are present on polysomes, it was found that polysomes in cell-free systems would not initiate protein synthesis without exogenous initiation factors. This suggested the presence of an inhibitor or regulator of protein synthesis. These studies show that fasting resulted in: (a) decreased amounts of polysomes; (b) disaggregation of polysomes to monosomes; (c) decreased capability of polysomes to synthesize nascent peptides and to initiate additional synthesis, apparently not related to concentration of initiation factors.     

Wounding of Aged Pea Epicotyls Enhances the Reinitiating Ability of Isolated Ribosomes

Total ribosomes (monosomes plus polysomes) isolated from woundedpea epicotyls are more efficient at supporting protein synthesisin a wheat germ S₃₀ system (containing wheat ribosomes) thanare total ribosomes from aged (control) pea tissue. This increasedefficiency is seen when enriched large polysomes, almost devoidof monosomes, are used to program a wheat germ S₃₀₀ system,from which the wheat germ ribosomes have been removed. Reactionsprimed by enriched polysomes from wounded tissue, but not agedtissue, continue for at least 30 min, suggesting that reinitiationis occurring during the reaction, albeit in the initial absenceof monosomes from wheat or pea. Wheat germ ribosomes, but notmonosomes from either aged or wounded pea tissue, are able totranslate pea poly(A) RNA and globin mRNA. Aurintricarboxylicacid reduces protein synthesis in a rather indiscriminate manner,whereas, pactamycin seems to have an inhibitory effect specificfor initiation, and it is much more effective on wounded thanon control tissue polysomes. We interpret these results to implythat polysomal ribosomes from wounded tissue are more efficientat initiation than are polysomal ribosomes from control tissueor than non-polysomal ribosomes (monosomes) from either tissue. (Received May 7, 1985; Accepted July 4, 1985)     

Fate of mRNA following disaggregation of brain polysomes after administration of (+)-lysergic acid diethylamide in vivo

Intravenous injection of (+)-lysergic acid diethylamide into young rabbits induced a transient brain-specific disaggregation of polysomes to monosomes. Investigation of the fate of mRNA revealed that brain poly(A+)mRNA was conserved. In particular, mRNA coding for brain-specific S100 protein was not degraded, nor was it released into free ribonucleoprotein particles. Following the (+)-lysergic acid diethylamide-induced disaggregation of polysomes, mRNA shifted from polysomes and accumulated on monosomes. Formation of a blocked monosome complex, which contained intact mRNA and 40-S plus 60-S ribosomal subunits but lacked nascent peptide chains, suggested that (+)-lysergic acid diethylamide inhibited brain protein synthesis at a specific stage of late initiation or early elongation.     

Accumulation of 70S Monoribosomes in Escherichia coli After Energy Source Shift-Down

When Escherichia coli is shifted from glucose-minimal to succinate-minimal medium, a transient inhibition of protein synthesis and a time-dependent redistribution of ribosomes from polysomes to 70S monosomes occurs. These processes are reversed by a shift-up with glucose. In a lysate made from a mixture of log-phase and down-shifted cells, the 70S monosomes are derived solely from the down-shifted cells and are therefore not produced by polysome breakage during preparation. This conclusion is supported by the absence of nascent proteins from the 70S peak. The monosomes are not dissociated by NaCl or by a crude ribosome dissociation factor, so they behave as "complexed" rather than "free" particles. When down-shifted cells are incubated with rifampin to block ribonucleic acid (RNA) synthesis, the 70S monosomes disappear with a half-life of 15 min. When glucose is also added this half-life decreases to 3 min. The 70S particles are stable in the presence of rifampin when chloramphenicol is added to block protein synthesis. We interpret these data to mean that the existence of the 70S monosomes depends on the continued synthesis of messenger RNA and their conversion to free ribosomes (which dissociate under our conditions) is a result of their participation in protein synthesis. Finally, a significant fraction of the RNA labeled during a brief pulse of (3)H-uracil is found associated with the 70S peak. These results are consistent with the hypothesis that the 70S monosomes are initiation complexes of single ribosomes and messenger RNA, which do not initiate polypeptide synthesis during a shift-down.     

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.     

Degradation of maternal poly(A)-containing RNA during early embryogenesis of an insect (Smittia spec., chironomidae,diptera)

Summary Eggs of the chironomid midgeSmittia spec. were shown to contain maternal rRNA, tRNA and poly(A)-containing RNA. The ribonucleoprotein spectrum consisted of monosomes, ribosomal subunits, and subribosomal particles, whereas polysomes could be detected only in small amounts. Poly(A)-containing RNA was found in different regions of the RNP spectrum, mainly between 15 S and 60 S. After labelling maternal RNA by feeding tritiated uridine to the larvae, the radioactivity associated with poly(A)-containing RNA accounted for about 4% of the label in the total RNA extracted from newly deposited eggs. About half of the radioactivity in the poly(A)-containing RNA was lost between egg deposition and an advanced blastoderm stage. The loss was accompanied by both a decrease in the size of the poly(A)-containing RNA molecules and a shift of poly(A)-containing RNP particles to less dense regions in sucrose gradients. Comparison with poly(A)-containing RNA synthesized by the embryo indicates that the reduction in size of maternal poly(A)-containing RNA is not artifactual but reflects its degradation after the formation of blastoderm.     

Dual effects of the ricin A chain on protein synthesis in rabbit reticulocyte lysate. Inhibition of initiation and translocation

Ricin A chain caused inhibition of protein synthesis by reticulocyte lysate with concomitant depurination of 28S rRNA. The partial reaction(s) of protein synthesis inhibited was investigated by following the appearance of [35S]methionine from initiator [35S]Met-tRNA into 40S ribosomal subunits, 80S monosomes and polysomes. Ricin A chain caused an accumulation of [35S]Met in monosomes which did not enter polysomes. In these respects the effects of the ricin A chain resembled those of diphtheria toxin, an inhibitor of elongation-factor-2-catalyzed translocation. This is consistent with the previously proposed site of action of ricin as an inhibitor of elongation. However, the inhibitory effects of the ricin A chain and diphtheria toxin are not equivalent because we observed that the rate of formation of the 80S initiation complex was reduced approximately sixfold with the ricin A chain relative to diphtheria toxin. Analysis of methionine-containing peptides bound to 80S monosomes in ricin-A-chain-inhibited and diphtheria-toxin-inhibited lysates, programmed with globin mRNA, revealed a predominance of Met-Val, suggesting that the elongation cycle is inhibited at the translocation step. Translocation was also implicated as the step blocked in both the ricin-A-chain-inhibited and diphtheria-toxin-inhibited lysates, by the finding that nascent peptide chains were unreactive towards puromycin. It is concluded that ricin-A-chain-modified ribosomes are deficient in two protein synthesis partial reactions: the formation of the 80S initiation complex during initiation and the translocation step of the elongation cycle.     

Subcellular distribution of ribosomal proteins in Dictyostelium discoideum

The distribution of ribosomal proteins in monosomes, polysomes, the postribosomal cytosol, and the nucleus was determined during steady-state growth in vegetative amoebae. A partitioning of previously reported cell-specific ribosomal proteins between monosomes and polysomes was observed. L18, one of the two unique proteins in amoeba ribosomes, was distributed equally among monosomes and polysomes. However S5, the other unique protein, was abundant in monosomes but barely visible in polysomes. Of the developmentally regulated proteins, D and S6 were detectable only in polysomes and S14 was more abundant in monosomes. The cytosol revealed no ribosomal proteins. On staining of the nuclear proteins with Coomassie blue, about 18, 7 from 40S subunit and 11 from 60S subunit, were identified as ribosomal proteins. By in vivo labeling of the proteins with [35S]methionine, 24 of the 34 small subunit proteins and 33 of the 42 large subunit proteins were localized in the nucleus. For the majority of the ribosomal proteins, the apparent relative stoichiometry was similar in nuclear preribosomal particles and in cytoplasmic ribosomes. However, in preribosomal particles the relative amount of four proteins (S11, S30, L7, and L10) was two- to four-fold higher and of eight proteins (S14, S15, S20, S34, L12, L27, L34, and L42) was two-to four-fold lower than that of cytoplasmic ribosomes.     

Structure and function of rat liver polysome populations. II. Characterization of polyadenylate-containing mRNA associated with subpopulations of membrane-bound particles

Poly(A)+RNA fractions prepared from free and loosely and tightly membrane-bound polysome populations (poly(A)+RNAfree, poly(A)+RNAloose, and poly(A)+RNAtight) were used to drive cDNA in homologous and heterologous hybridization reactions. A large fraction by mass of sequences was shared among the three poly(A)+RNA populations, but shared sequences exhibited distinct frequency distributions within the different populations. 13-15 in vitro translation products of poly(A)+RNAfree and poly(A)+RNAloose detected by gel electrophoresis were shared. Most of these were produced in different relative quantities by the two RNA populations. Five or six higher mol wt polypeptides were produced by poly(A)+RNAloose that were not detected as products of either poly(A)+free or poly(A)+RNAtight. We suggest that loosely bound polysomes may not be artifactually derived as reflected in their quantitatively distinct poly(A)+RNA population. Two tightly membrane-bound RNP fractions were prepared from rat liver on the basis of their release from or retention on purified rough microsomes or a crude membrane fraction after in vitro disaggregation of polysomes with high-salt and puromycin. Homologous and heterologous hybridizations involving their poly(A)+RNA fractions revealed that a large portion by mass of sequences was shared but that these sequences exhibited distinct frequency distributions in the two fractions. The RNA fractions produced exhibited distinct frequency distributions in the two fractions. The RNA fractions produced an identical set of in vitro translation products but individual polypeptides were produced in different relative quantities. This indicates that the two RNP fractions do not arise by any random artifactual process and suggests that they may represent functionally distinct populations.     

Localization of host poly(A)+ mRNA in the ribosome profile of mengovirus-infected Ehrlich ascites tumor cells

The distribution of poly(A)⁺ mRNA among polysomes, monosomes, and ribosome-free supernatant fractions after mengovirus infection of Ehrlich ascites tumor (EAT) cells was investigated employing sucrose gradient centrifugation of their corresponding postnuclear supernatants. Poly(A)⁺ mRNA was isolated from sucrose gradient fractions and quantitated in a cell-free protein synthesizing system from uninfected EAT cells. It was also localized by annealing [³H]-poly(U) to the poly(A)-tracts of mRNA present in the sucrose gradient fractions. Both experiments revealed a gradual shift of host poly(A)⁺ mRNA from large to small polysomes and monosomes, respectively, with the time postinfection. The greatest part of host template RNA appears to remain ribosome-bound and only a fraction seems to be detached from the ribosomes in the course of mengovirus infection. At the end of the infectious cycle, 8 h postinfection, approximately 70% of the poly(A)⁺ mRNA detected in uninfected cells is still biologically active, but not translated in vivo, in agreement with data from the [³H] poly(U) hybridization experiment.     

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.     

Characterization of monosomes produced by aflatoxin B1.

A single injection of 1.5 mg aflatoxin B1 per kg body weight produced approx. 70% disaggregation of rat liver polysomes into monosomes within 18 h. Isolated monosomes dissociated into 40 S subunits during centrifugation in linear sucrose gradients containing 0.3 M KCI. The 4 S to 5 S molar RNA ratio of the monosomes was calculated to be 0.6, indicating 0.6 tRNA and/or aminoacyl tRNA molecule per ribosome; no peptidyl tRNA was present. These results suggest that a single injection of affatoxin B1 produces monosomes which resemble runoff ribosomes.     

Specificity of factors isolated from free polysomes and microsomes on in vitro protein synthesis in plasmacytoma cells

A simple procedure for measuring chain initiation in complete in vitro systems is described. The capacity of free and membrane-bound polysomes prepared by a detergent technique or by nitrogen cavitation to incorporate radioactive amino acids into proteins was compared with the capacity of these polysomes to initiate polypeptide chains.The extent of amino acid incorporation by polysomes prepared by either of these methods did not differ significantly. However, chain initiation determined by measuring the incorporation of radioactive subunits into polysomes showed that polysomes prepared by the detergent technique were less effective than polysomes made by nitrogen cavitation in chain initiation.Crude initiation factors, prepared by washing either free polysomes or microsomes with 0.5 M KCl, stimulated chain initiation and amino acid incorporation by both types of polysomes. Free polysomes were stimulated almost to the same extent by factors isolated from free polysomes or microsomes. Membrane-bound polysomes on the other hand showed a specific requirement for microsomal factors.The extent of stimulation by crude polysomal factors was dependent on the concentration of high speed supernatant in the assay system.     

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

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

Mobilization of newly synthesized RNAs into polysomes inXenopus laevis embryos

Summary The mobilization of newly synthesized 18S and 28S rRNAs, 4S RNA and poly(A)⁺ RNA into polysomes was studied in isolated cells ofXenopus laevis embryos between cleavage and neurula stages. Throughout these stages, 4S RNA and poly(A)⁺ RNA were mobilized immediately following their appearance in the cytoplasm. 18S rRNA however, stayed in the ribosomal subunit fraction for about 30 min until the 28S rRNA appeared, when the two rRNAs were mobilized together at an equimolar ratio. This mobilization, at a 1:1 molar ratio, appeared to be realized at initiation monome formation. Thus, the efficiency of the mobilization of two newly synthesized rRNAs, shortly after their arrival at the cytoplasm, differed considerably but difference disappeared once steady state was reached.The contribution of newly synthesized 18S and 28S rRNAs to polysomes remains small throughout early development. around 3% of newly synthesized 4S RNA is polysomal which is the same distribution observed for unlabeled 4S RNA. Less than 10% of the newly synthesized cytoplasmic poly(A)⁺ RNA was mobilized into polysomes during cleavage, but in later stages the proportion increased to around 20%–25%. These results show that newly synthesized RNAs are utilized for protein synthesis at characteristic rates soon after they are synthesized during early embryonic development. On the basis of the data presented here and elsewhere we discuss quantitative aspects of the utilization of newly synthesized and maternal RNAs during early embryogenesis.     

The cytoplasmic distribution and characterization of poly(A)+RNA in oocytes and embryos of Drosophilia.

Using the presence of poly(A) tracts as a marker for mRNA, we have examined the distribution of this class of RNA between polysomes and free RNP particles. This has been done in mature oocytes and in embryos aged for various times from fertilization through to hatching of a larva. The proportion of ribosomes that are in polysomes to those that are not has been calculated. In mature oocytes, 58% of the poly(A)⁺ RNA and 72% of the ribosomes are not in polysomes. By 1 hr, this drops to 51% of the poly(A)⁺ RNA and 48% of the ribosomes. By 7 hr, a plateau is reached: 30% of each are not in polysomes. The poly(A)⁺ RNA in the cytoplasm of oocytes and 1-hr embryos is found in particles with an average size of 50S and a range of 30–70S. The poly(A)⁺ RNA ranges in size from 7 to 40S, with an average size of 22S. The polyA from this RNA is 50–200 nucleotides long with an average of 115 nucleotides. These data have allowed us to calculate that 1–2% of the total RNA is poly(A)⁺ RNA.     

RNA metabolism and poly(A) distribution in mouse liver following administration of dimethylnitrosamine

Dimethylnitrosamine (DMNA) strongly inhibited RNA synthesis in mouse liver under conditions when the nucleotide pattern, rate of nucleotide synthesis and phosphorylation ratio were unaffected. (An unidentified, probably non-nucleotide, component in the acid-soluble liver fraction was selectively reduced.) The inhibition of RNA synthesis was associated with a decrease in the RNA polymerase activity of isolated liver nuclei, well established already 45 min after DMNA administration. The reduced activity included both Mg2+- and Mn2+/(NH4)2SO4-stimulated polymerase functions. The inhibition in vivo involved the whole complement of RNA, including poly (A)-containing RNA and isolated poly(A) sequences. The transfer of labelled RNA from the nucleus to the cytoplasm was not impaired. There was no detachment of poly(A)-containing RNA from the microsomes, and the proportion of tightly membrane-bound microsomal RNA and poly(A) sequences was not reduced as determined by use of a flotation technique. No breakage or shortening of the poly(A) chains was indicated by sedimentation analysis.