Protein chain initiation in vitro by liver cell components from DMNA-treated rats.

The mechanism of inhibition of protein synthesis in rat liver after dimethylnitrosamine (DMNA) administration was studied at the level of peptide-chain initiation by use of initiation-dependent amino acid incorporating systems. Ribosomal monomers, poly(A)-concontaining loss of acticity due to the DMNA treatment. The poly(A) RNA from monosomes and polysomes, and crude initiation factors from microsomes were prepared 2 h after a single dose of DMNA (75 mg/kg), and their activities in the production of new protein chains determined under conditions of nearly linear response. Monosomes and crude initiation factors from DMNA-treated rats were at least as active as those from controls. Preparations of poly(A)-containing RNA had a consistently higher template activity when prepared from polysomes instead of monosomes. However, in neither case was there any ltaining RNA was methylated by DMNA to about the same extent as the 18S and 28S rRNA. The methylation was consistently somewhat higher in the RNA preparations from monosomes than in those from polysomes.     

Inhibition of cell-free protein synthesis by low-molecular-weight nuclear polyadenylate-containing ribonucleic acid species isolated from the lactating guinea pig.

1. Poly(A)-containing RNA was isolated from the nuclei of mammary gland, liver and brain of lactating guinea pigs. 2. Total nuclear poly(A)-containing RNA from mammary gland inhibited mRNA-directed protein synthesis by a wheat-germ cell-free system. It also inhibited the endogenous activity of the wheat-germ and other cell-free systems. It did not inhibit a wheat-germ cell-free system directed by poly(U). 3. Total nuclear poly(A)-containing RNA from liver and brain did not inhibit the mRNA-directed wheat-germ system. 4. Fractionation of the nuclear poly(A)-containing RNA revealed inhibitory activity in the less than 10 S fraction from mammary gland as well as that from liver and brain. 5. The mechanism of protein-synthesis inhibition appeared to be at the level of elongation. 6. The inhibitory activity could be reversed in a wheat-germ system by increasing the amount of S-30 supernatant. 7. The mechanism of inhibition of protein synthesis is discussed in relation to other RNA species known to inhibit such systems.     

Non-involvement of nucleolytic activities in the early effect of dimethylnitrosamine on the content of free mRNA in mouse liver.

1. After dimethylnitrosamine (DMNA) administration to mice, the content of poly(A)-containing RNA decreases rapidly in the postmicrosomal fraction of the liver. We report here that the loss of free mRNA is not a result of increased nucleolytic activity. On the contrary, a decreased activity of microsomal endonuclease, assayed by its effect on polyribosomal mRNA, was demonstrated already 15 min after the administration of DMNA at 37.5 mg/kg body wt. The loss of activity was more pronounced in the rough than in the smooth membranes. Total detergent-released microsomal nucleases, as assayed by use of labelled poly(U) as substrate, showed a less rapid decline. No corresponding increase in enzyme activities was observed in the postmicrosomal fraction. 2. The dimethylnitrosamine effect on the microsomal endonuclease was not accounted for by altered lysosomal contamination of the microsomal fraction. 3. No early effect of dimethylnitrosamine administration was found on the cytoplasmic ribonuclease inhibitor.     

Selective inhibition of initial polyadenylation in isolated nuclei by low levels of cordycepin 5"-triphosphate.

The effect of cordycepin 5'-triphosphate on poly(A) synthesis was investigated in isolated rat hepatic nuclei. Nuclei were incubated in the absence and presence of exogenous primer in order to distinguish the chromatin-associated poly(A) polymerase from the "free" enzyme (Jacob, S.T., Roe, F.J. and Rose, K.M. (1976) Biochem. J. 153, 733--735). The chromatin-bound enzyme, which adds adenylate residues onto the endogenous RNA, was selectively inhibited at low concentrations of cordycepin 5'-triphosphate, 50% inhibition being achieved at 2microng/ml. At least 80 times more inhibitor was required for 50% reduction in the "free" nuclear poly(A) polymerase activity. Inhibition of DNA-dependent RNA synthesis also required higher concentrations of the nucleotide analogue. These data not only offer a mechanism for the selective inhibition of initial polyadenylation of heterogeneous nuclear RNA in vivo by cordycepin, but also provide a satisfactory explanation for the indiscriminate effect of the inhibitor on partially purified or "free" poly(A) and RNA polymerases.     

Subcellular distribution of total poly (A) -containing RNA and ferritin-mRNA in the cytoplasm of rat liver.

Poly(A)-containing RNA was isolated from rat liver microsomes and from the post-microsomal supernatant fraction. Approximately 15% of total rat liver poly(A)-containing RNA was found to be present in the post-microsomal supernatant. The relative capacity for apoferritin synthesis of each poly(A)-containing RNA preparation was measured in a cell-free system derived from wheat germ. The post-microsomal supernatant fraction was found to be highly enriched with ferritin mRNA and accounted for 40–50% of the total ferritin-mRNA present in the cytoplasm of rat liver.     

Different binding of poly(A)-containing and poly(A)-free fractions of nuclear ribonucleic acid to ribosomes from rat liver.

Total nuclear RNA extracted from nuclei of rat liver cells by phenol/chloroform in the presence of sodium dodecyl sulphate was separated by combined gel filtration on Sepharose 4 B and affinity chromatography on poly(U) Sepharose into fractions differing in their molecular weights and contents of poly(A) sequences. The poly(A)-containing 45-S RNA became labelled most rapidly if rats were administered [3H] orotic acid. This fraction showed a high template activity when added to postmitochondrial supernatants of the Krebs ascites tumour. Fractions of nRNA, free of poly(A) sequences, had no stimulating effect on protein synthesis in this system. The 45-S RNA-containing poly(A) was readily bound to crude polyribosomes from rat liver at 0 degrees C and both ATP and GTP were necessary for this reaction. Sucrose gradient analyses provided evidence that this RNA species is bound predominantly to 80-S ribosomes. No binding was obtained with polyribosomes washed with 0.5 M KCl. The binding ability of washed polyribosomes was restored by the addition of the ribosomal wash fraction or rat liver cytosol. Crude polyribosomes bound significantly lower quantities of nRNA species free of poly(A) when compared with poly(A)-45-S RNA. The label was scattered through the whole ribosomal sedimentation pattern with no predominant peaks and the binding reaction required neither soluble factors nor nucleotide cofactors. The labelling kinetics and high template activity of poly(A)-45-S nRNA indicate that this fraction contains precursors of cytoplasmic mRNA. Requirements for soluble factors and nucleotide cofactors in the binding of this RNA species to 80-S ribosomes suggest that this binding, unlike that of other nRNA species, has a specific mechanism resembling that of mRNA binding during peptide initiation.     

Comparison of poly(A)-containing RNAs in different cell types of the lower eukaryote Schizophyllum commune

Poly(A)-containing RNAs were isolated from morphologically different cells of the fungus Schizophyllum commune. Using mRNA markers the number-average length of poly(A)-containing RNA in total RNA and in purified poly(A)-containing RNA was estimated as 1100 nucleotides. Number-average length of poly(A)-tracts was 33 nucleotides. 2.5% of total RNA is poly(A)-containing RNA and probably up to 7.5% are non-polyadenylated polydisperse RNA sequences. Saturation hybridization of poly(A)-containing RNA to gap-translated [3H]DNA resulted in 16% of the reactive single-copy DNA to become S1 nuclease resistant. It was found that purified poly(A)-containing RNA represented the entire RNA complexity, i.e. 10 000 different RNA sequences in S. commune. RNA sequences isolated from morphologically different mycelia and from fruiting and non-fruiting mycelia were identical for at least 90%.     

Differential effects of cordycepin triphosphate and 9 beta-D-arabinofuranosyladenine triphosphate on tRNA and 5 S RNA synthesis in isolated nuclei.

Although cordycepin 5'-triphosphate (3'-dATP), at low concentrations, preferentially inhibits chromatin-associated poly(A) synthesis in isolated nuclei, higher levels of the inhibitor prevent both rRNA (RNA polymerase I activity) and hnRNA (RNA polymerase II activity) synthesis in vitro (Rose, K.M., Bell, L.E. and Jacob, S.T. (1977) Nature 267, 178-180). The present studies demonstrate that this nucleotide can also inhibit tRNA and 5 S RNA synthesis (RNA polymerase III activity). At 50-200 microgram/ml, 3'-dATP inhibits incorporation of [3H]UTP into tRNA and 5 S RNA by approximately 65%, whereas the syntheses of these RNAs were completely blocked when [3H]GTP was used as the substrate. These data suggest the formation of poly(U) in the tRNA and 5 S RNA regions, which is resistant to 3'-dATP. In contrast, another ATP analog, Ara-ATP, which selectively inhibits poly(A) synthesis, does not block tRNA and 5 S RNA synthesis in isolated nuclei. The production of these RNA species in isolated nuclei is also insensitive to Ara-CTP and 2'-dATP. These data suggest that 3'-dATP exerts general inhibitory effects on RNA synthesis and further substantiate the conclusion that Ara-ATP is a selective inhibitor of the polyadenylation reaction in vitro.     

Isolation and characterization of poly(adenylic acid)-containing messenger ribonucleic acid from rat liver polysomes

Undegraded rat liver polysomes were obtained after homogenizing the tissue in a medium containing NH4Cl, heparine, and yeast tRNA. Purification of poly(A)-containing RNA from polysomal RNA was accomplished by affinity chromatography on oligo(dT)-cellulose columns. Poly(A)-containing RNA molecules were monitored by the formation of ribonuclease-resistant hybrids with [3H]poly(U). To improve the separation of messenger RNA and ribosomal RNA by oligo(dT)-cellulose it was found essential to dissociate the aggregates formed between both molecular species by heat treatment in the presence of dimethylsulfoxide (Me2SO) prior to chromatography. Sucrose gradient analysis under denaturing conditions showed that the preparations obtained were virtually free of ribosomal RNA. Poly(A)-containing RNA constituted approx. 2.2% of the total polysomal RNA and the number average size was 1500--1800 nucleotides, as judged by sedimentation analysis on sucrose density gradients containing Me2SO. Approximately 8.2% of the purified preparation obtained was able to anneal with [3H]poly(U); the number average nucleotide length of the poly(A) segment of the RNA population was calculated to be 133 adenylate residues. Based on these values, our preparations appear to be greater than 90% pure. The RNA fractions obtained after oligo(dT)-cellulose chromatography were used to direct the synthesis of liver polypeptides in a heterologous cell-free system derived from wheat-germ. The system was optimized with respect to monovalent and divalent cations, and presence of polyamines (spermine). More than 65% of the translational activity present in the unfractionated polysomal RNA was recovered in the final poly(A)-containing RNA fraction. However, about 25% of the activity was found to be associated with the unbound fraction which was essentially free of poly(A)-containing RNA. Immunoprecipitation analysis with a specific antiserum to rat serum albumin demonstrated that about 6--8% of the labeled synthetic products translated from the poly(A)-containing RNA sample corresponded to serum albumin. Analysis of the translation products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a heterogeneous distribution of molecular sizes ranging from 15 000 to greater than 70 000 daltons. Spermine not only increased the overall yield and extent of protein synthesis, but also resulted in higher yields of large protein products. Under optimal translation conditions a discrete peak representing about 7% of the total radioactivity was observed to migrate with rat serum albumin.     

Studies on ribonucleic acid and homopolyribonucleotide formation in neuronal, glial and liver nuclei

1. Various types of nuclear preparations, with different ratios of neuronal to glial nuclei, were isolated from guinea-pig cerebral grey matter and ox cerebral grey matter and white matter. Conditions appropriate for the separate assay of RNA and poly A formation were described. Comparative rates of RNA and poly A formation were studied in cerebral and liver nuclei. 2. RNA polymerase activity per nucleus is higher in neuronal nuclei than in glial nuclei. In liver nuclei, the activity is much lower than in cerebral nuclei. The physical relationship between RNA polymerase and deoxyribonucleoprotein seems to differ in neuronal, glial and liver nuclei. 3. Poly A polymerase activity in liver nuclei is selectively activated by Mn(2+) and inhibited by GTP, CTP and UTP. On a DNA basis, the activity in an aggregate enzyme is the same as in intact nuclei. Poly A polymerase activity per nucleus is much higher in liver nuclei than in neuronal nuclei. Glial nuclei show an intermediate activity. 4. It is suggested that, in neuronal nuclei, the synthesis of RNA is more prominent than that of poly A under conditions where both polymers are formed simultaneously. This contrasts with liver nuclei, where more poly A is made than RNA. 5. In neuronal nuclei, the rate of CTP incorporation is much higher than in glial and liver nuclei. This incorporation is most probably due to poly C synthesis.     

Activity of partially purified protein chain initiation factors from the livers of dimethylnitrosamine-treated rats

Partially purified polypeptide chain initiation factors were prepared from the 0.5 M KCl wash of rat liver microsomes. Their activities in connection with dimethylnitrosamine (DMNA)-induced inhibition of protein synthesis were studied by use of the following reactions: (1) poly(U)-directed binding of Phe-tRNA to ribosomes, (2) formation of a GTP-dependent ternary initiation complex with Met-tRNAf, (3) binding of Met-tRNAf to 40-S ribosomal subunits, (4) assembly of a Met-tRNAf containing 80-S ribosomal initiation complex and (5) ribosome-dependent GTPase activity. The inhibition of protein synthesis with DMNA was not associated with a loss of factor activity in any of these reactions. In the binding of Met-tRNAf to 40-S subunits there was a noticeable increase, probably related to the stability of the resulting complex. The Met-tRNA deacylase activity was also increased.     

Inhibition of hepatic deoxyribonucleic acid-dependent ribonucleic acid polymerases by the exotoxin of Bacillus thuringiensis in comparison with the effects of α-amanitin and cordycepin

The action of Bacillus thuringiensis exotoxin, a structural analogue of ATP, on mouse liver DNA-dependent RNA polymerases was studied and its effects were compared with those of alpha-amanitin and cordycepin. (1) Administration of exotoxin in vivo caused a marked decrease in RNA polymerase activity of isolated nuclei at various concentrations of Mg(2+), Mn(2+) and (NH(4))(2)SO(4). A similar action was recorded after addition of exotoxin to isolated nuclei from control or exotoxin-treated mice. (2) Chromatographic separation of nuclear RNA polymerases from mice treated in vivo with exotoxin showed a drastic decrease of the peak of nucleoplasmic RNA polymerase, whereas the peak of nucleolar RNA polymerase remained unaltered. The same effect was observed after administration of alpha-amanitin in vivo, but cordycepin did not alter the relative amounts of the two main RNA polymerase peaks. (3) Administration of exotoxin in vivo did not alter the template activity of isolated DNA or chromatin tested with different fractions of RNA polymerase from control or exotoxin-treated mice. (4) Addition of exotoxin to isolated liver RNA polymerases inhibited both enzyme fractions. However, the alpha-amanitin-sensitive RNA polymerase was also 50-100-fold more sensitive to exotoxin inhibition than was the alpha-amanitin-insensitive RNA polymerase. Kinetic analysis indicated the exotoxin produces a competitive inhibition with ATP on the nucleolar enzyme, but a mixed type of inhibition with nucleoplasmic enzyme. The results obtained indicate that the B. thuringiensis exotoxin inhibits liver RNA synthesis by affecting nuclear RNA polymerases, showing a preferential inhibition of the nucleoplasmic alpha-amanitin-sensitive RNA polymerase.     

Poly(adenylic acid)-containing and -deficient messenger RNA of mouse liver

RNA was isolated and fractionated into poly(A)-containing and -deficient classes by oligo(dT) chromatography. Approximately 99% of the poly(A) material bound to the oligo(dT); that which did not bind contained substantially shorter poly(A) chains. All RNA fractions retained an ability to initiate cell-free translation, with the poly(A)-deficient fraction containing half the total translational activity, i.e., mRNA. Two-dimensional polyacrylamide gel analysis of the cell-free translation products revealed three classes of mRNA: 1, mRNA preferentially containing poly(A), including the abundant liver mRNA species; 2, poly(A)-deficient mRNA, including many mid- and low-abundant mRNAs exhibiting less than 10% contamination in the poly(A)-containing fraction fraction; and 3, bimorphic species of mRNA proportioned between both the poly(A)-containing and -deficient fractions. Poly(A)-containing and bimorphic mRNA classes were further characterized by cDNA hybridizations. The capacity of various RNA fractions to prime cDNA synthesis was determined. Compared to total RNA, the poly(A)-containing RNA retained 70% of the priming capacity, while 20% was found in the poly(A)-deficient fraction. Poly(A)-containing, poly(A)-deficient, and total RNA fractions were hybridized to cDNAs synthesized from (+)poly(A)RNA. Poly(A)-containing RNA hybridized with an average R0t 1/2 approximately 20 times faster than total RNA. Poly(A)-deficient RNA hybridized with an average R0t 1/2 approximately 3-4 times slower than total RNA. These R0t 1/2 shifts indicated that in excess of three-quarters of the total hybridizable RNA was recovered in the poly(A)-containing fraction and that less than one-quarter was recovered in the poly(A)-deficient RNA fraction. Abundancy classes were less distinct in heterologous hybridizations. In all cases the extent of hybridization was similar, indicating that while the amount of various mRNA species varied among the RNA fractions, most hybridizing species of RNA were present in each RNA fraction. cDNA to the abundant class of mRNAs was purified and hybridized to both (+)- and (-)poly(A)RNA. Messenger RNA corresponding to the more abundant species was enriched in the poly(A)-containing fraction at least 2-fold over the less abundant species of mRNA, with less than 10% of the abundant mRNAs appearing inthe poly(A)-deficient fraction.     

Identification of mRNA in the slime mold Physarum polycephalum.

Using a differential extraction procedure which had previously been shown to yield one nucleic acid fraction enriched in cytoplasmic RNA and another enriched in nuclear RNA, we have been able to isolate two polyadenylated RNA populations from microplasmodia of Physarum polycephalum. The poly(A)-containing RNA from the cytoplasmic-enriched fraction accounts for approximately 1.2% of the cytoplasmic nucleic acid, has a number-average nucleotide size of 1339+/- 39 nucleotides, and has been shown, in a protein-synthesizing system in vitro, to be capable of directing the synthesis of peptides which have also been shown to be synthesized in vivo by microplasmodia. The poly(A)-containing RNA from the nuclear-enriched fraction has a number-average nucleotide size of 1533 +/- 104 nucleotides and represents a mixture of cytoplasmic and nuclear adenylated RNA molecules. Based upon these observations, we have identified the polyadenylated RNA isolated from the fraction enriched in cytoplasmic nuclei acid as Physarum poly(A)-containing messenger RNA.     

Primer specificity of ribosome-associated poly(A) polymerase from Ehrlich ascites tumour cells

The reaction product of the ribosomal poly(A) polymerase [ATP(UTP):RNA nucleotidyltransferase] is analyzed. Two systems are used in vitro: (a) isolated polyribosomes with endogenous enzyme and RNA primer and (b) purified enzyme with total polyribosomal RNA as primer. In the polyribosome system about 50% of the [3H]AMP label is in poly(A)-containing mRNA. This RNA displays a heterogeneous size ditribution in the range of 8--30 S with a maximum at about 14 S. Upon denaturation the maximum is shifted towards the 10-S zone. The poly(A) polymerase catalyzes the addition of 12--18 adenylate residues to pre-existing mRNA poly(A) sequences of 40--160 residues. The [3H]AMP incorporated into poly(A)-lacking RNA is mainly in a fraction with an electrophoretic mobility corresponding to 4-S RNA. In the purified enzyme system, specificity towards poly(A)-containing mRNA is lost to a considerable extent. Only 10% of the [3H]AMP label is retained by oligo(dT)-cellulose. The bulk of the product is in 18-S rRNA and heterogeneous small molecular weight RNA. We conclude that the ribosome-associated poly(A) polymerase is most likely the enzyme responsible for the cytoplasmic polyadenylation of poly(A)-containing mRNA in vivo.     

Polyadenylic acid-containing RNA and its polyadenylic acid sequences newly synthesized in isolated embryonic cells of Xenopus laevis.

Newly synthesized polyriboadenylic acid [poly(A)]-containing RNA and its poly(A) sequences were isolated and characterized in Xenopus embryonic cells. Upon sedimentation analysis, the poly(A)-containing RNA labeled for 30 min showed a very heterogeneous size distribution ranging from 9 to >40 S. After 5 hr of labeling, the profile became much less heterogeneous and the main component was distributed in the 9–28 S region. The average molecular weight of 6.5–7.0 × 10⁵ daltons was calculated for the 5-hr labeled RNA. This poly(A)-containing RNA, comprising about 10% of the total labeled RNA, was metabolically stable and accumulated linearly for 5 hr. Gel electrophoresis of the RNA revealed the presence of little or no free poly(A) sequences. Most of the poly(A) sequences, which were isolated from 30-min labeled poly(A)-containing RNA migrated as a single discrete component approximately 150 nucleotides long. In contrast, they were slightly smaller (130 nucleotides long) and more heterogeneous, when obtained from the poly(A)-containing RNA labeled for 5 hr. From these results, it may be likely that the embryonic poly(A)-containing RNA is similar in size to the steady-state population of the poly(A)-containing RNA reported to occur in vitellogenic oocytes and cultured kidney cells of the same species.     

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.