The effect of a new antibiotic, cryptosporiopsin, on RNA synthesis in L-cells.

The effect of cryptosporiopsin on RNA synthesis in L-cells was studied as part of an investigation on the mechanism of action and potential toxicity of the antibiotic in mammalian cells. RNA synthesis in vitro was tested in intact isolated L-cell nuclei, in conjunction with selective inhibitors of nucleolar and nucleoplasmic RNA synthetic activities; It was found that only the nucleoplasmic activity (polymerase II), was inhibited by cryptosporiopsin and that the drug showed no effect on the activity of the nucleolar enzyme (polymerase I). RNA synthesis in vivo was tested using double labelling with I114-C]guanine and [3-H]-uridine in an attempt at discriminating between G+C nucleolar trna and high A+U nucleoplasmic RNA synthesis. Results revealed that the uptake of these precursors into both types of RNA was inhibited by cryptosporiopsin in intact cells. Measurements of the nucleotide pools in these cells indicated that the antibiotic affects uptak and phosphorylation of nucleosides and nucleotides, especially the production of ATP; These results suggest that the uptake inhibition observed in vivo could be due, at least in part, to energy and/or precursor shortage.     

Deoxyribonucleic acid-dependent ribonucleic acid polymerases from murine spleen cells. Increased amounts of the nucleolar species in leukaemic tissue

1. In the spleens of infected mice, the Friend leukaemia virus induces a sharp increase in the ability of subsequently isolated nuclei to incorporate exogenous UTP into an acid-insoluble product. Inhibitor studies indicate that the incremental RNA synthesis proceeds from a DNA template and that both nucleolar and nucleoplasmic activities are involved. 2. The partially purified DNA-dependent RNA polymerases from control and virus-infected tissue are indistinguishable with respect to chromatographic mobility, dependence on bivalent cations, ionic strength, pH and their susceptibility to alpha-amanitin. The RNA polymerases of the murine spleen resemble the enzymes of other mammalian tissue in these properties. 3. A comparison of the amount of polymerase solubilized from normal and infected tissue correlates with the activity observed in assays of the respective nuclei. These experiments indicated that the increase in nucleolar RNA synthesis after infection is mediated by increased extractable polymerase I activity whereas the change in nucleoplasmic RNA synthesis results from an alteration of chromatin or a chromatin-associated factor.     

Decreased synthesis of nucleolar RNA in aging human cells in vitro

Normal human fibroblasts aged during serial passage in cell culture are known to display a reduction in total RNA synthetic activity. We have used “ghost monolayers”, which have been stripped of cytoplasm, in order to determine by autoradiographic techniques whether this decreased synthetic activity in aged cells could be localized to particular structures within the nuclei. Results indicated that the age-related decline in RNA synthesis was entirely due to decreased synthesis of nucleolar RNA, while nucleoplasmic synthesis was unchanged.     

RNA synthesis in newly isolated and cultivated mesophyll protoplasts

Tobacco leaf mesophyll protoplasts exhibit low incorporation of [3H]uridine and 32P into RNA, up to 12 h of cultivation, irrespective of the presence of phytohormones. After 24 h of cultivation a dramatic increase in RNA synthesis is observed; it is the highest in the heterogeneous nucleoplasmic RNA fraction. The protoplasts cultivated in the absence of phytohormones show lower incorporation of precursors.     

Effects of α-Amanitin <Emphasis Type="Italic">in vivo</Emphasis> on RNA Polymerase and Nuclear RNA Synthesis

The fungal toxin α-amanitin given in vivo selectively inhibits for 10–60 min a nucleoplasmic type of RNA polymerase in rat liver but the synthesis in vivo of all species of nuclear RNA remains blocked for several hours.     

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.     

Errors from using 3H-labeled ribonucleoside triphosphates to monitor nuclear RNA synthesis in vitro

The [3H]XTPs are used widely to monitor RNA synthesis in vitro. Recently, we discovered that they reflected only 40-45% of the true rate of nuclear RNA synthesis. Thus, when [8-14C]GTP was used, 1466 pmol [8-14C]GMP was incorporated per mg DNA/10 min. On the other hand, when [8-3H]GTP was used, only 564 pmol [8-3H]GMP was incorporated per mg DNA/10 min. There are three obvious factors that could have contributed to this greater than 2-fold difference in the apparent incorporation rate: commercial [8-3H]GTP sample was contaminated with substances causing the assay medium to be less efficient in RNA synthesis; 3H exchange occurred during acid washing of the [3H]RNA; and there was a greater quenching effect on [3H]RNA. Experiments were designed to test each of these alternatives. We are able to conclude that none of the above three are contributing factors. Our data also show that the 3H label was removed after it was incorporated into RNA. Similar differences were observed when 3H and 14C labeled pairs of ATP, UTP and CTP were compared. Furthermore, when nuclei were fractionated into nucleolar and nucleoplasmic fractions and carried out RNA synthesis, the loss of 3H label was observed mainly from the nucleoplasmic fraction.     

The action of α-amanitin in vivo on the synthesis and maturation of mouse liver ribonucleic acids

α-Amanitin acts in vitro and in vivo as a selective inhibitor of nucleoplasmic RNA polymerases. Treatment of mice with low doses of α-amanitin causes the following changes in the synthesis, maturation and nucleocytoplasmic transfer of liver RNA species. 1. The synthesis of the nuclear precursor of mRNA is strongly inhibited and all electrophoretic components are randomly affected. The labelling of cytoplasmic mRNA is blocked. These effects may be correlated with the rapid and lasting inhibition of nucleoplasmic RNA polymerase. 2. The synthesis and maturation of the nuclear precursor of rRNA is inhibited within 30min. (a) The initial effect is a strong (about 80%) inhibition of the early steps of 45S precursor rRNA maturation. (b) The synthesis of 45S precursor rRNA is also inhibited and the effect increases from about 30% at 30min to more than 70% at 150min. (c) The labelling of nuclear and cytoplasmic 28S and 18S rRNA is almost completely blocked. The labelling of nuclear 5S rRNA is inhibited by about 50%, but that of cytoplasmic 5S rRNA is blocked. (d) The action of α-amanitin on the synthesis of precursor rRNA cannot be correlated with the slight gradual decrease of nucleolar RNA polymerase activity (only 10–20% inhibition at 150min). (e) The inhibition of precursor rRNA maturation and synthesis precedes the ultrastructural lesions of the nucleolus detected by standard electron microscopy. 3. The synthesis of nuclear 4.6S precursor of tRNA is not affected by α-amanitin. However, the labelling of nuclear and cytoplasmic tRNA is decreased by about 50%, which indicates an inhibition of precursor tRNA maturation. The results of this study suggest that the synthesis and maturation of the precursor of rRNA and the maturation of the precursor of tRNA are under the control of nucleoplasmic gene products. The regulator molecules may be either RNA or proteins with exceedingly fast turnover.     

RNA metabolism in nuclei isolated from HeLa cells.

Nuclei with low cytoplasmic contamination, capable of synthesizing RNA for an extended period of time, were prepared from HeLa cells. Besides elongating RNA chains already initiated in vivo, the nuclear preparation initiates the synthesis of new RNA chains. This was shown by labelling the newly synthesized RNA with [gamma-32P]GTP and by detecting the presence of labelled guanosine tetraphosphate among the alkaline hydrolysis products of synthesized RNA. By synthesizing RNA in the presence of each of the four gamma-32P-labelled nucleoside triphosphates, it was possible to conclude that RNA chain synthesis starts predominantly with a purine base. Both nucleolar and nucleoplasmic RNAs are made. The nuclear preparation methylates the nucleolar RNA by utilizing S-adenosyl-L-methionine as a methyl-group donor.     

Effect of 2-allyl-2-isopropylacetamide on poly(adenylic acid)-containing ribonucleic acid.

A single administration of 2-allyl-2-isopropylacetamide, a porphyrinogenic drug, enhanced the 32P-labelling of nucleoplasmic as well as cytoplasmic poly(A)-containing RNA in rat liver. The synthesis of total microsomal RNA is only marginally increased under these conditions. The drug enhances the labelling of a variety of cytoplasmic poly(A)-containing RNA species, and this effect is counteracted by the simultaneous administration of haemin. 2-Allyl-2-isopropylacetamide also enhanced the release of RNA from the nucleus to the cytoplasm.     

The action of alpha-amanitin in vivo on the synthesis and maturation of mouse liver ribonucleic acids

alpha-Amanitin acts in vitro and in vivo as a selective inhibitor of nucleoplasmic RNA polymerases. Treatment of mice with low doses of alpha-amanitin causes the following changes in the synthesis, maturation and nucleocytoplasmic transfer of liver RNA species. 1. The synthesis of the nuclear precursor of mRNA is strongly inhibited and all electrophoretic components are randomly affected. The labelling of cytoplasmic mRNA is blocked. These effects may be correlated with the rapid and lasting inhibition of nucleoplasmic RNA polymerase. 2. The synthesis and maturation of the nuclear precursor of rRNA is inhibited within 30min. (a) The initial effect is a strong (about 80%) inhibition of the early steps of 45S precursor rRNA maturation. (b) The synthesis of 45S precursor rRNA is also inhibited and the effect increases from about 30% at 30min to more than 70% at 150min. (c) The labelling of nuclear and cytoplasmic 28S and 18S rRNA is almost completely blocked. The labelling of nuclear 5S rRNA is inhibited by about 50%, but that of cytoplasmic 5S rRNA is blocked. (d) The action of alpha-amanitin on the synthesis of precursor rRNA cannot be correlated with the slight gradual decrease of nucleolar RNA polymerase activity (only 10-20% inhibition at 150min). (e) The inhibition of precursor rRNA maturation and synthesis precedes the ultrastructural lesions of the nucleolus detected by standard electron microscopy. 3. The synthesis of nuclear 4.6S precursor of tRNA is not affected by alpha-amanitin. However, the labelling of nuclear and cytoplasmic tRNA is decreased by about 50%, which indicates an inhibition of precursor tRNA maturation. The results of this study suggest that the synthesis and maturation of the precursor of rRNA and the maturation of the precursor of tRNA are under the control of nucleoplasmic gene products. The regulator molecules may be either RNA or proteins with exceedingly fast turnover.     

Effects of alpha-amanitin on RNA synthesis by mouse embryos in culture

Investigations were conducted to test the effects of alpha-amanitin on RNA synthesis in preimplantation mouse embryos. Exposure of embryos in culture to 1-100 microgram/ml alpha-amanitin produced a dose- and time-dependence suppression of total RNA synthesis as measured by incorporation of [3H]uridine. Synthesis of polyadenylated RNA in blastocyst-stage embryos was abolished by alpha-amanitin-treatment at concentrations and exposure times that suppressed total RNA synthesis by less than 15%. DNA-dependent RNA polymerase activity was measured in lysates of embryos at several stages of preimplantation development. alpha-Amanitin suppressed total polymerase activity assayed under ionic conditions favorable to the detection of RNA polymerase II. Electrophoretic analyses revealed that preincubation of blastocysts in 100 microgram/ml alpha-amanitin reduced labelling of cytoplasmic 28S and 18S RNA by inhibition of both synthesis and maturation of nucleolar 45SrRNA-precursor. This action of alpha-amanitin on nucleolar RNA synthesis cannot be correlated with the minimal suppression of nucleolar RNA polymerase activity and suggests that the synthesis and processing of rRNA may be under control of nucleoplasmic gene products.     

Radioautographic comparison of RNA synthesis patterns in the epithelial cells of mouse pyloric antrum following 3H-uridine and 3H-orotic acid injections

RNA synthesis was examined in the epithelial cells of the mouse pyloric antrum using radioautography 20 min after injection of either 3H-uridine or 3H-orotic acid. The epithelium of the mouse antrum was known to invaginate into blind tubular units composed of mucous cells arranged from base to top into a gland, an isthmus, and a pit. These were subdivided into segments and, after radioautography, silver grains were counted over cell nuclei in each segment. Following 3H-uridine injection, silver grains were present over all nuclei but were more abundant over those of the isthmus than of the gland or the pit. When nuclei were examined in the electron microscope, nucleoplasmic as well as nucleolar silver grains were more numerous in the isthmus than in the pit or gland. Following 3H-orotic acid injection, silver grains were again present over all nuclei; but maximal incorporation appeared to be in pit cell nuclei where, by electron microscopy, it was mainly assigned to the nucleoplasm. When the incorporation was calculated per whole nucleus, however, it was less in pit cell than in isthmal cell nuclei. Even so, the proportion of label in pit cell nuclei was much greater than after 3H-uridine injection. The interpretation of these findings is based on the fact that isthmal cells are immature, whereas cells migrating from the isthmus to become gland or pit cells show increasing differentiation. The immature cells of the isthmus incorporate both uridine and orotic acid more effectively than do the differentiated cells of pit and gland. Since silver grain counts over nuclei provide an index of the rate of RNA synthesis, this synthesis proceeds more actively in the isthmus than in the pit or gland. This is true of ribosomal RNA synthesis, as shown by nucleolar grain counts, and of other RNA's synthesis, as shown by nucleoplasmic grain counts. It seems, however, that while uridine is involved in the synthesis of all types of RNA, orotic acid is mainly implicated in the synthesis of the heterogeneous RNA from which the messenger RNA arises.     

Quantitative analysis of rat liver nucleolar and nucleoplasmic ribosomal ribonucleic acids.

rRNA from detergent-purified nuclei was fractionated quantitatively, by two independent methods, into nucleolar and nucleoplasmic RNA fractions. The two RNA fractions were analysed by urea/agar-gel electrophoresis and the amount of pre-rRNA (precursor of rRNA) and rRNA components was determined. The rRNA constitutes 35% of total nuclear RNA, of which two-thirds are in nucleolar RNA and one-third in nucleoplasmic RNA. The identified pre-rRNA components (45 S, 41 S, 39 S, 36 S, 32 S and 21 S) are confined to the nucleolus and constitute about 70% of its rRNA. The remaining 30% are represented by 28 S and 18 S rRNA, in a molar ratio of 1.4. The bulk of rRNA in nucleoplasmic RNA is represented by 28 S and 18 S rRNA in a molar ratio close to 1.0. Part of the mature rRNA species in nucleoplasmic RNA originate from ribosomes attached to the outer nuclear membrane, which resist detergent treatment. The absolute amount of nuclear pre-rRNA and rRNA components was evaluated. The amount of 32 S and 21 S pre-rRNA (2.9 x 10(4) and 2.5 x 10(4) molecules per nucleus respectively) is 2-3-fold higher than that of 45 S, 41 S and 36 S pre-rRNA.     

THE ACTION OF α-AMANITIN ON RNA SYNTHESIS IN CHINESE HAMSTER OVARY CELLS : Ultrastructural and Biochemical Studies

α-Amanitin acts in vitro as a selective inhibitor of the nucleoplasmic form B RNA polymerases. Treatment of Chinese hamster ovary (CHO) cells with this drug leads principally to a severe fragmentation of the nucleoli. While the ultrastructural lesions induced by α-amanitin in CHO cells and in rat or mouse liver are quite similar, the results diverge concerning the effect on RNA synthesis. It has been shown that in rat or mouse liver α-amanitin blocks both extranucleolar and nucleolar RNA synthesis. Our autoradiographic and biochemical evidence indicates that in CHO cells high molecular weight extranucleolar RNA synthesis (HnRNA) is blocked by the α-amanitin treatment, whereas nucleolar RNA (preribosomal RNA) synthesis remains unaffected even several hours after the inhibition of extranucleolar RNA synthesis. Furthermore, the processing of this RNA as well as its transport to the cytoplasm seem only slightly affected by the treatment. Finally, under these conditions, the synthesis of the low molecular RNA species (4–5S) still occurs, though less actively. The results are interpreted as evidence for a selective impairment of HnRNA synthesis by α-amanitin in CHO cells.