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.     

A COMPARISON OF THE HETEROGENEOUS NUCLEAR RNA OF HELA CELLS IN DIFFERENT PERIODS OF THE CELL GROWTH CYCLE

HeLa cells synthesize heterogeneous nuclear RNA (HnRNA) in the G₁, S, and G₂ portions of the cell cycle. HnRNA prepared from these various periods was compared by RNA-DNA hybridization experiments. The results indicated that some of the HnRNA molecules were equivalent at all times in the cell cycle, but limitations in the sensitivity of the hydridization reactions, as well as in the spectrum of hybridizing molecules, restrict the conclusions that can be drawn from these comparisons.     

Characterization of RNA synthesis in mid-pachytene spermatocytes of the rat

In this paper the RNA synthesis in mid-pachytene spermatocytes of the rat has been studied. The results show that RNA synthesized by these cells is mostly high molecular weight RNA comparable to HnRNA of somatic cells. In comparison with the rapid metabolism of HnRNA in somatic cells, HnRNA in pachytene spermatocytes is stable and remains in the nucleus for a considerable time. About 30% of this HnRNA contains a poly(A) sequence, although no difference in the rate of metabolism between poly(A)+ and poly(A)− RNA was observed. Based on these results it is suggested that at least a part of the RNA which is synthesized by pachytene spermatocytes is stored in the cells and utilized later during spermatogenesis when the RNA synthesis of the spermatids ceases, but protein synthesis is still active for about 2 weeks.     

A COMPARISON BETWEEN HETEROGENEOUS NUCLEAR RNA AND POLYSOMAL MESSENGER RNA IN HELA CELLS BY RNA-DNA HYBRIDIZATION

Heterogeneous nuclear RNA (HnRNA) and mRNA from cytoplasmic polyribosomes of HeLa cells have been compared by RNA-DNA hybridization tests. 1 µg of HeLa cell DNA binds 0.05–0.10 µg of either HnRNA or mRNA. In addition, HeLa DNA that is preexposed to unlabeled HnRNA was found to have a reduced capacity to bind either HnRNA or mRNA. The results are compatible with considerable sequence similarity in the two types of RNA but, as is discussed, firm conclusions are precluded by imperfections of the hybridization reaction as presently employed.     

A tentative initiation inhibitor of chromosomal heterogeneous RNA synthesis

The nucleoside analogue 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole inhibits labelling of chromosomal, high molecular weight RNA in the salivary gland cells of Chironomus tentans but does not interfere with the synthesis of ribosomal RNA and chromosomal low molecular weight RNA. When DRB2 was added after an initial labelling period (pulse-chase experiment) the radioactivity diminished preferentially in the lower molecular weight region of the HnRNA spectrum. After short chase periods the activity decreased moderately, or even increased, in the higher molecular weight region of the spectrum (75–100 S). After prolonged chases there was an overall and similar reduction in the activity in the whole HnRNA distribution. If the glands were preincubated in DRB for a short period before exposure to radioactive precursors, the label was again diminished more in HnRNA of low molecular weight than in that of higher molecular weight. When α-amanitin or actinomycin D, both known to be inhibitors of RNA chain elongation, replaced DRB in pulse-chase experiments, labelling of HnRNA was depressed in all size classes to the same extent. The accumulated data suggest that DRB acts, in explanted salivary gland cells, at the polymerase level by interfering with the initiation of chromosomal HnRNA synthesis.     

On the size relationship between nuclear and cytoplasmic RNA in sea urchin embryos

The approximate sizes of heterogeneous nuclear (HnRNA) and cytoplasmic RNA of sea urchin embryos were determined by DMSO density gradient centrifugation and acrylamide-formamide gel electrophoresis. The data suggest that the sizes of these molecules are smaller than those estimated under nondenaturing conditions. The size of most of the nuclear RNA ranges from 0.5 to 3 × 10⁶ daltons, while that of the cytoplasmic RNA ranges from 0.1 to 2 × 10⁶ daltons. Both nuclear and cytoplasmic RNA of sea urchin embryos may have a minor fraction (5–10%) of very large species with molecular weights up to 4 to 5 × 10⁶ daltons.The idea that the size of HnRNA may be larger in organisms higher on the evolutionary scale is discussed.     

RNA synthesis in Chinese hamster cells. III. Non-coordinate increases during interphase in synthesis rates for informosomal, polysomal and heterogeneous nuclear RNAs.

Cultured Chinese hamster ovary cells were synchronized by mitotic selection. Relative synthesis rates for informosomal messenger-like RNA (mlRNA), polysomal messenger RNA (mRNA), and heterogeneous nuclear RNA (HnRNA) were estimated from the amount of labeled adenosine or uridine incorporated into these species in early and late interphase. The amounts of uridine incorporated into HnRNA, mRNA, and mlRNA during a pulse administered 9.75-10.75 h post-mitosis were 3.48 4.64, and 2.82 times the amounts incorporated 1.5-2.5 h post-mitosis. Adenosine incorporation values 9.5-11.0 h post-mitosis were 1.64 (HnRNA), 2.49 (mRNA), and 1.18 (mlRNA) times the 1.5-3.0 h values. The realitive incorporation into MRNA of large polysomes corresponded to incorporation into mRNA of smell polysomes. Thus, the synthesis rates of mRNA, mlRNA, and HnRNA increase during interphase in a noncoordinate fashion.     

The behaviour of heterogeneous nuclear ribonucleic acid in partially and completely denaturing conditions

It is shown that the heterogeneous nuclear RNA (HnRNA) synthesized in the presence of actinomycin and at low and high temperatures sediments in low-ionic-strength sucrose gradients between the rRNA components, similar to the unmethylated RNA synthesized under ;step-down' conditions. If the ionic strength is increased then the HnRNA sediments more rapidly than 28S rRNA, with a large proportion about the 45S precursor rRNA position. Initially this was thought to be due to aggregation of the HnRNA; however, centrifugation and electrophoresis in completely denaturing conditions suggest that the molecular weight of this species of RNA is very large The experiments reveal that HnRNA is conformationally unstable relative to the nucleolar RNA and that the slower sedimentation rate of HnRNA in 5mm-EDTA-Tris base-sucrose gradients reflects the greater expansion of the HnRNA relative to the nucleolar RNA. The implications of this finding are discussed.     

Effect of 5-bromodeoxyuridine on heterogeneous nuclear RNA in rat hepatoma cells.

Heterogeneous nuclear RNA HnRNA) was isolated from untreated and 5-bromodeoxyuridine (BrdUrd) treated hepatoma tissue culture (HTC) cells. analysis of this RNA by either electrophoresis on polyacrylamide-agarose gels or centrifugation in sucrose gradients demonstrated that BrdUrd caused a shift in the labeled HnRNA population toward a smaller size distribution. This effect was produced by concentrations of BrdUrd which specifically lower the level of the differentiated enzyme tyrosine aminotransferase, but do not greatly affect cell growth. Differential binding to oligo(dT) cellulose was used to fractionate HnRNA further into classes containing poly(A) (alpha), oligo(A) (beta) or neither category of A-rich sequences (gamma). BrdUrd did not alter the relative rates of uridine incorporation into the three classes. The shift in the labeled HnRNA population due to BrdUrd was observed in all three subclasses of HnRNA.     

Repetitive sequences and hairpin-like structures in giant RNA of pigeon erythroid cells

In giant molecules (>45 S) of HnRNA from pigeon bone marrow and peripheral blood erythroid cells a correlation is demonstrated between the amounts of hairpin-like structures and the sequences transcribed from the DNA repetitions. The same correlation is observed in the >45 S poly(A)⁺ and poly(A)^- subfractions.Abbreviations HnRNA heterogeneous nuclear RNA - poly(A)⁺ RNA RNA molecules containing polyadenylic acid sequences - poly(A)^- RNA RNA molecules which do not contain polyadenylic acid sequences - dsRNA double-stranded RNA - SDS sodium dodecylsulphate     

Rates of RNA chain growth in developing sea urchin embryos

The average time necessary to add a nucleotide onto growing RNA chains (step time) has been determined for several developmental stages of Strongylocentrotus purpuratus embryos. One procedure involved quantitation of isolated nucleosides derived from the 3′ end of newly synthesized chains plus total rates of nucleotide incorporation. The former values provide the number of growing chains so that rates of incorproation per RNA chain may be calculated. A second procedure involves determinations of the times required for RNA molecules of given size classes to become fully labeled. These times may be equated to the synthetic times for the particular size class [Bremer, H., and Yuan, D. (1968). RNA chain growth-rate in Escherichia coli. J. Mol. Biol.38, 163–180]. The step times for blastula and pluteus embryos is 6–9 nucleotides/sec at 15°C, which would result in an average synthetic time for heterogeneous nuclear RNA (HnRNA) molecules of about 2 × 10⁶ daltons of 12–18 min. The half-life of HnRNA in this species of sea urchins is 15–20 min, implying a transient existence for large HnRNA molecules.     

Effect of neuromediating and neuroblockading hormones on the RNA synthesis in the rat liver nucleus

It was shown that rRNA and HnRNA synthesis in rat liver nuclei does not change-within 30 min after intraperitoneal injection of acetylcholine (0.005 mg per 100 g of body weight) but decreases after injection of norepinephrine and epinephrine (0.05 mg per 100 g of body weight). The synthesis of rRNA (but not of HnRNA) increases after injection of hydrocortisone (2,5 mg per 100 g of body weight). The synthesis of HnRNA (but not of rRNA) increases after injection of ACTH1-24 (3 ME per 100 g of body weight) and oxytocin (1 ME per 100 g of body weight). The synthesis of rRNA decreases after injection of propranolol and atropine (0.5 mg per 100 g of body weight). At the same time, the synthesis of HnRNA does not change thereby. The inhibitory effect of propranolol and atropine was corrected by electrostimulation of hypothalamus. The content of cAMP and Ca2+ and the phosphorylation degree of nuclear proteins are increased after stimulation of hypothalamus. The phosphorylation of nuclear proteins is increased by 10(-8)-10(-6) M cAMP. The synthesis of RNA in liver nuclei is increased by 10(-6) M cAMP only after addition of cytosol. In this case the activity of RNA-polymerase II increases in a greater degree than that of RNA-polymerase I + III. It is assumed that the regulatory mechanisms of rRNA and HnRNA synthesis are different. The role of hypothalamus electrostimulation, neurotransmitters, hormones, and cAMP in the mechanisms of RNA synthesis in rat liver nuclei is discussed.     

The action of cordycepin on nascent nuclear RNA and poly(A) synthesis in regenerating liver.

Following a 5 min pulse of [5- 3H]orotic acid via the protal vein, the specific radioactivity of non-poly(A)heterogeneous nuclear RNA (HnRNA) reaches a peak at 12 h after partial hepatectomy. In contrast, poly(A)-HnRNA was maximally elevated only at 2 h after operation. After intraportal injection of cordycepin (3'-deoxyadenosine) 1 min before [5-3H]orotic acid, a dose-dependent inhibition of nuclear HnRNA and rRNA occurred. Fractionation of HnRNA on poly(U)-Sepharose following 20 mg/kg of cordycepin revealed that a 65% reduction occurred in the labeling of poly(A)-HnRNA while non-polyactivity of UTP in control and cordycepin-treated animals indicated no significant alterations in these parameters. Assessment of poly(A) size using poly(A)-HnRNA annealed with oligo(dT)10 as template primer for Escherichia coli DNA polymerase I, showed that 20 mg/kg of cordycepin inhibited nuclear polyadenylylation by 43%; no alteration in the binding of poly(A)-HnRNA to Millipore filters occurred at this dose of cordycepin. These results indicate that cordycepin is a non-selective inhibitor of nuclear RNA and poly(A)synthesis in regenerating rat liver.     

Initiation of poliovirus plus-strand RNA synthesis in a membrane complex of infected HeLa cells.

An in vitro poliovirus RNA-synthesizing system derived from a crude membrane fraction of infected HeLa cells was used to analyze the mechanism of initiation of poliovirus plus-strand RNA synthesis. This system contains an activity that synthesizes the nucleotidyl proteins VPg-pU and VPg-pUpU. These molecules represent the 5'-terminal structure of nascent RNA molecules and of virion RNA. The membranous replication complex is also capable of synthesizing nucleotidyl proteins containing nine or more of the poliovirus 5'-proximal nucleotides as assayed by the formation of the RNase T1-resistant oligonucleotide VPg-pUUAAAACAGp or by fingerprint analysis of the in vitro-synthesized RNA. Incubation of preformed VPg-pUpU with unlabeled nucleoside triphosphates resulted in the formation of VPg-pUUAAAACAGp. This reaction, which appeared to be an elongation of VPg-pUpU, was stimulated by the addition of a soluble fraction (S-10) obtained from uninfected HeLa cells. Preformed VPg-pU could be chased into VPg-pUpU in the presence of UTP. Our data are consistent with a model that VPg-pU can function as a primer for poliovirus plus-strand RNA synthesis in the membranous replication complex and that the elongation reaction may be stimulated by a host cellular factor.