Effect of hypophysectomy on liver nuclear ribonucleic acid synthesis in aging rats.

Changes in RNA synthesis in liver nuclei were observed at different ages and after hypophysectomy and hormone replacement in female Sprague-Dawley rats. As determined by the incorporation of [3H]UMP into an acid-insoluble product, RNA synthesis decreased by about 75% in intact rats from 6 months to 24 months of age. This decline with age was not observed in liver nuclei from 24-month-old rats that had been hypophysectomized at 12 months and maintained on a minimal hormone-replacement therapy. Thyroid hormones and somatotropin (growth hormone) had an additive effect on RNA synthesis in liver nuclei from these hypophysectomized rats. The same hormones had no significant effect on intact, age-matched rats. With advancing age, nuclei of intact rats had an increase in the pool of free RNA polymerase and an apparent decrease in the enzyme activity bound to nuclear chromatin. There was no change in total enzyme with age. In hypophysectomized, hormone-treated rats, free RNA polymerase activity decreased and chromatin-bound activity increased. There was no difference in total nuclear RNA polymerase activity between operated or intact rats. However, the ratio of the bound to the free activity was different. These results suggest that the ability of RNA polymerase to bind to chromatin may be involved in the age-related decrease in liver nuclear RNA synthesis of intact rats.     

Regulation of influenza virus RNA polymerase activity by cellular and viral factors.

An in vitro RNA synthesis system mimicking replication of genomic influenza virus RNA was developed with nuclear extracts prepared from influenza virus-infected HeLa cells using exogenously added RNA templates. The RNA synthesizing activity was divided into two complementing fractions, i.e. the ribonucleoprotein (RNP) complexes and the fraction free of RNP, which could be replaced with RNP cores isolated from virions and nuclear extracts from uninfected cells, respectively. When nuclear extracts from uninfected cells were fractionated by phosphocellulose column chromatography, the stimulatory activity for RNA synthesis was further separated into two distinct fractions. One of them, tentatively designated RAF (RNA polymerase activating factor), stimulated RNA synthesis with either RNP cores or RNA polymerase and nucleocapsid protein purified from RNP cores as the enzyme source. In contrast, the other, designated PRF (polymerase regulating factor), functioned as an activator only when RNP cores were used as the enzyme source. Biochemical analyses revealed that PRF facilitates dissociation of RNA polymerase from RNP cores. Of interest is that virus-coded non-structural protein 1 (NS1), which has been thought to be involved in regulation of replication, counteracted PRF function. Roles of cellular factors and viral proteins, NS1 in particular, are discussed in terms of regulation of influenza virus RNA genome replication.     

An improved method for the quantitative isolation of rat liver nuclear RNA polymerases.

Rat liver nuclear RNA polymerases exist in two functional states, one of which is active towards the endogenous chromatin template (engaged enzyme), while the other is inactive (free enzyme) (Yu, F.L. (1974) Nature 251, 344-346). This paper reports the direct separation of these two populations of RNA polymerases from isolated rat liver nuclei by a simple extraction procedure. It is estimated that as much as 50% of the total nuclear RNA polymerase activity in normal rat liver may exist in the form of the free enzyme. Evidence is also presented to indicate that the free enzyme activity is easily lost when the nuclear isolation procedure involves the use of an isotonic buffer medium, or when the isolated nuclei are subjected to sonication as is required for the solubilization of the nuclear RNA polymerases by the conventional method. Based on these new findings, it is proposed that nuclei be isolated directly in hypertonic sucrose and that the free enzyme be extracted before the nuclei are subjected to sonication to solubilize the engaged enzyme. This method circumvents the loss of the free RNA polymerase population and, as a result, the total yield of the nuclear RNA polymerases is greatly increased. The possible functional role of the free RNA polymerase in gene expression is discussed.     

Differential changes of tightly chromatin-bound RNA polymerase II in starved and cycloheximide-treated rat liver nuclei

In order to investigate the physiological function of loosely and tightly chromatin-bound RNA polymerase IIin vivo (1), the changes of these enzyme activities in cycloheximide-treated or starved rat liver nuclei were studied. Total nuclear mRNA synthesis activity in starved rats was considerably decreased, but that of cycloheximide-treated rats was not affected significantly. In starved rats, tightly bound enzyme activity was much more repressed as compared with that of loosely bound enzyme. On the other hand, cycloheximidetreated rats showed the reverse relationship. Thus, mRNA synthesis activity in hepatic nuclei seems to be dependent on the tightly bound RNA polymerase II activity. However, the difference of nuclear mRNA synthesis in both cases can not be explained by the change of chromatin-bound enzyme activity of Yu (2).     

Glucocorticoids modify the rate of ribosomal RNA synthesis in rat thymus cells by regulating the polymerase elongation rate

The mechanism by which glucocorticoids inhibit RNA polymerase A activity, and hence rRNA synthesis, in rat thymus cells has been investigated. Studies of the intranuclear distribution of RNA polymerase A between chromatin bound ("engaged") and unbound ("free") forms revealed that the steroid-mediated inhibition of the activity of the "engaged" form of the enzyme was not accompanied by significant changes in "free" pool activity. In the presence of rifamycin AF/0-13, an inhibitor of re-initiation of RNA polymerase A, the rate of [3H]UMP incorporation into RNA was slower in nuclei from steroid-treated cells than in those from control cells, although in both conditions similar plateau levels of UMP incorporation were attained. Direct measurements of the numbers of transcribing RNA polymerase A molecules and of elongation rates showed that the inhibition of pre-rRNA synthesis was the result of a decrease in enzyme elongation rate; no significant change was observed in the number of transcribing enzymes. The steroid-induced inhibition of pre-rRNA synthesis was selectively abolished by mild proteolysis of nuclei, suggesting the involvement of a labile, regulatory glucocorticoid-induced protein. It is concluded that glucocorticoid treatment of rat thymus cells decreases 45S rRNA synthesis primarily by decreasing the polyribonucleotide elongation rate of RNA polymerase A, possibly by modification of the enzyme.     

Alterations of protein synthesis in arbovirus-infected L cells

Lust, George (Fort Detrick, Frederick, Md.). Alterations of protein synthesis in arbovirus-infected L cells. J. Bacteriol. 91:1612-1617. 1966.-Cellular protein synthesis and ribonucleic acid (RNA) synthesis in mouse L cells were markedly depressed 1 hr after infection with Venezuelan equine encephalomyelitis virus. Host RNA and protein synthesis were inhibited more rapidly by the virus infection than by actinomycin D. In cells infected 4 hr, a cytoplasmic RNA polymerase was demonstrated which was absent in uninfected cells. At this time, deoxyribonucleic acid-directed RNA synthesis catalyzed by the nuclear RNA polymerase was inhibited in vitro in enzyme preparations from nuclei of virus-infected cells. For optimal activity, the cytoplasmic RNA polymerase required the four nucleoside triphosphates, Mg(++), and RNA. The enzyme was insensitive to actinomycin D and deoxyribonuclease, indicating that it catalyzed RNA-directed RNA synthesis. Attempts to purify the induced polymerase further were unsuccessful. Fresh preparations had to be used because the enzymatic activity was unstable.     

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.     

RNA polymerase I from higher plants. Evidence for allosteric regulation and interaction with a nuclear phosphatase activity controlled NTP pool.

RNA polymerase I was isolated from parsley cells grown in suspension culture and from soybean hypocotyls. Kinetic studies of the enzyme revealed that RNA polymerase I is an allosteric regulated enzyme. The enzyme activity was influenced by nucleoside triphosphates (NTP) and divalent cations. NTP exceeding a 1:1 ratio of these two components acted as allosteric inhibitors, contrary to free divalent cations, which had promotive effects on the RNA polymerase I. Furthermore, isolated nuclei from parsley exhibited a powerful nucleoside triphosphatase (NTPase) activity. Contrary to RNA polymerase I, this enzyme was stimulated by NTP exceeding the 1:1 ratio of NTP and divalent cations. Free divalent cations had an inhibitory effect. Assuming that a causal connection of these two processes does exist, a possible role of this NTPase would be the control of NTP pools in relation to divalent cations and thus regulating RNA synthesis.     

Heterogeneous nuclear RNA promotes synthesis of (2'',5'')oligoadenylate and is cleaved by the (2'',5'')oligoadenylate-activated endoribonuclease.

Heterogeneous nuclear RNA contains double-stranded regions that are not found in mRNA and that may serve as recognition elements for processing enzymes. The double-stranded regions of heterogeneous nuclear RNA prepared from HeLa cells promoted the synthesis of (2',5')oligoadenylate [(2',5')oligo(A) or (2'5')An] when incubated with (2',5')An polymerase. This enzyme is present in elevated levels in interferon-treated cells, and labeled heterogeneous nuclear RNA incubated with extracts of these cells is preferentially cleaved, since mRNA included in the same incubations is not appreciably degraded. The cleavage of heterogenous nuclear RNA is caused by the synthesis of (2'5')An and by a "localized" activation of the (2',5')An-dependent endonuclease, since it was enhanced by ATP, the substrate of the (2',5')An polymerase, and inhibited by 2'-dATP and ethidium bromide. Both of these compounds suppress the synthesis of (2',5')An, the first by competitive inhibition and the latter by intercalating into double-stranded RNA. The possible role of double-stranded regions and of the (2',5')An polymerase-endonuclease system in the processing of heterogeneous nuclear RNA is discussed.     

Vaccinia virus RNA polymerase associated with nuclei of infected HeLa cells.

Though vaccinia virus DNA and RNA replication take place predominantly in the cytoplasm of an infected cell, virus formation requires the presence of a functional nucleus in a yet undefined manner. When the nuclei from cells infected for 3 h are isolated and purified, they are found to synthesize five times more RNA in vitro than do corresponding nuclei from noninfected cells. Fifty percent of the RNA synthesized in vitro by nuclei from infected cells is vaccinia specific, and this vaccinia RNA synthesis is resistant to alpha-amanitin concentrations up to 100 micrograms/ml. Furthermore, when the RNA polymerase activities of these nuclei are separated on DEAE-Sephadex columns, 56% of the total nuclear enzyme activity is found to be the vaccinia-specific RNA polymerase known to be alpha-amanitin resistant. The nucleus associated vaccinia RNA polymerase represents 18% of the total cellular vaccinia RNA polymerase. This synthesis of vaccinia RNA in the nucleus may explain the nuclear requirement for vaccinia virus maturation.     

Selective Modulation of RNA Polymerase I Activity during Growth Transitions in the Soybean Seedling

RNA polymerase I and II activities were measured in tissues of the soybean (Glycina max, var. Wayne) hypocotyl where dramatic changes in the relative level of RNA synthesis are associated with normal and auxin-induced growth transitions. When assayed in isolated nuclei, the activity of RNA polymerase I changed much more than the activity of RNA polymerase II during these growth transitions. The activity of RNA polymerase I expressed in the nuclei generally showed a positive correlation with the relative level of RNA synthesis (i.e. accumulation) of that tissue. Following solubilization of the RNA polymerases from these isolated nuclei and fractionation of them on DEAE-cellulose, the activity of RNA polymerase I relative to that of RNA polymerase II showed smaller changes during these growth transitions than when assayed in the nuclei. Thus, these data indicate that the activity of RNA polymerase I is significantly modulated in the nucleus, up or down depending upon the growth state, during growth transitions in the soybean in addition to lesser changes which occur in the apparent level of the enzyme.     

RNA Polymerase III of <Emphasis Type="Italic">Blastocladiella emersonii</Emphasis> is Mitochondrial

MULTIPLE RNA polymerases have been shown to exist in a wide variety of eukaryotic organisms^1–5. Two nuclear polymerases have been found in all the cells studied, each with a specific location and a specific function: the DEAE fraction I enzyme is located in the nucleolus and may be involved in the synthesis of ribosomal RNA^1,2,5,6; the DEAE fraction II enzyme is located in the non-nucleolar nucleoplasm and functions in the synthesis of DNA-like RNA^2–5,7. The DEAE fraction III enzyme was reported to exist in sea urchin¹, the aquatic fungus B. emersonii⁵ and to be present sometimes in rat liver preparations^1,8. Although there have been some reports that polymerase III is nuclear, Horgen and Griffin⁵ showed that the enzyme was sensitive to the prokaryotic RNA polymerase inhibitor rifampicin. They suggested that the fraction III enzyme may be mitochondrial, formed as the result of organelle contamination in their crude nuclear preparations. The results of this study show that the DEAE fraction III enzyme in B. emersonii is a mitochondrial enzyme, most likely functioning in the synthesis of mitochondrial RNA. The rifampicin sensitivity of the enzyme is further evidence of a prokaryotic origin of mitochondria^9,10.     

Conservation of RNA polymerase during maturation of the Rana pipiens oocyte.

DNA-dependent RNA polymerase was extracted from oocytes of the frog, Rana pipiens. The bulk of the enzyme activity was present in the germinal vesicle and the amounts of each major form of such activity did not significantly change during oocyte maturation. Therefore, either nuclear polymerase activity is conserved after breakdown of the oocyte nucleus during maturation or, alternatively, de novo synthesis of the enzymes must occur during oocyte maturation concomitant with degradation. We have measured rates of protein synthesis in oocytes and determined a maximum rate of synthesis for RNA polymerases. Our kinetic studies show that no more than 20, 10, and 5% of RNA polymerases type I, IIa, and IIb, respectively, could be synthesized during steroid-induced oocyte maturation. These results thus show that the bulk of RNA polymerase accumulates in the germinal vesicle during oogenesis, is dispersed into the cytoplasm during maturation, and, since only limited synthesis seems to be occurring, the polymerase is available during embryogenesis.