NUCLEAR-CYTOSOL INTERACTIONS THAT FACILITATE RELEASE OF RNA FROM MOUSE BRAIN NUCLEI

Abstract— Mouse brain nuclei were incubated in vitro under conditions that primarily lead to the synthesis of radioactive polydisperse and messengerlike nuclear RNA. After incubation the effects of Mg² concentrations, nucleoside triphosphate levels and brain cytosol were examined with regard to their ability to influence the release of RNA from brain nuclei. The presence of 8 mM -MgCl₂ and a total of 0.3 mM-nuclcoside triphosphates during the labelling procedure allowed only a minimal amount of RNA to be released. However, when the MgCl₂ was decreased to 2 mM and the nucleoside triphosphates were increased to 1 mM, a stimulation of RNA release was observed. The addition of unfractionated brain cytosol under these conditions resulted in an inhibition of RNA release.
G-100 Sephadex filtration removed detectable RNase activity from the cytosol preparations and allowed the identification of fractions that were able to facilitate nuclear RNA release by 3-fold. The fractions that stimulated release did not have detectable levels of RNase, protease or DNA-dependenl RNA polymerase. Under conditions that provided maximum nuclear RNA release by both labelled mouse brain and neuroblastoma nuclei, no release of DNA could be measured. The cytosol fractions that facilitated RNA release did not have a high affinity for nuclear RNA or an ability to stimulate nuclear RNA synthesis. However, other components in the cytosol were shown to stimulate RNA metabolism in isolated mouse brain nuclei and to have a relatively high binding affinity to nuclear RNA. Further purification of the RNA release components in the brain cytosol by DEAF. Sephadex chromatography allowed an increase in specific activity of at least 40-fold. The thermal lability, effective filtration size, and solubility in phenol suggested that the cytosol factors that facilitiated nuclear RNA release were associated with cellular proteins.     

RNA METABOLISM IN ISOLATED MOUSE BRAIN NUCLEI DURING EARLY POSTNATAL DEVELOPMENT

—RNA metabolism in isolated brain nuclei has been shown to be dramatically altered during early postnatal brain development. The present study involved an examination of the RNA products synthesized by nuclei at various stages of postnatal neural maturation. In all cases, the majority of the RNA appeared to be heterodisperse, non-ribosomal and non-tRNA in nature. In comparison to the RNA isolated from nuclei of neonatal tissue, the RNA from nuclei of 12-day and 30-day-old mouse brain was found to be of smaller molecular weight. Despite the heterodisperse nature of these RNA molecules, the addition of α-amanitin did not completely inhibit nuclear synthesis. An investigation of RNA synthesis in isolated neuronal and glial cell nuclei revealed that nucleic acid metabolism in these respective cell populations had different and distinct developmental patterns. Preparations enriched with glial cell nuclei were found to be most active at birth and then decreased in activity (3–4-fold) during neural maturation. On the other hand, the rate of RNA synthesis in fractions enriched in neuronal cell nuclei was observed to increase dramatically in activity (4–5-fold) until 14 days of age. From 14 days of age until adulthood, RNA synthetic activity remained essentially the same.     

In vitro RNA synthesis by intact rat brain nuclei

The characteristics of DNA-dependent RNA polymerase activity in intact rat cerebral cortex nuclei tested at low ionic strength are presented. The system was most dependent on the presence of spermidine and an ATP-generating system and to a lesser extent on Mg2+ and K+ for maximal incorporation. Substitution of Mg2+ by Mn2+ or of K+ by Na+ resulted in substantially less activity than under the optimum conditions described. Maximal incorporation was about 10 per cent that of brain nuclear systems of high ionic strength. In addition, the labelling patterns of the in vitro RNA products were shown to be very similar to those found in vivo. The stability of isolated nuclei toward degradation of RNA synthetic capacity and products formed was much greater than that of a similar liver system.     

The relationship between DNA synthesis and the synthesis of nuclear proteins in rat brain during development

—The incorporation of [³H]thymidine into nuclear DNA of rat brain progressively increased from birth until the 8th postnatal day and it was lowest in the adult brain. When isolated nuclei from brain cells were separated into a neuronal- and a glial-rich fraction (composed of glial and neuroblast nuclei in young animals), the specific radioactivity of the DNA was higher in the glial-rich fraction at all ages investigated. The incorporation of [³H]leucine into proteins of rat brain was considerably higher in the 8-than in the 1-day-old rat. The greatest difference in the rate of protein synthesis between 8- and 1-day-old brain occurred in the nuclear proteins, especially those associated with DNA. There was an accumulation of protein and RNA in nuclei from brain cells from birth to the 8th postnatal day. The increased content of proteins occurred primarily in the fraction soluble in buffered saline (nuclear sap).     

An assay of RNA synthesis in hepatic nuclei from control and Streptococcus pneumoniae-infected rats

Hepatic nuclei were isolated, purified, and partially characterized from control and Streptococcus pneumoniae-infected rats. Biochemical and morphologic examination showed little contamination by other cell organelles. An in vitro system for the incorporation of 2-[14C]uridine-5'-triphosphate into ribonucleic acid (RNA) was developed and characterized. Although the stimulatory effects of cytosol on incorporation of labeled precursors into RNA have been previously reported, nuclei isolated from the livers of S. pneumoniae-infected rats were stimulated to a significantly greater extent than were nuclei isolated from the livers of control rats. In the presence of cytosol prepared from either control or infected rats, the increased incorporation of labeled precursor into RNA by nuclei isolated from infected rats was observed over broad pH and temperature ranges. The increased activity of infected nuclei was eliminated when albumin was substituted for cytosol, and could not be accounted for by differences in endogenous precursor pool size. These results are consistent with other infection-induced alterations in hepatic RNA and protein synthesis.     

Effect of brain cytosol proteins of embryonal and adult animals on RNA-polymerase activity of isolated brain nuclei]

Cytosol and its fractions obtained by the precipitation with ammonium sulphate and ion-exchange chromatography have been studied for their effect on the RNA-polymerase activity of isolated nuclei. We observed the discrepancies in the action of total cytosol of embryonal, newborn or adult animals on the label's incorporation in RNA. It was found that some fractions increased DNA-polymerase activity of isolated nuclei in cattle embryonal cytosol. The same fractions obtained from adult cytosol did not act in such a way. It was found that most fractions obtained from cytosol of adult brain inhibited the RNA-polymerase activity of brain nuclei.     

Age-related characteristics of RNA transport through the nuclear membrane

The effect of cytosol and ATP-regenerating system on RNA, transport was studied in isolated liver nuclei of adult and old rats. The stimulating effect of cytosol was found not to depend on the age of animals. The release of RNA from old rat liver nuclei activated by the ATP-regenerating system was more expressed compared to adult rats. It is assumed that the age changes of energy-delivering system of the RNA transport through nuclear membrane may be conditioned by the deficit of endogenous energetic substrates in the hepatic cells of old animals.     

Some features of nucleo-cytoplasmic RNA transport from isolated nuclei

Messenger RNA is released preferentially from isolated rat liver nuclei in the presence of the ATP-generating system and cytosol. The release is suppressed by spermidine, while cytoplasmic RNase inhibitor was ineffective and PCMB like some other thiol-blocking agents inhibitory. Cytoplasmic SOD added to the system strongly suppressed RNA release. A similar effect could be obtained by anaerobiosis due to addition of SMP. In both cases the inhibition is reversed by cyanide.In contrast to normal liver where the generation of superoxide radicals takes place almost exclusively in microsomes and is coupled with the oxidation of NADPH, in mouse ascites hepatoma 22a the generation of superoxide radicals occurs mainly in the nuclear envelope and is coupled with the oxidation of both NADPH and NADH and inhibited by cyanide.Abbreviations PCMB p-Chloromercuri benzoate - SMP Submitochondrial particles - SOD Superoxide dismutase     

Differential effect of ATP on RNA and DNA release from nuclei of normal and neoplastic liver.

Nuclei isolated from differentiated normal liver, moderately differentiated Hepatoma 5123 and poorly differentiated Novikoff hepatoma have been compared with respect to ATP-dependence of messenger-like RNA release and resistance to lysis (DNA release) in a cell-free system containing homologous cytosol. The release of RNA from the nuclei of liver, Hepatoma 5123D and the Novikoff hepatoma was totally ATP-dependent, partially ATP-dependent and ATP-independent, repectively. The sensitivity of the nuclear RNA transport in the presence of ATP to beryllium nitrate, an inhibitor of a nuclear pore phosphatase, paralleled their ATP-dependence. Although RNA release from the nuclei of both liver and Novikoff hepatoma has an absolute requirement for cytosol proteins, the structural integrity of liver, but not Novikoff hepatoma nuclei in the presence of ATP, is dependent on macromolecules in the cytosol.     

Cell-free Synthesis of the Myelin Basic Proteins in a Wheat Germ System Programmed with Brain Messenger RNA

Poly A(+) messenger RNA (mRNA) was isolated from the brains of 3-week-old mice and translated in a cell-free system derived from wheat germ. Maximal stimulation of the system by brain mRNA was observed at a relatively low K+ concentration (45 mM) and low mRNA concentration (1-10 microgram/ml). The translational system was dependent on an energy-generating system and stimulated by the addition of spermidine and transfer RNA. Under optimal conditions, incorporation was linear for almost 45 min, but the overall stimulation with brain mRNA was relatively low (about twofold). In spite of the low stimulation, analysis of the translation products indicated that in the presence of brain mRNA polypeptides which co-chromatographed and co-electrophoresed with the two mouse myelin basic proteins could be detected. In control experiments with liver poly A(+) mRNA, which stimulated the translational system to a greater extent than brain mRNA, no such polypeptides could be detected. In this system the ratio of synthesis of small myelin basic protein to large myelin basic protein was found to be about 4.0, which correlates well with that found in vivo.     

CHANGES IN NUCLEAR RNA OF BRAIN INDUCED BY OLFACTION IN CATFISH

Abstract— —Studies were undertaken to correlate the changes in the synthesis of brain nuclear RNA during olfactory stimulation in saltwater catfish ( Galeichthys felis ). Catfish allowed to swim for 1 hr in sea water containing morpholine (10⁻⁴ M) showed an increase in brain nuclear RNA and a change in base ratios in contrast to controls in plain sea water. These changes in brain nuclear RNA were reversed within 24 hr to the levels of unstimulated controls when morpholine stimulated fish were transferred to fresh sea water.
In a split-brain preparation in an isolated catfish head, one naris was washed with morpholine in sea water (10⁻⁶ M), while the other naris was washed with plain sea water. The stimulated half of the brain, compared to the unstimulated half, showed the same changes in nuclear RNA as those noted in free swimming catfish. Brain cytoplasmic fractions did not exhibit any changes in RNA following olfactory stimulation. Amyl acetate, shrimp extract, and extracts from red fish skin as odorants also elicited changes in brain nuclear RNA. With each odorant there was an increase in amount of RNA and also a change in base ratio, where the base ratio changes were different for each odorant tested. With camphor as an odorant, there was an increase in brain nuclear RNA, while with menthol as an odorant, there was a decrease in brain nuclear RNA. In both instances the base ratio of the RNA did not change in contrast to the controls. These studies suggest that olfactory stimulants affect a change in content and character of the RNA in brain nuclei, whereas irritants to the olfactory epithelium change the content of brain nuclear RNA but do not alter the base ratio.     

Effect of undernutrition on DNA and RNA synthesis in subcellular fractions from different regions of the developing rat brain

The effect of undernutrition on the incorporation of [methyl-³H]thymidine into DNA and of 5-[³H]uridine into RNA of cerebral hemispheres, cerebellum, and brain stem was studied in vivo and in vitro in rats. The labeling of DNA from nuclei and mitochondria and of RNA from nuclei, mitochondria, microsomes, and soluble fractions, was also measured in vitro. The results demonstrate that nucleic acid synthesis is impaired and delayed during undernutrition. Specific effects were observed for the different brain regions and subcellular fractions: at 10 days nuclear and mitochondrial DNA and RNA synthesis was impaired, whereas at 30 days only the mitochondrial nucleic acid synthesis was affected.The delay of DNA and RNA labeling, caused by undernutrition, was most evident in the cerebellum, probably due to its intense cell proliferation during postnatal development. The specific sensitivity of mitochondria as compared to other subcellular fractions, may be due to the intense biogenesis and/or turnover of nucleic acids in brain mitochondria not only during postnatal development, but also in the adult animal.