The in vivo equivalence of a cell-free system for RNA processing and transport

The equivalence of messenger RNA released (transported) from isolated rat liver nuclei to three selected media, with messenger RNA normally released to liver cytoplasm $\text{in vivo}$, has been evaluated by competitive DNA: RNA hybridization. Near normal nuclear restriction was exhibited by nuclei in media fortified with ATP, salts, spermidine and dialyzed cytosol. The RNA transport in the latter system was markedly inhibited by colchicine as was also the transport of RNA $\text{in vivo}$. Both nuclear restriction and sensitivity of the RNA transport to colchicine in media lacking spermidine and cytosol deviated significantly from the $\text{in vivo}$ norm. The results emphasize the importance of establishing the $\text{in vivo}$ equivalence in cell-free systems designed to study RNA synthesis, processing and transport.     

Characterization of a messenger RNA transport protein

A cytoplasmic protein which facilitates the energy-dependent transport of mRNA from isolated nuclei to a specified medium has been further characterized, since it could have relevance to the mechanism of mRNA nucleo-cytoplasmic transport in vivo. This protein is now shown, by cDNA hybridization analysis using appropriate recombinant probes, to be obligatory for the transport of alpha 2u-globulin and albumin mRNA from male rat liver nuclei. It is concentrated in the cytoplasm. When isolated under conditions where they retain nuclear proteins, the nuclei contain less than 2% of the total mRNA transport activity. Approx. 20% is recovered in the cytosol, while the rest (80%) copurifies with the messenger ribonucleoproteins in the polyribosome fraction. The protein is eluted from the poly A-messenger ribonucleoproteins between 0.25 and 0.50 M NaCl. The activities of the cytosolic- and messenger ribonucleoprotein-derived transport proteins were mutually additive below saturation of the transport system. Further, the activities of both fractions were increased when they were fortified with the catalytic subunit of the cAMP-dependent protein kinase in the presence of ATP. On the other hand, protein kinase-induced thiophosphorylation of the protein with ATP[S] decreased transport activity. The molecular weight of the transport protein from either cell compartment as judged by molecular sieving is approx. 35,000. It has now been purified 2000-fold and requires manganese ions and serum albumin for stabilization of activity. The highly purified transport factor from the cytosol is tentatively assigned a molecular weight of 32,000 by SDS-polyacrylamide gel electrophoresis.     

Regulated transport of messenger ribonucleic acid from isolated liver nuclei by nucleic acid binding proteins

Rat liver nucleocytosolic messenger ribonucleic acid (mRNA) transport is shown to be regulated by proteins with a high affinity for nucleic acids. In the cell-free system described, the energy-dependent transport of all RNA classes [transfer RNA (tRNA), mRNA, and ribosomal RNA (rRNA)] exhibited a dependence upon the availability of discrete minor sets of cytosol proteins. In addition to having a different level of saturation, only the mRNA "transport protein" activities are increased by adenosine cyclic 3',5'-phosphate (cAMP), an effect most likely mediated by a cAMP-dependent protein kinase. The mRNA transport proteins were isolated from cytosol by precipitation with streptomycin sulfate followed by deoxyribonucleic acid (DNA)-cellulose affinity chromatography, or from oligo-(thymidylate)-cellulose bound cytoplasmic messenger ribonucleoprotein (mRNP) particles by high-salt extraction. Either method yielded a protein fraction which exhibited a 1000-fold increase in mRNA transport activity as compared to cytosol. Over one-half of the mRNA transport activity is associated with the mRNP of the cell. A partial homology between the cytosol and mRNP-derived proteins was demonstrated by polyacrylamide gel electrophoresis. One major (20 000 daltons) and several minor proteins (23 000, 52 000, 54 000, and 72 000 daltons) were in common. Nuclear 4-5S exited from in vitro incubated nuclei in three phases, according to their differential in vivo rates of labeling and intranuclear pool sizes. The amount of nuclear RNA transported in vitro as mRNA (about 1.0%) agrees wtih the in vivo estimates. Additional evidence for in vivo equivalence was provided by the physicochemical characterization and bioassay of the RNA. The transported mRNA sedimented in urea-sucrose gradients as an 8-18S heterodisperse product. This RNA initiated cell-free translation with the synthesis of precursor peptides as diverse in size as those for albumin and alpha 2U-globulin. The relative abundancies of various transported mRNAs were different than the corresponding abundancies of liver cytoplasmic mRNAs.     

Insulin-modulated transport of RNA from isolated live nuclei

The addition of 3 × 10^?7m insulin to a cell-free RNA transport system caused an increase of 50% in the release of messenger-like RNA from 30-min prelabeled rat liver nuclei. Insulin concentrations above 1.2 × 10^?6m inhibited RNA release. These hormonal effects were not observed when nuclei were prepared from the insulin-resistant Zucker rat (fa/fa), while the level of stimulation in the heterozygote was approximately one-half that observed with normal liver nuclei. Nuclei prelabeled for 120 min and releasing predominantly ribosomal RNA also did not respond to insulin added to the cell-free system. The hormone appears to affect primarily mRNA transport rather than processing.     

Ribonucleic acid synthesis in isolated rat liver nuclei under conditions of ribonucleic acid processing and transport.

A cell-free system is described which permits a significant and prolonged synthesis of RNA in isolated rat liver nuclei, under conditions previously demonstrated to support normal nuclear processing and transport of both rRNA and mRNA. The system contains cytosol but not (NH4)2SO4 or other non-physiological components. Evidence is presented for cytosol factors which stimulate ribosomal, and to a lesser degree, non-ribosomal RNA synthesis.     

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.     

Analysis of nuclear RNA processing and transport by temperature perturbation of a cell-free system from mammalian cells

The similarity of the Arrhenius plots relating temperature to messenger RNA (mRNA) transport from intact and membrane-denuded rat liver nuclei demonstrates that the ATP and cytosol-dependent transport is independent of the lipid phase of the nuclear membrane. This temperature dependence of RNA release was confirmed for alpha 2u-globulin mRNA by use of a recombinant DNA probe. Ribosomal RNA (rRNA) release showed a similar temperature dependence, suggesting that both mRNA and rRNA share a common temperature-sensitive step. The kinetics of RNA release at different temperatures suggest that RNA transport from mammalian cell nuclei is a rate-controlled rather than a graded unlocking phenomenon. The processing of mRNA precursors also exhibits a temperature dependence as shown by the linear increase in the ratio of total alpha 2u-globulin RNA to alpha 2u-globulin precursor as a function of time at 30 degrees C but not at 14 degrees C in spite of residual transport at the lower temperature. This temperature dependence of mRNA processing was confirmed by Northern blot analysis of the nuclear RNA following a 45 min incubation. Thus, both the processing and transport of RNA show temperature-sensitive steps when analyzed in cell-free systems derived from mammalian cells.     

Vitamin E up-regulates arachidonic acid release and phospholipase A2 in megakaryocytes

The alteration in calcium transport in the liver nuclei of rats orally administered carbon tetrachloride (CCl4) was investigated. Rats received a single oral administration of CCl₄(5, 10, and 25%, 1.0ml/100 g body weight), and 5, 24 and 48 h later the animals were sacrificed. The administration of CCl₄ (25%) caused a remarkable elevetion of calcium content in the liver tissues and the nuclei of rats. Liver nuclear Ca2+-ATPase activity was markedly decreased by CCl₄ (25%) administration. The presence of dibutyryl cyclic AMP(10^-4 and 10^-3 M) or inositol 1,4,5-trisphosphate (10^-6 and 10^-5 M) in the enzyme reaction mixture caused a significant decrease in Ca²⁺-ATPase activity in the liver nuclei obtained from normal rat, while the enzyme activity was significantly increased by calmodulin (1.0 and 2.0 g/ml). These signaling factor's effects were completely impaired in the liver nuclei obtained from CCl₄ (25%)-administered rats. DNA fragmentation in the liver nuclei obtained from CCl₄ -administered rats was significantly decreased by the presence of EGTA (2 mM) in the reaction mixture, suggesting that the endogenous calcium activates nuclear DNA fragmentation. The present study demonstrates that calcium transport system in the liver nuclei is impaired by liver injury with CCl₄ administration in rats.     

A special estrogen-binding protein in the rat liver: the effect of nuclear accumulation of estradiol-receptor complexes in vitro

The accumulation of [3H]estradiol-receptor complexes by liver nuclei after preliminary incubation of the hormone with rat liver cytosol was studied. It was demonstrated that addition to female rat liver cytosol of a purified preparation of the unusual estrogen-binding protein (UEBP) from male rat liver causes a dose-dependent inhibition of subsequent accumulation of specifically bound [3H]estradiol in the nuclei. Addition to male rat liver cytosol of 1.5 microM 2 alpha-hydroxytestosterone, testosterone, 1-dehydrotestosterone, 5 alpha-androstane-3 alpha, 17 beta-diol and 5 alpha-dihydrotestosterone, i. e. compounds possessing marked affinity for UEBP, resulted in a 2-5-fold increase of the subsequent nuclear accumulation of estrogen-receptor complexes. The use of UEBP-deficient female rat liver cytosol revealed that the afore-mentioned steroids are ineffective with respect to estrogen reception. It is concluded that UEBP of male rat liver is capable of modulating estrogen reception.     

trans activation of rat phosphoenolpyruvate carboxykinase (GTP) gene expression by micro-coinjection of rat liver mRNA in Xenopus laevis oocytes.

To study the liver-specific trans activation of the rat phosphoenolpyruvate carboxykinase (PEPCK) gene, the PEPCK promoter was linked to a reporter gene and was microinjected into Xenopus laevis oocytes alone or in conjunction with rat liver poly(A)+ RNA. The rat liver mRNA markedly enhanced the expression of the PEPCK-chimeric construct. This effect appeared to be sequence specific, as it was dependent on the presence of the intact promoter. Moreover, the RNA effect was limited to mRNA preparations from PEPCK-expressing tissues only. Finally, microinjection of size-fractionated liver mRNA revealed that the trans-acting factor(s) is encoded by RNA of 1,600 to 2,000 nucleotides, providing a direct bioassay for the gene(s) involved in this tissue-specific trans-activation process.     

Minimal methylation of yeast messenger RNA

Most, if not all, yeast mRNAs are capped at their 5-terminus by m⁷G. Apart from m⁷G no other methylated nucleotides could be detected in poly (A)⁺ mRNA isolated from yeast polysomes.Abbreviations used poly (A)⁺ mRNA messenger RNA containing poly (A) - poly (A)^- RNA RNA lacking poly (A) - m⁷G N₇-methyl guanosine - N_m any 2-0 methylated nucleoside - mN any basemethylated nucleoside     

On the altered nucleocytoplasmic transport "in vitro" of rapidly labelled RNA, in the presence of cytosol or serum from tumor-bearing rats

Enhanced nucleocytoplasmic RNA transport has been demonstrated by incubating normal rat liver nuclei in presence of cytosols originating from the poorly differentiated, fast-growing hepatoma HW-165, in the linear phase of tumor growth. The effect of hepatoma HW-165 cytosol was reduced or suppressed in presence of small amounts of normal liver cytosol: on the other hand, several polypeptides of molecular weight 20,000 to 40,000 daltons were hardly detectable in hepatoma HW-165 cytosol, both arguments indicating that potentially regulatory proteins should be absent or present in reduced concentration in hepatoma HW-165 cytosol. No modification of RNA release was observed in presence of cytosols originating from the thymus of RNA virus (BL/F)-infected rats, whatever be the time after inoculation. Attempts were made to use the nuclear restriction assay, supplemented with plasma or serum of various origins, as a biochemical marker of neoplasia. In a first series of assays, including 80 cancer patients and 12 healthy controls, the RNA transport activity was stimulated by the serum of patients bearing various tumors (lung cancer, cancer of the respiratory tract, uterine cervix...), except in a few cases of mammary carcinoma, where values equivalent to or lower than the controls were obtained.     

Transport of functional messenger RNA from liver nuclei in a reconstituted cell-free system

The reliability of a reconstituted cell-free system for messenger RNA processing and transport, consisting of isolated nuclei in fortified cytosol, has been evaluated in terms of the functionality and regulated release of the transported product. The poly(A) messenger RNA transport in vitro formed appropriate initiation complexes with ribosomes in an optimized translation system and had template activity comparable to that transported in vivo. The intra-nuclear origin of this messenger RNA is supported by pulse-labeling studies, its transport from detergent-treated nuclei and the absence of the release under non-transport conditions. Serum albumin was identified by immunoprecipitation and electrophoresis as one of the products synthesized when the transported RNA was translated in vitro. The transport of messenger RNA in the cell-free system was dependent on specific cytosol (soluble cytoplasmic) proteins. These proteins, which constitutes less than 0.1% of the total cytosol proteins, are precipitated wtih streptomycin with high specificity.     

Effects of sex hormones on enzymic esterification of 2-(N-hydroxyacetamido)-fluorene by rat liver cytosol

1. Enzymic esterifications of 2-(N-hydroxyacetamido)fluorene and several other hydroxamic acids by liver cytosol were studied. Determination of 2-acetamido-3-methylthiofluorene was used for the assay. 2. With rat liver cytosol, requirement for ATP, Mg²⁺ and SO₄²⁻ suggested formation of phosphate and sulphate esters of 2-(N-hydroxyacetamido)fluorene. 3. Rats showed sex and age differences in their activity. Liver from adult male rat was at least twice as active as liver from adult female rat. No such sex differences were found in mice, hamsters and guinea pigs. 4. Administration of testosterone (300μg/day) subcutaneously for 8 days increased the activity in the female rat by 100%, whereas diethylstilboestrol (100μg/day) had no effect. In the male rat diethylstilboestrol treatment decreased the activity by 60%, whereas testosterone pretreatment was without any effect. 5. Among various endocrine ablations such as adrenalectomy, castration, adrenalectomy–castration and hypophysectomy in the adult male rat, hypophysectomy was found to be the most effective in decreasing the activity of the liver to about 50% of control values. 6. Like 2-(N-hydroxyacetamido)fluorene, various other N-hydroxy derivatives of 2-acetamido-7-fluorofluorene, 2-acetamidophenanthrene, 4-acetamidobiphenyl and 4-acetamidostilbene were also shown to be esterified to different extents by rat liver cytosol.     

Hormonal regulation of the hepatic messenger RNA levels for alpha2u globulin.

The messenger RNA rat alpha2u globulin has been identified and quantitated in a cell-free translational system derived from Krebs II ascites cells. Hepatic tissue of the mature male rats which normally produce alpha2u globulin was also found to contain a high level of alpha2u mRNA. Approximately 1.6 per cent of all poly(A) containing RNA of the adult male rat liver could be accounted for alpha2u messenger activity. Female rats do not produce alpha2u globulin and no alpha2u mRNA activity could be detected in the poly(A) containing RNA fraction obtained from the livers of these animals. However, androgen treatment to spayed female rats was found to induce the parallel appearance to both alpha2u globulin and its corresponding mRNA. Both hypophysectomy and adrenalectomy which are known to reduce the level of alpha2u globulin in the urine of male rats were found also to reduce the hepatic level of alpha2u mRNA. The results indicate that hormonal control of alpha2u globulin synthesis in rat liver is achieved primarily through regulation of its translatable mRNA level and that more than one hormone may participate in this regulation.