Energy requirement and kinetics of transport of poly(A)-free histone mRNA compared to poly(A)-rich mRNA from isolated L-cell nuclei

ATP-promoted efflux of poly(A)-rich RNA from isolated nuclei of prelabeled mouse lymphoma L5178y cells has an activation energy of 51.5 kJ/mol, similar to that found for the nuclear envelope nucleoside triphosphatase (48.1 kJ/mol) assumed to be involved in mediating nucleocytoplasmic transport of at least some RNA. Here we show that efflux of two specific poly(A)-rich mRNAs (actin and beta-tubulin) from isolated L-cell nuclei is almost totally dependent on the presence of ATP, while efflux of poly(A)-free histone mRNA (H4, H2B, and H1) also occurs to a marked extent in the absence of this nucleotide. Measurements of temperature dependence of transport rate revealed an activation energy of 56.1 kJ/mol for actin mRNA, while the activation energy for histone-H4-mRNA efflux was in the same range as that found for ATP-induced release of RNA from demembranated nuclei (about 15-20 kJ/mol). Addition of nonhydrolyzable nucleotide analogs of ATP to the in vitro system used for measurement of RNA transport did not result in release of nonhistone mRNA (actin), but enhanced the efflux of H4 mRNA to approximately the same extent as ATP. Although not absolutely required, addition of ATP stimulated the rate of export of histone mRNA about twofold. Only the poly(A)-rich RNA, but not the poly(A)-free RNA, released from isolated nuclei was found to compete with poly(A) for the nuclear envelope mRNA-binding site, indicating the mechanism of transport for both RNA classes to be distinct. Export of both nonhistone and histone mRNA was found to be inhibited by a monoclonal antibody against a p60 nuclear-pore-complex antigen. This antibody had no effect on the nucleoside triphosphatase, mediating transport of poly(A)-rich mRNA.     

Nucleocytoplasmic transport of RNA. The effect of 3''-deoxyadenosine triphosphate on RNA release from isolated nuclei.

The addition of 3'-deoxyadenosine (cordycepin) to cells in culture results in the inhibition of the appearance of mRNA in the cytoplasm through a mechanism thought to involve the inhibition of polyadenylate synthesis. I studied the effect of 3'-deoxyadenosine triphosphate, the physiologically active form of 3'-deoxyadenosine, on RNA release from isolated nuclei. Nuclei were isolated from baby-hamster kidney (BHK) fibroblasts that had been given a short pulse of radioactive uridine or adenosine in the presence of a low concentration of actinomycin D before harvest. RNA release from the isolated nuclei under the appropriate incubation conditions was time-, temperature- and ATP-dependent. 3'-Deoxyadenosine triphosphate inhibited RNA release from the isolated nuclei. However, RNA that was restricted to the nuclei during incubation with the drug could be chased out of the nuclei if the incubation medium was replaced with medium containing only ATP. The chased poly(A)+ (polyadenylated) RNA had shortened poly(A) tracts, indicating that poly(A)+ RNA with shortened poly(A) tracts can be transported out of the nucleus. An experiment was designed to test the effect of 3'-deoxyadenosine triphosphate on the release of poly(A)+ RNA at drug concentrations which caused 33 or 64% inhibition of RNA release. The release of poly(A)+ RNA and poly(A)- RNA (not polyadenylated) was equally inhibited by the drug. Thus, although 3'-deoxyadenosine triphosphate does inhibit release of RNA from the nucleus, it would appear that the drug does so through a mechanism independent of the inhibition of polyadenylation. The process that is inhibited must be one that is common to both poly(A)+ and poly(A)- RNA. The possibility that 3'-deoxyadenosine triphosphate inhibits a reaction at the nuclear membrane or nuclear pore complex is considered.     

Functional dissection of nuclear envelope mRNA translocation system: effects of phorbol ester and a monoclonal antibody recognizing cytoskeletal structures

Unidirectional transport of poly(A)-containing mRNA [poly(A)+ mRNA] through the nuclear envelope pore complex is thought to be an energy (ATP or GTP)-dependent process which involves a nuclear envelope nucleoside triphosphatase (NTPase). In the intact envelope, this enzyme is regulatable by poly(A) binding and by poly(A)-dependent phosphorylation/dephosphorylation of other components of the mRNA translocation system, which are as yet unidentified. Monoclonal antibodies (mAbs) were elicited against the poly(A) binding nuclear envelope fraction isolated from rat liver. The mAbs were screened for their modulatory effects on mRNA transport in vitro. One stable clone decreased the efflux of rapidly labeled RNA and of one specific mRNA (ovalbumin) from isolated nuclei. It increased the binding of poly(A) to the envelope and increased the maximal catalytic rate of the NTPase, but it did not alter the apparent Km of the enzyme or the extent of its stimulation by poly(A). The nuclear envelope-associated protein kinase that down-regulates the NTPase was inhibited by the antibody, while other protein kinases were not affected. Because both the NTPase and mRNA efflux were inhibited by the tumor promoter, 12-O-tetradecanoylphorbol 13-acetate, the sensitive kinase is probably protein kinase C. Protein kinase C was found to be associated with the isolated nuclear envelope. The antibody reacted with both a Mr 83,000 and a Mr 65,000 nuclear envelope polypeptide from rat liver and other tissues. By immunofluorescence microscopy in CV-1 cells, the antibody localized to the nuclear envelope and, in addition, to cytoplasmic filaments which show some superposition with the microfilament network.     

The role of protein phosphokinase and protein phosphatase during the nuclear envelope nucleoside triphosphatase reaction

The activities of nuclear envelope-associated protein phosphokinase and protein phosphatase were determined in nuclear ghosts from liver and oviduct of quails. The protein kinase was found to be inhibited by poly(A) by 75%. During the kinase reaction proteins with molecular weights of 106 000 and 64 000 were phosphorylated. The phosphoprotein phosphatase from liver was stimulated to 190% by poly(A), whereas only a slight enhancing effect by this polymer was determined with the oviduct enzyme (to 125%). Comparative determinations of the nuclear ghost-associated enzyme activities revealed the following values (in nmol P_i/min per 10⁸ ghosts); oviduct: phosphokinase, 0.015; phosphatase, 0.004 and nucleoside triphosphatase, 39.4; and liver: phosphokinase, 0.044; phosphatase, 0.012 and nucleoside triphosphatase, 11.7. These data indicate that phosphorylation/dephosphorylation proceeds independently of the nucleoside triphosphatase cycle. This assumption is supported by analytical results revealing that no marked dephosphorylation occurs after poly(A) binding to the nuclear envelope. Moreover, stoichiometrical data showed a nearly 1:1 molar ratio between ATP-binding and phosphorylation of nuclear envelope protein. From these findings a new model for the nucleoside triphosphatase-mediated poly(A)(+)mRNA efflux from nuclei is deducted, proposing phosphokinase and phosphatase only to modulate the affinity of the ‘carrier structure’ for poly(A) (+)mRNA, but not to constitute the nucleoside triphosphatase.     

Studies on protein kinases involved in regulation of nucleocytoplasmic mRNA transport.

The rate of energy-dependent nucleoside triphosphatase (NTPase)-mediated nucleocytoplasmic translocation of poly(A)-containing mRNA [poly(A)+mRNA] across the nuclear envelope is thought to be regulated by poly(A)-sensitive phosphorylation and dephosphorylation of nuclear-envelope protein. Studying the phosphorylation-related inhibition of the NTPase, we found that phosphorylation of one polypeptide of rat liver envelopes by endogenous NI- and NII-like protein kinase was particularly sensitive to poly(A). This polypeptide (106 kDa) was also phosphorylated by nuclear-envelope-bound Ca2+-activated and phospholipid-dependent protein kinase (protein kinase C). Activation of kinase C by tumour-promoting phorbol esters resulted in inhibition of nuclear-envelope NTPase activity and in a concomitant decrease of mRNA (actin) efflux rate from isolated rat liver nuclei. Protein kinase C, but not nuclear envelope NI-like or NII-like protein kinase, was found to be solubilized from the envelope by Triton X-100, whereas the presumable poly(A)-binding site [the 106 kDa polypeptide, representing the putative carrier for poly(A)+mRNA transport] remained bound to this structure. RNA efflux from detergent-treated nuclei lost its susceptibility to phorbol esters. Addition of purified protein kinase C to these nuclei restored the effect of the tumour promoters. Protein kinase C was found to bind also to isolated rat liver nuclear matrices in the absence but not in the presence of ATP. The NII-like nuclear-envelope protein kinase co-purified together with the 106 kDa polypeptide which specifically binds to poly(A) in an ATP-labile linkage.     

Purification and characterization of the major nucleoside triphosphatase from rat liver nuclear envelopes

Nuclear envelopes contain a nucleoside triphosphatase. Hydrolysis of ATP or GTP by this enzyme parallels energy-dependent efflux of poly(A)-containing mRNA from nuclei in vitro. Nucleoside triphosphatase has been purified from highly purified preparations of nuclear envelopes from rat liver by three successive affinity steps. The essentially homogeneous enzyme has an apparent molecular weight of 40,000 as checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and displays a rather broad substrate specificity. ATP and GTP are hydrolyzed at nearly equal rates, whereas UTP and CTP are only half as active as substrates. For optimal activity, a one-to-one ratio of a divalent cation (Mg2+, Mn2+, or Ca2+) and the nucleoside triphosphate substrate, an alkaline pH and a temperature of 34 degrees C are required. In contrast to the enzyme associated with nuclear envelopes which is stimulated by synthetic poly(A) and the poly(A) segment of the natural poly(A)-containing mRNA, homogeneous nucleoside triphosphatase is unable to be modulated by this polynucleotide species.     

Phospholipase A2 inhibits nuclear nucleoside triphosphatase activity and mRNA export in isolated nuclei from rat liver

The present study is undertaken to investigate whether the phospholipase A(2) (PLA(2)) influences mRNA nucleocytoplasmic transport evaluated by nucleoside triphosphatase (NTPase) activity and mRNA export in isolated hepatic nuclear envelope. Isolated hepatic nuclei from rat liver were exposed to PLA(2) (10(-5) approximately 10(-2)/ml) with or without incorporation of nuclei with phosphatidylcholine (PC) liposome. Messenger RNA exports and NTPase activities of nuclear membrane were assayed using ATP and GTP as substrates. We found that the RNA efflux, evaluated by [3H] uridine, was potently decreased in a concentration-dependent manner, by incubation of hepatic nuclei with PLA(2), regardless using ATP or GTP as substrates. The PC content in nuclear membrane was also decreased by PLA(2)-treatment. The PC was incorporated into the nuclear membrane by addition of phospholipid liposomes into the incubation mixture. PC incorporation into the nuclear membrane did not alter mRNA export. However this resulted in a significant increase in mRNA export rate in PLA(2)-treated group. Messenger RNA export rate in PLA(2) (10(-3) unit/mL)- treated nuclear membrane was positively correlated with level of PC incorporation, both using ATP and GTP as substrates. The activity of nucleoside triphosphatase, a nuclear membrane-associated enzyme, showed parallel variations with mRNA transport. It is concluded that nuclear PLA(2) plays a regulatory role in RNA transport, which can be antagonized by exogenous PC. These might be pathophysiologically significance, although the mechanisms by which this effect takes place remain to be clarified.     

Influence of nucleotides, cations and nucleoside triphosphatase inhibitors on the release of ribonucleic acid from isolated rat liver nuclei.

The reasons underlying reported discrepancies in the effects of ATP, ADP, adenosine 5'-[beta gamma-methylene]triphosphate, AMP + PPi, P-chloromercuribenzoate and F- on RNA efflux from isolated rat liver nuclei and on nuclear envelope nucleoside triphosphatase activity were investigated. The stimulatory effect of ADP was attributed to myokinase activity associated with the nuclei; this activity was eluted on repeated washing with nuclear incubation medium. In the absence of Ca2+ and Mn2+, ATP, adenosine 5'[beta gamma-methylene]triphosphate and AMP +PPi were found to promote release of both DNA and RNA. In the presence of 0.5 mM-Ca2+ and 9.3 mM-Mn2+, only ATP promoted RNA efflux to a significant extent. In the absence of spermidine, Ca2+ and Mn2+, nuclei released large quantities of DNA and RNA into the medium; this effect was promoted by p-chloromereuribenzoate. In the presence of the three cations, however, p-chloromercuribenzoate inhibited RNA efflux. F- caused a slight leakage of DNA from nuclei. The results are discussed in terms of models for the effects of ATP and analogues on RNA efflux and nuclear stability.     

Evidence for a direct interaction of Rev protein with nuclear envelop mRNA-translocation system.

The interaction of the Rev protein from human immunodeficiency virus type 1 (HIV-1) with the nucleocytoplasmic mRNA-transport system was investigated. In gel-shift assay, the recombinant Rev protein used in this study selectively bound to the Rev-responsive element (RRE) region of HIV-1 env-specific RNA. Nitrocellulose-filter-binding studies and Northern/Western-blotting experiments revealed an association constant of approximately 1 x 10(10) M-1. The Rev protein also strongly bound to isolated nuclear envelopes from H9 cells, containing the poly(A)-binding site (= mRNA carrier) and the nucleoside triphosphatase (= NTPase), which are thought to be involved in nuclear export of poly(A)-rich mRNA. Binding of 125I-Rev to a 110-kDa nuclear-envelope protein, the putative mRNA carrier, could be demonstrated in in vitro experiments. Both efflux of cellular poly(A)-rich RNA, such as actin RNA [but not efflux of poly(A)-free RNA] from isolated nuclei and the nuclear-envelope NTPase activity were strongly inhibited by Rev protein. On the other hand, transport of viral env RNA, containing the Rev-responsive element, was increased in the presence of Rev. Studying the release of RNA from closed nuclear-envelope vesicles containing entrapped RNA, the action of Rev was found to occur at the level of translocation of RNA through the nuclear pore. Evidence is presented that Rev down-regulates the NTPase-driven transport of mRNA lacking the RRE, most likely via binding to the mRNA carrier within the envelope. In contrast to the efflux of RRE-free RNA, ATP-dependent efflux of RRE-containing RNA from resealed nuclear-envelope vesicles was found to be increased, if the RNA was entrapped in the vesicles together with Rev protein. In addition, it was found that phosphorylated Rev, which is transported together with RRE-containing RNA out of the vesicles, becomes dephosphorylated during transport. In the vesicle experiments it is demonstrated for the first time that a protein selectively channels a specific mRNA across the nuclear-envelope pore complex.     

Differential changes of nuclear-envelope-associated enzyme activities involved in nucleocytoplasmic mRNA transport in the developing rat brain and liver

Nucleocytoplasmic transport of rat liver mRNA is thought to be regulated by a nucleoside triphosphatase whose activity in the intact nuclear envelope is stimulated by the 3'poly(A) tail of poly(A)+ mRNA. In contrast to the liver mRNA, the mRNA from rat brain contains a great population of poly(A)- mRNA's that does not appear until after birth. Measurements of the nuclear-envelope-associated enzyme activities involved in mRNA transport, and their dependence on endogenous (isolated cytoplasmic mRNA-transport-stimulating proteins) and exogenous (poly(A), lectins, and neoglycoproteins) factors during prenatal and postnatal rat brain and liver development, revealed marked organ-dependent differences paralleling the appearance of the poly(A)- mRNA unique in the brain.     

Cordycepin disrupts the microtubule networks and arrests Nil 8 hamster fibroblasts at the onset of mitosis

The nucleoside analogue 3'-deoxyadenosine (cordycepin) arrests dividing cells at the onset of mitosis in prometaphase. The microtubules in the arrested prometaphase cells depolymerize to two small asters. A minimum of 80 micrograms/ml cordycepin or 20 micrograms/ml cordycepin in combination with 2 micrograms/ml of the deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenosine (EHNA) to inhibit its degradation is required to see these effects. Analysis of cell extracts by high-pressure liquid chromatography indicates that cordycepin enters the cells rapidly and is phosphorylated to 3'-dATP. The intracellular concentration rises almost linearly from 0.7 mM after 15 min to 7 mM by 210 min. Concomitantly the ATP concentration shows a rapid drop from the 4 mM present in controls. However, the direct reduction of ATP levels does not mimic the same rapid effects of cordycepin on the microtubules. In addition, similar effects are not produced by a variety of other adenosine analogues with alterations in the 2' and 3' ribose positions. Although other pharmacological reagents arrest cells at the onset of mitosis, cordycepin is unusual because of the collapse of the microtubule networks to two small asters that radiate from the microtubule-organizing center. 3'-dATP can replace the requirement for ATP or GTP in the vitro polymerization of microtubules from microtubule protein: however, at limiting concentrations of nucleotide it requires approximately two times the concentration of 3'-dATP as ATP to support an equivalent level of microtubule polymerization. This suggests that the effects of cordycepin in vivo may be the result of the depletion of cellular ATP pools and the altered ability of 3'dATP to substitute for ATP-dependent reactions. Current experiments are testing this hypothesis.     

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.     

Cordycepin rapidly collapses the intermediate filament networks into juxtanuclear caps in fibroblasts and epidermal cells

The nucleoside analog 3'-deoxyadenosine (cordycepin) rapidly collapses the intermediate filaments into juxtanuclear caps in interphase fibroblasts and keratinocytes. A minimum of 80 micrograms/ml cordycepin or 20 micrograms/ml cordycepin in combination with 2 micrograms/ml of the deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenosine (EHNA) to inhibit its degradation is required to see these effects. This is the same concentration required for cordycepin to arrest cells at the onset of mitosis and depolymerize the microtubules to small asters. Cordycepin enters the cells rapidly and is phosphorylated to 3'-dATP with a concomitant drop in ATP levels. However, the direct reduction of ATP levels does not mimic the same rapid effects of cordycepin on either the intermediate filaments or microtubules. In addition, similar effects are not produced by a variety of other adenosine analogs with alterations in the 2'- and 3'-ribose positions. Although other pharmacological reagents result in alterations of the fibroblastic intermediate filaments, cordycepin is unusual because of the rapidity with which the fibroblastic intermediate filaments collapse into the juxtanuclear caps. The juxtanuclear caps have a morphology different from that of the perinuclear bundles of intermediate filaments that arise after long-term depolymerization of the microtubules. The keratin fibers in the epidermal cells retract to a perinuclear ring when treated with cordycepin.     

Ribonucleic acid stimulation of mammalian liver nuclear-envelope nucleoside triphosphatase. A possible enzymic marker for the nuclear envelope.

1. The specific activity of rat and pig liver nuclear-envelope nucleoside triphosphatase (EC 3.6.1.3) decreases when the system is depleted of RNA. The activity can be restored by adding high concentrations of yeast RNA to the assay medium. 2. Exogenous RNA also increases the activity of the enzyme in control envelopes (not RNA-depleted). The effect appears to be largely specific for poly(A) and poly(G); it is not stimulated by rRNA or tRNA preparations, ribonuclease-hydrolysed RNA, AMP, or double- or single-stranded DNA. 3. Inhibitors of the enzyme, in concentrations at which half-maximal inhibition of the enzyme is achieved, do not affect the percentage stimulation of the enzyme by yeast RNA. 4. The simulation is abolished by the inclusion of 150 mM-KCl or -NaCl in the assay medium, but not by increasing the assay pH to 8.5. 5. The results are discussed in the light of the possible role of the nucleoside triphosphatase in vivo in nucleo-cytoplasmic ribonucleoprotein translocation. 6. It is proposed that poly(G)-stimulated Mg2+-activated adenosine triphosphatase activity should be adopted as an enzymic marker for the nuclear envelope.     

The effect of antimycin A on cytochromes b561, b566, and their relationship to ubiquinone and the iron-sulfer centers S-1 (+N-2) and S-3.

Three nuclear RNA polymerases and one poly(A) polymerase were isolated from the yeast, Saccharomyces cerevisiae. The ability of cordycepin triphosphate to inhibit each was determined. RNA polymerase II was significantly more sensitive to this compound than the other polymerases. RNA polymerase I was relatively insensitive, being inhibited less than 20% by 40 μm cordycepin triphosphate. The calculated apparent K_i values of RNA polymerases II and III and poly(A) polymerase were, respectively, 0.3, 3.0, and 4.6 μm. Inhibition was competitive with regard to ATP. These data do not support the idea that, in yeast, poly(A) addition to preformed RNA in vivo is the primary site of cordycepin action.     

Ara-ATP impairs 3''-end processing of pre-mRNAs by inhibiting both cleavage and polyadenylation.

Previous studies have demonstrated that Ara-ATP can inhibit poly(A) polymerase activity by competing with ATP. To elucidate the mechanism of action of this compound, its effect on the cleavage and polyadenylation of two specific substrates, SV40L and adenovirus L3 pre-mRNAs, was studied in HeLa nuclear extracts. Unlike cordycepin 5' triphosphate, Ara-ATP inhibited both cleavage and poly(A) addition. Addition of poly(A) polymerase fraction devoid of any other factors required for the processing reactions overcame the inhibitory effect on cleavage as well as polyadenylation of pre-mRNAs. These data suggest that Ara-ATP inhibits both cleavage and polyadenylation reactions by interacting with the ATP-binding site on poly(A) polymerase, the activity of which is essential for the cleavage reaction. Ara-ATP also blocked formation of the post-cleavage and polyadenylation-specific complexes, which further confirmed the inhibitory effect of the ATP analog on the two tightly coupled 3'-end processing reactions.     

Effect of cordycepin(3'-deoxyadenosine) on excision repair of 5,6-dihydroxy-dihydrothymine-type products from the DNA of Micrococcus radiodurans

Cordycepin(3'-deoxyadenosine), a nucleoside analog, has been shown to enhance radiation-induced cell killing. In an effort to elucidate the possible mechanism for enhancement of cell killing, the effect of cordycepin on the excision repair of radiation-induced 5,6-dihydroxy-dihydrothymine-type (t') products from the DNA of wild type Micrococcus radiodurans was investigated. The capacity of M. radiodurans to excise nondimeric (t') products from its DNA was significantly impaired after cordycepin treatment. The results suggest that the increased radiation sensitivity of cordycepin-treated cells could be due to alterations in cellular processes that repair DNA damage.     

Modified messenger ribonucleic acid release from isolated hepatic nuclei after inhibition of polyadenylate formation

Treatment of rats with Cordycepin (3'-deoxyadenosine), an inhibitor of nuclear poly(A) synthesis, selectively decreased the energy and cytosol-dependent release of a portion of the mRNA species from the isolated hepatic nuclei in a cell-free system by approximately 40%. An analysis of the differential binding of the transported mRNA containing poly(A) tracts to nitrocellulose filters, and to cellulose or poly(U)-Sepharose columns suggested that the poly(A) tracts in the mRNA transported from the isolated hepatic nuclei of Cordycepin-treated rats, are decreased in size. This size decrease was confirmed through an analysis of the average size of the poly(A) tracts, released from the messengers by ribonuclease activity, on sucrose density gradients.