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 46-kDa nucleoside triphosphatase of rat liver nuclear scaffold represents the N-terminal portion of lamins A/C

The major nucleoside triphosphatase (NTPase) of rat liver nuclear scaffold (NS) or envelope, which is thought to participate in nucleocytoplasmic transport, has been identified via photoaffinity labeling as a 46-kDa polypeptide. This 46-kDa protein was purified by SDS-polyacrylamide gel electrophoresis and cleaved with trypsin. The resulting peptides were purified by HPLC and five were microsequenced. All five peptides appear to be derived from the N-terminal region of lamins A/C. Subsequent experiments with photolabeled NS showed that the 46-kDa polypeptide was selectively immunoprecipitated by antiserum specific to lamins A/C and by affinity-purified anti-lamin antibodies. Photolabeling of nuclei prepared in the presence of protease inhibitors showed predominant labeling of the 46-kDa polypeptide, suggesting that it is an integral nuclear constituent and not an artifact produced during NS preparation. Use of protease inhibitors throughout purification of NS increased the specificity of photolabeling of the 46-kDa band by significantly reducing photolabeling of smaller molecular weight components, which arise by proteolysis. Anti-lamin antibodies also produced a significant inhibition of NTPase activity in NS. These results suggest that the N-terminal portion of lamins A/C represents the 46-kDa NTPase, which, according to previous reports, may participate in RNA transport.     

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.     

Newborn human skin fibroblasts senesce in vitro without acquiring adult growth factor requirements

Nuclear-envelope nucleoside triphosphatase activity (NTPase), an enzymatic activity thought to participate in RNA transport, was localized in rat liver in situ after brief perfusion with 3% paraformaldehyde. Reaction product was distributed along the nucleoplasmic side of the nuclear envelope (NE) in heterochromatin, was only occasionally found at nuclear pores, and nuclear deposition was selectively blocked by inhibitors of NE NTPase activity. Our results suggest that NTPases, which are active in the NE and which participate in RNA transport, are not specifically associated with nuclear-pore complexes.     

RNA metabolism in isolated nuclei: processing and transport of immunoglobulin light chain sequences

Transport of prelabeled RNA from isolated myeloma nuclei is studied using conditions that permit RNA synthesis. Cytosol and spermidine are not required to maintain nuclear stability and inhibited RNA release. Omission of ATP or GTP decreased release 25 to 40%. The stimulatory effect of ATP or GTP is not due to hydrolysis of the triphosphates by the nuclear envelope NTPase, since addition of quercetin (an inhibitor of this NTPase) has no effect on the quantity of RNA released. The size distribution and percentage of poly A-containing species released from nuclei incubated with or without ATP or the other rNTPs are identical. Hybridization analysis of nuclear RNA before the transport assay revealed mature and precursor k light chain mRNA sequences. Following the transport assay, a significant fraction of k mRNA precursors is chased into mature k mRNA which is found both in nuclear-retained and released RNA.     

缺血／再灌注损伤对心肌细胞核膜NTPase活性和mRNA出核转运的影响

目的：观察家兔心肌缺血／再灌注（I／R）损伤对心肌细胞核膜核苷三磷酸酶（NTPase）动力学及mRNA出核转运的变化。方法：制备家兔离体心脏I／R模型,密度梯度离心法制备心肌细胞核膜,按Tiffany等的方法测定NTPase活性及mRNA的转运率。结果：心肌细胞核膜NTPase在以ATP或GTP为底物时,与对照组比较,I／R组最大反应速率（Vmax)分别降低26％及22％（P＜0．01）。Km值分别升高54％及72％（P＜0．01）;而缺血组Vmax及Km值较对照组无明显差异。以ATP或GTP启动反应时I／R组10min内心肌细胞核mRNA转出率分别较对照组降低27％及32％（P＜0．01）;而缺血组与对照组相比并无显著性差异（P＞0．05）。结论：心肌缺血／再灌注损伤时,心肌细胞核膜受损,核膜上NTPase活性降低,导致细胞核膜mRNA出核受阻,从而可能影响蛋白质的合成表达。     

Photoaffinity labeling of the major nucleosidetriphosphatase of rat liver nuclear envelope

We employed the photoaffinity probe 8-azido-adenosine 5'-triphosphate (aATP) to identify the nuclear envelope (NE) nucleosidetriphosphatase activity (NTPase) implicated in control of RNA transport. The photoprobe was hydrolyzed at rates comparable to those for ATP, with a Michaelis constant of 0.225 mM. Photolabeling was dependent upon UV irradiation (300-nm max) and was not affected by quercetin. Unlabeled ATP or GTP competed with [32P]aATP in photolabeling experiments, and UTP was a less effective competitor, paralleling the substrate specificity of the NTPase. Incubation of NE with aATP led to a UV, time, and concentration dependent irreversible inactivation of NTPase. The inactivation could be blocked by ATP or GTP. Polyacrylamide gel electrophoresis and autoradiography of photolabeled NE showed selective, UV-dependent labeling of a 46-kDa protein with both [gamma-32P]aATP and [alpha-32P]aATP. This band was not labeled with [gamma-32P]ATP. Since the NE NTPase implicated in RNA transport is modulated by RNA, we examined the effects of RNA on the labeling process. Removal of RNA from the NE preparations (by RNase/DNase digestion) reduced NTPase by 30-40% and eliminated photolabeling of the 46-kDa band. Addition of yeast RNA to such preparations increased NTPase activity to control levels and selectively reinstated photolabeling of the 46-kDa band. These results suggest that the 46-kDa protein represents the major NTPase implicated in RNA transport.     

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.     

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.     

Action of insulin at the nuclear envelope

Summary Insulin binding sites are present on purified nuclear envelopes from liver and other tissues, and EM autoradiographs and other types of studies indicate that insulin can enter intact target cells and interact with several types of intracellular membranes, including the nuclear envelope. More recent studies indicate that insulin has direct effects on both mRNA efflux from isolated nuclei and nuclear envelope NTPase, the enzyme that regulates mRNA efflux. These studies raise the possibility, therefore, that insulin regulates mRNA levels in target cells by directly influencing nuclear membrane functions as NTPase. Since insulin does not dramatically elevate mRNA levels for all proteins, the question arises as to how insulin selectively increases mRNA for specific mRNAs. One possibility is that there is targeting of specific mRNA molecules for specific pore complexes and that insulin may only influence a certain fraction of the nuclear pores. Thus, continued investigation is needed concerning the role of polypeptide hormones such as insulin in nucleocytoplasmic exchange.     

Ribonucleic acid efflux from isolated mouse liver nuclei is altered by diet and genotypically determined change in nuclear envelope composition

Differences in immunological abnormalities like autoimmunity, abnormal T cell proliferative disorders and accelerated ageing occur between MRL/Mp-lpr/lpr(lpr/lpr) and MRL/Mp-+/+(+/+) mice as a consequence of one gene. The present study was designed to assess the effect of these differences in genotype and diet on the composition and function of the liver nuclear envelope. Mice of both strains were fed nutritionally adequate diets differing only in fatty acid composition for 4 weeks. Phospholipid fatty acid composition of the liver nuclear envelope was determined and the effect of altering the lipid composition of the nuclear membrane on nucleoside-triphosphatase (NTPase) activity, ribonucleic acid (RNA) efflux and binding of L-triiodothyronine (L-T3) was determined. Strain of mouse and level of dietary linoleic acid exhibited significant effects on the phospholipid fatty acid composition of the nuclear envelope. Levels of 18:1(n - 9) and 18:2(n - 6) were lower and 20:4(n - 6) content was higher in nuclear envelope phospholipids of lpr/lpr mice compared with mice of the +/+ strain. Mice fed the high linoleic acid diet exhibited higher levels of 18:0, 18:2(n - 6) and 20:4(n - 6) and lower levels of 16:0 and 18:1(n - 9) in liver nuclear envelope phospholipids, compared with mice fed the low linoleic acid diet. These changes in membrane composition were reflected in alteration of NTPase activity and efflux of RNA from isolated mouse liver nuclei. Nucleoside triphosphatase activity and efflux of ribonucleic acid from isolated nuclei were significantly higher in livers of the lpr/lpr strain. NTPase activity and RNA efflux from isolated nuclei were higher in the high linoleic acid fed group compared with the low linoleic acid group. A single class of binding sites for L-T3 was present in liver nuclear envelopes of these mice and Kd values were not influenced by strain or dietary linoleic acid levels. Nuclear envelopes prepared from +/+ animals exhibited a significantly higher number of binding sites for L-T3 compared with the lpr/lpr group. These observations indicate that the single gene difference characterizing lpr/lpr mice from +/+ mice results in alterations in the composition and function of the nuclear envelope. This genetic difference also alters the response of this membrane to dietary factors known to modulate characteristics and functions of the nuclear envelope.     

Differential effect of insulin and epidermal growth factor on the mRNA translocation system and transport of specific poly(A+) mRNA and poly(A-) mRNA in isolated nuclei

The efficiency of efflux of rapidly labeled poly(A)-containing mRNA from isolated rat liver nuclei was found to be modulated by insulin and epidermal growth factor (EGF) in a biphasic but opposite way. At physiological concentrations (10 pM insulin and 1 pM EGF), maximal stimulation of the transport rate by insulin (to 137%) and maximal inhibition by EGF (to 69%) were obtained; at higher concentrations (greater than 100 pM and greater than 10 pM, respectively), the amount of poly(A)-containing mRNA released into the postnuclear supernatant was nearly identical with the level found in untreated nuclei (= 100%). Using mRNA entrapped into closed nuclear envelope (NE) vesicles as a model system, it was found that the modulation of nuclear efflux of mRNA by the two growth factors occurs at the level of translocation through the nuclear pore. The NE nucleoside-triphosphatase (NTPase) activity, which is thought to mediate nucleocytoplasmic transport of at least some mRNAs, responded to insulin and EGF in the same manner as the mRNA transport rate. The increase in NTPase activity caused by insulin and the decrease in NTPase activity caused by EGF were found to be due to changes of the maximal catalytic rate; the Michaelis constant of the enzyme remained almost constant. Investigating the effect of the two growth factors on transport of specific mRNAs, poly(A)-containing actin mRNA was found to display the same alteration in efflux rate as rapidly labeled, total poly(A)-containing mRNA. In contrast, efflux of histone H4 mRNA, which lacks a 3'-poly(A) sequence, decreased in response to insulin and reached minimum levels at the same concentration at which maximum levels of actin mRNA transport rate were obtained. Studying the mechanism of action of insulin and EGF on NE mRNA translocation system, insulin was found to cause an enhancement of NE-associated phosphoprotein phosphatase activity, resulting in a dephosphorylation of the NE poly(A) binding site (= mRNA carrier) and, hence, in a decrease in its affinity to poly(A) [the poly(A) binding affinity of the poly(A)-recognizing mRNA carrier within the envelope is increased after phosphorylation]. EGF, on the other hand, stimulated the protein kinase, which phosphorylates the carrier, and, hence increased the NE poly(A) binding affinity. Because the stage of phosphorylation of the mRNA carrier (which is coupled with the NTPase within the intact NE structure) is inversely correlated with the activity of the NTPase, an enhancement of poly(A)-containing mRNA transport rate by insulin and an inhibition by EGF are observed.     

RNA metabolism in isolated nuclei: effect of temperature on RNA transport from intact and membrane-denuded nuclei

The kinetics of RNA transport from intact (both inner and outer nuclear membranes present) and membrane-denuded myeloma nuclei were monitored at temperatures between 10 and 37 degrees C. A linear rate for RNA transport was calculated and the log of RNA transported from membrane-denuded nuclei was greater than that transported from intact nuclei and ii) RNA transport from both nuclear preparations exhibited straight line Arrhenius plots. We conclude the nuclear envelope (or a nuclear matrix element) modulates the amount of RNA transported from nuclei and that nuclear membrane thermal phase transitions do not alter the apparent energy of activation for the transport process.     

Analysis of the internal nuclear matrix. Oligomers of a 38 kD nucleolar polypeptide stabilized by disulfide bonds

When rat liver nuclei are treated with the sulfhydryl cross-linking reagent sodium tetrathionate (NaTT) prior to nuclease treatment and extraction with 1.6 M NaCl, residual nucleoli and an extensive non-chromatin intranuclear network remain associated with the nuclear envelope. Subsequent treatment of this structure with 1 M NaCl containing 20 mM dithiothreitol (DTT) solubilizes the intranuclear material, while the nuclear envelope remains structurally intact. We have isolated and partially characterized a major polypeptide of the disulfide-stabilized internal nuclear matrix. The polypeptide, which has an apparent molecular mass 38 kD and isoelectric point 5.3, has been localized to the nucleolus of rat liver nuclei by indirect immunofluorescence using a specific polyclonal chicken antiserum. Based on its molecular mass, isoelectric point, intracellular localization and amino acid composition, the 38 kD polypeptide appears to be analogous to the nucleolar phosphoprotein B23 described by Prestayko et al. (Biochemistry 13 (1974) 1945) [20]. Immunologically related polypeptides have likewise been localized to the nucleoli of both hamster and human tissue culture cell lines as well as the cellular slime mold Physarum polycephalum. By immunoblotting, a single 38 kD polypeptide is recognized by the antiserum in rat, mouse, hamster and human cell lines. The antiserum has been utilized to investigate the oligomeric structure of the 38 kD polypeptide and the nature of its association with the rat liver nuclear matrix. By introducing varying numbers of disulfide bonds, we have found that the 38 kD polypeptide becomes incorporated into the internal nuclear matrix in a two-step process. Soluble disulfide-bonded homodimers of the polypeptide are first formed and then are rendered salt-insoluble by more extensive disulfide cross-linking.     

Partial purification of a nucleoside triphosphatase from the inner membrane of the chloroplast envelope of pea

A Mg2+-NTPase has been partially purified from the inner membrane of the pea chloroplast envelope. Isolated envelope membranes were solubilized with Triton X-100 and fractionated by DEAE-Sephadex chromatography, followed by ultrafiltration and sucrose density gradient centrifugation. An approximate 35-fold increase in the specific activity of the vanadate and sodium fluoride sensitive NTPase was obtained. Analysis of the partially purified NTPase by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a single 37-kDa polypeptide that appeared to be associated with the activity. In support of this identification, it is demonstrated that the 37-kDa polypeptide can be photolabeled with 8-azido-ATP.     

Proteins from rat liver cytosol which stimulate mRNA transport. Purification and interactions with the nuclear envelope mRNA translocation system

Two polysome-associated proteins with particular affinities for poly(A) have been purified from rat liver. These proteins stimulate the efflux of mRNA from isolated nuclei in conditions under which such efflux closely stimulates mRNA transport in vivo, and they are therefore considered as mRNA-transport-stimulatory proteins. Their interaction with the mRNA-translocation system in isolated nuclear envelopes has been studied. The results are generally consistent with the most recently proposed kinetic model of mRNA translocation. One protein, P58, has not been described previously. It inhibits the protein kinase that down-regulates the NTPase, it enhances the NTPase activity in both the presence and the absence of poly(A) and it seems to increase poly(A) binding in unphosphorylated, but not in phosphorylated, envelopes. The other protein, P31, which probably corresponds to the 35,000-Mr factor described by Webb and his colleagues, enhances the binding of poly(A) to the mRNA-binding site in the envelope, thus stimulating the phosphoprotein phosphatase and, in consequence, the NTPase. The possible physiological significance of these two proteins is discussed.     

Purification of a glucose-binding protein from rat liver nuclei. Evidence for a role in targeting of nuclear mRNP to nuclear pore complex.

A nuclear carbohydrate-binding protein with a molecular mass of 67 kDa (CBP67), which is specific for glucose residues, was purified to essential homogeneity from rat liver nuclear extracts. This protein could also be isolated from nuclear ribonucleoprotein (RNP) complexes by extraction in the presence of 0.6 M or 2 M NaCl, but it was absent in polysomal RNP complex. The binding of the purified protein, which has an isoelectric point of 7.3, to glucose-containing glycoconjugates depends on the presence of Ca2+ and Mg2+. Using closed nuclear envelope vesicles as a system to study nuclear transport of RNA, it was shown that both entrapped polysomal mRNA and nuclear RNA precursors are readily exported from the vesicles in an ATP-dependent manner. The transport was unidirectional and strongly promoted by the poly(A) segment attached to these RNAs. In contrast, nuclear RNP complexes entrapped into the vesicles together with glucose-conjugated bovine serum albumin or nucleoplasmin, or bird nest glycoprotein, were not exported into the extravesicular space. However, transport of nuclear RNP complexes could be achieved in the presence of glucose or after co-addition of a glucose-recognizing lectin from Pellina semitubulosa. In Western blots, radioiodinated CBP67 binds to an 80-kDa polypeptide both in isolated rat liver nuclear envelopes and pore-complex laminae. From these results we postulate that CBP67 may direct nuclear RNP complexes to the nuclear pore.     

Isolation and characterization of the nuclear matrix from the male xenopus laevis following estrogen administration: Kinetics of [3H] uridine incorporation

At various times following estorgen administration, the nuclear matrix was isolated from the liver of male Xenopus laevis by sucrose gradient centrifugation of nuclei treated with a high-salt buffer and DNase I in the presence of a proteolytic inhibitor (PMSC - phenylmethyl sulfonyl chloride). Electron micrographs of the nuclear matrix demonstrate a sponge-like network attached to a well-defined inner envelope with a ribosome-free outer envelope. Chemical analyses show that the HSB-DNase-treated nuclei consist of 16% DNA, 2% RNA, and 82% protein, a composition that is consistent with that of nuclear matrices isolated from other species. The specific activity of the matrix-associated RNA following estrogen treatment appears to be maximally enhanced after 5 h and decreases until approximately 12 h, when the activity begins to increase again.