Changes of nuclear membrane lipid composition affect RNA nucleocytoplasmic transport.

We have previously shown that the nuclear membrane fluidity of rat liver, measured by fluorescence anisotropy of two probes, is higher in the hydrophobic core, with respect to the bilayer surface, in newborn rats compared to adult rats. The aim of the present research is to investigate whether the nuclear membrane fluidity influences RNA nucleocytoplasmic transport. To this end two experimental models were used: the fluidity of nuclear membrane isolated from adult rats was increased by a choline base exchange reaction, which is known to be accompanied by an increase of phosphatidylcholine unsaturated fatty acids, whereas that of nuclear membrane isolated from newborn rats was decreased by incubation with dimyristoylphosphatidylcholine-cholesterol liposomes. The RNA efflux, evaluated by using [3H]uridine, significantly increased in the adult nuclear membrane submitted to choline base exchange reaction, whereas a strong decrease in the newborn nuclear membrane enriched with cholesterol was found. The activity of nucleoside triphosphatase, a nuclear membrane-associated enzyme which is correlated with mRNA transport, showed parallel variations. Therefore, for the first time, we have provided evidence that the nuclear membrane fluidity plays a regulatory role in RNA nucleocytoplasmic transport, although the mechanism by which this effect takes place remains to be clarified.     

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.     

Role of the nuclear envelope in synthesis, processing, and transport of membrane glycoproteins

The outer nuclear membrane is morphologically similar to rough endoplasmic reticulum. The presence of ribosomes bound to its cytoplasmic surface suggests that it could be a site of synthesis of membrane glycoproteins. We have examined the biogenesis of the vesicular stomatitis virus G protein in the nuclear envelope as a model for the biogenesis of membrane glycoproteins. G protein was present in nuclear membranes of infected Friend erythroleukemia cells immediately following synthesis and was transported out of nuclear membranes to cytoplasmic membranes with a time course similar to transport from rough endoplasmic reticulum (t 1/2 = 5-7 min). Temperature-sensitive mutations in viral membrane proteins which block transport of G protein from endoplasmic reticulum also blocked transport of G protein from the nuclear envelope. Friend erythroleukemia cells and NIH 3T3 cells differed in the fraction of newly synthesized G protein found in nuclear membranes, apparently reflecting the relative amount of nuclear membrane compared to endoplasmic reticulum available for glycoprotein synthesis. Nuclear membranes from erythroleukemia cells appeared to have the enzymatic activities necessary for cleavage of the signal sequence and core glycosylation of newly synthesized G protein. Signal peptidase activity was detected by the ability of detergent-solubilized membranes of isolated nuclei to correctly remove the signal sequence of human preplacental lactogen. RNA isolated from the nuclear envelope was highly enriched for G protein mRNA, suggesting that G protein was synthesized on the outer nuclear membrane rather than redistributing to nuclear membranes from endoplasmic reticulum before or during cell fractionation. These results suggest a mechanism for incorporation of membrane glycoproteins into the nuclear envelope and suggest that in some cell types the nuclear envelope is a major source of newly synthesized membrane glycoproteins.     

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.     

Fluorescence photobleaching analysis of nuclear transport: dynamic evidence for auxiliary channels in detergent-treated nuclei

Nuclear transport experiments were performed on isolated rat liver nuclei to examine the permeability of membrane and detergent-free peripheral nuclear lamina. The transport of 64K molecular weight fluorescent-derivatized dextrans was measured by using the technique of fluorescence redistribution after photobleaching. Results of these experiments provide evidence for transport pathways that appear to be functionally distinct from nuclear pore complex channels. The suggestion is made that these supplemental pathways are embedded in the peripheral nuclear lamina and are normally masked by the inner nuclear membrane.     

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.     

Expansion of the nucleoplasmic reticulum requires the coordinated activity of lamins and CTP:phosphocholine cytidylyltransferase alpha

The nucleoplasmic reticulum (NR), a nuclear membrane network implicated in signaling and transport, is formed by the biosynthetic and membrane curvature-inducing properties of the rate-limiting enzyme in phosphatidylcholine synthesis, CTP:phosphocholine cytidylyltransferase (CCT) alpha. The NR is formed by invagination of the nuclear envelope and has an underlying lamina that may contribute to membrane tubule formation or stability. In this study we investigated the role of lamins A and B in NR formation in response to expression and activation of endogenous and fluorescent protein-tagged CCTalpha. Similarly to endogenous CCTalpha, CCT-green fluorescent protein (GFP) reversibly translocated to nuclear tubules projecting from the NE in response to oleate, a lipid promoter of CCT membrane binding. Coexpression and RNA interference experiments revealed that both CCTalpha and lamin A and B were necessary for NR proliferation. Expression of CCT-GFP mutants with compromised membrane-binding affinity produced fewer nuclear tubules, indicating that the membrane-binding function of CCTalpha promotes the expansion of the NR. Proliferation of atypical bundles of nuclear membrane tubules by a CCTalpha mutant that constitutively associated with membranes revealed that expansion of the double-bilayer NR requires the coordinated assembly of an underlying lamin scaffold and induction of membrane curvature by CCTalpha.     

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.     

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 transport in isolated myeloma nuclei. Transport from membrane- denuded nuclei

Nuclei prepared from MOPC-21 cells were treated with the nonionic detergents Triton X-100 or Nonidet P-40. Chemical analysis revealed that nearly 90% of the nuclear phospholipid was removed by detergent treatment. The membrane-denuded nuclei remained intact with preservation of nuclear pore complexes as demonstrated by electron microscopy. Ribonucleic acid transport from detergent-treated nuclei proceeded at the same rate and to the same extent as in control nuclei. Normal nuclear restriction of nucleic acids was unaltered by removal of the nuclear membranes. The effect of temperature on transport of RNA from freshly isolated myeloma nuclei with intact nuclear envelopes was studied. No temperature transition was associated with the transport process. These data indicate that the transport of macromolecules from isolated myeloma nuclei is independent of the nuclear membrane.     

Chemical factors that influence nucleocytoplasmic transport: a fluorescence photobleaching study

The technique of fluorescence redistribution after photobleaching was used to measure the translocation rate of fluorescein-labeled dextrans across the nuclear pore complex in isolated rat liver nuclei. A transport assay system was established that could monitor the effect of biologically active molecules, e.g., ATP, GTP, cAMP on the translocation process. The results show that ATP, phosphoinositides, RNA, and insulin can enhance transport rates from 195 to 432%. It was further demonstrated that concanavalin A, but not wheat germ or soybean agglutinin, can block dextran transport completely. The effectors of dextran transport are similar to substances demonstrated to effect the efflux of RNA from isolated nuclei. A model for translocation through the nuclear pore is now presented that incorporates data from protein influx and RNA efflux experiments into a single pathway controlled by ATP.     

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.     

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.     

Characterization of the N-terminal segment used by the barley yellow dwarf virus movement protein to promote interaction with the nuclear membrane of host plant cells

The barley yellow dwarf virus movement protein (BYDV-MP) requires its N-terminal sequence to promote the transport of viral RNA into the nuclear compartment of host plant cells. Here, graphical analysis predicts that this sequence would form a membrane interactive amphiphilic alpha-helix. Confirming this prediction, NT1, a peptide homologue of the BYDV-MP N-terminal sequence, was found to be alpha-helical (65%) in the presence of vesicles mimics of the nuclear membrane. The peptide increased the fluidity of these nuclear membrane mimics (rise in wavenumber of circa 0.5-1.0 cm(-1)) and induced surface pressure changes of 2 mN m(-1) in lipid monolayers with corresponding compositions. Taken with isotherm analysis these results suggest that BYDV-MP forms an N-terminal amphiphilic alpha-helix, which partitions into the nuclear membrane primarily through thermodynamically stable associations with the membrane lipid headgroup region. We speculate that these associations may play a role in targeting of the nuclear membrane by BYDM-MP.     

Identification of a novel signal sequence that targets transmembrane proteins to the nuclear envelope inner membrane

Herpesvirus maturation requires translocation of glycoprotein B homologue from the endoplasmic reticulum to the inner nuclear membrane. Glycoprotein B of human cytomegalovirus was used in this context as a model protein. To identify a specific signal sequence within human cytomegalovirus glycoprotein B acting in a modular fashion, coding sequences were recombined with reporter proteins. Immunofluorescence and cell fractionation demonstrated that a short sequence element within the cytoplasmic tail of human cytomegalovirus glycoprotein B was sufficient to translocate the membrane protein CD8 to the inner nuclear membrane. This carboxyl-terminal sequence had no detectable nuclear localization signal activity for soluble beta-Galactosidase and could not be substituted by the nuclear localization signal of SV40 T antigen. For glycoprotein B of herpes simplex virus, a carboxyl-terminal element with comparable properties was found. Further experiments showed that the amino acid sequence DRLRHR of human cytomegalovirus glycoprotein B (amino acids 885-890) was sufficient for nuclear envelope translocation. Single residue mutations revealed that the arginine residues in positions 4 and 6 of the DRLRHR sequence were essential for its function. These results support the view that transmembrane protein transport to the inner nuclear membrane is controlled by a mechanism different from that of soluble proteins.     

Effect of colchicine on mammalian liver nuclear envelope and on nucleo-cytoplasmic RNA transport

The binding of colchicine to nuclear envelopes was studied in order to elucidate the mechanism whereby this compound inhibits nucleocytoplasmic RNA transport. The results suggest that a single class of colchicine-binding site (dissociation constant=approx. 0.7 mM, concentration=approx. 330 nmol colchicine/mg protein) is localised in the nuclear periphery (pore-lamina) and that binding to these sites effects a constriction of the pore-complexes with concomitant inhibition of RNA egress and disordering of the nuclear membrane phospholipid bilayers.     

The packaging signal of MLV is an integrated module that mediates intracellular transport of genomic RNAs

Packaging of MLV genomes requires four cis-acting stem-loops. Stem-loops A and B are self-complementary and bind Gag in their dimeric form, while the C and D elements mediate loop-loop interactions that facilitate RNA dimerization. Packaging also requires nuclear export of viral genomes, and their cytoplasmic transport toward the plasma membrane. For MLV, this is mediated by Gag and Env, and occurs on endosomal vesicles. Here, we report that MLV Psi acts at several steps during the transport of genomic RNAs. First, deletion of stem-loop B or C leads to the accumulation of genomic RNAs in the nucleus, suggesting that these elements are involved in export. Second, in chronically infected cells, mutation of the C and D loops impairs endosomal transport. This suggests that RNA dimerization is essential for vesicular transport, consistent with its proposed requirement for Gag binding. Surprisingly, deletion of stem-loop A blocks vesicular transport, whereas removal of stem-loop B has no effects. This suggests that stem-loop A has unique functions in packaging, not predicted from previous in vitro analyses. Finally, in packaging cells that do not express any Psi-containing RNA, endosomal RNA transport becomes sequence-independent. This non-specific activity of Gag likely promotes packaging of cellular mRNAs.