Conformational variability of nucleo-cytoplasmic transport factors

The transport of macromolecules between the nucleus and cytoplasm of eukaryotic cells is largely mediated by a single family of transport factors, the karyopherin or importin beta-like family. Structural and biochemical evidence suggests conformational flexibility of these modular HEAT-repeat proteins is crucial for their regulation. Here we use small angle x-ray scattering to assess the extent of conformational variation within a set of nuclear import and export factors. The study reveals that importin beta, transportin, and the exportin Xpo-t share a similar S-like superhelical conformation in their unbound state. There are no obvious differences in the overall structures that might generally distinguish nuclear export from nuclear import mediators. Two other members of the family, the exportins Cse1 and Xpo1, possess a significantly more globular conformation, indicating that the extended S-like architecture is not a hallmark of all karyopherins. Binding of RanGTP/cargo to importin beta, transportin, and Xpo-t triggers distinct conformational responses, suggesting that even closely related karyopherins employ different mechanisms of conformational regulation and that cargo and nuclear pore-interacting surfaces of the different receptors may be unique.     

Some features of nucleo-cytoplasmic RNA transport from isolated nuclei

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

Polyadenylated H3 histone transcripts and H3 histone variants in alfalfa

Histone H3 mRNAs were found in polyA(+) fractions of total RNA prepared from alfalfa plants, calli and somatic embryos. The sequence analysis of cDNAs revealed the presence of a polyA tail on independent alfalfa H3 mRNAs. A highly conserved sequence motif AAUGAAA identified about 20bp upstream from the 3' ends of the alfalfa H3 cDNAs was suggested to be one of the possible regulatory elements in the 3' end formation and polyadenylation. Three out of the four analysed H3 cDNAs have more than 97% homology with a genomic clone and encode the same protein. While the fourth represents a minor species with only 78.8% homology to the coding region of the genomic clone and encodes a H3 histone with four amino acid replacements. On the basis of compilation analysis we suggest a consensus sequence for plant H3 histones which differs from that of animal's by four amino acid changes.     

Importance of mammalian nuclear-envelope nucleoside triphosphatase in nucleo-cytoplasmic transport of ribonucleoproteins.

The nucleoside triphosphate-stimulated efflux of RNA from isolated nuclei was studied under a range of conditions, and the effects of these conditions on the process were compared with the properties of the nucleoside triphosphatase located in the pore complex. A marked similarity between the rate of efflux and the rate of nucleoside triphosphate hydrolysis was apparent, in terms of substrate specificity, sensitivity to treatment with insolubilized trypsin, kinetics and the effects of increased ionic strength and of many inhibitors. These results are taken, in view of earlier evidence, to suggest that the activity of the nucleoside triphosphatase is a prerequisite for nucleo-cytoplasmic RNA transport in vivo. There are some indications that the nuclear-envelope lipid is also involved in regulating the efflux process.     

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.     

Association of AUUUA-binding protein with A+U-rich mRNA during nucleo-cytoplasmic transport.

Resealed nuclear envelope (NE) vesicles from rat liver containing entrapped exogenous RNA were used to study the effect of adenosine+uridine binding factor (AUBF), present in cytosolic cell extracts, on ATP-dependent transport of A+U-rich RNA (AU+RNA) and A+U-free RNA (AU-RNA) across the NE. This factor specifically binds to A+U-rich sequences present in the 3' untranslated regions of lymphokine and cytokine mRNAs, containing overlapping AUUUA boxes (granulocyte-macrophage colony stimulating factor, interleukin-3). Addition of AUBF to the extravesicular compartment markedly increased the efflux of the in vitro transcribed, capped and polyadenylated AU+ RNAs. Export of entrapped AU- control RNA, such as beta-globin RNA, was not affected by AUBF, in contrast to chimeric AU+ beta-globin RNA containing the A+U-rich sequence of human interferon-alpha mRNA (6 reiterated AUUUA motifs). Competition experiments revealed that AUBF enhances the affinity of poly(A)-containing AU+ RNAs to the NE poly(A)-binding component (poly(A)-recognizing mRNA carrier p106), and thereby accelerates nuclear export of these RNAs. We could demonstrate that AUBF added to the extravesicular space forms stable complexes with polyadenylated AU+ RNA with relative molecular masses of about 45,000, 62,000 and 70,000 inside the vesicles or during ATP-dependent export. In addition we determined that AUBF may affect mRNA stability by protecting A+U-rich RNA against degradation by trans-acting, nuclear matrix-associated and A+U-specific endoribonuclease V.     

Translocation biosensors to study signal-specific nucleo-cytoplasmic transport, protease activity and protein-protein interactions

Regulated nucleo-cytoplasmic transport is crucial for cellular homeostasis and relies on protein interaction networks. In addition, the spatial division into the nucleus and the cytoplasm marks two intracellular compartments that can easily be distinguished by microscopy. Consequently, combining the rules for regulated nucleo-cytoplasmic transport with autofluorescent proteins, we developed novel cellular biosensors composed of glutathione S-transferase, mutants of green fluorescent protein and rational combinations of nuclear import and export signals. Addition of regulatory sequences resulted in three classes of biosensors applicable for the identification of signal-specific nuclear export and import inhibitors, small molecules that interfere with protease activity and compounds that prevent specific protein-protein interactions in living cells. As a unique feature, our system exploits nuclear accumulation of the cytoplasmic biosensors as the reliable readout for all assays. Efficacy of the biosensors was systematically investigated and also demonstrated by using a fully automated platform for high throughput screening (HTS) microscopy and assay analysis. The introduced modular biosensors not only have the potential to further dissect nucleo-cytoplasmic transport pathways but also to be employed in numerous screening applications for the early stage evaluation of potential drug candidates.     

Senescence-related functional nuclear barrier by down-regulation of nucleo-cytoplasmic trafficking gene expression

One of the characteristic natures of senescent cells is the hypo- or irresponsiveness not only to growth factors but also to apoptotic stress. In the present study, we confirmed the inhibition of nuclear translocation of activated p-ERK1/2 and NF-kB p50 in response to growth stimuli or LPS in the senescent human diploid fibroblasts. In order to elucidate the underlying mechanism for the senescence-associated hypo-responsiveness, we carried out the comparison study for gene expression profiles through microarray analysis. In consequence, we observed the vast reduction in expression of nucleo-cytoplasmic trafficking genes in senescent cells, when compared with those in young cells. Expression levels of several nucleoporins, karyopherin α, karyopherin β, Ran, and Ran-regulating factors were confirmed to be down-regulated in senescent HDFs by using RT-PCR and Western blot methods. Taken together, these data suggest the operation of certain senescence-associated functional nuclear barriers by down-regulation of the nucleo-cytoplasmic trafficking genes in the senescent cells.     

Metazoan replication-dependent histone mRNAs: a distinct set of RNA polymerase II transcripts

Metazoan replication-dependent histone mRNAs are the only eukaryotic mRNAs that lack polyA tails. The genes for the five histone proteins have remained physically linked during evolution. Expression of histone mRNAs and histone proteins requires a unique set of factors, and may be coordinated by association of the histone genes with Cajal bodies. Recently several novel factors, including components of the U7 snRNP, as well as proteins involved in regulation of histone gene expression, have been described.     

Intimate contacts of mitochondria with nuclear envelope as a potential energy gateway for nucleo-cytoplasmic mRNA transport

The aim of the present study was to show that close contacts of mitochondria with nuclear envelope need not be just an accidental situation in the cell, but that such contacts could serve for flow of energy from a place of origin directly to a place of consumption. Mitochondria in close proximity to the nuclear envelope can be found virtually in all metabolically active cells. We used transmission electron microscopy to demonstrate this entity in different leukemia cells of human origin (patient's blood) and in mouse leukemia cell line. At high resolution, not only close proximity but even fusion of mitochondrial and nuclear membranes can be seen. Based on available data about mRNA transport through the nuclear pore complex and observed contacts of mitochondria with nuclei, we hypothesize that such contacts can provide a gateway for energy delivery to power mRNA export from the nucleus to the cytoplasm. Hence the lumen of the nuclear envelope can serve for transport or even storage of macroergic molecules in a manner similar to sarcoplasmic reticulum in fast-twitch skeletal muscles.     

Molecular characterization of a ChaC K+ transport regulator gene and its transcripts in the glassy-winged sharpshooter,Homalodisca coagulata

Four mRNA variants in pooled cDNA samples of the glassy-winged sharpshooter, Homalodisca coagulata, encoding a putative regulator of cation transporters were identified. RT-PCR showed that the hc-chaC variants were expressed during different stages of insect development and in different tissues and sexes. Structural analysis of the hc-chaC gene indicated that intron and exon sequences of the mRNA variants were identical, and similar to chaC-like genes of other insects. The Hc-ChaC protein (22.5 kDa) contained the conserved FGYGSL K⁺-binding motif near its amino-terminus, and the carboxy-terminal region contained two coiled-coil motifs, which had similarity to the PDZ domain present in well-characterized Na⁺/H⁺ exchanger regulatory factors. Our analyses suggest that Hc-ChaC is a regulator of K⁺ transporters such as K⁺/H⁺ antiporters.