Marie-Christine Dabauvalle

Cell type-specific differences in protein composition of nuclear pore complex-lamina structures in oocytes and erythrocytes of Xenopus laevis☆

Abstract Nuclear envelopes from oocytes of Xenopus laevis are rich in pore complexes and contain a major polypeptide of apparent molecular weight (Mr) 68,000. A rapid extraction procedure using buffer containing 1% ( v v ) Triton X-100 and 1.0 m -KCl allows the preparation of insoluble nuclear envelope skeletons showing only residual pore complex structures, with some interconnecting filament material, and one major polypeptide; i.e. that of Mr 68,000. This skeletal protein, which is not found in nuclear contents, reveals, on two-dimensional gel electrophoresis, a series of distinct isoelectric variants focusing in the pH range from 6.4 to 6.6. In living oocytes, this protein is continuously synthesized, as demonstrated by incorporation of labelled amino acids, and phosphorylated, A similar prominent skeletal protein has been found in nuclear envelopes of oocytes of other amphibia; however, slight but significant differences in electrophoretic mobility can be noted between different amphibian species. For comparison, nucleocortical lamina structures containing few pore complexes have been isolated, using similar extraction procedures, from various somatic cells of X. laevis, including erythrocytes. Laminae from these cells contain two major polypeptides, one (LI) of Mr 72,000 focusing at approximately pH 5.35 and another (LII) of Mr 69,000 focusing in several variants between pH 6.20 and 6.35. Similarly extracted “pore complex-lamina” fractions from rat liver contain a polypeptide of similar size and electrical charge as protein LI from Xenopus and, in addition, two other polypeptides (Mr values: 74,000 and 62,000) both focusing between pH 6.6 and 6.9. It is concluded that the pore complex-lamina structure of the oocyte nucleus is assembled by only one major protein of Mr 68,000. The results also show that the protein composition of this insoluble nucleocortical structure can be different in different cells of the same organism. The compositional differences of these nuclear envelope skeletons are discussed in relation to the relative proportions of pore complex and interporous (lamina) material in the nuclear envelopes of the specific cells. It is suggested that the Mr 68,000 protein predominant in oocyte nuclear envelopes contributes, as an architectural component, to the formation of the highly organized nuclear pore complex.

Expression of CD83 Is Regulated by HuR via a Novel cis-Active Coding Region RNA Element

Dendritic cells are the most potent of the antigen-presenting cells and are characterized by surface expression of CD83. Here, we show that the coding region of CD83 mRNA contains a novel cis-acting structured RNA element that binds to HuR, a member of the ELAV family of AU-rich element RNA-binding proteins. Transient transfection of mammalian cells demonstrated that this CD83 mRNA-derived element acts as a post-transcriptional regulatory element in cells overexpressing HuR. Notably, binding of HuR to the CD83 post-transcriptional regulatory element did not affect mRNA stability. Using RNA interference, we show that HuR mediated efficient expression of CD83. In particular, HuR was required for cytoplasmic accumulation of CD83 transcripts. Likewise, inhibition of the CRM1 nuclear export pathway by leptomycin B or overexpression of a defective form of the nucleoporin Nup214/CAN diminished cytoplasmic CD83 mRNA levels. In summary, the data presented demonstrate that the HuR-CRM1 axis affects the nucleocytoplasmic translocation of CD83 mRNA under regular physiological conditions.

Role of nuclear material in the early cell cycle of xenopus embryos

Activated Xenopus eggs show periodic surface contraction waves and oscillations in endogenous protein phosphorylation, MPF, and kinase activities timed with the cleavage cycle of control fertilized eggs. In this paper, we show that in activated eggs lacking the material that originates from the oocyte nucleus, MPF and kinase oscillations occur in the absence of surface contraction waves. Two mitotic phosphoproteins (M116 and M46), previously described by 32P labeling in nucleated eggs, are no longer detected in the enucleated eggs. We conclude that a cytoplasmic temporal control of MPF and kinase activities is likely to be the essential cell cycle oscillator. The oocyte nuclear components normally stored in the cytoplasm of the embryos are not involved in the clock although they appear to be required for the generation of surface contraction waves.

Assembly of nuclear pore complexes mediated by major vault protein.

During interphase growth of eukaryotic cells, nuclear pore complexes (NPCs) are continuously incorporated into the intact nuclear envelope (NE) by mechanisms that are largely unknown. De novo formation of NPCs involves local fusion events between the inner and outer nuclear membrane, formation of a transcisternal membranous channel of defined diameter and the coordinated assembly of hundreds of nucleoporins into the characteristic NPC structure. Here we have used a cell-free system based on Xenopus egg extract, which allows the experimental separation of nuclear-membrane assembly and NPC formation. Nuclei surrounded by a closed double nuclear membrane, but devoid of NPCs, were first reconstituted from chromatin and a specific membrane fraction. Insertion of NPCs into the preformed pore-free nuclei required cytosol containing soluble nucleoporins or nucleoporin subcomplexes and, quite unexpectedly, major vault protein (MVP). MVP is the main component of vaults, which are ubiquitous barrel-shaped particles of enigmatic function. Our results implicate MVP, and thus also vaults, in NPC biogenesis and provide a functional explanation for the association of a fraction of vaults with the NE and specifically with NPCs in intact cells.

Functional Organization of the Amphibian Oocyte Nucleus

Nuclei of amphibian oocytes (“germinal vesicles”) can grow to considerable size. For example, the nucleus of a mature Xenopus laevis oocyte has a diameter of about 0.5 mm and, after isolation, can be seen with the naked eye. This exceptional size (for comparison, the diameter of somatic cell nuclei usually ranges from 5 to 30 μm), the ease and rapidity with which they can be manually isolated, and the occurrence of lampbrush chromosomes and amplified nucleoli makes amphibian nuclei choice material not only for studies of chromosome structure and genetic activity at different levels of resolution, but also for analysis of the biochemical composition of individual nuclei and nuclear components as well. A single manually isolated nucleus from an amphibian oocyte is sufficient to analyze its major protein constituents by gel electrophoresis, and a few nuclei provide sufficient RNA for analysis by gel electrophoresis or electron microscopic spreading methods. In addition, the size of amphibian oocyte nuclei greatly facilitates the introduction of certain substances by microinjection. In fact, Xenopus oocytes are presently used in numerous laboratories as “living test tubes” to study the expression of cloned DNA sequences after microinjection into their nuclei with the aim of identifying those DNA sequences necessary for transcriptional initiation and termination events, as well as the factors involved in gene regulation.

Interaction of eukaryotic initiation factor 5A (eIF-5A) with nuclear pore complexes

The HIV-I Rev tram-activator protein is essential for the nuclear export of viral mRNAs. In the nucleus, Rev binds to the Rev Response Element (RRE) RNA target sequence, multimerizes and interacts with cellular cofactor(s), including the nucleoporin-like protein hRlP/Rab, CRMI and elF-SA. In particular, mutants of eiF-5A block nuclear export of Rev and thereby virus replication in human cells. Furthermore, immunological studies have demonstrated that eIF-SA is present both in the cytoplasm and the nucleus of mammalian ceils. In view of the proposed involvement of eIF-5A in RNA export processes, it was reasonable to assume that this factor inrcracts directly with nuclear pore complexes. We have studied the distribution of eIF-SA by a combination of immunofluorescence microscopy, immunogold EM and microinjection experiments using cultured mammalian cells (Vera) and Xenopus oocytes. in Vero cells, double-latreling experiments using antibodies reacting with nuc!eoporins (e.g. ~62) and eiF-SA demonstrated colocalizUion of eIF-SA with nuclear pore complexes. By immunogold EM, eIF-SA was found to be enriched at the nucteoplasmic face of the nuclear Pore complexes. In a second series of experiments, we demonstrated biochemically the presence of elF-5A in amphibian oocytes by Western analysis. Moreover, anti-elF-5A antibodies brightly stained the nuclear envelope in cryosections of Xenopus oocytes. At the EM level, eIF-SA was specifically enriched at the filaments extending from the inner ring of the Pore complexes into the nucleoplasm. To identify Potential interaction partners of elF-SA, we used a nitmcellulose overlay assay with nuclear envelope proteins of Xenopus oocytes. Several reacting bands were detected, including the nucleoporin ~62. Finally we provide direct evidence for an active nucleo/cytoplasmic transpoit of eIF5A: When we expressed (-Gal/eIF-5A fusion proteins in Vero cells. the protein became specifically enriched in the nucleus. Furthermore, micminiectiti of GST-eIF-SA fusion orotein into Vero cell nuclei resulted in the &pid translocation of the protein to the cytoplasm. This export was inhibited in the presence of CiTP(S, by tow temperature and the presence of Leptomycin B (an inhibitor of Rev export thaf binds CRMI). In sum, our data show that eIF-SA is able to actively enter and leave the cell nucleus. Moreover, eIF-SA interacts directly with components of rhe nuclear Pore complex. Thus, eIF-SA has characteristics that are typical for nuclear exmn factors.