The nucleoporin Nup88 is interacting with nuclear lamin A

Nuclear pore complexes (NPCs) are embedded in the nuclear envelope (NE) and mediate bidirectional nucleocytoplasmic transport. Their spatial distribution in the NE is organized by the nuclear lamina, a meshwork of nuclear intermediate filament proteins. Major constituents of the nuclear lamina are A- and B-type lamins. In this work we show that the nuclear pore protein Nup88 binds lamin A in vitro and in vivo. The interaction is mediated by the N-terminus of Nup88, and Nup88 specifically binds the tail domain of lamin A but not of lamins B1 and B2. Expression of green fluorescent protein-tagged lamin A in cells causes a masking of binding sites for Nup88 antibodies in immunofluorescence assays, supporting the interaction of lamin A with Nup88 in a cellular context. The epitope masking disappears in cells expressing mutants of lamin A that are associated with laminopathic diseases. Consistently, an interaction of Nup88 with these mutants is disrupted in vitro. Immunoelectron microscopy using Xenopus laevis oocyte nuclei further revealed that Nup88 localizes to the cytoplasmic and nuclear face of the NPC. Together our data suggest that a pool of Nup88 on the nuclear side of the NPC provides a novel, unexpected binding site for nuclear lamin A.     

In vitro and histological investigation of antitumor effect of some triazole compounds in colon cancer cell line

1,2,4-Triazoles are used as antifungal, antibacterial, antimicrobial, and antioxidant against some oxidative radical species. Recently, many 1,2,4-triazoles continue to be synthesized. In this study, the effect of the 1,2,4-triazole derivatives on human colon cancer (HT29) was investigated in vitro and in vivo in rats. MTT test was applied to in in vitro experiments. For in vivo study, rats were divided into seven groups as follows: Control group (negative control), azoxymethane (AOM), AOM + cisplatin 15, AOM + L1, AOM + L2, AOM + L3, and AOM + L4. To create colon cancer, the AOM injection was injected subcutaneously at a dose of 15 mg/kg, three times (once weekly). The in vivo studies were completed at 28 weeks. It was found that the 1,2,4-triazole derivatives reduced the cell viability (P < 0.05). In all animals in the experimental groups, mild dysplasia was detected in 100% of the colon mucosal epithelium. Severe dysplasia and adenocarcinoma were observed in L1 groups. As a result, this study determined that the 1,2,4-triazole derivatives exhibit antitumor activity.     

RanBP3 influences interactions between CRM1 and its nuclear protein export substrates

We investigated the role of RanBP3, a nuclear member of the Ran-binding protein 1 family, in CRM1-mediated protein export in higher eukaryotes. RanBP3 interacts directly with CRM1 and also forms a trimeric complex with CRM1 and RanGTP. However, RanBP3 does not bind to CRM1 like an export substrate. Instead, it can stabilize CRM1–export substrate interaction. Nuclear RanBP3 stimulates CRM1-dependent protein export in permeabilized cells. These data indicate that RanBP3 functions by a novel mechanism as a cofactor in recognition and export of certain CRM1 substrates. In vitro, RanBP3 binding to CRM1 affects the relative affinity of CRM1 for different substrates.     

Herpes simplex virus type 1 entry into host cells: reconstitution of capsid binding and uncoating at the nuclear pore complex in vitro

During entry, herpes simplex virus type 1 (HSV-1) releases its capsid and the tegument proteins into the cytosol of a host cell by fusing with the plasma membrane. The capsid is then transported to the nucleus, where it docks at the nuclear pore complexes (NPCs), and the viral genome is rapidly released into the nucleoplasm. In this study, capsid association with NPCs and uncoating of the viral DNA were reconstituted in vitro. Isolated capsids prepared from virus were incubated with cytosol and purified nuclei. They were found to bind to the nuclear pores. Binding could be inhibited by pretreating the nuclei with wheat germ agglutinin, anti-NPC antibodies, or antibodies against importin beta. Furthermore, in the absence of cytosol, purified importin beta was both sufficient and necessary to support efficient capsid binding to nuclei. Up to 60 to 70% of capsids interacting with rat liver nuclei in vitro released their DNA if cytosol and metabolic energy were supplied. Interaction of the capsid with the nuclear pore thus seemed to trigger the release of the viral genome, implying that components of the NPC play an active role in the nuclear events during HSV-1 entry into host cells.     

TAP (NXF1) belongs to a multigene family of putative RNA export factors with a conserved modular architecture

Vertebrate TAP (also called NXF1) and its yeast orthologue, Mex67p, have been implicated in the export of mRNAs from the nucleus. The TAP protein includes a noncanonical RNP-type RNA binding domain, four leucine-rich repeats, an NTF2-like domain that allows heterodimerization with p15 (also called NXT1), and a ubiquitin-associated domain that mediates the interaction with nucleoporins. Here we show that TAP belongs to an evolutionarily conserved family of proteins that has more than one member in higher eukaryotes. Not only the overall domain organization but also residues important for p15 and nucleoporin interaction are conserved in most family members. We characterize two of four human TAP homologues and show that one of them, NXF2, binds RNA, localizes to the nuclear envelope, and exhibits RNA export activity. NXF3, which does not bind RNA or localize to the nuclear rim, has no RNA export activity. Database searches revealed that although only one p15 (nxt) gene is present in the Drosophila melanogaster and Caenorhabditis elegans genomes, there is at least one additional p15 homologue (p15-2 [also called NXT2]) encoded by the human genome. Both human p15 homologues bind TAP, NXF2, and NXF3. Together, our results indicate that the TAP-p15 mRNA export pathway has diversified in higher eukaryotes compared to yeast, perhaps reflecting a greater substrate complexity.     

Identification of two novel RanGTP-binding proteins belonging to the importin beta superfamily

Nucleo-cytoplasmic transport comprises a large number of distinct pathways, many of which are defined by members of the importin beta superfamily of nuclear transport receptors. These transport receptors all directly interact with RanGTP to modulate the compartment-specific binding of their transport substrates. To identify new members of the importin beta family, we used affinity chromatography on immobilized RanGTP and isolated Ran-binding protein (RanBP) 16 from HeLa cell extracts. RanBP16 and its close human homologue, RanBP17, are distant members of the importin beta family. Like the other members of the transport receptor superfamily, RanBP16 interacts with the nuclear pore complex and is able to enter the nucleus independent of energy and additional nuclear transport receptors.     

The C-terminal domain of TAP interacts with the nuclear pore complex and promotes export of specific CTE-bearing RNA substrates

Messenger RNAs are exported from the nucleus as large ribonucleoprotein complexes (mRNPs). To date, proteins implicated in this process include TAP/Mex67p and RAE1/Gle2p and are distinct from the nuclear transport receptors of the beta-related, Ran-binding protein family. Mex67p is essential for mRNA export in yeast. Its vertebrate homolog TAP has been implicated in the export of cellular mRNAs and of simian type D viral RNAs bearing the constitutive transport element (CTE). Here we show that TAP is predominantly localized in the nucleoplasm and at both the nucleoplasmic and cytoplasmic faces of the nuclear pore complex (NPC). TAP interacts with multiple components of the NPC including the nucleoporins CAN, Nup98, Nup153, p62, and with three major NPC subcomplexes. The nucleoporin-binding domain of TAP comprises residues 508-619. In HeLa cells, this domain is necessary and sufficient to target GFP-TAP fusions to the nuclear rim. Moreover, the isolated domain strongly competes multiple export pathways in vivo, probably by blocking binding sites on the NPC that are shared with other transport receptors. Microinjection experiments implicate this domain in the export of specific CTE-containing RNAs. Finally, we show that TAP interacts with transportin and with two proteins implicated in the export of cellular mRNAs: RAE1/hGle2 and E1B-AP5. The interaction of TAP with nucleoporins, its direct binding to the CTE RNA, and its association with two mRNP binding proteins suggest that TAP is an RNA export mediator that may bridge the interaction between specific RNP export substrates and the NPC.     

An in vitro nuclear disassembly system reveals a role for the RanGTPase system and microtubule-dependent steps in nuclear envelope breakdown

During prophase, vertebrate cells disassemble their nuclear envelope (NE) in the process of NE breakdown (NEBD). We have established an in vitro assay that uses mitotic Xenopus laevis egg extracts and semipermeabilized somatic cells bearing a green fluorescent protein-tagged NE marker to study the molecular requirements underlying the dynamic changes of the NE during NEBD by live microscopy. We applied our in vitro system to analyze the role of the Ran guanosine triphosphatase (GTPase) system in NEBD. Our study shows that high levels of RanGTP affect the dynamics of late steps of NEBD in vitro. Also, inhibition of RanGTP production by RanT24N blocks the dynamic rupture of nuclei, suggesting that the local generation of RanGTP around chromatin may serve as a spatial cue in NEBD. Furthermore, the microtubule-depolymerizing drug nocodazole interferes with late steps of nuclear disassembly in vitro. High resolution live cell imaging reveals that microtubules are involved in the completion of NEBD in vivo by facilitating the efficient removal of membranes from chromatin.     

Nuclear export and cytoplasmic maturation of ribosomal subunits

Based on the characterization of ribosome precursor particles and associated trans-acting factors, a biogenesis pathway for the 40S and 60S subunits has emerged. After nuclear synthesis and assembly steps, pre-ribosomal subunits are exported through the nuclear pore complex in a Crm1- and RanGTP-dependent manner. Subsequent cytoplasmic biogenesis steps of pre-60S particles include the facilitated release of several non-ribosomal proteins, yielding fully functional 60S subunits. Cytoplasmic maturation of 40S subunit precursors includes rRNA dimethylation and pre-rRNA cleavage, allowing 40S subunits to achieve translation competence. We review current knowledge of nuclear export and cytoplasmic maturation of ribosomal subunits.