Atomic structure of the nuclear pore complex targeting domain of a Nup116 homologue from the yeast, Candida glabrata

The nuclear pore complex (NPC), embedded in the nuclear envelope, is a large, dynamic molecular assembly that facilitates exchange of macromolecules between the nucleus and the cytoplasm. The yeast NPC is an eightfold symmetric annular structure composed of ～456 polypeptide chains contributed by ～30 distinct proteins termed nucleoporins. Nup116, identified only in fungi, plays a central role in both protein import and mRNA export through the NPC. Nup116 is a modular protein with N‐terminal “FG” repeats containing a Gle2p‐binding sequence motif and a NPC targeting domain at its C‐terminus. We report the crystal structure of the NPC targeting domain of Candida glabrata Nup116, consisting of residues 882–1034 [CgNup116(882–1034)], at 1.94 Å resolution. The X‐ray structure of CgNup116(882–1034) is consistent with the molecular envelope determined in solution by small‐angle X‐ray scattering. Structural similarities of CgNup116(882–1034) with homologous domains from Saccharomyces cerevisiae Nup116, S. cerevisiae Nup145N, and human Nup98 are discussed. Proteins 2012; © 2012 Wiley Periodicals, Inc.     

Novel vertebrate nucleoporins Nup133 and Nup160 play a role in mRNA export.

RNA undergoing nuclear export first encounters the basket of the nuclear pore. Two basket proteins, Nup98 and Nup153, are essential for mRNA export, but their molecular partners within the pore are largely unknown. Because the mechanism of RNA export will be in question as long as significant vertebrate pore proteins remain undiscovered, we set out to find their partners. Fragments of Nup98 and Nup153 were used for pulldown experiments from Xenopus egg extracts, which contain abundant disassembled nuclear pores. Strikingly, Nup98 and Nup153 each bound the same four large proteins. Purification and sequence analysis revealed that two are the known vertebrate nucleoporins, Nup96 and Nup107, whereas two mapped to ORFs of unknown function. The genes encoding the novel proteins were cloned, and antibodies were produced. Immunofluorescence reveals them to be new nucleoporins, designated Nup160 and Nup133, which are accessible on the basket side of the pore. Nucleoporins Nup160, Nup133, Nup107, and Nup96 exist as a complex in Xenopus egg extracts and in assembled pores, now termed the Nup160 complex. Sec13 is prominent in Nup98 and Nup153 pulldowns, and we find it to be a member of the Nup160 complex. We have mapped the sites that are required for binding the Nup160 subcomplex, and have found that in Nup98, the binding site is used to tether Nup98 to the nucleus; in Nup153, the binding site targets Nup153 to the nuclear pore. With transfection and in vivo transport assays, we find that specific Nup160 and Nup133 fragments block poly[A]+ RNA export, but not protein import or export. These results demonstrate that two novel vertebrate nucleoporins, Nup160 and Nup133, not only interact with Nup98 and Nup153, but themselves play a role in mRNA export.     

Nucleoporin domain topology is linked to the transport status of the nuclear pore complex

Nuclear pore complexes (NPCs) facilitate macromolecular exchange between the nucleus and cytoplasm of eukaryotic cells. The vertebrate NPC is composed of approximately 30 different proteins (nucleoporins), of which around one third contain phenylalanine-glycine (FG)-repeat domains that are thought to mediate the main interaction between the NPC and soluble transport receptors. We have recently shown that the FG-repeat domain of Nup153 is flexible within the NPC, although this nucleoporin is anchored to the nuclear side of the NPC. By using domain-specific antibodies, we have now mapped the domain topology of Nup214 in Xenopus oocytes and in human somatic cells by immuno-EM. We have found that whereas Nup214 is anchored to the cytoplasmic side of the NPC via its N-terminal and central domain, its FG-repeat domain appears flexible, residing on both sides of the NPC. Moreover, the spatial distribution of the FG-repeat domains of both Nup153 and Nup214 shifts in a transport-dependent manner, suggesting that the location of FG-repeat domains within the NPC correlates with cargo/receptor interactions and that they concomitantly move with cargo through the central pore of the NPC.     

黄鳝Nup93基因的分子克隆及其在性腺和肾的显著表达

核孔蛋白（Nucleoporins,Nups）是核孔复合体（Nuclear pore complexes,NPC）的重要组成成分,核孔复合体可以控制细胞内信号分子在核质问的双向转运,从而控制基因表达、细胞增殖和分化。在构建的黄鳝精巢SMART cDNA文库中,采用差异筛选的方法得到黄鳝核孔蛋白家族中Nup93基因的3’端片段,根据此段序列设计引物,使用兼并PCR和5＇RACE方法克隆得到此基因的全长cDNA。序列比对显示该基因与酵母Nic96、斑马鱼Nup93和人类Nup93的同源性分别为36．5％、94．6％和90．5％。进化树分析显示,黄鳝Nup93与其他鱼类的Nup93归为一支。采用荧光定量PCR方法对不同性别黄鳝的性腺和其他组织内该基因的表达作定量分析发现,Nup93在性腺和肾中的表达量远高于其他组织,而且表达量存在一定的性别差异。这一结果提示Nup93可能与性腺发育相关。     

Binding of the Mex67p/Mtr2p heterodimer to FXFG, GLFG, and FG repeat nucleoporins is essential for nuclear mRNA export

It is not known how Mex67p and Mtr2p, which form a heterodimer essential for mRNA export, transport mRNPs through the nuclear pore. Here, we show that the Mex67p/Mtr2p complex binds to all of the repeat types (GLFG, FXFG, and FG) found in nucleoporins. For this interaction, complex formation between Mex67p and Mtr2p has to occur. MEX67 and MTR2 also genetically interact with different types of repeat nucleoporins, such as Nup116p, Nup159p, Nsp1p, and Rip1p/Nup40p. These data suggest a model in which nuclear mRNA export requires the Mex67p/Mtr2p heterodimeric complex to directly contact several repeat nucleoporins, organized in different nuclear pore complex subcomplexes, as it carries the mRNP cargo through the nuclear pore.     

Gradient of increasing affinity of importin beta for nucleoporins along the pathway of nuclear import

Nuclear import and export signals on macromolecules mediate directional, receptor-driven transport through the nuclear pore complex (NPC) by a process that is suggested to involve the sequential binding of transport complexes to different nucleoporins. The directionality of transport appears to be partly determined by the nucleocytoplasmic compartmentalization of components of the Ran GTPase system. We have analyzed whether the asymmetric localization of discrete nucleoporins can also contribute to transport directionality. To this end, we have used quantitative solid phase binding analysis to determine the affinity of an importin beta cargo complex for Nup358, the Nup62 complex, and Nup153, which are in the cytoplasmic, central, and nucleoplasmic regions of the NPC, respectively. These nucleoporins are proposed to provide progressively more distal binding sites for importin beta during import. Our results indicate that the importin beta transport complex binds to nucleoporins with progressively increasing affinity as the complex moves from Nup358 to the Nup62 complex and to Nup153. Antibody inhibition studies support the possibility that importin beta moves from Nup358 to Nup153 via the Nup62 complex during import. These results indicate that nucleoporins themselves, as well as the nucleocytoplasmic compartmentalization of the Ran system, are likely to play an important role in conferring directionality to nuclear protein import.     

Dimerization and direct membrane interaction of Nup53 contribute to nuclear pore complex assembly

Nuclear pore complexes (NPCs) fuse the two membranes of the nuclear envelope (NE) to a pore, connecting cytoplasm and nucleoplasm and allowing exchange of macromolecules between these compartments. Most NPC proteins do not contain integral membrane domains and thus it is largely unclear how NPCs are embedded and anchored in the NE. Here, we show that the evolutionary conserved nuclear pore protein Nup53 binds independently of other proteins to membranes, a property that is crucial for NPC assembly and conserved between yeast and vertebrates. The vertebrate protein comprises two membrane binding sites, of which the C‐terminal domain has membrane deforming capabilities, and is specifically required for de novo NPC assembly and insertion into the intact NE during interphase. Dimerization of Nup53 contributes to its membrane interaction and is crucial for its function in NPC assembly.     

Core Components of the Nuclear Pore Bind Distinct States of Chromatin and Contribute to Polycomb Repression

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Chromatin organization in relation to the nuclear periphery

In the limited space of the nucleus, chromatin is organized in a dynamic and non-random manner. Three ways of chromatin organization are compaction, formation of loops and localization within the nucleus. To study chromatin localization it is most convenient to use the nuclear envelope as a fixed viewpoint. Peripheral chromatin has both been described as silent chromatin, interacting with the nuclear lamina, and active chromatin, interacting with nuclear pore proteins. Current data indicate that the nuclear envelope is a reader as well as a writer of chromatin state, and that its influence is not limited to the nuclear periphery.     

Exo70-Mediated Recruitment of Nucleoporin Nup62 at the Leading Edge of Migrating Cells is Required for Cell Migration

Nucleoporin Nup62 localizes at the central channel of the nuclear pore complex and is essential for nucleocytoplasmic transport. Through its FG-repeat domain, Nup62 regulates nuclear pore permeability and binds nuclear transport receptors. Here, we report that Nup62 interacts directly with Exo70 and colocalizes with Exo70 at the leading edge of migrating cells. Nup62 binds the N-terminal domain of Exo70 through its coiled-coil domain but not through its FG-repeat domain. Selective inhibition of leading edge Nup62 using RNA interference significantly reduces cell migration. Furthermore, Exo70 recruits Nup62 at the plasma membrane and at filopodia. Removal of the Exo70-binding domain of Nup62 prevents leading edge localization of Nup62. Analogous to Exo70, Nup62 cycles between the plasma membrane and the perinuclear recycling compartment. Altogether, we propose that Nup62 not solely regulates access to the cell nucleus, but additionally functions in conjunction with Exo70, a key regulator of exocytosis and actin dynamics, at the leading edge of migrating cells.     

Identification of nucleoporin 93 (Nup93) that mediates antiviral innate immune responses

RIG-I-like receptors (RLRs) are cytoplasmic sensors for viral RNA that elicit antiviral innate immune responses. RLR signaling culminates in the activation of the protein kinase TBK1, which mediates phosphorylation and nuclear translocation of IRF3 that regulates expression of type I interferon genes. Here, we found that Nucleoporin 93 (Nup93), components of nuclear pore complex (NPC), plays an important role in RLR-mediated antiviral responses. Nup93-deficient RAW264.7 macrophage cells exhibited decreased expression of Ifnb1 and Cxcl10 genes after treatment with a synthetic RLR agonist stimulation as well as Newcastle Disease Virus infection. Silencing Nup93 in murine primary macrophages and embryonic fibroblasts also resulted in reduced expression of these genes. IRF3 nuclear translocation during RLR signaling was impaired in Nup93-deficient RAW264.7 cells. Notably, the activation of TBK1 during RLR signaling was also decreased in Nup93-deficient cells. We found that Nup93 formed a complex with TBK1, and Nup93 overexpression enhanced TBK1-mediated IFNβ promoter activation. Taken together, our findings suggest that Nup93 regulates antiviral innate immunity by enhancing TBK1 activity and IRF3 nuclear translocation.     

The Nup153-Nup50 Protein Interface and Its Role in Nuclear Import

Interactions between Nup50 and soluble transport factors underlie the efficiency of certain nucleocytoplasmic transport pathways. The platform on which these interactions take place is important to building a complete understanding of nucleocytoplasmic trafficking. Nup153 is the nucleoporin that provides this scaffold for Nup50. Here, we have delineated requirements for the interaction between Nup153 and Nup50, revealing a dual interface. An interaction between Nup50 and a region in the unique N-terminal region of Nup153 is critical for the nuclear pore localization of Nup50. A second site of interaction is at the distal tail of Nup153 and is dependent on importin α. Both of these interactions involve the N-terminal domain of Nup50. The configuration of the Nup153-Nup50 partnership suggests that the Nup153 scaffold provides not just a means of pore targeting for Nup50 but also serves to provide a local environment that facilitates bringing Nup50 and importin α together, as well as other soluble factors involved in transport. Consistent with this, disruption of the Nup153-Nup50 interface decreases efficiency of nuclear import.     

The Nup2 meiotic-autonomous region relieves inhibition of Nup60 to promote progression of meiosis and sporulation in Saccharomyces cerevisiae

During meiosis, chromosomes undergo dramatic changes in structural organization, nuclear positioning, and motion. Although the nuclear pore complex has been shown to affect genome organization and function in vegetative cells, its role in meiotic chromosome dynamics has remained largely unexplored. Recent work in the budding yeast Saccharomyces cerevisiae demonstrated that the mobile nucleoporin Nup2 is required for normal progression through meiosis I prophase and sporulation in strains where telomere-led chromosome movement has been compromised. The meiotic-autonomous region, a short fragment of Nup2 responsible for its role in meiosis, was shown to localize to the nuclear envelope via Nup60 and to bind to meiotic chromosomes. To understand the relative contribution these 2 activities have on meiotic-autonomous region function, we first carried out a screen for meiotic-autonomous region mutants defective in sporulation and found that all the mutations disrupt interaction with both Nup60 and meiotic chromosomes. Moreover, nup60 mutants phenocopy nup2 mutants, exhibiting similar nuclear division kinetics, sporulation efficiencies, and genetic interactions with mutations that affect the telomere bouquet. Although full-length Nup60 requires Nup2 for function, removal of Nup60’s C-terminus allows Nup60 to bind meiotic chromosomes and promotes sporulation without Nup2. In contrast, binding of the meiotic-autonomous region to meiotic chromosomes is completely dependent on Nup60. Our findings uncover an inhibitory function for the Nup60 C-terminus and suggest that Nup60 mediates recruitment of meiotic chromosomes to the nuclear envelope, while Nup2 plays a secondary role counteracting the inhibitory function in Nup60’s C-terminus.     

MicroRNA-145: a promising biomarker for hepatocellular carcinoma (HCC)

It has been demonstrated that microRNA-145 (miR-145) is downregulated in patients with hepatocellular carcinoma (HCC) compared with healthy controls. The mechanisms for miR-145 in HCC will become potential in future researches.     

Down-modulation of nucleoporin RanBP2/Nup358 impaired chromosomal alignment and induced mitotic catastrophe

Chromosomal missegregation is a common feature of many human tumors. Recent studies have indicated a link between nucleoporin RanBP2/Nup358 and chromosomal segregation during mitosis; however, the molecular details have yet to be fully established. Observed through live cell imaging and flow cytometry, here we show that RNA interference-mediated knockdown of RanBP2 induced G2/M phase arrest, metaphase catastrophe and mitotic cell death. Furthermore, RanBP2 down-modulation disrupted importin/karyopherin β1 as well as the expression and localization of the Ran GTPase activating protein 1. We found that N-terminal of RanBP2 interacted with the N-terminal of importin β1. Moreover, at least a portion of RanBP2 partially localizes at the centrosome during mitosis. Notably, we also found that GTPase Ran is also involved in the regulation of RanBP2–importin β1 interaction. Overall, our results suggest that mitotic arrest and the following cell death were caused by depletion of RanBP2. Our findings point to a crucial role for RanBP2 in proper mitotic progression and faithful chromosomal segregation.     

Structure and assembly of the Nup84p complex

The Nup84p complex consists of five nucleoporins (Nup84p, Nup85p, Nup120p, Nup145p-C, and Seh1p) and Sec13p, a bona fide subunit of the COPII coat complex. We show that a pool of green fluorescent protein-tagged Sec13p localizes to the nuclear pores in vivo, and identify sec13 mutant alleles that are synthetically lethal with nup85Delta and affect the localization of a green fluorescent protein-Nup49p reporter protein. In the electron microscope, sec13 mutants exhibit structural defects in nuclear pore complex (NPC) and nuclear envelope organization. For the assembly of the complex, Nup85p, Nup120p, and Nup145p-C are essential. A highly purified Nup84p complex was isolated from yeast under native conditions and its molecular mass was determined to be 375 kD by quantitative scanning transmission electron microscopy and analytical ultracentrifugation, consistent with a monomeric complex. Furthermore, the Nup84p complex exhibits a Y-shaped, triskelion-like morphology 25 nm in diameter in the transmission electron microscope. Thus, the Nup84p complex constitutes a paradigm of an NPC structural module with distinct composition, structure, and a role in nuclear mRNA export and NPC bio- genesis.