RNA Export: Searching for MRNA Identity

Current Biology : CB. Jul, 2002  |  Pubmed ID: 12121638

Efficient eukaryotic gene expression hinges on the ability of mRNA to travel from the nucleus to its cytoplasmic destination. Recent work lends insight into features that allow diverse mRNAs to be recognized by shared export machinery.

Domain-specific Antibodies Reveal Multiple-site Topology of Nup153 Within the Nuclear Pore Complex

Journal of Structural Biology. Oct-Dec, 2002  |  Pubmed ID: 12490173

Nup153, one of the best characterized nuclear pore complex proteins (nucleoporins), plays a critical role in the import of proteins into the nucleus as well as in the export of RNAs and proteins from the nucleus. Initially an epitope of Nup153 was found to reside at the distal ring of the NPC, whereas more recently another epitope was localized to the nuclear ring moiety of the NPC. In an effort to more definitively determine the location of Nup153 within the 3-D architecture of the NPC we have generated domain-specific antibodies against distinct domains of Xenopus Nup153. With this approach we have found that the N-terminal domain is exposed at the nuclear ring of the NPC, whereas the zinc-finger domain of Nup153 is exposed at the distal ring of the NPC. In contrast, the C-terminal domain of Nup153 is not restricted to one particular subdomain of the NPC but rather appears to be highly flexible. Exogenous epitope-tagged hNup153 incorporated into Xenopus oocyte NPCs further underscored these findings. Our data illustrate that multiple domain-specific antibodies are essential to understanding the topology of a nucleoporin within the context of the NPC. Moreover, this approach has revealed new clues to the mechanisms by which Nup153 may contribute to nucleocytoplasmic transport.

The COPI Complex Functions in Nuclear Envelope Breakdown and is Recruited by the Nucleoporin Nup153

Developmental Cell. Sep, 2003  |  Pubmed ID: 12967567

Nuclear envelope breakdown is a critical step in the cell cycle of higher eukaryotes. Although integral membrane proteins associated with the nuclear membrane have been observed to disperse into the endoplasmic reticulum at mitosis, the mechanisms involved in this reorganization remain to be fully elucidated. Here, using Xenopus extracts, we report a role for the COPI coatomer complex in nuclear envelope breakdown, implicating vesiculation as an important step. We have found that a nuclear pore protein, Nup153, plays a critical role in directing COPI to the nuclear membrane at mitosis and that this event provides feedback to other aspects of nuclear disassembly. These results provide insight into how key steps in nuclear division are orchestrated.

The RNA Binding Domain Within the Nucleoporin Nup153 Associates Preferentially with Single-stranded RNA

RNA (New York, N.Y.). Jan, 2004  |  Pubmed ID: 14681581

The nuclear pore protein Nup153 is important for the transport of protein and RNA between the nucleus and cytoplasm. Recently, a novel RNA binding domain (RBD) was mapped within the N-terminal region of Nup153; however, the determinants of RNA association were not characterized. Here we have tested a range of RNAs with different general features to better understand targets recognized by this domain. We have found that the RBD associates with single-stranded RNA with little sequence preference. These results provide new information about a novel RNA binding domain and suggest new models to consider for the contribution of Nup153 to nucleocytoplasmic transport.

Distinct Functional Domains Within Nucleoporins Nup153 and Nup98 Mediate Transcription-dependent Mobility

Molecular Biology of the Cell. Apr, 2004  |  Pubmed ID: 14718558

Despite the apparent overall structural stability of the nuclear pore complex during interphase, at least two nucleoporins have been shown to move dynamically on and off the pore. It is not yet certain what contribution nucleoporin mobility makes to the process of nuclear transport or how such mobility is regulated. Previously, we showed that Nup98 dynamically interacts with the NPC as well as bodies within the nucleus in a transcription-dependent manner. We have extended our studies of dynamics to include Nup153, another mobile nucleoporin implicated in RNA export. In both cases, we found that although only one domain is essential for NPC localization, other regions of the protein significantly affect the stability of association with the pore. Interestingly, like Nup98, the exchange of Nup153 on and off the pore is inhibited when transcription by Pol I and Pol II is blocked. We have mapped the regions required to link Nup98 and Nup153 mobility to transcription and found that the requirements differ depending on which polymerases are inhibited. Our data support a model whereby transcription of RNA is coupled to nucleoporin mobility, perhaps ultimately linking transport of RNAs to a cycle of remodeling at the nuclear pore basket.

Nucleocytoplasmic Transport: Integrating MRNA Production and Turnover with Export Through the Nuclear Pore

Molecular and Cellular Biology. Apr, 2004  |  Pubmed ID: 15060131

Nucleoporin Domain Topology is Linked to the Transport Status of the Nuclear Pore Complex

Journal of Molecular Biology. Aug, 2005  |  Pubmed ID: 16045929

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.

Versatility at the Nuclear Pore Complex: Lessons Learned from the Nucleoporin Nup153

Chromosoma. Nov, 2005  |  Pubmed ID: 16133350

The vertebrate pore protein Nup153 plays pivotal roles in nuclear pore function. In addition to being important to pore architecture, Nup153 is a key participant in both import and export. The scope of Nup153 function also extends beyond the canonical view of the pore as a trafficking gateway. During the transition into mitosis, Nup153 directs proteins involved in membrane remodeling to the nuclear envelope. As cells exit mitosis, Nup153 is recruited to the chromosomal surface, where nuclear pores are formed anew in a complicated process still under much experimental scrutiny. In addition, Nup153 is targeted for protease cleavage during apoptosis and in response to certain viral infections, providing molecular insight into pore reconfiguration during cell response. Overall, the versatile nature of Nup153 underscores an emerging view of the nuclear pore at the nexus of many key cellular processes.

Nuclear Envelope Breakdown is Coordinated by Both Nup358/RanBP2 and Nup153, Two Nucleoporins with Zinc Finger Modules

Molecular Biology of the Cell. Feb, 2006  |  Pubmed ID: 16314393

When higher eukaryotic cells transition into mitosis, the nuclear envelope, nuclear pore complexes, and nuclear lamina are coordinately disassembled. The COPI coatomer complex, which plays a major role in membrane remodeling at the Golgi, has been implicated in the process of nuclear envelope breakdown and requires interactions at the nuclear pore complex for recruitment to this new site of action at mitosis. Nup153, a resident of the nuclear pore basket, was found to be involved in COPI recruitment, but the molecular nature of the interface between COPI and the nuclear pore has not been fully elucidated. To better understand what occurs at the nuclear pore at this juncture, we have probed the role of the nucleoporin Nup358/RanBP2. Nup358 contains a repetitive zinc finger domain with overall organization similar to a region within Nup153 that is critical to COPI association, yet inspection of these two zinc finger domains reveals features that also clearly distinguish them. Here, we found that the Nup358 zinc finger domain, but not a zinc finger domain from an unrelated protein, binds to COPI and dominantly inhibits progression of nuclear envelope breakdown in an assay that robustly recapitulates this process in vitro. Moreover, the Nup358 zinc finger domain interferes with COPI recruitment to the nuclear rim. Consistent with a role for this pore protein in coordinating nuclear envelope breakdown, Nup358-specific antibodies impair nuclear disassembly. Significantly, targeting either Nup153 or Nup358 for inhibition perturbs nuclear envelope breakdown, supporting a model in which these nucleoporins play nonredundant roles, perhaps contributing to COPI recruitment platforms on both the nuclear and cytoplasmic faces of the pore. We found that an individual zinc finger is the minimal interface for COPI association, although tandem zinc fingers are optimal. These results provide new information about the critical components of nuclear membrane remodeling and lay the foundation for a better understanding of how this process is regulated.

The Nuclear Envelope: Form and Reformation

Current Opinion in Cell Biology. Feb, 2006  |  Pubmed ID: 16364623

The membrane system that encloses genomic DNA is referred to as the nuclear envelope. However, with emerging roles in signaling and gene expression, these membranes clearly serve as more than just a physical barrier separating the nucleus and cytoplasm. Recent progress in our understanding of nuclear envelope architecture and composition has also revealed an intriguing connection between constituents of the nuclear envelope and human disease, providing further impetus to decipher this cellular structure and the dramatic remodeling process it undergoes with each cell division.

Studying Nuclear Disassembly in Vitro Using Xenopus Egg Extract

Methods (San Diego, Calif.). Aug, 2006  |  Pubmed ID: 16879978

Xenopus egg extract provides an extremely powerful approach in the study of cell cycle regulated aspects of nuclear form and function. Each egg contains enough membrane and protein components to support multiple rounds of cell division. Remarkably, incubation of egg extract with DNA in the presence of an energy regeneration system is sufficient to induce formation of a nuclear envelope around DNA. In addition, these in vitro nuclei contain functional nuclear pore complexes, which form de novo and are capable of supporting nucleocytoplasmic transport. Mitotic entry can be induced by the addition of recombinant cyclin to an interphase extract. This initiates signaling that leads to disassembly of the nuclei. Thus, this cell-free system can be used to decipher events involved in mitotic remodeling of the nuclear envelope such as changes in nuclear pore permeability, dispersal of membrane, and disassembly of the lamina. Both general mechanisms and individual players required for orchestrating these events can be identified via biochemical manipulation of the egg extract. Here, we describe a procedure for the assembly and disassembly of in vitro nuclei, including the production of Xenopus egg extract and sperm chromatin DNA.

Changes in Nucleoporin Domain Topology in Response to Chemical Effectors

Journal of Molecular Biology. Oct, 2006  |  Pubmed ID: 16962132

Nucleoporins represent the molecular building blocks of nuclear pore complexes (NPCs), which mediate facilitated macromolecular trafficking between the cytoplasm and nucleus of eukaryotic cells. Phenylalanine-glycine (FG) repeat motifs are found in about one-third of the nucleoporins, and they provide major binding or docking sites for soluble transport receptors. We have shown recently that localization of the FG-repeat domains of vertebrate nucleoporins Nup153 and Nup214 within the NPC is influenced by its transport state. To test whether chemical effectors, such as calcium and ATP, influence the localization of the FG-repeat domains of Nup153 and Nup214 within the NPC, we performed immuno-electron microscopy of Xenopus oocyte nuclei using domain-specific antibodies against Nup153 and Nup214, respectively. Ca2+ and ATP are known to induce conformational changes in the NPC architecture, especially at the cytoplasmic face, but also at the nuclear basket of the NPC. We have found concentrations of calcium in the micromolar range or 1 mM ATP in the surrounding buffer leaves the spatial distribution of the FG-repeat of Nup153 and Nup214 largely unchanged. In contrast, ATP depletion, calcium store depletion by EGTA or thapsigargin, and high concentrations of divalent cation (i.e. 2 mM Ca2+ and 2 mM Mg2+) constrain the distribution of the FG-repeats of Nup153 and Nup214. Our data suggest that the location of the FG-repeat domains of Nup153 and Nup214 is sensitive to chemical changes within the near-field environment of the NPC.

Sequence Preference in RNA Recognition by the Nucleoporin Nup153

The Journal of Biological Chemistry. Mar, 2007  |  Pubmed ID: 17242408

The vertebrate nuclear pore protein Nup153 contains a novel RNA binding domain. This 150-amino acid region was previously found to bind preferentially to a panel of mRNAs when compared with structured RNAs, such as tRNA, U snRNA, and double-stranded RNA. The ability to broadly recognize mRNA led to the conclusion that the Nup153 RNA binding domain confers a general affinity for single-stranded RNA. Here, we have probed Nup153 RNA recognition to decipher how this unique RNA binding domain discriminates between potential targets. We first mapped the binding determinant within an RNA fragment that associates relatively robustly with the Nup153 RNA binding domain. We next designed synthetic RNA oligonucleotides to systematically delineate the features within this minimal RNA fragment that are key to Nup153 RNA-binding domain binding and demonstrated that the binding preferences of Nup153 do not reflect general preferences of an mRNA/single-stranded RNA-binding protein. We further found that the association between Nup153 and a cellular mRNA can be attributed to an interaction with specific subregions of the RNA. These results indicate that Nup153 can discriminate between mRNA and other classes of RNA transcripts due in part to direct recognition of a loose sequence motif. This information adds a new dimension to the interfaces that can contribute to recognition in mRNA export cargo selection and fate.

Molecular Characterization of the Ran-binding Zinc Finger Domain of Nup153

The Journal of Biological Chemistry. Jun, 2007  |  Pubmed ID: 17426026

The nuclear pore complex is the gateway for selective traffic between the nucleus and cytoplasm. To learn how building blocks of the pore can create specific docking sites for transport receptors and regulatory factors, we have studied a zinc finger module present in multiple copies within the nuclear pores of higher eukaryotes. All four zinc fingers of human Nup153 were found to bind the small GTPase Ran with dissociation constants ranging between 5 and 40 mum. In addition a fragment of Nup153 encompassing the four tandem zinc fingers was found to bind Ran with similar affinity. NMR structural studies revealed that a representative Nup153 zinc finger adopts the same zinc ribbon structure as the previously characterized Npl4 NZF module. Ran binding was mediated by a three-amino acid motif (Leu(13)/Val(14)/Asn(25)) located within the two zinc coordination loops. Nup153 ZnFs bound GDP and GTP forms of Ran with similar affinities, indicating that this interaction is not influenced by a nucleotide-dependent conformational switch. Taken together, these studies elucidate the Ran-binding interface on Nup153 and, more broadly, provide insight into the versatility of this zinc finger binding module.

Biology and Biophysics of the Nuclear Pore Complex and Its Components

International Review of Cell and Molecular Biology. 2008  |  Pubmed ID: 18544502

Nucleocytoplasmic exchange of proteins and ribonucleoprotein particles occurs via nuclear pore complexes (NPCs) that reside in the double membrane of the nuclear envelope (NE). Significant progress has been made during the past few years in obtaining better structural resolution of the three-dimensional architecture of NPC with the help of cryo-electron tomography and atomic structures of domains from nuclear pore proteins (nucleoporins). Biophysical and imaging approaches have helped elucidate how nucleoporins act as a selective barrier in nucleocytoplasmic transport. Nucleoporins act not only in trafficking of macromolecules but also in proper microtubule attachment to kinetochores, in the regulation of gene expression and signaling events associated with, for example, innate and adaptive immunity, development and neurodegenerative disorders. Recent research has also been focused on the dynamic processes of NPC assembly and disassembly that occur with each cell cycle. Here we review emerging results aimed at understanding the molecular arrangement of the NPC and how it is achieved, defining the roles of individual nucleoporins both at the NPC and at other sites within the cell, and finally deciphering how the NPC serves as both a barrier and a conduit of active transport.

The Nucleoporin Nup153 Has Separable Roles in Both Early Mitotic Progression and the Resolution of Mitosis

Molecular Biology of the Cell. Mar, 2009  |  Pubmed ID: 19158386

Accurate inheritance of genomic content during cell division is dependent on synchronized changes in cellular organization and chromosome dynamics. Elucidating how these events are coordinated is necessary for a complete understanding of cell proliferation. Previous in vitro studies have suggested that the nuclear pore protein Nup153 is a good candidate for participating in mitotic coordination. To decipher whether this is the case in mammalian somatic cells, we reduced the levels of Nup153 in HeLa cells and monitored consequences on cell growth. Reduction of Nup153 resulted in a delay during the late stages of mitosis accompanied by an increase in unresolved midbodies. Depletion of Nup153 to an even lower threshold led to a pronounced defect early in mitosis and an accumulation of cells with multilobed nuclei. Although global nucleocytoplasmic transport was not significantly altered under these depletion conditions, the FG-rich region of Nup153 was required to rescue defects in late mitosis. Thus, this motif may play a specialized role as cells exit mitosis. Rescue of the multilobed nuclei phenotype, in contrast, was independent of the FG-domain, revealing two separable roles for Nup153 in the execution of mitosis.

Defects in Nuclear Pore Assembly Lead to Activation of an Aurora B-mediated Abscission Checkpoint

The Journal of Cell Biology. Nov, 2010  |  Pubmed ID: 21098116

Correct assembly of nuclear pore complexes (NPCs), which directly and indirectly control nuclear environment and architecture, is vital to genomic regulation. We previously found that nucleoporin 153 (Nup 153) is required for timely progression through late mitosis. In this study, we report that disruption of Nup 153 function by either small interfering RNA-mediated depletion or expression of a dominant-interfering Nup 153 fragment results in dramatic mistargeting of the pore basket components Tpr and Nup 50 in midbody-stage cells. We find a concomitant appearance of aberrantly localized active Aurora B and an Aurora B-dependent delay in abscission. Depletion of Nup 50 is also sufficient to increase the number of midbody-stage cells and, likewise, triggers distinctive mislocalization of Aurora B. Together, our results suggest that defects in nuclear pore assembly, and specifically the basket structure, at this time of the cell cycle activate an Aurora B-mediated abscission checkpoint, thereby ensuring that daughter cells are generated only when fully formed NPCs are present.

Protein Arginine Methyltransferase 5 Accelerates Tumor Growth by Arginine Methylation of the Tumor Suppressor Programmed Cell Death 4

Cancer Research. Aug, 2011  |  Pubmed ID: 21700716

Programmed cell death 4 (PDCD4) has been described as a tumor suppressor, with high expression correlating with better outcomes in a number of cancer types. Yet a substantial number of cancer patients with high PDCD4 in tumors have poor survival, suggesting that oncogenic pathways may inhibit or change PDCD4 function. Here, we explore the significance of PDCD4 in breast cancer and identify protein arginine methyltransferase 5 (PRMT5) as a cofactor that radically alters PDCD4 function. Specifically, we find that coexpression of PDCD4 and PRMT5 in an orthotopic model of breast cancer causes accelerated tumor growth and that this growth phenotype is dependent on both the catalytic activity of PRMT5 and a site of methylation within the N-terminal region of PDCD4. In agreement with the xenograft model, elevated PDCD4 expression was found to correlate with worse outcome within the cohort of breast cancer patients whose tumors contain higher levels of PRMT5. These results reveal a new cofactor for PDCD4 that alters its tumor suppressor functions and point to the utility of PDCD4/PRMT5 status as both a prognostic biomarker and a potential target for chemotherapy.

Coordinating Postmitotic Nuclear Pore Complex Assembly with Abscission Timing

Nucleus (Austin, Tex.). Jul, 2011  |  Pubmed ID: 21941107

Cells divide and accurately inherit genomic and cellular content through synchronized changes in cellular organization and chromosome dynamics. Although DNA segregation, nuclear reformation, and cytokinesis/abscission temporally overlap, little is known about how these distinct events are coordinated to ensure accurate cell division. Recently, we found that disruption of postmitotic nuclear pore complex assembly, an essential aspect of the newly forming nuclear envelope, triggers an Aurora B-dependent delay in abscission. This delay is further characterized by mislocalized, aberrantly active Aurora B in the cytoplasm of midbody-stage cells. These results support a model in which an Aurora B-mediated abscission checkpoint provides surveillance of nuclear pore complex formation to ensure that elements of nuclear architecture are fully formed before daughter cells are physically separated. Here we discuss the process of nuclear pore complex assembly, describe potential mechanisms that may explain how this process could be coordinated with abscission, and postulate why such a checkpoint mechanism may exist.

The Nuclear Envelope Environment and Its Cancer Connections

Nature Reviews. Cancer. Feb, 2012  |  Pubmed ID: 22337151

Because of the association between aberrant nuclear structure and tumour grade, nuclear morphology is an indispensible criterion in the current pathological assessment of cancer. Components of the nuclear envelope environment have central roles in many aspects of cell function that affect tumour development and progression. As the roles of the nuclear envelope components, including nuclear pore complexes and nuclear lamina, are being deciphered in molecular detail there are opportunities to harness this knowledge for cancer therapeutics and biomarker development. In this Review, we summarize the progress that has been made in our understanding of the nuclear envelope and the implications of changes in this environment for cancer biology.