RESUMO
The nuclear pore complex is the primary conduit for nuclear import and export of molecules. In this issue, Gu et al. uncover a novel mechanism in which immune signaling and programmed cell death require nuclear pore rearrangement and release of sequestered cyclin-dependent kinase inhibitors to elicit immunity and death.
Assuntos
Transporte Ativo do Núcleo Celular , Poro Nuclear/metabolismo , Núcleo Celular/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Humanos , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Transdução de SinaisRESUMO
Nuclear pore complexes (NPCs) create large conduits for cargo transport between the nucleus and cytoplasm across the nuclear envelope (NE)1-3. These multi-megadalton structures are composed of about thirty different nucleoporins that are distributed in three main substructures (the inner, cytoplasmic and nucleoplasmic rings) around the central transport channel4-6. Here we use cryo-electron tomography on DLD-1 cells that were prepared using cryo-focused-ion-beam milling to generate a structural model for the human NPC in its native environment. We show that-compared with previous human NPC models obtained from purified NEs-the inner ring in our model is substantially wider; the volume of the central channel is increased by 75% and the nucleoplasmic and cytoplasmic rings are reorganized. Moreover, the NPC membrane exhibits asymmetry around the inner-ring complex. Using targeted degradation of Nup96, a scaffold nucleoporin of the cytoplasmic and nucleoplasmic rings, we observe the interdependence of each ring in modulating the central channel and maintaining membrane asymmetry. Our findings highlight the inherent flexibility of the NPC and suggest that the cellular environment has a considerable influence on NPC dimensions and architecture.
Assuntos
Modelos Estruturais , Poro Nuclear/química , Linhagem Celular Tumoral , Núcleo Celular/química , Citoplasma/química , Tomografia com Microscopia Eletrônica , Humanos , Complexo de Proteínas Formadoras de Poros Nucleares/químicaRESUMO
Many tumors express meiotic genes that could potentially drive somatic chromosome instability. While germline cohesin subunits SMC1B, STAG3, and REC8 are widely expressed in many cancers, messenger RNA and protein for RAD21L subunit are expressed at very low levels. To elucidate the potential of meiotic cohesins to contribute to genome instability, their expression was investigated in human cell lines, predominately in DLD-1. While the induction of the REC8 complex resulted in a mild mitotic phenotype, the expression of the RAD21L complex produced an arrested but viable cell pool, thus providing a source of DNA damage, mitotic chromosome missegregation, sporadic polyteny, and altered gene expression. We also found that genomic binding profiles of ectopically expressed meiotic cohesin complexes were reminiscent of their corresponding specific binding patterns in testis. Furthermore, meiotic cohesins were found to localize to the same sites as BORIS/CTCFL, rather than CTCF sites normally associated with the somatic cohesin complex. These findings highlight the existence of a germline epigenomic memory that is conserved in cells that normally do not express meiotic genes. Our results reveal a mechanism of action by unduly expressed meiotic cohesins that potentially links them to aneuploidy and chromosomal mutations in affected cells.
Assuntos
Expressão Ectópica do Gene , Neoplasias , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Instabilidade Cromossômica/genética , Proteínas Cromossômicas não Histona , Segregação de Cromossomos , Proteínas de Ligação a DNA/metabolismo , Humanos , Masculino , Meiose/genética , Neoplasias/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , RNA Mensageiro , CoesinasRESUMO
Nuclear speckles are non-membrane-bound organelles known as storage sites for messenger RNA (mRNA) processing and splicing factors. More recently, nuclear speckles have also been implicated in splicing and export of a subset of mRNAs, including the influenza virus M mRNA that encodes proteins required for viral entry, trafficking, and budding. However, little is known about how nuclear speckles are assembled or regulated. Here, we uncovered a role for the cellular protein kinase TAO2 as a constituent of nuclear speckles and as a factor required for the integrity of these nuclear bodies and for their functions in pre-mRNA splicing and trafficking. We found that a nuclear pool of TAO2 is localized at nuclear speckles and interacts with nuclear speckle factors involved in RNA splicing and nuclear export, including SRSF1 and Aly/Ref. Depletion of TAO2 or inhibition of its kinase activity disrupts nuclear speckle structure, decreasing the levels of several proteins involved in nuclear speckle assembly and splicing, including SC35 and SON. Consequently, splicing and nuclear export of influenza virus M mRNA were severely compromised and caused a disruption in the virus life cycle. In fact, low levels of TAO2 led to a decrease in viral protein levels and inhibited viral replication. Additionally, depletion or inhibition of TAO2 resulted in abnormal expression of a subset of mRNAs with key roles in viral replication and immunity. Together, these findings uncovered a function of TAO2 in nuclear speckle formation and function and revealed host requirements and vulnerabilities for influenza infection.
Assuntos
Núcleo Celular , Salpicos Nucleares , Proteínas Quinases , Splicing de RNA , Transporte Ativo do Núcleo Celular , Núcleo Celular/enzimologia , Células HeLa , Humanos , Proteínas Quinases/metabolismo , RNA/metabolismo , RNA Mensageiro/metabolismo , Fatores de Processamento de Serina-Arginina/genéticaRESUMO
The in vivo characterization of the exact copy number and the specific function of each composite protein within the nuclear pore complex (NPC) remains both desirable and challenging. Through the implementation of live-cell high-speed super-resolution single-molecule microscopy, we first quantified the native copies of nuclear basket (BSK) proteins (Nup153, Nup50, and Tpr) prior to knocking them down in a highly specific manner via an auxin-inducible degron strategy. Second, we determined the specific roles that BSK proteins play in the nuclear export kinetics of model messenger RNA (mRNA) substrates. Finally, the three-dimensional (3D) nuclear export routes of these mRNA substrates through native NPCs in the absence of specific BSK proteins were obtained and further validated via postlocalization computational simulations. We found that these BSK proteins possess the stoichiometric ratio of 1:1:1 and play distinct roles in the nuclear export of mRNAs within live cells. The absence of Tpr from the NPC predominantly reduces the probability of nuclear mRNAs entering the NPC for export. Complete depletion of Nup153 and Nup50 results in an mRNA nuclear export efficiency decrease of approximately four folds. mRNAs can gain their maximum successful export efficiency as the copy number of Nup153 increased from zero to only half the full complement natively within the NPC. Lastly, the absence of Tpr or Nup153 seems to alter the 3D export routes of mRNAs as they pass through the NPC. However, the removal of Nup50 alone has almost no impact upon mRNA export route and kinetics.
Assuntos
Núcleo Celular/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/fisiologia , Proteínas Nucleares/metabolismo , Transporte de RNA , RNA Mensageiro/metabolismo , Transporte Ativo do Núcleo Celular , Núcleo Celular/genética , Humanos , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas Nucleares/genética , RNA Mensageiro/genéticaRESUMO
One of the hallmarks of cancer is chromosome instability (CIN), which leads to aneuploidy, translocations, and other chromosome aberrations. However, in the vast majority of human tumors the molecular basis of CIN remains unknown, partly because not all genes controlling chromosome transmission have yet been identified. To address this question, we developed an experimental high-throughput imaging (HTI) siRNA assay that allows the identification of novel CIN genes. Our method uses a human artificial chromosome (HAC) expressing the GFP transgene. When this assay was applied to screen an siRNA library of protein kinases, we identified PINK1, TRIO, IRAK1, PNCK, and TAOK1 as potential novel genes whose knockdown induces various mitotic abnormalities and results in chromosome loss. The HAC-based assay can be applied for screening different siRNA libraries (cell cycle regulation, DNA damage response, epigenetics, and transcription factors) to identify additional genes involved in CIN. Identification of the complete spectrum of CIN genes will reveal new insights into mechanisms of chromosome segregation and may expedite the development of novel therapeutic strategies to target the CIN phenotype in cancer cells.
Assuntos
Instabilidade Cromossômica/genética , Cromossomos Humanos/genética , Proteínas Quinases/genética , RNA Interferente Pequeno/genética , Aneuploidia , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/genética , Linhagem Celular Tumoral , Cromossomos Artificiais Humanos/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Quinases Associadas a Receptores de Interleucina-1/genética , Mitose/genética , Proteínas Quinases/isolamento & purificação , Proteínas Serina-Treonina Quinases/genética , RNA de Cadeia Dupla/genética , Transgenes , Translocação Genética/genéticaRESUMO
Mitosis is the stage of the cell cycle during which replicated chromosomes must be precisely divided to allow the formation of two daughter cells possessing equal genetic material. Much of the careful spatial and temporal organization of mitosis is maintained through post-translational modifications, such as phosphorylation and ubiquitination, of key cellular proteins. Here, we will review evidence that sumoylation, conjugation to the SUMO family of small ubiquitin-like modifiers, also serves essential regulatory roles during mitosis. We will discuss the basic biology of sumoylation, how the SUMO pathway has been implicated in particular mitotic functions, including chromosome condensation, centromere/kinetochore organization and cytokinesis, and what cellular proteins may be the targets underlying these phenomena.
Assuntos
Cromossomos/metabolismo , Mitose , Transdução de Sinais , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Ubiquitina-Proteína Ligases/metabolismo , Animais , Cromossomos/química , Cromossomos/genética , Citocinese , Humanos , Conformação de Ácido NucleicoRESUMO
Despite numerous studies on specific sumoylated transcriptional regulators, the global role of SUMO on chromatin in relation to transcription regulation remains largely unknown. Here, we determined the genome-wide localization of SUMO1 and SUMO2/3, as well as of UBC9 (encoded by UBE2I) and PIASY (encoded by PIAS4), two markers for active sumoylation, along with Pol II and histone marks in proliferating versus senescent human fibroblasts together with gene expression profiling. We found that, whereas SUMO alone is widely distributed over the genome with strong association at active promoters, active sumoylation occurs most prominently at promoters of histone and protein biogenesis genes, as well as Pol I rRNAs and Pol III tRNAs. Remarkably, these four classes of genes are up-regulated by inhibition of sumoylation, indicating that SUMO normally acts to restrain their expression. In line with this finding, sumoylation-deficient cells show an increase in both cell size and global protein levels. Strikingly, we found that in senescent cells, the SUMO machinery is selectively retained at histone and tRNA gene clusters, whereas it is massively released from all other unique chromatin regions. These data, which reveal the highly dynamic nature of the SUMO landscape, suggest that maintenance of a repressive environment at histone and tRNA loci is a hallmark of the senescent state. The approach taken in our study thus permitted the identification of a common biological output and uncovered hitherto unknown functions for active sumoylation at chromatin as a key mechanism that, in dynamically marking chromatin by a simple modifier, orchestrates concerted transcriptional regulation of a network of genes essential for cell growth and proliferation.
Assuntos
Proliferação de Células , Cromatina/genética , Cromatina/metabolismo , Regulação da Expressão Gênica , Genes Essenciais , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Animais , Ciclo Celular , Linhagem Celular , Senescência Celular , Perfilação da Expressão Gênica , Histonas/genética , Histonas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Proteínas Inibidoras de STAT Ativados/genética , Proteínas Inibidoras de STAT Ativados/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA de Transferência/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Sumoilação , Transcrição Gênica , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismoAssuntos
Proteína SUMO-1/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sumoilação , Enzimas de Conjugação de Ubiquitina/metabolismo , Meiose , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Enzimas de Conjugação de Ubiquitina/químicaRESUMO
DNA Topoisomerase IIα (TopoIIα) decatenates sister chromatids, allowing their segregation in mitosis. Without the TopoIIα Strand Passage Reaction (SPR), chromosome bridges and ultra-fine DNA bridges (UFBs) arise in anaphase. The TopoIIα C-terminal domain is dispensable for the SPR in vitro but essential for mitotic functions in vivo. Here, we present evidence that the Chromatin Tether (ChT) within the CTD interacts with specific methylated nucleosomes and is crucial for high-fidelity chromosome segregation. Mutation of individual αChT residues disrupts αChT-nucleosome interaction, induces loss of segregation fidelity and reduces association of TopoIIα with chromosomes. Specific methyltransferase inhibitors reducing histone H3 or H4 methylation decreased TopoIIα at centromeres and increased segregation errors. Methyltransferase inhibition did not further increase aberrant anaphases in the ChT mutants, indicating a functional connection. The evidence reveals novel cellular regulation whereby TopoIIα specifically interacts with methylated nucleosomes via the αChT to ensure high-fidelity chromosome segregation.
RESUMO
Nuclear export of influenza A virus (IAV) mRNAs occurs through the nuclear pore complex (NPC). Using the Auxin-Induced Degron (AID) system to rapidly degrade proteins, we show that among the nucleoporins localized at the nucleoplasmic side of the NPC, TPR is the key nucleoporin required for nuclear export of influenza virus mRNAs. TPR recruits the TRanscription and EXport complex (TREX)-2 to the NPC for exporting a subset of cellular mRNAs. By degrading components of the TREX-2 complex (GANP, Germinal-center Associated Nuclear Protein; PCID2, PCI domain containing 2), we show that influenza mRNAs require the TREX-2 complex for nuclear export and replication. Furthermore, we found that cellular mRNAs whose export is dependent on GANP have a small number of exons, a high mean exon length, long 3' UTR, and low GC content. Some of these features are shared by influenza virus mRNAs. Additionally, we identified a 45 nucleotide RNA signal from influenza virus HA mRNA that is sufficient to mediate GANP-dependent mRNA export. Thus, we report a role for the TREX-2 complex in nuclear export of influenza mRNAs and identified RNA determinants associated with the TREX-2-dependent mRNA export.
Assuntos
Transporte Ativo do Núcleo Celular , Influenza Humana , Orthomyxoviridae , Transporte de RNA , Humanos , Transporte Ativo do Núcleo Celular/genética , Núcleo Celular/metabolismo , Influenza Humana/metabolismo , Poro Nuclear/genética , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/metabolismo , Orthomyxoviridae/genética , Transporte de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The Ran GTPase controls multiple cellular processes, including nuclear transport, mitotic checkpoints, spindle assembly and post-mitotic nuclear envelope reassembly. Here we examine the mitotic function of Crm1, the Ran-GTP-binding nuclear export receptor for leucine-rich cargo (bearing nuclear export sequence) and Snurportin-1 (ref. 3). We find that Crm1 localizes to kinetochores, and that Crm1 ternary complex assembly is essential for Ran-GTP-dependent recruitment of Ran GTPase-activating protein 1 (Ran-GAP1) and Ran-binding protein 2 (Ran-BP2) to kinetochores. We further show that Crm1 inhibition by leptomycin B disrupts mitotic progression and chromosome segregation. Analysis of spindles within leptomycin B-treated cells shows that their centromeres were under increased tension. In leptomycin B-treated cells, centromeres frequently associated with continuous microtubule bundles that spanned the centromeres, indicating that their kinetochores do not maintain discrete end-on attachments to single kinetochore fibres. Similar spindle defects were observed in temperature-sensitive Ran pathway mutants (tsBN2 cells). Taken together, our findings demonstrate that Crm1 and Ran-GTP are essential for Ran-BP2/Ran-GAP1 recruitment to kinetochores, for definition of kinetochore fibres and for chromosome segregation at anaphase. Thus, Crm1 is a critical Ran-GTP effector for mitotic spindle assembly and function in somatic cells.
Assuntos
Carioferinas/metabolismo , Cinetocoros/metabolismo , Mitose/fisiologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Fuso Acromático/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Linhagem Celular Tumoral , Segregação de Cromossomos/fisiologia , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Células HeLa , Humanos , Carioferinas/antagonistas & inibidores , Carioferinas/genética , Cinetocoros/ultraestrutura , Microtúbulos/genética , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação/fisiologia , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Transporte Proteico/fisiologia , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/genética , Fuso Acromático/genética , Fuso Acromático/ultraestrutura , Proteína ran de Ligação ao GTP/genética , Proteína Exportina 1RESUMO
During mitosis, the inner centromeric region (ICR) recruits protein complexes that regulate sister chromatid cohesion, monitor tension, and modulate microtubule attachment. Biochemical pathways that govern formation of the inner centromere remain elusive. The kinetochore protein Bub1 was shown to promote assembly of the outer kinetochore components, such as BubR1 and CENP-F, on centromeres. Bub1 was also implicated in targeting of Shugoshin (Sgo) to the ICR. We show that Bub1 works as a master organizer of the ICR. Depletion of Bub1 from Xenopus laevis egg extract or from HeLa cells resulted in both destabilization and displacement of chromosomal passenger complex (CPC) from the ICR. Moreover, soluble Bub1 controls the binding of Sgo to chromatin, whereas the CPC restricts loading of Sgo specifically onto centromeres. We further provide evidence that Bub1 kinase activity is pivotal for recruitment of all of these components. Together, our findings demonstrate that Bub1 acts at multiple points to assure the correct kinetochore formation.
Assuntos
Núcleo Celular/metabolismo , Centrômero/metabolismo , Mitose/fisiologia , Proteínas Quinases/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/genética , Centrômero/ultraestrutura , Segregação de Cromossomos/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Células HeLa , Humanos , Masculino , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Oócitos/citologia , Oócitos/fisiologia , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases , Espermatozoides , Fuso Acromático/metabolismo , Fuso Acromático/ultraestrutura , XenopusRESUMO
SUMOs (small ubiquitin-like modifiers) are ubiquitin-related proteins that become covalently conjugated to cellular target proteins that are involved in a variety of processes. Frequently, this modification has a key role in regulating the activities of those targets and, thus, their cellular functions. SUMO conjugation is a highly dynamic process that can be rapidly reversed by the action of members of the Ubl (ubiquitin-like protein)-specific protease (Ulp) family. The same family of enzymes is also responsible for maturation of newly synthesized SUMOs prior to their initial conjugation. Recent advances in structural, biochemical and cell biological analysis of Ulp/SENPs reveal their high degree of specificity towards SUMO paralogs, in addition to discrimination between processing, deconjugation and chain-editing reactions. The dissimilar sub-nuclear localization patterns of Ulp/SENPs and phenotypes of Ulp/SENP mutants further indicate that different Ulp/SENPs have distinct and non-redundant roles.
Assuntos
Cisteína Endopeptidases/fisiologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/fisiologia , Ubiquitinas/fisiologia , Sequência de Aminoácidos , Animais , Cisteína Endopeptidases/genética , Endopeptidases/genética , Endopeptidases/fisiologia , Evolução Molecular , Regulação da Expressão Gênica , Humanos , Modelos Biológicos , Peptídeo Hidrolases/fisiologia , Transporte Proteico/fisiologia , Proteínas Repressoras/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologiaRESUMO
Over the last decade, the use of auxin-inducible degrons (AID) to control the stability of target proteins has revolutionized the field of cell biology. AID-mediated degradation helps to overcome multiple hurdles that have been encountered in studying multisubunit protein complexes, like the nuclear pore complex (NPC), using classical biochemical and genetic methods. We have used the AID system for acute depletion of individual members of the NPC, called nucleoporins, in order to distinguish their roles both within established NPCs and during NPC assembly.Here, we describe a protocol for CRISPR/Cas9-mediated gene targeting of genes with the AID tag. As an example, we describe a step-by-step protocol for targeting of the NUP153 gene. We also provide recommendations for screening strategies and integration of the sequence encoding the Transport Inhibitor Response 1 (TIR1) protein, a E3-Ubiquitin ligase subunit necessary for AID-dependent protein degradation. In addition, we discuss applications of the NUP-AID system and functional assays for analysis of NUP-AID tagged cell lines.
Assuntos
Marcação de Genes , Ácidos Indolacéticos , Complexo de Proteínas Formadoras de Poros Nucleares , Sistemas CRISPR-Cas , Marcação de Genes/métodos , Ácidos Indolacéticos/farmacologia , Poro Nuclear , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas , ProteóliseRESUMO
Field emission scanning electron microscopy (FESEM) is a well-established technique for acquiring three-dimensional surface images of nuclear pore complexes (NPCs). We present an optimized protocol for the exposure of mammalian cell nuclei and direct surface imaging of nuclear envelopes by FESEM, allowing for a detailed morphological comparison of individual NPCs, without the need for averaging techniques. This provides a unique high resolution tool for studying the effects of cellular stress, specific genetic manipulations and inherited diseases on the ultrastructure of human NPCs.
Assuntos
Membrana Nuclear , Poro Nuclear , Animais , Núcleo Celular/ultraestrutura , Humanos , Imageamento Tridimensional , Mamíferos , Microscopia Eletrônica de Varredura , Membrana Nuclear/ultraestrutura , Poro Nuclear/metabolismoRESUMO
Assembly of the nucleus following mitosis requires rapid and coordinate recruitment of diverse constituents to the inner nuclear membrane. We have identified an unexpected role for the nucleoporin Nup153 in promoting the continued addition of a subset of nuclear envelope (NE) proteins during initial expansion of nascent nuclei. Specifically, disrupting the function of Nup153 interferes with ongoing addition of B-type lamins, lamin B receptor, and SUN1 early in telophase, after the NE has initially enclosed chromatin. In contrast, effects on lamin A and SUN2 were minimal, pointing to differential requirements for the ongoing targeting of NE proteins. Further, distinct mistargeting phenotypes arose among the proteins that require Nup153 for NE targeting. Thus, disrupting the function of Nup153 in nuclear formation reveals several previously undescribed features important for establishing nuclear architecture: 1) a role for a nuclear basket constituent in ongoing recruitment of nuclear envelope components, 2) two functionally separable phases of NE formation in mammalian cells, and 3) distinct requirements of individual NE residents for continued targeting during the expansion phase of NE reformation.
Assuntos
Membrana Nuclear , Complexo de Proteínas Formadoras de Poros Nucleares , Animais , Núcleo Celular/metabolismo , Cromatina/metabolismo , Lamina Tipo A/metabolismo , Mamíferos/metabolismo , Proteínas de Membrana/metabolismo , Mitose , Membrana Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismoRESUMO
Nuclear clearance of the RNA-binding protein TDP-43 is a hallmark of neurodegeneration and an important therapeutic target. Our current understanding of TDP-43 nucleocytoplasmic transport does not fully explain its predominantly nuclear localization or mislocalization in disease. Here, we show that TDP-43 exits nuclei by passive diffusion, independent of facilitated mRNA export. RNA polymerase II blockade and RNase treatment induce TDP-43 nuclear efflux, suggesting that nuclear RNAs sequester TDP-43 in nuclei and limit its availability for passive export. Induction of TDP-43 nuclear efflux by short, GU-rich oligomers (presumably by outcompeting TDP-43 binding to endogenous nuclear RNAs), and nuclear retention conferred by splicing inhibition, demonstrate that nuclear TDP-43 localization depends on binding to GU-rich nuclear RNAs. Indeed, RNA-binding domain mutations markedly reduce TDP-43 nuclear localization and abolish transcription blockade-induced nuclear efflux. Thus, the nuclear abundance of GU-RNAs, dictated by the balance of transcription, pre-mRNA processing, and RNA export, regulates TDP-43 nuclear localization.
Assuntos
Esclerose Lateral Amiotrófica , RNA Nuclear , Transporte Ativo do Núcleo Celular , Esclerose Lateral Amiotrófica/metabolismo , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , RNA Nuclear/metabolismoRESUMO
INTRODUCTION The subcellular compartmentalization of eukaryotic cells requires selective transport of folded proteins and protein-nucleic acid complexes. Embedded in nuclear envelope pores, which are generated by the circumscribed fusion of the inner and outer nuclear membranes, nuclear pore complexes (NPCs) are the sole bidirectional gateways for nucleocytoplasmic transport. The ~110-MDa human NPC is an ~1000-protein assembly that comprises multiple copies of ~34 different proteins, collectively termed nucleoporins. The symmetric core of the NPC is composed of an inner ring encircling the central transport channel and outer rings formed by Yshaped coat nucleoporin complexes (CNCs) anchored atop both sides of the nuclear envelope. The outer rings are decorated with compartmentspecific asymmetric nuclear basket and cytoplasmic filament nucleoporins, which establish transport directionality and provide docking sites for transport factors and the small guanosine triphosphatase Ran. The cytoplasmic filament nucleoporins also play an essential role in the irreversible remodeling of messenger ribonucleoprotein particles (mRNPs) as they exit the central transport channel. Unsurprisingly, the NPC's cytoplasmic face represents a hotspot for diseaseassociated mutations and is commonly targeted by viral virulence factors. RATIONALE Previous studies established a near-atomic composite structure of the human NPC's symmetric core by combining (i) biochemical reconstitution to elucidate the interaction network between symmetric nucleoporins, (ii) crystal and single-particle cryo-electron microscopy structure determination of nucleoporins and nucleoporin complexes to reveal their three-dimensional shape and the molecular details of their interactions, (iii) quantitative docking in cryo-electron tomography (cryo-ET) maps of the intact human NPC to uncover nucleoporin stoichiometry and positioning, and (iv) cellbased assays to validate the physiological relevance of the biochemical and structural findings. In this work, we extended our approach to the cytoplasmic filament nucleoporins to reveal the near-atomic architecture of the cytoplasmic face of the human NPC. RESULTS Using biochemical reconstitution, we elucidated the protein-protein and protein-RNA interaction networks of the human and Chaetomium thermophilum cytoplasmic filament nucleoporins, establishing an evolutionarily conserved heterohexameric cytoplasmic filament nucleoporin complex (CFNC) held together by a central heterotrimeric coiledcoil hub that tethers two separate mRNPremodeling complexes. Further biochemical analysis and determination of a series of crystal structures revealed that the metazoanspecific cytoplasmic filament nucleoporin NUP358 is composed of 16 distinct domains, including an Nterminal Sshaped αhelical solenoid followed by a coiledcoil oligomerization element, numerous Raninteracting domains, an E3 ligase domain, and a Cterminal prolylisomerase domain. Physiologically validated quantitative docking into cryo-ET maps of the intact human NPC revealed that pentameric NUP358 bundles, conjoined by the oligomerization element, are anchored through their Nterminal domains to the central stalk regions of the CNC, projecting flexibly attached domains as far as ~600 Å into the cytoplasm. Using cellbased assays, we demonstrated that NUP358 is dispensable for the architectural integrity of the assembled interphase NPC and RNA export but is required for efficient translation. After NUP358 assignment, the remaining 4-shaped cryoET density matched the dimensions of the CFNC coiledcoil hub, in close proximity to an outer-ring NUP93. Whereas the N-terminal NUP93 assembly sensor motif anchors the properly assembled related coiledcoil channel nucleoporin heterotrimer to the inner ring, biochemical reconstitution confirmed that the NUP93 assembly sensor is reused in anchoring the CFNC to the cytoplasmic face of the human NPC. By contrast, two C. thermophilum CFNCs are anchored by a divergent mechanism that involves assembly sensors located in unstructured portions of two CNC nucleoporins. Whereas unassigned cryoET density occupies the NUP358 and CFNC binding sites on the nuclear face, docking of the nuclear basket component ELYS established that the equivalent position on the cytoplasmic face is unoccupied, suggesting that mechanisms other than steric competition promote asymmetric distribution of nucleoporins. CONCLUSION We have substantially advanced the biochemical and structural characterization of the asymmetric nucleoporins' architecture and attachment at the cytoplasmic and nuclear faces of the NPC. Our nearatomic composite structure of the human NPC's cytoplasmic face provides a biochemical and structural framework for elucidating the molecular basis of mRNP remodeling, viral virulence factor interference with NPC function, and the underlying mechanisms of nucleoporin diseases at the cytoplasmic face of the NPC. [Figure: see text].
Assuntos
Citoplasma , Proteínas Fúngicas , Complexo de Proteínas Formadoras de Poros Nucleares , Poro Nuclear , Transporte de RNA , RNA Mensageiro , Chaetomium , Microscopia Crioeletrônica , Citoplasma/química , Proteínas Fúngicas/química , Humanos , Chaperonas Moleculares/química , Poro Nuclear/química , Complexo de Proteínas Formadoras de Poros Nucleares/química , Conformação Proteica , RNA Mensageiro/metabolismoRESUMO
PIASy is a small ubiquitin-related modifier (SUMO) ligase that modifies chromosomal proteins in mitotic Xenopus egg extracts and plays an essential role in mitotic chromosome segregation. We have isolated a novel SUMO-2/3-modified mitotic chromosomal protein and identified it as poly(ADP-ribose) polymerase 1 (PARP1). PARP1 was robustly conjugated to SUMO-2/3 on mitotic chromosomes but not on interphase chromatin. PIASy promotes SUMOylation of PARP1 both in egg extracts and in vitro reconstituted SUMOylation assays. Through tandem mass spectrometry analysis of mitotically SUMOylated PARP1, we identified a residue within the BRCA1 C-terminal domain of PARP1 (lysine 482) as its primary SUMOylation site. Mutation of this residue significantly reduced PARP1 SUMOylation in egg extracts and enhanced the accumulation of species derived from modification of secondary lysine residues in assays using purified components. SUMOylation of PARP1 did not alter in vitro PARP1 enzyme activity, poly-ADP-ribosylation (PARylation), nor did inhibition of SUMOylation of PARP1 alter the accumulation of PARP1 on mitotic chromosomes, suggesting that SUMOylation regulates neither the intrinsic activity of PARP1 nor its localization. However, loss of SUMOylation increased PARP1-dependent PARylation on isolated chromosomes, indicating SUMOylation controls the capacity of PARP1 to modify other chromatin-associated proteins.