Methylated histones on mitotic chromosomes promote topoisomerase IIα function for high fidelity chromosome segregation.

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.

Influenza virus mRNAs encode determinants for nuclear export via the cellular TREX-2 complex.

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.

Ectopic expression of meiotic cohesin generates chromosome instability in cancer cell line.

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.

A role for Nup153 in nuclear assembly reveals differential requirements for targeting of nuclear envelope constituents.

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.

Nuclear RNA binding regulates TDP-43 nuclear localization and passive nuclear export.

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.

Architecture of the cytoplasmic face of the nuclear pore.

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].

Nuclear speckle integrity and function require TAO2 kinase.

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.

Analysis of Nucleoporin Function Using Inducible Degron Techniques.

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.

High-Resolution Imaging and Analysis of Individual Nuclear Pore Complexes.

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.

Association of RanGAP to nuclear pore complex component, RanBP2/Nup358, is required for pupal development in Drosophila.

Ran's GTPase-activating protein (RanGAP) is tethered to the nuclear envelope (NE) in multicellular organisms. We investigated the consequences of RanGAP localization in human tissue culture cells and Drosophila. In tissue culture cells, disruption of RanGAP1 NE localization surprisingly has neither obvious impacts on viability nor nucleocytoplasmic transport of a model substrate. In Drosophila, we identified a region within nucleoporin dmRanBP2 required for direct tethering of dmRanGAP to the NE. A dmRanBP2 mutant lacking this region shows no apparent growth defects during larval stages but arrests at the early pupal stage. A direct fusion of dmRanGAP to the dmRanBP2 mutant rescues this arrest, indicating that dmRanGAP recruitment to dmRanBP2 per se is necessary for the pupal ecdysis sequence. Our results indicate that while the NE localization of RanGAP is widely conserved in multicellular organisms, the targeting mechanisms are not. Further, we find a requirement for this localization during pupal development.

The cellular environment shapes the nuclear pore complex architecture.

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.

Distinct roles of nuclear basket proteins in directing the passage of mRNA through the nuclear pore.

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.

Thyroid Hormone Receptor Is Essential for Larval Epithelial Apoptosis and Adult Epithelial Stem Cell Development but Not Adult Intestinal Morphogenesis during Xenopus tropicalis Metamorphosis.

Vertebrate postembryonic development is regulated by thyroid hormone (T3). Of particular interest is anuran metamorphosis, which offers several unique advantages for studying the role of T3 and its two nuclear receptor genes, TRα and TRß, during postembryonic development. We have recently generated TR double knockout (TRDKO) Xenopus tropicalis animals and reported that TR is essential for the completion of metamorphosis. Furthermore, TRDKO tadpoles are stalled at the climax of metamorphosis before eventual death. Here we show that TRDKO intestine lacked larval epithelial cell death and adult stem cell formation/proliferation during natural metamorphosis. Interestingly, TRDKO tadpole intestine had premature formation of adult-like epithelial folds and muscle development. In addition, T3 treatment of premetamorphic TRDKO tadpoles failed to induce any metamorphic changes in the intestine. Furthermore, RNA-seq analysis revealed that TRDKO altered the expression of many genes in biological pathways such as Wnt signaling and the cell cycle that likely underlay the inhibition of larval epithelial cell death and adult stem cell development caused by removing both TR genes. Our data suggest that liganded TR is required for larval epithelial cell degeneration and adult stem cell formation, whereas unliganded TR prevents precocious adult tissue morphogenesis such as smooth-muscle development and epithelial folding.

Nucleoporin TPR is an integral component of the TREX-2 mRNA export pathway.

Nuclear pore complexes (NPCs) are important for cellular functions beyond nucleocytoplasmic trafficking, including genome organization and gene expression. This multi-faceted nature and the slow turnover of NPC components complicates investigations of how individual nucleoporins act in these diverse processes. To address this question, we apply an Auxin-Induced Degron (AID) system to distinguish roles of basket nucleoporins NUP153, NUP50 and TPR. Acute depletion of TPR causes rapid and pronounced changes in transcriptomic profiles. These changes are dissimilar to shifts observed after loss of NUP153 or NUP50, but closely related to changes caused by depletion of mRNA export receptor NXF1 or the GANP subunit of the TRanscription-EXport-2 (TREX-2) mRNA export complex. Moreover, TPR depletion disrupts association of TREX-2 subunits (GANP, PCID2, ENY2) to NPCs and results in abnormal RNA transcription and export. Our findings demonstrate a unique and pivotal role of TPR in gene expression through TREX-2- and/or NXF1-dependent mRNA turnover.

PICH regulates the abundance and localization of SUMOylated proteins on mitotic chromosomes.

Proper chromosome segregation is essential for faithful cell division and if not maintained results in defective cell function caused by the abnormal distribution of genetic information. Polo-like kinase 1-interacting checkpoint helicase (PICH) is a DNA translocase essential for chromosome bridge resolution during mitosis. Its function in resolving chromosome bridges requires both DNA translocase activity and ability to bind chromosomal proteins modified by the small ubiquitin-like modifier (SUMO). However, it is unclear how these activities cooperate to resolve chromosome bridges. Here, we show that PICH specifically disperses SUMO2/3 foci on mitotic chromosomes. This PICH function is apparent toward SUMOylated topoisomerase IIα (TopoIIα) after inhibition of TopoIIα by ICRF-193. Conditional depletion of PICH using the auxin-inducible degron (AID) system resulted in the retention of SUMO2/3-modified chromosomal proteins, including TopoIIα, indicating that PICH functions to reduce the association of these proteins with chromosomes. Replacement of PICH with its translocase-deficient mutants led to increased SUMO2/3 foci on chromosomes, suggesting that the reduction of SUMO2/3 foci requires the remodeling activity of PICH. In vitro assays showed that PICH specifically attenuates SUMOylated TopoIIα activity using its SUMO-binding ability. Taking the results together, we propose a novel function of PICH in remodeling SUMOylated proteins to ensure faithful chromosome segregation.

RanBP1 controls the Ran pathway in mammalian cells through regulation of mitotic RCC1 dynamics.

The Ran GTPase plays critical roles in multiple cellular processes including interphase nucleocytoplasmic transport and mitotic spindle assembly. During mitosis in mammalian cells, GTP-bound Ran (Ran-GTP) is concentrated near mitotic chromatin while GDP-bound Ran (Ran-GDP) is more abundant distal to chromosomes. This pattern spatially controls spindle formation because Ran-GTP locally releases spindle assembly factors (SAFs), such as Hepatoma Up-Regulated Protein (HURP), from inhibitory interactions near chromosomes. Regulator of Chromatin Condensation 1 (RCC1) is Ran's chromatin-bound exchange factor, and RanBP1 is a conserved Ran-GTP-binding protein that has been implicated as a mitotic regulator of RCC1 in embryonic systems. Here, we show that RanBP1 controls mitotic RCC1 dynamics in human somatic tissue culture cells. In addition, we observed the re-localization of HURP in metaphase cells after RanBP1 degradation, consistent with the idea that altered RCC1 dynamics functionally modulate SAF activities. Together, our findings reveal an important mitotic role for RanBP1 in human somatic cells, controlling the spatial distribution and magnitude of mitotic Ran-GTP production and thereby ensuring the accurate execution of Ran-dependent mitotic events. ABBREVIATIONS: AID: Auxin-induced degron; FLIP: Fluorescence loss in photobleaching; FRAP: Fluorescence recovery after photobleaching; GDP: guanosine diphosphate; GTP: guanosine triphosphate; HURP: Hepatoma Up-Regulated Protein; NE: nuclear envelope; NEBD: Nuclear Envelope Breakdown; RanBP1: Ran-binding protein 1; RanGAP1: Ran GTPase-Activating Protein 1; RCC1: Regulator of Chromatin Condensation 1; RRR complex: RCC1/Ran/RanBP1 heterotrimeric complex; SAF: Spindle Assembly Factor; TIR1: Transport Inhibitor Response 1 protein; XEE: Xenopus egg extract.

IRBIT Directs Differentiation of Intestinal Stem Cell Progeny to Maintain Tissue Homeostasis.

The maintenance of the intestinal epithelium is ensured by the controlled proliferation of intestinal stem cells (ISCs) and differentiation of their progeny into various cell types, including enterocytes (ECs) that both mediate nutrient absorption and provide a barrier against pathogens. The signals that regulate transition of proliferative ISCs into differentiated ECs are not fully understood. IRBIT is an evolutionarily conserved protein that regulates ribonucleotide reductase (RNR), an enzyme critical for the generation of DNA precursors. Here, we show that IRBIT expression in ISC progeny within the Drosophila midgut epithelium cells regulates their differentiation via suppression of RNR activity. Disruption of this IRBIT-RNR regulatory circuit causes a premature loss of intestinal tissue integrity. Furthermore, age-related dysplasia can be reversed by suppression of RNR activity in ISC progeny. Collectively, our findings demonstrate a role of the IRBIT-RNR pathway in gut homeostasis.

A novel assay to screen siRNA libraries identifies protein kinases required for chromosome transmission.

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.