Inhibition of RAN attenuates influenza a virus replication and nucleoprotein nuclear export.

Nuclear export of the viral ribonucleoprotein (vRNP) is a critical step in the influenza A virus (IAV) life cycle and may be an effective target for the development of anti-IAV drugs. The host factor ras-related nuclear protein (RAN) is known to participate in the life cycle of several viruses, but its role in influenza virus replication remains unknown. In the present study, we aimed to determine the function of RAN in influenza virus replication using different cell lines and subtype strains. We found that RAN is essential for the nuclear export of vRNP, as it enhances the binding affinity of XPO1 toward the viral nuclear export protein NS2. Depletion of RAN constrained the vRNP complex in the nucleus and attenuated the replication of various subtypes of influenza virus. Using in silico compound screening, we identified that bepotastine could dissociate the RAN-XPO1-vRNP trimeric complex and exhibit potent antiviral activity against influenza virus both in vitro and in vivo. This study demonstrates the important role of RAN in IAV replication and suggests its potential use as an antiviral target.

Association of RAN and RANBP2 Gene Polymorphisms With Glioma Susceptibility in Chinese Children.

BACKGROUND: Glioma is the most prevalent pediatric central nervous system malignancy. RAN, member RAS oncogene family (RAN), is a key signaling molecule that regulates the polymerization of microtubules during mitosis. RAN binding protein 2 (RANBP2) is involved in DNA replication, mitosis, metabolism, and tumorigenesis. The effects of RAN and RANBP2 gene polymorphisms on glioma susceptibility in Chinese children are currently unknown. AIMS: This study aimed to evaluate the association between RAN and RANBP2 gene polymorphisms and glioma susceptibility in Chinese children. METHODS AND RESULTS: We recruited 191 patients with glioma and 248 children without cancer for this case-control study. Polymerase chain reaction-based TaqMan was applied to gene sequencing and typing. Logistic regression model-calculated odds ratio and 95% confidence interval were used to verify whether the gene polymorphisms (RAN rs56109543 C>T, rs7132224 A>G, rs14035 C>T, and RANBP2 rs2462788 C>T) influence glioma susceptibility. Based on age, gender, tumor subtype, and clinical stage, stratified analyses of risk and protective genotypes were conducted. p values for mutant genotype analyses were all >0.05, indicating no significant correlation between these gene polymorphisms and glioma risk. CONCLUSION: RAN and RANBP2 gene polymorphisms were not found to be statistically significantly associated with glioma susceptibility in Chinese children. Other potential functional gene polymorphism loci of RAN and RANBP2 will need to be evaluated in the search for novel glioma biomarkers.

Roles of Tubulin Concentration during Prometaphase and Ran-GTP during Anaphase of Caenorhabditis elegans Meiosis.

In many animal species, the oocyte meiotic spindle, which is required for chromosome segregation, forms without centrosomes. In some systems, Ran-GEF on chromatin initiates spindle assembly. We found that in Caenorhabditis elegans oocytes, endogenously-tagged Ran-GEF dissociates from chromatin during spindle assembly but re-associates during meiotic anaphase. Meiotic spindle assembly occurred after auxin-induced degradation of Ran-GEF, but anaphase I was faster than controls and extrusion of the first polar body frequently failed. In search of a possible alternative pathway for spindle assembly, we found that soluble tubulin concentrates in the nuclear volume during germinal vesicle breakdown. We found that the concentration of soluble tubulin in the metaphase spindle region is enclosed by ER sheets which exclude cytoplasmic organelles including mitochondria and yolk granules. Measurement of the volume occupied by yolk granules and mitochondria indicated that volume exclusion would be sufficient to explain the concentration of tubulin in the spindle volume. We suggest that this concentration of soluble tubulin may be a redundant mechanism promoting spindle assembly near chromosomes.

Nucleocytoplasmic transport rates are regulated by cellular processes that modulate GTP availability.

Nucleocytoplasmic transport (NCT), the facilitated diffusion of cargo molecules between the nucleus and cytoplasm through nuclear pore complexes (NPCs), enables numerous fundamental eukaryotic cellular processes. Ran GTPase uses cellular energy in the direct form of GTP to create a gradient across the nuclear envelope (NE) that drives the majority of NCT. We report here that changes in GTP availability resulting from altered cellular physiology modulate the rate of NCT, as monitored using synthetic and natural cargo, and the dynamics of Ran itself. Cell migration, cell spreading, and/or modulation of the cytoskeleton or its connection to the nucleus alter GTP availability and thus rates of NCT, regulating RNA export and protein synthesis. These findings support a model in which changes in cellular physiology that alter GTP availability can regulate the rate of NCT, impacting fundamental cellular processes that extensively utilize NCT.

Glucose hypometabolism prompts RAN translation and exacerbates C9orf72-related ALS/FTD phenotypes.

The most prevalent genetic cause of both amyotrophic lateral sclerosis and frontotemporal dementia is a (GGGGCC)n nucleotide repeat expansion (NRE) occurring in the first intron of the C9orf72 gene (C9). Brain glucose hypometabolism is consistently observed in C9-NRE carriers, even at pre-symptomatic stages, but its role in disease pathogenesis is unknown. Here, we show alterations in glucose metabolic pathways and ATP levels in the brains of asymptomatic C9-BAC mice. We find that, through activation of the GCN2 kinase, glucose hypometabolism drives the production of dipeptide repeat proteins (DPRs), impairs the survival of C9 patient-derived neurons, and triggers motor dysfunction in C9-BAC mice. We also show that one of the arginine-rich DPRs (PR) could directly contribute to glucose metabolism and metabolic stress by inhibiting glucose uptake in neurons. Our findings provide a potential mechanistic link between energy imbalances and C9-ALS/FTD pathogenesis and suggest a feedforward loop model with potential opportunities for therapeutic intervention.

CFDP1 regulates the stability of pericentric heterochromatin thereby affecting RAN GTPase activity and mitotic spindle formation.

The densely packed centromeric heterochromatin at minor and major satellites is comprised of H3K9me2/3 histones, the heterochromatin protein HP1α, and histone variants. In the present study, we sought to determine the mechanisms by which condensed heterochromatin at major and minor satellites stabilized by the chromatin factor CFDP1 affects the activity of the small GTPase Ran as a requirement for spindle formation. CFDP1 colocalized with heterochromatin at major and minor satellites and was essential for the structural stability of centromeric heterochromatin. Loss of CENPA, HP1α, and H2A.Z heterochromatin components resulted in decreased binding of the spindle nucleation facilitator RCC1 to minor and major satellite repeats. Decreased RanGTP levels as a result of diminished RCC1 binding interfered with chromatin-mediated microtubule nucleation at the onset of mitotic spindle formation. Rescuing chromatin H2A.Z levels in cells and mice lacking CFDP1 through knock-down of the histone chaperone ANP32E not only partially restored RCC1-dependent RanGTP levels but also alleviated CFDP1-knockout-related craniofacial defects and increased microtubule nucleation in CFDP1/ANP32E co-silenced cells. Together, these studies provide evidence for a direct link between condensed heterochromatin at major and minor satellites and microtubule nucleation through the chromatin protein CFDP1.

γ-TuRC asymmetry induces local protofilament mismatch at the RanGTP-stimulated microtubule minus end.

The γ-tubulin ring complex (γ-TuRC) is a structural template for de novo microtubule assembly from α/ß-tubulin units. The isolated vertebrate γ-TuRC assumes an asymmetric, open structure deviating from microtubule geometry, suggesting that γ-TuRC closure may underlie regulation of microtubule nucleation. Here, we isolate native γ-TuRC-capped microtubules from Xenopus laevis egg extract nucleated through the RanGTP-induced pathway for spindle assembly and determine their cryo-EM structure. Intriguingly, the microtubule minus end-bound γ-TuRC is only partially closed and consequently, the emanating microtubule is locally misaligned with the γ-TuRC and asymmetric. In the partially closed conformation of the γ-TuRC, the actin-containing lumenal bridge is locally destabilised, suggesting lumenal bridge modulation in microtubule nucleation. The microtubule-binding protein CAMSAP2 specifically binds the minus end of γ-TuRC-capped microtubules, indicating that the asymmetric minus end structure may underlie recruitment of microtubule-modulating factors for γ-TuRC release. Collectively, we reveal a surprisingly asymmetric microtubule minus end protofilament organisation diverging from the regular microtubule structure, with direct implications for the kinetics and regulation of nucleation and subsequent modulation of microtubules during spindle assembly.

RANBP17 Overexpression Restores Nucleocytoplasmic Transport and Ameliorates Neurodevelopment in Induced DYT1 Dystonia Motor Neurons.

DYT1 dystonia is a debilitating neurological movement disorder, and it represents the most frequent and severe form of hereditary primary dystonia. There is currently no cure for this disease due to its unclear pathogenesis. In our previous study utilizing patient-specific motor neurons (MNs), we identified distinct cellular deficits associated with the disease, including a deformed nucleus, disrupted neurodevelopment, and compromised nucleocytoplasmic transport (NCT) functions. However, the precise molecular mechanisms underlying these cellular impairments have remained elusive. In this study, we revealed the genome-wide changes in gene expression in DYT1 MNs through transcriptomic analysis. We found that those dysregulated genes are intricately involved in neurodevelopment and various biological processes. Interestingly, we identified that the expression level of RANBP17, a RAN-binding protein crucial for NCT regulation, exhibited a significant reduction in DYT1 MNs. By manipulating RANBP17 expression, we further demonstrated that RANBP17 plays an important role in facilitating the nuclear transport of both protein and transcript cargos in induced human neurons. Excitingly, the overexpression of RANBP17 emerged as a substantial mitigating factor, effectively restoring impaired NCT activity and rescuing neurodevelopmental deficits observed in DYT1 MNs. These findings shed light on the intricate molecular underpinnings of impaired NCT in DYT1 neurons and provide novel insights into the pathophysiology of DYT1 dystonia, potentially leading to the development of innovative treatment strategies.

Cryo-EM of the Nucleosome Core Particle Bound to Ran-RCC1 Reveals a Dynamic Complex.

Ras-related nuclear protein (Ran) is a member of the Ras superfamily of small guanosine triphosphatases (GTPases) and a regulator of multiple cellular processes. In healthy cells, the GTP-bound form of Ran is concentrated at chromatin, creating a Ran•GTP gradient that provides the driving force for nucleocytoplasmic transport, mitotic spindle assembly, and nuclear envelope formation. The Ran•GTP gradient is maintained by the regulator of chromatin condensation 1 (RCC1), a guanine nucleotide exchange factor that accelerates GDP/GTP exchange in Ran. RCC1 interacts with nucleosomes, which are the fundamental repeating units of eukaryotic chromatin. Here, we present a cryo-EM analysis of a trimeric complex composed of the nucleosome core particle (NCP), RCC1, and Ran. While the contacts between RCC1 and Ran in the complex are preserved compared with a previously determined structure of RCC1-Ran, our study reveals that RCC1 and Ran interact dynamically with the NCP and undergo rocking motions on the nucleosome surface. Furthermore, the switch 1 region of Ran, which plays an important role in mediating conformational changes associated with the substitution of GDP and GTP nucleotides in Ras family members, appears to undergo disorder-order transitions and forms transient contacts with the C-terminal helix of histone H2B. Nucleotide exchange assays performed in the presence and absence of NCPs are not consistent with an active role for nucleosomes in nucleotide exchange, at least in vitro. Instead, the nucleosome stabilizes RCC1 and serves as a hub that concentrates RCC1 and Ran to promote efficient Ran•GDP to Ran•GTP conversion.

Nuclear export of circular RNA.

Circular RNAs (circRNAs), which are increasingly being implicated in a variety of functions in normal and cancerous cells1-5, are formed by back-splicing of precursor mRNAs in the nucleus6-10. circRNAs are predominantly localized in the cytoplasm, indicating that they must be exported from the nucleus. Here we identify a pathway that is specific for the nuclear export of circular RNA. This pathway requires Ran-GTP, exportin-2 and IGF2BP1. Enhancing the nuclear Ran-GTP gradient by depletion or chemical inhibition of the major protein exporter CRM1 selectively increases the nuclear export of circRNAs, while reducing the nuclear Ran-GTP gradient selectively blocks circRNA export. Depletion or knockout of exportin-2 specifically inhibits nuclear export of circRNA. Analysis of nuclear circRNA-binding proteins reveals that interaction between IGF2BP1 and circRNA is enhanced by Ran-GTP. The formation of circRNA export complexes in the nucleus is promoted by Ran-GTP through its interactions with exportin-2, circRNA and IGF2BP1. Our findings demonstrate that adaptors such as IGF2BP1 that bind directly to circular RNAs recruit Ran-GTP and exportin-2 to export circRNAs in a mechanism that is analogous to protein export, rather than mRNA export.

Mechanism of exportin retention in the cell nucleus.

Exportin receptors are concentrated in the nucleus to transport essential cargoes out of it. A mislocalization of exportins to the cytoplasm is linked to disease. Hence, it is important to understand how their containment within the nucleus is regulated. Here, we have studied the nuclear efflux of exportin2 (cellular apoptosis susceptibility protein or CAS) that delivers karyopherinα (Kapα or importinα), the cargo adaptor for karyopherinß1 (Kapß1 or importinß1), to the cytoplasm in a Ran guanosine triphosphate (RanGTP)-mediated manner. We show that the N-terminus of CAS attenuates the interaction of RanGTPase activating protein 1 (RanGAP1) with RanGTP to slow GTP hydrolysis, which suppresses CAS nuclear exit at nuclear pore complexes (NPCs). Strikingly, a single phosphomimetic mutation (T18D) at the CAS N-terminus is sufficient to abolish its nuclear retention and coincides with metastatic cellular behavior. Furthermore, downregulating Kapß1 disrupts CAS nuclear retention, which highlights the balance between their respective functions that is essential for maintaining the Kapα transport cycle. Therefore, NPCs play a functional role in selectively partitioning exportins in the cell nucleus.

Oxidative stress and signaling through EGFR and PKA pathways converge on the nuclear transport factor RanBP1.

Nuclear protein trafficking requires the soluble transport factor RanBP1. The subcellular distribution of RanBP1 is dynamic, as the protein shuttles between the nucleus and cytoplasm. To date, the signaling pathways regulating RanBP1 subcellular localization are poorly understood. During interphase, RanBP1 resides mostly in the cytoplasm. We show here that oxidative stress concentrates RanBP1 in the nucleus, and our study defines the underlying mechanisms. Specifically, RanBP1's cysteine residues are not essential for its oxidant-induced relocation. Furthermore, our pharmacological approaches uncover that signaling mediated by epidermal growth factor receptor (EGFR) and protein kinase A (PKA) control RanBP1 localization during stress. In particular, pharmacological inhibitors of EGFR or PKA diminish the oxidant-dependent relocation of RanBP1. Mutant analysis identified serine 60 and tyrosine 103 as regulators of RanBP1 nuclear accumulation during oxidant exposure. Taken together, our results define RanBP1 as a target of oxidative stress and a downstream effector of EGFR and PKA signaling routes. This positions RanBP1 at the intersection of important cellular signaling circuits.

Nuclear transport proteins: structure, function, and disease relevance.

Proper subcellular localization is crucial for the functioning of biomacromolecules, including proteins and RNAs. Nuclear transport is a fundamental cellular process that regulates the localization of many macromolecules within the nuclear or cytoplasmic compartments. In humans, approximately 60 proteins are involved in nuclear transport, including nucleoporins that form membrane-embedded nuclear pore complexes, karyopherins that transport cargoes through these complexes, and Ran system proteins that ensure directed and rapid transport. Many of these nuclear transport proteins play additional and essential roles in mitosis, biomolecular condensation, and gene transcription. Dysregulation of nuclear transport is linked to major human diseases such as cancer, neurodegenerative diseases, and viral infections. Selinexor (KPT-330), an inhibitor targeting the nuclear export factor XPO1 (also known as CRM1), was approved in 2019 to treat two types of blood cancers, and dozens of clinical trials of are ongoing. This review summarizes approximately three decades of research data in this field but focuses on the structure and function of individual nuclear transport proteins from recent studies, providing a cutting-edge and holistic view on the role of nuclear transport proteins in health and disease. In-depth knowledge of this rapidly evolving field has the potential to bring new insights into fundamental biology, pathogenic mechanisms, and therapeutic approaches.

RANBP1, a member of the nuclear-cytoplasmic trafficking-regulator complex, is the terminal-striking point of the SGK1-dependent Th17+ pathological differentiation.

The Th17+ arrangement is critical for orchestrating both innate and acquired immune responses. In this context, the serum and glucocorticoid regulated kinase 1 (SGK1) exerts a key role in the governance of IL-23R-dependent Th17+ maturation, through the phosphorylation-dependent control of FOXO1 localization. Our previous work has shown that some of the SGK1-key functions are dependent on RAN-binding protein 1 (RANBP1), a terminal gene in the nuclear transport regulation. Here, we show that RANBP1, similarly to SGK1, is modulated during Th17+ differentiation and that RANBP1 fluctuations mediate the SGK1-dependent effects on Th17+ maturation. RANBP1, as the final effector of the SGK1 pathway, affects FOXO1 transport from the nucleus to the cytoplasm, thus enabling RORγt activation. In this light, RANBP1 represents the missing piece, in an essential and rate-limiting manner, underlying the Th17+ immune asset.

Microtubule binding of the human augmin complex is directly controlled by importins and Ran-GTP.

Mitotic spindle assembly during cell division is a highly regulated process. Ran-GTP produced around chromosomes controls the activity of a multitude of spindle assembly factors by releasing them from inhibitory interaction with importins. A major consequence of Ran-GTP regulation is the local stimulation of branched microtubule nucleation around chromosomes, which is mediated by the augmin complex (composed of the eight subunits HAUS1-HAUS8), a process that is crucially important for correct spindle assembly. However, augmin is not known to be a direct target of the Ran-GTP pathway, raising the question of how its activity is controlled. Here, we present the in vitro reconstitution of Ran-GTP-regulated microtubule binding of the human augmin complex. We demonstrate that importins directly bind to augmin, which prevents augmin from binding to microtubules. Ran-GTP relieves this inhibition. Therefore, the augmin complex is a direct target of the Ran-GTP pathway, suggesting that branching microtubule nucleation is directly regulated by the Ran-GTP gradient around chromosomes in dividing cells.

Investigation of the association of the RAN (rs14035) and XPO5 (rs11077) polymorphisms with venous thromboembolism.

INTRODUCTION: Venous thromboembolism (VTE) is the third most common hemostatic disease worldwide. Studies have reported a role for microRNA (miRNA) in the homeostasis and development of VTE. The ras-related nuclear protein (RAN) and exportin 5 (XPO5) genes are involved in miRNA biogenesis, as both regulate the transport of pre-miRNA from the nucleus to the cytoplasm. Therefore, the aim of the current study is to examine the association between RAN (rs14035) and XPO5 (rs11077) single nucleotide polymorphisms (SNPs) and VTE. METHODS: The study sample consisted of 300 subjects (150 patients and 150 age and sex matched controls). The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and tetra-primer amplification refractory mutation system (T-ARMS) techniques were used to genotype rs14035 and rs11077, respectively. RESULTS: The results showed that there was a significant association between the XPO5 rs11077 and the risk of VTE (P < 0.05). Subjects with AC (OR: 2.08, CI:1.26-3.44) and CC (OR: 1.77, CI: 0.88-3.55) genotypes were at increased risk of the developing VTE. Regarding RAN gene, no association was found between rs14035 and VTE (P > 0.05). In addition, no associations were found between XPO5 rs11077 and RAN rs14035 genotypes with blood cell parameters (P > 0.05). As for the demographic characteristics, the results indicated a strong association between family history and body mass index (BMI) with the risk of VTE (P < 0.01). CONCLUSION: The XPO5 rs11077, BMI and family history might contribute to the development of VTE in Jordan.

Tat-RAN attenuates brain ischemic injury in hippocampal HT-22 cells and ischemia animal model.

Oxidative stress plays a key role in the pathogenesis of neuronal injury, including ischemia. Ras-related nuclear protein (RAN), a member of the Ras superfamily, involves in a variety of biological roles, such as cell division, proliferation, and signal transduction. Although RAN reveals antioxidant effect, its precise neuroprotective mechanisms are still unclear. Therefore, we investigated the effects of RAN on HT-22 cell which were exposed to H2O2-induced oxidative stress and ischemia animal model by using the cell permeable Tat-RAN fusion protein. We showed that Tat-RAN transduced into HT-22 cells, and markedly inhibited cell death, DNA fragmentation, and reactive oxygen species (ROS) generation under oxidative stress. This fusion protein also controlled cellular signaling pathways, including mitogen-activated protein kinases (MAPKs), NF-κB, and apoptosis (Caspase-3, p53, Bax and Bcl-2). In the cerebral forebrain ischemia animal model, Tat-RAN significantly inhibited both neuronal cell death, and astrocyte and microglia activation. These results indicate that RAN significantly protects against hippocampal neuronal cell death, suggesting Tat-RAN will help to develop the therapies for neuronal brain diseases including ischemic injury.

Augmin is a Ran-regulated spindle assembly factor.

Mitotic spindles are composed of microtubules (MTs) that must nucleate at the right place and time. Ran regulates this process by directly controlling the release of spindle assembly factors (SAFs) from nucleocytoplasmic shuttle proteins importin-αß and subsequently forms a biochemical gradient of SAFs localized around chromosomes. The majority of spindle MTs are generated by branching MT nucleation, which has been shown to require an eight-subunit protein complex known as augmin. In Xenopus laevis, Ran can control branching through a canonical SAF, TPX2, which is nonessential in Drosophila melanogaster embryos and HeLa cells. Thus, how Ran regulates branching MT nucleation when TPX2 is not required remains unknown. Here, we use in vitro pulldowns and total internal reflection fluorescence microscopy to show that augmin is a Ran-regulated SAF. We demonstrate that augmin directly interacts with both importin-α and importin-ß through two nuclear localization sequences on the Haus8 subunit, which overlap with the MT-binding site. Moreover, we show that Ran controls localization of augmin to MTs in both Xenopus egg extract and in vitro. Our results demonstrate that RanGTP directly regulates augmin, which establishes a new way by which Ran controls branching MT nucleation and spindle assembly both in the absence and presence of TPX2.

Ran GTPase and Its Importance in Cellular Signaling and Malignant Phenotype.

Ran is a member of the Ras superfamily of proteins, which primarily regulates nucleocytoplasmic trafficking and mediates mitosis by regulating spindle formation and nuclear envelope (NE) reassembly. Therefore, Ran is an integral cell fate determinant. It has been demonstrated that aberrant Ran expression in cancer is a result of upstream dysregulation of the expression of various factors, such as osteopontin (OPN), and aberrant activation of various signaling pathways, including the extracellular-regulated kinase/mitogen-activated protein kinase (ERK/MEK) and phosphatidylinositol 3-kinase/Protein kinase B (PI3K/Akt) pathways. In vitro, Ran overexpression has severe effects on the cell phenotype, altering proliferation, adhesion, colony density, and invasion. Therefore, Ran overexpression has been identified in numerous types of cancer and has been shown to correlate with tumor grade and the degree of metastasis present in various cancers. The increased malignancy and invasiveness have been attributed to multiple mechanisms. Increased dependence on Ran for spindle formation and mitosis is a consequence of the upregulation of these pathways and the ensuing overexpression of Ran, which increases cellular dependence on Ran for survival. This increases the sensitivity of cells to changes in Ran concentration, with ablation being associated with aneuploidy, cell cycle arrest, and ultimately, cell death. It has also been demonstrated that Ran dysregulation influences nucleocytoplasmic transport, leading to transcription factor misallocation. Consequently, patients with tumors that overexpress Ran have been shown to have a higher malignancy rate and a shorter survival time compared to their counterparts.