NEMF mutations in mice illustrate how Importin-ß specific nuclear transport defects recapitulate neurodegenerative disease hallmarks.

Pathological disruption of Nucleocytoplasmic Transport (NCT), such as the mis-localization of nuclear pore complex proteins (Nups), nuclear transport receptors, Ran-GTPase, and RanGAP1, are seen in both animal models and in familial and sporadic forms of amyotrophic lateral sclerosis (ALS), frontal temporal dementia and frontal temporal lobar degeneration (FTD\FTLD), and Alzheimer's and Alzheimer's Related Dementias (AD/ADRD). However, the question of whether these alterations represent a primary cause, or a downstream consequence of disease is unclear, and what upstream factors may account for these defects are unknown. Here, we report four key findings that shed light on the upstream causal role of Importin-ß-specific nuclear transport defects in disease onset. First, taking advantage of two novel mouse models of NEMF neurodegeneration (NemfR86S and NemfR487G) that recapitulate many cellular and biochemical aspects of neurodegenerative diseases, we find an Importin-ß-specific nuclear import block. Second, we observe cytoplasmic mis-localization and aggregation of multiple proteins implicated in the pathogenesis of ALS/FTD and AD/ADRD, including TDP43, Importin-ß, RanGap1, and Ran. These findings are further supported by a pathological interaction between Importin-ß and the mutant NEMFR86S protein in cytoplasmic accumulations. Third, we identify similar transcriptional dysregulation in key genes associated with neurodegenerative disease. Lastly, we show that even transient pharmaceutical inhibition of Importin-ß in both mouse and human neuronal and non-neuronal cells induces key proteinopathies and transcriptional alterations seen in our mouse models and in neurodegeneration. Our convergent results between mouse and human neuronal and non-neuronal cellular biology provide mechanistic evidence that many of the mis-localized proteins and dysregulated transcriptional events seen in multiple neurodegenerative diseases may in fact arise primarily from a primary upstream defect in Importin- ß nuclear import. These findings have critical implications for investigating how sporadic forms of neurodegeneration may arise from presently unidentified genetic and environmental perturbations in Importin-ß function.

RCC1 regulation of subcellular protein localization via Ran GTPase drives pancreatic ductal adenocarcinoma growth.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy, with limited therapeutic options. Here, we evaluated the role of regulator of chromosome condensation 1 (RCC1) in PDAC. RCC1 functions as a guanine exchange factor for GTP-binding nuclear protein Ran (Ran) GTPase and is involved in nucleocytoplasmic transport. RCC1 RNA expression is elevated in PDAC tissues compared to normal pancreatic tissues and correlates with poor prognosis. RCC1 silencing by RNAi and CRISPR-Cas9 knockout (KO) results in reduced proliferation in 2-D and 3-D cell cultures. RCC1 knockdown (KD) reduced migration and clonogenicity, enhanced apoptosis, and altered cell cycle progression in human PDAC and murine cells from LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1-Cre (KPC) tumors. Mechanistically, RCC1 KO shows widespread transcriptomic alterations including regulation of PTK7, a co-receptor of the Wnt signaling pathway. RCC1 KD disrupted subcellular Ran localization and the Ran gradient. Nuclear and cytosolic proteomics revealed altered subcellular proteome localization in Rcc1 KD KPC-tumor-derived cells and several altered metabolic biosynthesis pathways. In vivo, RCC1 KO cells show reduced tumor growth potential when injected as sub-cutaneous xenografts. Finally, RCC1 KD sensitized PDAC cells to gemcitabine chemotherapy treatment. This study reveals the role of RCC1 in pancreatic cancer as a novel molecular vulnerability that could be exploited to enhance therapeutic response.

HASTY-mediated miRNA dynamics modulate nitrogen starvation-induced leaf senescence in Arabidopsis.

Nitrogen (N) deficiency responses are essential for plant survival and reproduction. Here, via an expression genome-wide association study (eGWAS), we reveal a mechanism that regulates microRNA (miRNA) dynamics necessary for N deficiency responses in Arabidopsis. Differential expression levels of three NAC transcription factor (TF) genes involved in leaf N deficiency responses among Arabidopsis accessions are most significantly associated with polymorphisms in HASTY (HST), which encodes an importin/exportin family protein responsible for the generation of mature miRNAs. HST acts as a negative regulator of N deficiency-induced leaf senescence, and the disruption and overexpression of HST differently modifies miRNA dynamics in response to N deficiency, altering levels of miRNAs targeting transcripts. Interestingly, N deficiency prevents the interaction of HST with HST-interacting proteins, DCL1 and RAN1, and some miRNAs. This suggests that HST-mediated regulation of miRNA dynamics collectively controls regulations mediated by multiple N deficiency response-associated NAC TFs, thereby being central to the N deficiency response network.

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.

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.

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.

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.

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.

Matrix metalloproteinase 2 is a target of the RAN-GTP pathway and mediates migration, invasion and metastasis in human breast cancer.

RAS-related nuclear protein(RAN) is a nuclear shuttle and normally regulates events in the cell cycle. When overexpressed in cultured cells, it causes increases in cell migration/invasion in vitro and its overexpression is associated with early breast cancer patient deaths in vivo. However, the underlying mechanism is unknown. The effect of RAN overexpression on potential targets MMP2, ATF3, CXCR3 was investigated by Real-Time PCR/Western blots in the triple receptor negative breast cancer(TRNBC) cell line MDA-MB231 and consequent biological effects were measured by cell adhesion, cell migration and cell invasion assays. Results showed that knockdown of RAN lead to a reduction of MMP2 and its potential regulators ATF3 and CXCR3. Moreover, knockdown of ATF3 or CXCR3 downregulated MMP2 without affecting RAN, indicating that RAN regulates MMP2 through ATF3 and CXCR3. Knockdown of RAN and MMP2 reduced cell adhesion, cell migration and cell growth in agar, whilst overexpression of MMP2 reversed the knockdown of RAN. Furthermore, immunohistochemical staining for RAN and MMP2 are positively associated with each other in the same tumour and separately with patient survival times in breast cancer specimens, suggesting that a high level of RAN may be a pre-requisite for MMP2 overexpression and metastasis. Moreover, positive immunohistochemical staining for both RAN and MMP-2 reduces further patient survival times over that for either protein separately. Our results suggest that MMP2 expression can stratify progression of breast cancers with a high and low incidence of RAN, both RAN and MMP2 in combination can be used for a more accurate patient prognosis. SIMPLE SUMMARY: Ran is an important regulator of normal cell growth and behaviour. We have established in cell line models of breast cancer (BC) a molecular pathway between RAN and its protein-degrading effector MMP-2 and properties related to metastasis in culture. Using immunohistochemistry (IHC) staining of primary BCs, we have shown that RAN and MMP-2 are on their own significantly associated with patient demise from metastatic BC. Moreover, when staining for MMP-2 is added to that for RAN in the primary tumours, there is a significant decrease in patient survival time over that for either protein alone. Thus a combination of staining for RAN and MMP2 is an excellent marker for poor prognosis in breast cancer.

The importin beta superfamily member RanBP17 exhibits a role in cell proliferation and is associated with improved survival of patients with HPV+ HNSCC.

BACKGROUND: More than twenty years after its discovery, the role of the importin beta superfamily member Ran GTP-binding protein (RanBP) 17 is still ill defined. Previously, we observed notable RanBP17 RNA expression levels in head and neck squamous cell carcinoma (HNSCC) cell lines with disruptive TP53 mutations. METHODS: We deployed HNSCC cell lines as well as cell lines from other tumor entities such as HCT116, MDA-MB-231 and H460, which were derived from colon, breast and lung cancers respectively. RNAi was used to evaluate the effect of RanBP17 on cell proliferation. FACS analysis was used for cell sorting according to their respective cell cycle phase and for BrdU assays. Immunocytochemistry was deployed for colocalization studies of RanBP17 with Nucleolin and SC35 (nuclear speckles) domains. TCGA analysis was performed for prognostic assessment and correlation analysis of RanBP17 in HNSCC patients. RESULTS: RNAi knockdown of RanBP17, significantly reduced cell proliferation in HNSCC cell lines. This effect was also seen in the HNSCC unrelated cell lines HCT116 and MDA-MB-231. Similarly, inhibiting cell proliferation with cisplatin reduced RanBP17 in keratinocytes but lead to induction in tumor cell lines. A similar observation was made in tumor cell lines after treatment with the EGFR kinase inhibitor AG1478. In addition to previous reports, showing colocalization of RanBP17 with SC35 domains, we observed colocalization of RanBP17 to nuclear bodies that are distinct from nucleoli and SC35 domains. Interestingly, for HPV positive but not HPV negative HNSCC, TCGA data base analysis revealed a strong positive correlation of RanBP17 RNA with patient survival and CDKN2A. CONCLUSIONS: Our data point to a role of RanBP17 in proliferation of HNSCC and other epithelial cells. Furthermore, RanBP17 could potentially serve as a novel prognostic marker for HNSCC patients. However, we noted a major discrepancy between RanBP17 RNA and protein expression levels with the used antibodies. These observations could be explained by the presence of additional RanBP17 splice isoforms and more so of non-coding circular RanBP17 RNA species. These aspects need to be addressed in more detail by future studies.

Mog1 deficiency promotes cardiac contractile dysfunction and isoproterenol-induced arrhythmias associated with cardiac fibrosis and Cx43 remodeling.

Our earlier studies identified MOG1 as a Nav1.5-binding protein that promotes Nav1.5 intracellular trafficking to plasma membranes. Genetic studies have identified MOG1 variants responsible for cardiac arrhythmias. However, the physiological functions of MOG1 in vivo remain incompletely characterized. In this study, we generated Mog1 knockout (Mog1-/-) mice. Mog1-/- mice did not develop spontaneous arrhythmias at the baseline, but exhibited a prolongation of QRS duration. Mog1-/- mice treated with isoproterenol (ISO), but not with flecainide, exhibited an increased risk of arrhythmias and even sudden death. Mog1-/- mice had normal cardiac morphology, however, LV systolic dysfunction was identified and associated with an increase in ventricular fibrosis. Whole-cell patch-clamping and Western blotting analysis clearly demonstrated the normal cardiac expression and function of Nav1.5 in Mog1-/- mice. Further RNA-seq and iTRAQ analysis identified critical pathways and genes, including extracellular matrix (Mmp2), gap junction (Gja1), and mitochondrial components that were dysregulated in Mog1-/- mice. RT-qPCR, Western blotting, and immunofluorescence assays revealed reduced cardiac expression of Gja1 in Mog1-/- mice. Dye transfer assays confirmed impairment of gap-junction function; Cx43 gap-junction enhancer ZP123 decreased arrhythmia inducibility in ISO-treated Mog1-/- mice. Transmission electron microscopy analysis revealed abnormal sarcomere ultrastructure and altered mitochondrial morphology in Mog1-/- mice. Mitochondrial dynamics was found to be disturbed, and associated with a trend toward increased mitochondrial fusion in Mog1-/- mice. Meanwhile, the level of ATP supply was increased in the hearts of Mog1-/- mice. These results indicate that MOG1 plays an important role in cardiac electrophysiology and cardiac contractile function.

Mechanistic convergence across initiation sites for RAN translation in fragile X associated tremor ataxia syndrome.

Repeat associated non-AUG (RAN) translation of CGG repeats in the 5'UTR of FMR1 produces toxic proteins that contribute to fragile X-associated tremor/ataxia syndrome (FXTAS) pathogenesis. The most abundant RAN product, FMRpolyG, initiates predominantly at an ACG upstream of the repeat. Accurate FMRpolyG measurements in FXTAS patients are lacking. We used data-dependent acquisition and parallel reaction monitoring (PRM) mass spectrometry coupled with stable isotope labeled standard peptides to identify signature FMRpolyG fragments in patient samples. Following immunoprecipitation, PRM detected FMRpolyG signature peptides in transfected cells, and FXTAS tissues and cells, but not in controls. We identified two amino-terminal peptides: an ACG-initiated Ac-MEAPLPGGVR and a GUG-initiated Ac-TEAPLPGGVR, as well as evidence for RAN translation initiation within the CGG repeat itself in two reading frames. Initiation at all sites increased following cellular stress, decreased following eIF1 overexpression and was eIF4A and M7G cap-dependent. These data demonstrate that FMRpolyG is quantifiable in human samples and FMR1 RAN translation initiates via similar mechanisms for near-cognate codons and within the repeat through processes dependent on available initiation factors and cellular environment.

Ran GTPase is an independent prognostic marker in malignant melanoma which promotes tumour cell migration and invasion.

AIMS: Ran GTPase is involved in nucleocytoplasmic shuttling of proteins and is overexpressed in several cancers. The expression of Ran in malignant melanoma (MM) and its functional activity have not been described and were investigated in this study. METHODS: The prognostic value of Ran expression was tested in a series of 185 primary cutaneous MM cases using immunohistochemistry. The functional activity of Ran was investigated in the two melanoma cell lines. Ran expression was knocked down using two siRNAs and the effect on the expression of the c-Met oncogene, a potential downstream target of Ran, was tested. Functional effects of Ran knockdown on cell motility and cell proliferation were also assessed. RESULTS: Positive Ran expression was seen in 12.4% of MM and was associated with advanced clinical stage and greater Breslow thickness. Positive expression was an independent marker of shorter overall survival (p=0.023). Knockdown of Ran results in decreased expression of c-Met and the downstream c-met signalling targets ERK1/2. There was a significant reduction in cell migration (p<0.001) and cell invasion (p<0.001). c-Met knockdown decreased the expression of Ran through MAPK and PI3K-AKT in A375 cell line, inhibited the cell viability and migration of both A375 and G361 melanoma cell lines while invasion was enhanced. CONCLUSION: Ran is a poor prognostic marker in cutaneous MM. It upregulates expression of the oncogene c-Met and, possibly through this, it promotes cell motility which may in turn promote metastasis.

RAN and YBX1 are required for cell proliferation and IL-4 expression and linked to poor prognosis in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is one of the most common malignancies in the world, with a high mortality rate. RAN is a member of the Ras GTPase family and is overexpressed in a range of cancers, however, the relationship between RAN and OSCC is rarely reported. In this study, we found that RAN is overexpressed in OSCC tissues. RAN inhibition retarded OSCC cell proliferation and led to apoptosis and cell cycle arrest. Knockdown of RAN inhibited tumor growth in vivo. Strikingly, we found that RAN and oncogene Y-box binding protein-1 (YBX1) are positively associated with the immune infiltrates of CD4+ Th2 cells in multiple types of cancer, and can promote IL-4 expression. IL-4 treatment can partially rescue RAN knockdown-induced cell apoptosis in OSCC cells. Moreover, overexpression of RAN could rescue cell growth inhibition caused by knockdown of YBX1. Furthermore, patients with low expression of both RAN and YBX1 had better overall survival than others. Collectively, these findings indicate that RAN is a target of YBX1. RAN and YBX1 are required for cell proliferation and IL-4 expression. RAN and YBX1 are co-expressed and can serve as potential co-biomarkers for poor prognosis in OSCC.

Overexpression of a small GTP-binding protein Ran1 in Arabidopsis leads to promoted elongation growth and enhanced disease resistance against P. syringae DC3000.

Plants resist infection through an innate immune response, which is usually associated with slowing of growth. The molecular mechanisms underlying the trade-off between plant growth and defense remain unclear. The present study reveals that growth/defense trade-offs mediated by gibberellin (GA) and salicylic acid (SA) signaling pathways are uncoupled during constitutive overexpression of transgenic AtRAN1 and AtRAN1Q72L (active, GTP-locked form) Arabidopsis plants. It is well known that the small GTP-binding protein Ran (a Ras-related nuclear protein) functions in the nucleus-cytoplasmic transport of proteins. Although there is considerable evidence indicating that nuclear-cytoplasmic partitioning of specific proteins can participate in hormone signaling, the role of Ran-dependent nuclear transport in hormone signaling is not yet fully understood. In this report, we used a combination of genetic and molecular methods to reveal whether AtRAN1 is involved in both GA and SA signaling pathways. Constitutively overexpressed AtRAN1 promoted both elongation growth and the disease resistance response, whereas overexpression of AtRAN1Q72L in the atran2atran3 double mutant background clearly inhibited elongation growth and the defense response. Furthermore, we found that AtRAN1 coordinated plant growth and defense by promoting the stability of the DELLA protein RGA in the nucleus and by modulating NPR1 nuclear localization. Interestingly, genetically modified rice (Oryza sativa) overexpressing AtRAN1 exhibited increased plant height and yield per plant. Altogether, the ability to achieve growth/defense trade-offs through AtRAN1 overexpression provides an approach to maximizing crop yield to meet rising global food demands.

TNPO2 variants associate with human developmental delays, neurologic deficits, and dysmorphic features and alter TNPO2 activity in Drosophila.

Transportin-2 (TNPO2) mediates multiple pathways including non-classical nucleocytoplasmic shuttling of >60 cargoes, such as developmental and neuronal proteins. We identified 15 individuals carrying de novo coding variants in TNPO2 who presented with global developmental delay (GDD), dysmorphic features, ophthalmologic abnormalities, and neurological features. To assess the nature of these variants, functional studies were performed in Drosophila. We found that fly dTnpo (orthologous to TNPO2) is expressed in a subset of neurons. dTnpo is critical for neuronal maintenance and function as downregulating dTnpo in mature neurons using RNAi disrupts neuronal activity and survival. Altering the activity and expression of dTnpo using mutant alleles or RNAi causes developmental defects, including eye and wing deformities and lethality. These effects are dosage dependent as more severe phenotypes are associated with stronger dTnpo loss. Interestingly, similar phenotypes are observed with dTnpo upregulation and ectopic expression of TNPO2, showing that loss and gain of Transportin activity causes developmental defects. Further, proband-associated variants can cause more or less severe developmental abnormalities compared to wild-type TNPO2 when ectopically expressed. The impact of the variants tested seems to correlate with their position within the protein. Specifically, those that fall within the RAN binding domain cause more severe toxicity and those in the acidic loop are less toxic. Variants within the cargo binding domain show tissue-dependent effects. In summary, dTnpo is an essential gene in flies during development and in neurons. Further, proband-associated de novo variants within TNPO2 disrupt the function of the encoded protein. Hence, TNPO2 variants are causative for neurodevelopmental abnormalities.

Nuclear lipid droplets and nuclear damage in Caenorhabditis elegans.

Fat stored in the form of lipid droplets has long been considered a defining characteristic of cytoplasm. However, recent studies have shown that nuclear lipid droplets occur in multiple cells and tissues, including in human patients with fatty liver disease. The function(s) of stored fat in the nucleus has not been determined, and it is possible that nuclear fat is beneficial in some situations. Conversely, nuclear lipid droplets might instead be deleterious by disrupting nuclear organization or triggering aggregation of hydrophobic proteins. We show here that nuclear lipid droplets occur normally in C. elegans intestinal cells and germ cells, but appear to be associated with damage only in the intestine. Lipid droplets in intestinal nuclei can be associated with novel bundles of microfilaments (nuclear actin) and membrane tubules that might have roles in damage repair. To increase the normal, low frequency of nuclear lipid droplets in wild-type animals, we used a forward genetic screen to isolate mutants with abnormally large or abundant nuclear lipid droplets. Genetic analysis and cloning of three such mutants showed that the genes encode the lipid regulator SEIP-1/seipin, the inner nuclear membrane protein NEMP-1/Nemp1/TMEM194A, and a component of COPI vesicles called COPA-1/α-COP. We present several lines of evidence that the nuclear lipid droplet phenotype of copa-1 mutants results from a defect in retrieving mislocalized membrane proteins that normally reside in the endoplasmic reticulum. The seip-1 mutant causes most germ cells to have nuclear lipid droplets, the largest of which occupy more than a third of the nuclear volume. Nevertheless, the nuclear lipid droplets do not trigger apoptosis, and the germ cells differentiate into gametes that produce viable, healthy progeny. Thus, our results suggest that nuclear lipid droplets are detrimental to intestinal nuclei, but have no obvious deleterious effect on germ nuclei.