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.

Nuclear pore dysfunction and disease: a complex opportunity.

The separation of genetic material from bulk cytoplasm has enabled the evolution of increasingly complex organisms, allowing for the development of sophisticated forms of life. However, this complexity has created new categories of dysfunction, including those related to the movement of material between cellular compartments. In eukaryotic cells, nucleocytoplasmic trafficking is a fundamental biological process, and cumulative disruptions to nuclear integrity and nucleocytoplasmic transport are detrimental to cell survival. This is particularly true in post-mitotic neurons, where nuclear pore injury and errors to nucleocytoplasmic trafficking are strongly associated with neurodegenerative disease. In this review, we summarize the current understanding of nuclear pore biology in physiological and pathological contexts and discuss potential therapeutic approaches for addressing nuclear pore injury and dysfunctional nucleocytoplasmic transport.

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.

Analysis of the effects of importin α1 on the nuclear translocation of IL-1α in HeLa cells.

Interleukin-1α (IL-1α), a cytokine released by necrotic cells, causes sterile inflammation. On the other hand, IL-1α is present in the nucleus and also regulates the expression of many proteins. A protein substrate containing a classical nuclear localization signal (cNLS) typically forms a substrate/importin α/ß complex, which is subsequently transported to the nucleus. To the best of our knowledge, no study has directly investigated whether IL-1α-which includes cNLS-is imported into the nucleus in an importin α/ß-dependent manner. In this study, we noted that all detected importin α subtypes interacted with IL-1α. In HeLa cells, importin α1-mediated nuclear translocation of IL-1α occurred at steady state and was independent of importin ß1. Importin α1 not only was engaged in IL-1α nuclear transport but also concurrently functioned as a molecule that regulated IL-1α protein level in the cell. Furthermore, we discussed the underlying mechanism of IL-1α nuclear translocation by importin α1 based on our findings.

Therapeutic targeting of nuclear export and import receptors in cancer and their potential in combination chemotherapy.

Systemic modalities are crucial in the management of disseminated malignancies and liquid tumours. However, patient responses and tolerability to treatment are generally poor and those that enter remission often return with refractory disease. Combination therapies provide a methodology to overcome chemoresistance mechanisms and address dose-limiting toxicities. A deeper understanding of tumorigenic processes at the molecular level has brought a targeted therapy approach to the forefront of cancer research, and novel cancer biomarkers are being identified at a rapid rate, with some showing potential therapeutic benefits. The Karyopherin superfamily of proteins is soluble receptors that mediate nucleocytoplasmic shuttling of proteins and RNAs, and recently, nuclear transport receptors have been recognized as novel anticancer targets. Inhibitors against nuclear export have been approved for clinical use against certain cancer types, whereas inhibitors against nuclear import are in preclinical stages of investigation. Mechanistically, targeting nucleocytoplasmic shuttling has shown to abrogate oncogenic signalling and restore tumour suppressor functions through nuclear sequestration of relevant proteins and mRNAs. Hence, nuclear transport inhibitors display broad spectrum anticancer activity and harbour potential to engage in synergistic interactions with a wide array of cytotoxic agents and other targeted agents. This review is focussed on the most researched nuclear transport receptors in the context of cancer, XPO1 and KPNB1, and highlights how inhibitors targeting these receptors can enhance the therapeutic efficacy of standard of care therapies and novel targeted agents in a combination therapy approach. Furthermore, an updated review on the therapeutic targeting of lesser characterized karyopherin proteins is provided and resistance to clinically approved nuclear export inhibitors is discussed.

Nuclear envelope budding: Getting large macromolecular complexes out of the nucleus.

Transport of macromolecules from the nucleus to the cytoplasm is essential for nearly all cellular and developmental events, and when mis-regulated, is associated with diseases, tumor formation/growth, and cancer progression. Nuclear Envelope (NE)-budding is a newly appreciated nuclear export pathway for large macromolecular machineries, including those assembled to allow co-regulation of functionally related components, that bypasses canonical nuclear export through nuclear pores. In this pathway, large macromolecular complexes are enveloped by the inner nuclear membrane, transverse the perinuclear space, and then exit through the outer nuclear membrane to release its contents into the cytoplasm. NE-budding is a conserved process and shares many features with nuclear egress mechanisms used by herpesviruses. Despite its biological importance and clinical relevance, little is yet known about the regulatory and structural machineries that allow NE-budding to occur in any system. Here we summarize what is currently known or proposed for this intriguing nuclear export process.

A functional and structural comparative analysis of large tumor antigens reveals evolution of different importin α-dependent nuclear localization signals.

Nucleocytoplasmic transport regulates the passage of proteins between the nucleus and cytoplasm. In the best characterized pathway, importin (IMP) α bridges cargoes bearing basic, classical nuclear localization signals (cNLSs) to IMPß1, which mediates transport through the nuclear pore complex. IMPα recognizes three types of cNLSs via two binding sites: the major binding site accommodates monopartite cNLSs, the minor binding site recognizes atypical cNLSs, while bipartite cNLSs simultaneously interact with both major and minor sites. Despite the growing knowledge regarding IMPα-cNLS interactions, our understanding of the evolution of cNLSs is limited. We combined bioinformatic, biochemical, functional, and structural approaches to study this phenomenon, using polyomaviruses (PyVs) large tumor antigens (LTAs) as a model. We characterized functional cNLSs from all human (H)PyV LTAs, located between the LXCXE motif and origin binding domain. Surprisingly, the prototypical SV40 monopartite NLS is not well conserved; HPyV LTA NLSs are extremely heterogenous in terms of structural organization, IMPα isoform binding, and nuclear targeting abilities, thus influencing the nuclear accumulation properties of full-length proteins. While several LTAs possess bipartite cNLSs, merkel cell PyV contains a hybrid bipartite cNLS whose upstream stretch of basic amino acids can function as an atypical cNLS, specifically binding to the IMPα minor site upon deletion of the downstream amino acids after viral integration in the host genome. Therefore, duplication of a monopartite cNLS and subsequent accumulation of point mutations, optimizing interaction with distinct IMPα binding sites, led to the evolution of bipartite and atypical NLSs binding at the minor site.

Evolutionary, structural and functional insights in nuclear organisation and nucleocytoplasmic transport in trypanosomes.

One of the remarkable features of eukaryotes is the nucleus, delimited by the nuclear envelope (NE), a complex structure and home to the nuclear lamina and nuclear pore complex (NPC). For decades, these structures were believed to be mainly architectural elements and, in the case of the NPC, simply facilitating nucleocytoplasmic trafficking. More recently, the critical roles of the lamina, NPC and other NE constituents in genome organisation, maintaining chromosomal domains and regulating gene expression have been recognised. Importantly, mutations in genes encoding lamina and NPC components lead to pathogenesis in humans, while pathogenic protozoa disrupt the progression of normal development and expression of pathogenesis-related genes. Here, we review features of the lamina and NPC across eukaryotes and discuss how these elements are structured in trypanosomes, protozoa of high medical and veterinary importance, highlighting lineage-specific and conserved aspects of nuclear organisation.

[Structural and mechanical plasticity of the nuclear pore]. / Plasticité structurelle et mécanique du pore nucléaire.

The nuclear pore, which can be seen as the gateway to the cell nucleus, is central to many processes including gene regulation. It is a complex and dynamic structure composed of more than 30 proteins present in multiple copies that allows the selective and directional transport of RNA and proteins. As shown by recent studies, it is able to adapt its overall structure to the state of the cell. These results suggest that the structural and mechanical plasticity of the nuclear pore is important for its function but also in the development of cancer or viral infections.

Title: Plasticité structurelle et mécanique du pore nucléaire. Abstract: Le pore nucléaire, qui peut être vu comme la porte (d'entrée et de sortie) du noyau cellulaire, joue un rôle central dans de nombreux processus, dont la régulation génique. C'est une structure complexe et dynamique. Il est composé de plus de trente protéines présentes en de multiples copies. C'est sur lui que repose le transport sélectif et orienté des ARN et des protéines. Des études récentes montrent qu'il est susceptible d'adapter sa structure globale à l'état de la cellule. La plasticité structurelle et mécanique du pore nucléaire apparaît ainsi importante pour son fonctionnement, mais aussi dans le développement de maladies comme le cancer ou les infections virales.

Changing the guard-nuclear pore complex quality control.

The integrity of the nuclear envelope depends on the function of nuclear pore complexes (NPCs), transport channels that control macromolecular traffic between the nucleus and cytosol. The central importance of NPCs suggests the existence of quality control (QC) mechanisms that oversee their assembly and function. In this perspective, we emphasize the challenges associated with NPC assembly and the need for QC mechanisms that operate at various stages of an NPC's life. This includes cytosolic preassembly QC that helps enforce key nucleoporin-nucleoporin interactions and their ultimate stoichiometry in the NPC in addition to mechanisms that monitor aberrant fusion of the inner and outer nuclear membranes. Furthermore, we discuss whether and how these QC mechanisms may operate to sense faulty mature NPCs to facilitate their repair or removal. The so far uncovered mechanisms for NPC QC provide fertile ground for future research that not only benefits a better understanding of the vital role that NPCs play in cellular physiology but also how loss of NPC function and/or these QC mechanisms might be an input to aging and disease.

Nuclear pore complex and nucleocytoplasmic transport disruption in neurodegeneration.

Nuclear pore complexes (NPCs) play a critical role in maintaining the equilibrium between the nucleus and cytoplasm, enabling bidirectional transport across the nuclear envelope, and are essential for proper nuclear organization and gene regulation. Perturbations in the regulatory mechanisms governing NPCs and nuclear envelope homeostasis have been implicated in the pathogenesis of several neurodegenerative diseases. The ESCRT-III pathway emerges as a critical player in the surveillance and preservation of well-assembled, functional NPCs, as well as nuclear envelope sealing. Recent studies have provided insights into the involvement of nuclear ESCRT-III in the selective reduction of specific nucleoporins associated with neurodegenerative pathologies. Thus, maintaining quality control of the nuclear envelope and NPCs represents a pivotal element in the pathological cascade leading to neurodegenerative diseases. This review describes the constituents of the nuclear-cytoplasmic transport machinery, encompassing the nuclear envelope, NPC, and ESCRT proteins, and how their structural and functional alterations contribute to the development of neurodegenerative diseases.

Targeting Proteins in Nucleus through Dual-Regulatory Pathways Acting in Cytoplasm.

Nuclear proteins have been regarded as attractive targets for exploiting therapeutic agents. However, those agents cannot efficiently pass through nuclear pores and it is also difficult to overcome the crowded nuclear environment to react with proteins. Herein, we propose a novel strategy acting in the cytoplasm to regulate nuclear proteins based on their signaling pathways, instead of directly entering into nuclei. A multifunctional complex PKK-TTP/hs carries human telomerase reverse transcriptase (hTERT) small interfering RNA (defined as hs) for gene silencing in the cytoplasm, which reduced the import of nuclear protein. At the same time, it could generate reactive oxygen species (ROS) under light irradiation, which raised the export of nuclear proteins by promoting proteins translocation. Through this dual-regulatory pathway, we successfully reduced nuclear protein (hTERT proteins) in vivo (42.3%). This work bypasses the challenge of directly entering into the nucleus and provides an effective strategy for regulating nuclear proteins.

Protein transport from pre- and postsynapse to the nucleus: Mechanisms and functional implications.

The extreme length of neuronal processes poses a challenge for synapse-to-nucleus communication. In response to this challenge several different mechanisms have evolved in neurons to couple synaptic activity to the regulation of gene expression. One of these mechanisms concerns the long-distance transport of proteins from pre- and postsynaptic sites to the nucleus. In this review we summarize current evidence on mechanisms of transport and consequences of nuclear import of these proteins for gene transcription. In addition, we discuss how information from pre- and postsynaptic sites might be relayed to the nucleus by this type of long-distance signaling. When applicable, we highlight how long-distance protein transport from synapse-to-nucleus can provide insight into the pathophysiology of disease or reveal new opportunities for therapeutic intervention.

Self-regulation of the nuclear pore complex enables clogging-free crowded transport.

Nuclear pore complexes (NPCs) are the main conduits for macromolecular transport into and out of the nucleus of eukaryotic cells. The central component of the NPC transport mechanism is an assembly of intrinsically disordered proteins (IDPs) that fills the NPC channel. The channel interior is further crowded by large numbers of simultaneously translocating cargo-carrying and free transport proteins. How the NPC can efficiently, rapidly, and selectively transport varied cargoes in such crowded conditions remains ill understood. Past experimental results suggest that the NPC is surprisingly resistant to clogging and that transport may even become faster and more efficient as the concentration of transport protein increases. To understand the mechanisms behind these puzzling observations, we construct a computational model of the NPC comprising only a minimal set of commonly accepted consensus features. This model qualitatively reproduces the previous experimental results and identifies self-regulating mechanisms that relieve crowding. We show that some of the crowding-alleviating mechanisms-such as preventing saturation of the bulk flux-are "robust" and rely on very general properties of crowded dynamics in confined channels, pertaining to a broad class of selective transport nanopores. By contrast, the counterintuitive ability of the NPC to leverage crowding to achieve more efficient single-molecule translocation is "fine-tuned" and relies on the particular spatial architecture of the IDP assembly in the NPC channel.

Nuclear envelope budding and its cellular functions.

The nuclear pore complex (NPC) has long been assumed to be the sole route across the nuclear envelope, and under normal homeostatic conditions it is indeed the main mechanism of nucleo-cytoplasmic transport. However, it has also been known that e.g. herpesviruses cross the nuclear envelope utilizing a pathway entitled nuclear egress or envelopment/de-envelopment. Despite this, a thread of observations suggests that mechanisms similar to viral egress may be transiently used also in healthy cells. It has since been proposed that mechanisms like nuclear envelope budding (NEB) can facilitate the transport of RNA granules, aggregated proteins, inner nuclear membrane proteins, and mis-assembled NPCs. Herein, we will summarize the known roles of NEB as a physiological and intrinsic cellular feature and highlight the many unanswered questions surrounding these intriguing nuclear events.

Nuclear import of IRF11 via the importin α/ß pathway is essential for its antiviral activity.

Interferon regulatory factor 11 (IRF11), an intriguing IRF member found only in fish species, has recently been shown to have antiviral properties that are dependent on its nuclear entry and DNA binding affinity. However, the mechanisms by which IRF11 enters the nucleus are unknown. In the present study, we found orthologs of IRF11 in lamprey and lancelet species by combining positional, phylogenetic and structural comparison data, showing that this gene has an ancient origin. The IRF11 gene (AjIRF11) from the Japanese eel, Anguilla japonica, was subsequently characterized, and it was found that AjIRF11 has antiviral activities against spring viremia of carp virus (SVCV), which are accomplished by regulating the production of type I IFN and IFN-stimulated genes. In addition to its known DNA binding residues in the α3 helix, two residues in Loop 1, His40 and Trp46, are also involved in DNA binding and activation of the IFN promoter. Using immunofluorescence microscopy and site-directed mutagenesis analysis, we confirmed that full nuclear localization of AjIRF11 requires the bipartite nuclear localization sequence (NLS) spanning residues 75 to 101, as well as the monopartite NLS situated between residues 119 and 122. Coimmunoprecipitation assays confirmed that AjIRF11 interacts with importin α via its NLSs and can also bind to importin ß directly, implying that IRF11 can be imported to the nucleus by one or more transport receptors.

Analysis of Tau/Nucleoporin Interactions by Surface Plasmon Resonance Spectroscopy.

Tau, a soluble and predominantly neuronal protein, is best known for its microtubule (MT)-binding function in the cytosol, where it decisively contributes to stability as well as modulation of MT dynamics. In Alzheimer's disease and other tauopathies, Tau is altered into forming intracellular neurofibrillary tangles; additionally, also a mislocalization from the cytosol to the nucleus has been observed where interactions of Tau with the nucleus become possible. Using surface plasmon resonance (SPR), it was recently shown that Tau can directly interact with certain nucleoporins (e.g., Nup98), components of the nuclear pore complex (NPC). The NPC constitutes large regulated pores in the nuclear envelope that facilitate the bidirectional exchange of proteins, nucleic acids, and other biomolecules between the inner section of the nucleus and the cytosol, the nucleocytoplasmic transport. The mechanism of Tau/Nup interactions is as yet unknown, and a systematic interaction analysis of Tau with different Nups can be of high value to decipher the molecular binding mechanism of Tau to Nups. SPR is a useful tool to analyze binding affinities and kinetic parameters in a label-free environment. While one interaction partner is immobilized on a sensor chip, the second is supplied within a constant flow of buffer. Binding of mobile molecules to immobilized ones changes the refractive index of the medium close to the sensor surface with the signal being proportional to the bound mass. In this chapter, we describe the application of the SPR technique for the investigation of Tau binding to nucleoporins.

Tau-FG-nucleoporin98 interaction and impaired nucleocytoplasmic transport in Alzheimer's disease.

An emerging pathophysiology associated with the neurodegenerative Alzheimer's disease (AD) is the impairment of nucleocytoplasmic transport (NCT). The impairment can originate from damage to the nuclear pore complex (NPC) or other factors involved in NCT. The phenylalanine-glycine nucleoporins (FG-Nups) form a crucial component of the NPC, which is central to NCT. Recent discoveries have highlighted that the neuropathological protein tau is involved in direct interactions with the FG-Nups and impairment of the NCT process. Targeting such interactions may lead to the identification of novel interaction inhibitors and offer new therapeutic alternatives for the treatment of AD. This review highlights recent findings associated with impaired NCT in AD and the interaction between tau and the FG-Nups.

The Role of Nucleoporins in Cardiac Tissue Development and Disease.

Nuclear pore complexes (NPCs) are intricate intracellular structures composed of approximately 30 nuclear pore proteins (NUPs) that regulate the transport of materials between the nucleus and cytoplasm in eukaryotic cells. The heart is a crucial organ for sustaining the vital functions of the body, pumping blood rich in nutrients and energy to all organs and tissues. Recent studies have shown that NPCs play pivotal roles not only in normal cardiac physiological processes such as myocardial cell proliferation and differentiation but also in various pathological processes such as ischemic and hypoxic myocardial injury. Due to their mass and complicated nature, the structures of NPCs have been challenging to identify by the scientific community. With the development of cryo-electron microscopy and advanced sampling techniques, researchers have made significant progress in understanding the structures of NPCs. This review aims to summarize the latest research on the structural aspects of NPCs and their roles in cardiac physiology and pathology, increase the understanding of the intricate mechanisms of NPC actions, provide valuable insights into the pathogenesis of heart diseases and describe the development of potential novel therapeutic strategies.

The chaperone DNAJB6 surveils FG-nucleoporins and is required for interphase nuclear pore complex biogenesis.

Biogenesis of nuclear pore complexes (NPCs) includes the formation of the permeability barrier composed of phenylalanine-glycine-rich nucleoporins (FG-Nups) that regulate the selective passage of biomolecules across the nuclear envelope. The FG-Nups are intrinsically disordered and prone to liquid-liquid phase separation and aggregation when isolated. How FG-Nups are protected from making inappropriate interactions during NPC biogenesis is not fully understood. Here we find that DNAJB6, a molecular chaperone of the heat shock protein network, forms foci in close proximity to NPCs. The number of these foci decreases upon removal of proteins involved in the early steps of interphase NPC biogenesis. Conversely, when this process is stalled in the last steps, the number of DNAJB6-containing foci increases and these foci are identified as herniations at the nuclear envelope. Immunoelectron tomography shows that DNAJB6 localizes inside the lumen of the herniations arising at NPC biogenesis intermediates. Loss of DNAJB6 results in the accumulation of cytosolic annulate lamellae, which are structures containing partly assembled NPCs, a feature associated with disturbances in NPC biogenesis. We find that DNAJB6 binds to FG-Nups and can prevent the aggregation of the FG region of several FG-Nups in cells and in vitro. Together, our data show that the molecular chaperone DNAJB6 provides quality control during NPC biogenesis and is involved in the surveillance of native intrinsically disordered FG-Nups.