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Importance: HIV-infected host cells impose varied degrees of regulation on viral replication, from very high to abortive. Proliferation of HIV in astrocytes is limited when compared to immune cells, such as CD4+ T lymphocytes. Understanding such differential regulation is one of the key questions in the field as these cells permit HIV persistence and rebound viremia, challenging HIV treatment and clinical cure. This study focuses on understanding the molecular mechanism behind such cell-specific disparities. We show that one of the key mechanisms is the regulation of heterogenous nuclear ribonucleoprotein A2, a host factor involved in alternative splicing and RNA processing, by HIV-1 Tat in CD4+ T lymphocytes, not observed in astrocytes. This regulation causes an increase in the levels of unspliced/partially spliced viral RNA and nuclear export of Rev-RNA complexes which results in high viral propagation in CD4+ T lymphocytes. The study reveals a new mechanism imposed by HIV on host cells that determines the fate of infection., Competing Interests: The authors declare no conflict of interest.

Importance: 3'UTRs can affect gene transcription and post-transcriptional regulation in multiple ways, further influencing the function of proteins in a unique manner. Recently, ALV-J has been mutating and evolving rapidly, especially the 3'UTR of viral genome. Meanwhile, clinical symptoms caused by ALV-J have changed significantly. In this study, we found that the ALV-J strains containing △-r-TM-type 3'UTR are the most abundant. By constructing ALV-J infectious clones and subgenomic vectors containing different 3'UTRs, we prove that 3'UTRs directly affect viral tissue preference and can promote virus replication as an enhancer. ALV-J strain containing 3'UTR of △-r-TM proliferated fastest in primary cells. All five forms of 3'UTRs can assist intron-containing viral mRNA nuclear export, with similar efficiency. ALV-J mRNA half-life is not influenced by different 3'UTRs. Our results dissect the roles of 3'UTR on regulating viral replication and pathogenicity, providing novel insights into potential anti-viral strategies., Competing Interests: The authors declare no conflict of interest.

Trypanosoma cruzi is a parasitic protozoa causative of Chagas disease. As part of our interest in studying the basic biology of this microorganism, this work reports our observations related to the characterization of motifs and structural domains present in two fibrillarin isoforms (TcFib1 and TcFib2) that were found to be necessary for the nuclear targeting of these nucleolar proteins. Previous characterization of these proteins indicated that they share 68.67% of identical amino acids and are both expressed as nucleolar proteins in T. cruzi epimastigotes. Using an approach based on the transfection of recombinant genes encoding fluorescent fibrillarin-EGFP fusion proteins, this study found evidence for the presence of 4 motifs or protein domains that help target these proteins to the nucleus: The GAR domain and carboxyl terminus in both TcFibs, as well as two lysines and a computationally predicted cNLS in TcFib1. As a distinctive feature, the GAR domain of TcFib2 proved to be essential for the nuclear localization of this protein paralog. Such a difference between TcFib1 and Tcfib2 nuclear localization signals can be explained as the presence of two partially related nuclear import pathways for the two fibrillarin homologues in this organism., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest associated with this publication, and there has been no significant financial support for this work that could have influenced its outcome., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The cyclin-dependent kinase 1 (Cdk1)-cyclin B (CycB) complex plays critical roles in cell-cycle regulation. Before Drosophila male meiosis, CycB is exported from the nucleus to the cytoplasm via the nuclear porin 62kD (Nup62) subcomplex of the nuclear pore complex. When this export is inhibited, Cdk1 is not activated, and meiosis does not initiate. We investigated the mechanism that controls the cellular localization and activation of Cdk1. Cdk1-CycB continuously shuttled into and out of the nucleus before meiosis. Overexpression of CycB, but not that of CycB with nuclear localization signal sequences, rescued reduced cytoplasmic CycB and inhibition of meiosis in Nup62 -silenced cells. Full-scale Cdk1 activation occurred in the nucleus shortly after its rapid nuclear entry. Cdk1-dependent centrosome separation did not occur in Nup62- silenced cells, whereas Cdk1 interacted with Cdk-activating kinase and Twine/Cdc25C in the nuclei of Nup62- silenced cells, suggesting the involvement of another suppression mechanism. Silencing of roughex rescued Cdk1 inhibition and initiated meiosis. Nuclear export of Cdk1 ensured its escape from inhibition by a cyclin-dependent kinase inhibitor. The complex re-entered the nucleus via importin β at the onset of meiosis. We propose a model regarding the dynamics and activation mechanism of Cdk1-CycB to initiate male meiosis.

The human aryl hydrocarbon receptor (AHR) undergoes continuous shuttling between nucleus and cytoplasm. Binding to exogenous or endogenous ligands promotes its rapid nuclear import. The proposed mechanism for the ligand-dependent import is based on exposing the bipartite nuclear localisation signal (NLS) to members of the importin (IMP) superfamily. Among this, the molecular interactions involved in the basal import still need to be clarified. Utilizing fluorescently fused AHR variants, we recapitulated and characterized AHR localization and nucleo-cytoplasmic shuttling in living cells. Analysis of AHR variants carrying NLS point mutations demonstrated a mandatory role of first ( 13 RKRRK 17 ) and second ( 37 KR-R 40 ) NLS segments on the basal import of AHR. Further experiments indicated that ligand-induced import is mainly regulated through the first NLS, while the second NLS is supportive but not essential. Additionally, applying IMPα/β specific inhibitors, ivermectin (IVM) and importazole (IPZ), slowed down the ligand-induced import and, correspondingly, decreased the basal nuclear accumulation of the receptor. In conclusion, our data show that ligand-induced and basal nuclear entry of AHR rely on the same mechanism but are controlled uniquely by the two NLS components., (© 2023. The Author(s).)

In eukaryotic cells, the spatial distribution between cytoplasm and nucleus is essential for cell homeostasis. This dynamic distribution is selectively regulated by the nuclear pore complex (NPC), which allows the passive or energy-dependent transport of proteins between these two compartments. Viruses possess many strategies to hijack nucleocytoplasmic shuttling for the benefit of their viral replication. Here, we review how viruses interfere with the karyopherin CRM1 that controls the nuclear export of protein cargoes. We analyze the fact that the viral hijacking of CRM1 provokes are-localization of numerous cellular factors in a suitable place for specific steps of viral replication. While CRM1 emerges as a critical partner for viruses, it also takes part in antiviral and inflammatory response regulation. This review also addresses how CRM1 hijacking affects it and the benefits of CRM1 inhibitors as antiviral treatments.

Nuclear pore complexes are large multicomponent protein complexes that are embedded in the nuclear envelope, where they mediate nucleocytoplasmic transport. In addition to supporting transport, nuclear pore components, termed nucleoporins (Nups), can interact with chromatin and influence genome function. A subset of Nups can also localize to the nuclear interior and bind chromatin intranuclearly, providing an opportunity to investigate chromatin-associated functions of Nups outside of the transport context. This review focuses on the gene regulatory functions of such intranuclear Nups, with a particular emphasis on their identity as components of several chromatin regulatory complexes. Recent proteomic screens have identified Nups as interacting partners of active and repressive epigenetic machinery, architectural proteins, and DNA replication complexes, providing insight into molecular mechanisms via which Nups regulate gene expression programs. This review summarizes these interactions and discusses their potential functions in the broader framework of nuclear genome organization., (© 2023 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The Drosophila melanogaster embryo, an elongated multi-nucleated cell, is a classical model system for eukaryotic development and morphogenesis. Recent work has shown that bulk cytoplasmic flows, driven by cortical contractions along the walls of the embryo, enable the uniform spreading of nuclei along the anterior-posterior axis necessary for proper embryonic development. Here, we propose two mathematical models to characterize cytoplasmic flows driven by tangential cortical contractions in elongated cells. Assuming Newtonian fluid flow at low Reynolds number in a spheroidal cell, we first compute the flow field exactly, thereby bypassing the need for numerical computations. We then apply our results to recent experiments on nuclear transport in cell cycles 4-6 of Drosophila embryo development. By fitting the cortical contractions in our model to measurements, we reveal that experimental cortical flows enable near-optimal axial spreading of nuclei. A second mathematical approach, applicable to general elongated cell geometries, exploits a long-wavelength approximation to produce an even simpler solution, with errors below [Formula: see text] compared with the full model. An application of this long-wavelength result to transport leads to fully analytical solutions for the nuclear concentration that capture the essential physics of the system, including optimal axial spreading of nuclei.

Resistance to anti-androgen therapy for metastatic prostate cancer is a major clinical problem. Sema3C promotes resistance to androgen withdrawal via its receptor, PlexinB1. Activation of PlexinB1 promotes the ligand-independent nuclear translocation of the androgen receptor (AR), which may contribute to resistance to androgen deprivation therapy. However, the mechanism by which PlexinB1 promotes nuclear translocation is unclear. We show here that PlexinB1 and B2 regulate nuclear import by acting as GTPase activating proteins (GAPs) for the small RasGTPase Ran, a key regulator of nuclear trafficking. Purified PlexinB1/B2 protein catalyses the hydrolysis of RanGTP, and mutations in the GAP domain of PlexinB1 inhibit this activity. Activation of PlexinB1/B2 with Sema4D decreases the levels of RanGTP, while PlexinB1 or B2 depletion increases the levels of activated Ran in the cell. Ran directly associates with B-type plexins in a GTP-dependent manner. Sema4D is internalised by endocytosis, and PlexinB1 and Ran display overlapping patterns of expression. Furthermore, Sema4D/PlexinB1-induced AR nuclear translocation is dependent on the GAP domain of PlexinB1 and is blocked by the expression of non-functional Ran mutants. Depletion of PlexinB1 decreases the nuclear/cytoplasmic ratio of Ran, indicative of a higher RanGTP/GDP ratio. Plexins may promote the growth of androgen-independent prostate cancer through their activity as RanGAPs., (© 2023. The Author(s).)

Dual-specificity phosphatase 8 (DUSP8) is a MAPK phosphatase that dephosphorylates and inactivates the kinase JNK. DUSP8 is highly expressed in T cells; however, the in vivo role of DUSP8 in T cells remains unclear. Using T cell-specific Dusp8 conditional KO (T-Dusp8 cKO) mice, mass spectrometry analysis, ChIP-Seq, and immune analysis, we found that DUSP8 interacted with Pur-α, stimulated interleukin-9 (IL-9) gene expression, and promoted Th9 differentiation. Mechanistically, DUSP8 dephosphorylated the transcriptional repressor Pur-α upon TGF-β signaling, leading to the nuclear export of Pur-α and subsequent IL-9 transcriptional activation. Furthermore, Il-9 mRNA levels were induced in Pur-α-deficient T cells. In addition, T-Dusp8-cKO mice displayed reduction of IL-9 and Th9-mediated immune responses in the allergic asthma model. Reduction of Il-9 mRNA levels in T cells and allergic responses of T-Dusp8-cKO mice was reversed by Pur-α knockout. Remarkably, DUSP8 protein levels and the DUSP8-Pur-α interaction were indeed increased in the cytoplasm of T cells from people with asthma and patients with atopic dermatitis. Collectively, DUSP8 induces TGF-β-stimulated IL-9 transcription and Th9-induced allergic responses by inhibiting the nuclear translocation of the transcriptional repressor Pur-α. DUSP8 may be a T-cell biomarker and therapeutic target for asthma and atopic dermatitis.

Alpha-synuclein (SNCA) accumulation plays a central role in the pathogenesis of Parkinson's disease. Determining and interfering with the mechanisms that control SNCA expression is one approach to limiting disease progression. Currently, most of our understanding of SNCA regulation is protein-based. Post-transcriptional mechanisms directly regulating SNCA mRNA expression via its 3' untranslated region (3'UTR) were investigated here. Mass spectrometry of proteins pulled down from murine brain lysates using a biotinylated SNCA 3'UTR revealed multiple RNA-binding proteins, of which HNRNPD/AUF1 was chosen for further analysis. AUF1 bound both proximal and distal regions of the SNCA 3'UTR, but not the 5'UTR or CDS. In the nucleus, AUF1 attenuated SNCA pre-mRNA maturation and was indispensable for the export of SNCA transcripts. AUF1 destabilized SNCA transcripts in the cytosol, primarily those with shorter 3'UTRs, independently of microRNAs by recruiting the CNOT1-CNOT7 deadenylase complex to trim the polyA tail. Furthermore, AUF1 inhibited SNCA mRNA binding to ribosomes. These data identify AUF1 as a multi-tasking protein regulating maturation, nucleocytoplasmic shuttling, stability and translation of SNCA transcripts.

The subcellular compartmentalisation of eukaryotic cells requires selective exchange between the cytoplasm and the nucleus. Intact nucleocytoplasmic transport is vital for normal cell function and mutations in the executing machinery have been causally linked to human disease. Central players in nucleocytoplasmic exchange are nuclear pore complexes (NPCs), which are built from ~30 distinct proteins collectively termed nucleoporins. Aberrant nucleoporin expression was detected in human cancers and autoimmune diseases since quite some time, while it was through the increasing use of next generation sequencing that mutations in nucleoporin genes associated with mainly rare hereditary diseases were revealed. The number of newly identified mutations is steadily increasing, as is the number of diseases. Mutational hotspots have emerged: mutations in the scaffold nucleoporins seemingly affect primarily inner organs, such as heart, kidney, and ovaries, whereas genetic alterations in peripheral, cytoplasmic nucleoporins affect primarily the central nervous system and development. In this review, we summarise latest insights on altered nucleoporin function in the context of human hereditary disorders, with a focus on those where mechanistic insights are beginning to emerge., (© 2023 Federation of European Biochemical Societies.)

Importance: Itaconate derivates, as well as the naturally produced metabolite, have been proposed as antivirals against influenza virus. Here, the mechanism behind the antiviral effects of exogenous 4-octyl itaconate (4-OI), a derivative of itaconate, against the influenza A virus replication is demonstrated. The data indicate that 4-OI targets the cysteine at position 528 of the CRM1 protein, resulting in inhibition of the nuclear export of viral ribonucleoprotein complexes in a similar manner as previously described for other selective inhibitors of nuclear export. These results postulate a mechanism not observed before for this immuno-metabolite derivative. This knowledge is helpful for the development of derivatives of 4-OI as potential antiviral and anti-inflammatory therapeutics., Competing Interests: Y.Z. and M.R. are employees and hold stock in AbbVie.

Importance: The architecture of sub-nuclear structures of eucaryotic cells is often changed during the infectious cycle of many animal and plant viruses. Cajal bodies (CBs) form a major sub-nuclear structure whose functions may include the regulation of cellular RNA metabolism. During the lifecycle of human adenovirus 5 (Ad5), CBs are reorganized from their spherical-like structure into smaller clusters termed microfoci. The mechanism of this reorganization and its significance for virus replication has yet to be established. Here we show that the major CB protein, p80-coilin, facilitates the nuclear export of Ad5 transcripts. Depletion of p80-coilin by RNA interference led to lowered levels of viral proteins and infectious virus. p80-coilin was found to form a complex with the viral L4-22K protein in Ad5-infected cells and in some reorganized microfoci. These findings assign a new role for p80-coilin as a potential regulator of infection by a human DNA virus., Competing Interests: The authors declare no conflict of interest.

Lymph node metastases are commonly observed in diverse malignancies where they promote cancer progression and poor outcomes, although the molecular basis is incompletely understood. Thyroid cancer is the most prevalent endocrine neoplasm characterized by high frequency of lymph node metastases. Here, we uncover an inflammatory cytokines-controlled epigenetic program during thyroid cancer progression. LNCPTCTS acts as a novel tumor suppressive lncRNA with remarkably decreased expression in thyroid cancer specimens, especially in metastatic lymph nodes. Inflammatory cytokines TNFα or CXCL10, which are released from tumor microenvironment (TME), impair binding capabilities of the transcription factor (TF) EGR1 to the LNCPTCTS promoter and reduce the lncRNA expression in cells. Notably, LNCPTCTS binds to eEF1A2 protein and facilitates the interaction between eEF1A2 and Snail, which promotes Snail nucleus export via the RanGTP-Exp5-aa-tRNA-eEF1A2 complex. Loss of LNCPTCTS in tumors leads to accumulation of Snail in the nucleus, suppressed transcription of E-cadherin and PEBP1, reduced E-cadherin and PEBP1 protein levels, and activated epithelial-mesenchymal transition and MAPK signaling. Our results reveal what we believe to be a novel paradigm between TME and epigenetic reprogram in cancer cells which drives lymph node metastases, therefore illuminating the suitability of LNCPTCTS as a targetable vulnerability in thyroid cancer., Competing Interests: Declaration of competing interest The authors declared no potential conflicts of interests., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Nucleoporins (NUPs) comprise nuclear pore complexes, gateways for nucleocytoplasmic transport. As primary human keratinocytes switch from the progenitor state towards differentiation, most NUPs are strongly downregulated, with NUP93 being the most downregulated NUP in this process. To determine if this NUP downregulation is accompanied by a reduction in nuclear pore numbers, we leveraged Stochastic Optical Reconstruction Microscopy. No significant changes in nuclear pore numbers were detected using three independent NUP antibodies; however, NUP reduction in other subcellular compartments such as the cytoplasm was identified. To investigate how NUP reduction influences keratinocyte differentiation, we knocked down NUP93 in keratinocytes in the progenitor-state culture condition. NUP93 knockdown diminished keratinocytes' clonogenicity and epidermal regenerative capacity, without drastically affecting nuclear pore numbers or permeability. Using transcriptome profiling, we identified that NUP93 knockdown induces differentiation genes related to both mechanical and immune barrier functions, including the activation of known NF-κB target genes. Consistently, keratinocytes with NUP93 knockdown exhibited increased nuclear localization of the NF-κB p65/p50 transcription factors, and increased NF-κB reporter activity. Taken together, these findings highlight the gene regulatory roles contributed by differential NUP expression levels in keratinocyte differentiation, independent of nuclear pore numbers., (© 2023. Springer Nature Limited.)

Nucleocytoplasmic transport of transcription factors is vital for normal cellular function, and its breakdown is a major contributing factor in many diseases. The glucocorticoid receptor (GR) is an evolutionarily conserved, ligand-dependent transcription factor that regulates homeostasis and response to stress and is an important target for therapeutics in inflammation and cancer. In unstimulated cells, the GR resides in the cytoplasm bound to other molecules in a large multiprotein complex. Upon stimulation with endogenous or synthetic ligands, GR translocation to the cell nucleus occurs, where the GR regulates the transcription of numerous genes by direct binding to glucocorticoid response elements or by physically associating with other transcription factors. While much is known about molecular mechanisms underlying GR function, the spatial organization of directionality of GR nucleocytoplasmic transport remains less well characterized, and it is not well understood how the bidirectional nucleocytoplasmic flow of GR is coordinated in stimulated cells. Here, we use two-foci cross-correlation in a massively parallel fluorescence correlation spectroscopy (mpFCS) system to map in live cells the directionality of GR translocation at different positions along the nuclear envelope. We show theoretically and experimentally that cross-correlation of signals from two nearby observation volume elements (OVEs) in an mpFCS setup presents a sharp peak when the OVEs are positioned along the trajectory of molecular motion and that the time position of the peak corresponds to the average time of flight of the molecule between the two OVEs. Hence, the direction and velocity of nucleocytoplasmic transport can be determined simultaneously at several locations along the nuclear envelope. We reveal that under ligand-induced GR translocation, nucleocytoplasmic import/export of GR proceeds simultaneously but at different locations in the cell nucleus. Our data show that mpFCS can characterize in detail the heterogeneity of directional nucleocytoplasmic transport in a live cell and may be invaluable for studies aiming to understand how the bidirectional flow of macromolecules through the nuclear pore complex (NPC) is coordinated to avoid intranuclear transcription factor accretion/abatement.

MicroRNAs (miRNAs) are small non-coding RNAs first discovered in Caenorhabditis elegans. The let-7 miRNA is highly conserved in sequence, biogenesis and function from C. elegans to humans. During miRNA biogenesis, XPO5-mediated nuclear export of pre-miRNAs is a rate-limiting step and, therefore, might be critical for the quantitative control of miRNA levels, yet little is known about how this is regulated. Here we show a novel role for lipid kinase PPK-1/PIP5K1A (phosphatidylinositol-4-phosphate 5-kinase) in regulating miRNA levels. We found that C. elegans PPK-1 functions in the lin-28/let-7 heterochronic pathway, which regulates the strict developmental timing of seam cells. In C. elegans and human cells, PPK-1/PIP5K1A regulates let-7 miRNA levels. We investigated the mechanism further in human cells and show that PIP5K1A interacts with nuclear export protein XPO5 in the nucleus to regulate mature miRNA levels by blocking the binding of XPO5 to pre-let-7 miRNA. Furthermore, we demonstrate that this role for PIP5K1A is kinase-independent. Our study uncovers the novel finding of a direct connection between PIP5K1A and miRNA biogenesis. Given that miRNAs are implicated in multiple diseases, including cancer, this new finding might lead to a novel therapeutic opportunity., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The assembly of cortical circuits involves the generation and migration of interneurons from the ventral to the dorsal forebrain 1-3 , which has been challenging to study at inaccessible stages of late gestation and early postnatal human development 4 . Autism spectrum disorder and other neurodevelopmental disorders (NDDs) have been associated with abnormal cortical interneuron development 5 , but which of these NDD genes affect interneuron generation and migration, and how they mediate these effects remains unknown. We previously developed a platform to study interneuron development and migration in subpallial organoids and forebrain assembloids 6 . Here we integrate assembloids with CRISPR screening to investigate the involvement of 425 NDD genes in human interneuron development. The first screen aimed at interneuron generation revealed 13 candidate genes, including CSDE1 and SMAD4. We subsequently conducted an interneuron migration screen in more than 1,000 forebrain assembloids that identified 33 candidate genes, including cytoskeleton-related genes and the endoplasmic reticulum-related gene LNPK. We discovered that, during interneuron migration, the endoplasmic reticulum is displaced along the leading neuronal branch before nuclear translocation. LNPK deletion interfered with this endoplasmic reticulum displacement and resulted in abnormal migration. These results highlight the power of this CRISPR-assembloid platform to systematically map NDD genes onto human development and reveal disease mechanisms., (© 2023. The Author(s).)

Ran-binding protein 2 (RANBP2)/Nup358 is a nucleoporin and a key component of the nuclear pore complex. Through its multiple functions (e.g., SUMOylation, regulation of nucleocytoplasmic transport) and subcellular localizations (e.g., at the nuclear envelope, kinetochores, annulate lamellae), it is involved in many cellular processes. RANBP2 dysregulation or mutation leads to the development of human pathologies, such as acute necrotizing encephalopathy 1, cancer, neurodegenerative diseases, and it is also involved in viral infections. The chromosomal region containing the RANBP2 gene is highly dynamic, with high structural variation and recombination events that led to the appearance of a gene family called RANBP2 and GCC2 Protein Domains (RGPD), with multiple gene loss/duplication events during ape evolution. Although RGPD homoplasy and maintenance during evolution suggest they might confer an advantage to their hosts, their functions are still unknown and understudied. In this review, we discuss the appearance and importance of RANBP2 in metazoans and its function-related pathologies, caused by an alteration of its expression levels (through promotor activity, post-transcriptional, or post-translational modifications), its localization, or genetic mutations., (© 2023 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Development of colorectal cancer (CRC) involves activation of Kirsten rat sarcoma viral oncogene homolog (KRAS) signaling. However, the post-transcriptional regulation of KRAS has yet to be fully characterized. Here, we found that the colorectal neoplasia differentially expressed (CRNDE)/heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1) axis was notably elevated in CRC and was strongly associated with poor prognosis of patients, while also significantly promoting CRC cell proliferation and metastasis both in vitro and in vivo. Furthermore, CRNDE maintained the stability of hnRNPA2B1 protein by inhibiting E3 ubiquitin ligase TRIM21 mediated K63 ubiquitination-dependent protein degradation. CRNDE/hnRNPA2B1 axis facilitated the nuclear export and translation of KRAS mRNA, which specifically activated the MAPK signaling pathway, eventually accelerating the malignant progression of CRC. Our findings provided insight into the regulatory network for stable hnRNPA2B1 protein expression, and the molecular mechanisms by which the CRNDE/hnRNPA2B1 axis mediated KRAS nucleocytoplasmic transport and translation, deeply underscoring the bright future of hnRNPA2B1 as a promising biomarker and therapeutic target for CRC. By hindering hnRNPA2B1 from binding to the E3 ubiquitin ligase TRIM21, whose mediated ubiquitin-dependent degradation was thereby inhibited, CRNDE protected the stability of hnRNPA2B1's high protein expression in CRC. Supported by the high level of the oncogenic molecule CRNDE, hnRNPA2B1 bound to KRAS mRNA and promoted KRAS mRNA nucleus export to enter the ribosomal translation program, subsequently activating the MAPK signaling pathway and ultimately accelerating the malignant progression of CRC., (© 2023. The Author(s).)

Monoubiquitylation is a principal mechanism driving nuclear translocation of the protein PTEN (phosphatase and tensin homolog deleted on chromosome ten). In this study, we describe a novel mechanism wherein the protein CHIP (C-terminus of Hsc70-interacting protein) mediates PTEN monoubiquitylation, leading to its nuclear import. Western blot analysis revealed a rise in both nuclear and total cellular PTEN levels under monoubiquitylation-promoting conditions, an effect that was abrogated by silencing CHIP expression. We established time-point kinetics of CHIP-mediated nuclear translocation of PTEN using immunocytochemistry and identified a role of karyopherin α1 (KPNA1) in facilitating nuclear transport of monoubiquitylated PTEN. We further established a direct interaction between CHIP and PTEN inside the nucleus, with CHIP participating in either polyubiquitylation or monoubiquitylation of nuclear PTEN. Finally, we showed that oxidative stress enhanced CHIP-mediated nuclear import of PTEN, which resulted in increased apoptosis, and decreased cell viability and proliferation, whereas CHIP knockdown counteracted these effects. To the best of our knowledge, this is the first report elucidating non-canonical roles for CHIP on PTEN, which we establish here as a nuclear interacting partner of CHIP., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

The ATPase p97 (also known as VCP, Cdc48) has crucial functions in a variety of important cellular processes such as protein quality control, organellar homeostasis, and DNA damage repair, and its de-regulation is linked to neuromuscular diseases and cancer. p97 is tightly controlled by numerous regulatory cofactors, but the full range and function of the p97-cofactor network is unknown. Here, we identify the hitherto uncharacterized FAM104 proteins as a conserved family of p97 interactors. The two human family members V CP nuclear c ofactor f amily member 1 and 2 (VCF1/2) bind p97 directly via a novel, alpha-helical motif and associate with p97-UFD1-NPL4 and p97-UBXN2B complexes in cells. VCF1/2 localize to the nucleus and promote the nuclear import of p97. Loss of VCF1/2 results in reduced nuclear p97 levels, slow growth, and hypersensitivity to chemical inhibition of p97 in the absence and presence of DNA damage, suggesting that FAM104 proteins are critical regulators of nuclear p97 functions., Competing Interests: MK, SM, GM, MK, CG, CS, UF, KH, AB No competing interests declared, (© 2023, Körner, Meyer et al.)

Dysregulation of nucleocytoplasmic shuttling impairs cellular homeostasis and promotes cancer development. KPNB1 is a member of karyopherin β family, mediating the transportation of proteins from the cytoplasm to the nucleus. In a variety of cancers, the expression of KPNB1 is upregulated to facilitate tumor growth and progression. Both downregulation of KPNB1 level and inhibition of KPNB1 activity prevent the entry of cancer-related transcription factors into the nucleus, subsequently suppressing the proliferation and metastasis of cancer cells. Currently, five KPNB1 inhibitors have been reported and exhibited good efficacy against cancer. This paper provides an overview of the role and mechanism of KPNB1 in different cancers and KPNB1-targeted anticancer compounds which hold promise for the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The transcription factor TATA-box binding protein (TBP) modulates gene expression in nuclei. This process requires the involvement of nuclear transport receptors, collectively termed karyopherin-β (Kap-β) in yeast, and various regulatory factors. In previous studies we showed that Kap114p, a Kap-β that mediates nuclear import of yeast TBP (yTBP), modulates yTBP-dependent transcription. However, how Kap114p associates with yTBP to exert its multifaceted functions has remained elusive. Here, we employ single-particle cryo-electron microscopy to determine the structure of Kap114p in complex with the core domain of yTBP (yTBP C ). Remarkably, Kap114p wraps around the yTBP C N-terminal lobe, revealing a structure resembling transcriptional regulators in complex with TBP, suggesting convergent evolution of the two protein groups for a common function. We further demonstrate that Kap114p sequesters yTBP away from promoters, preventing a collapse of yTBP dynamics required for yeast responses to environmental stress. Hence, we demonstrate that nuclear transport receptors represent critical elements of the transcriptional regulatory network., (© 2023. Springer Nature Limited.)

PGC-1α plays a central role in maintaining mitochondrial and energy metabolism homeostasis, linking external stimuli to transcriptional co-activation of genes involved in adaptive and age-related pathways. The carboxyl-terminus encodes a serine/arginine-rich (RS) region and an RNA recognition motif, however the RNA-processing function(s) were poorly investigated over the past 20 years. Here, we show that the RS domain of human PGC-1α directly interacts with RNA and the nuclear RNA export receptor NXF1. Inducible depletion of PGC-1α and expression of RNAi-resistant RS-deleted PGC-1α further demonstrate that its RNA/NXF1-binding activity is required for the nuclear export of some canonical mitochondrial-related mRNAs and mitochondrial homeostasis. Genome-wide investigations reveal that the nuclear export function is not strictly linked to promoter-binding, identifying in turn novel regulatory targets of PGC-1α in non-homologous end-joining and nucleocytoplasmic transport. These findings provide new directions to further elucidate the roles of PGC-1α in gene expression, metabolic disorders, aging and neurodegeneration., (© 2023. Springer Nature Limited.)

Using all-atom replica-exchange molecular dynamics simulations, we mapped the mechanisms of binding of the nuclear localization signal (NLS) sequence from Venezuelan equine encephalitis virus (VEEV) capsid protein to importin-α (impα) transport protein. Our objective was to identify the VEEV NLS sequence fragment that confers native, experimentally resolved binding to impα as well as to study associated binding energetics and conformational ensembles. The two selected VEEV NLS peptide fragments, KKPK and KKPKKE, show strikingly different binding mechanisms. The minNLS peptide KKPK binds non-natively and nonspecifically by adopting five diverse conformational clusters with low similarity to the x-ray structure 3VE6 of NLS-impα complex. Despite the prevalence of non-native interactions, the minNLS peptide still largely binds to the impα major NLS binding site. In contrast, the coreNLS peptide KKPKKE binds specifically and natively, adopting a largely homogeneous binding ensemble with a dominant, highly native-like conformational cluster. The coreNLS peptide retains most of native binding interactions, including π-cation contacts and a tryptophan cage. While KKPK binding is governed by a complex multistate free energy landscape featuring transitions between multiple binding poses, the coreNLS peptide free energy map is simple, exhibiting a single dominant native-like bound basin. We argue that the origin of the coreNLS peptide binding specificity is several electrostatic interactions formed by the two C-terminal amino acids, Lys10 and Glu11, with impα. The coreNLS sequence is then sufficient for native binding, but none of the amino acids flanking minNLS, including Lys10 and Glu11, are strictly necessary for the native pose. Our analyses indicate that the VEEV coreNLS sequence is virtually unique among human and viral proteins interacting with impα making it a potential target for VEEV-specific inhibitors., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Programmed cell death (PCD) is a process that occurs naturally in cells in response to different endogenous or exogenous factors and facilitated by specific proteins. The three common pathways are pyroptosis, necroptosis, and apoptosis. Each pathway has its own unique proteins, mechanisms, and byproducts. Dysregulated PCD can lead to abnormal growth of cells causing tumor growth, a hallmark feature of many cancer pathologies. Recently, the PCD pathways have been considered to be activated simultaneously in a combined nature defined as PANoptosis (pyroptosis, apoptosis, and necroptosis). An integral protein, Z-DNA binding protein 1 (ZBP1) aids in the initiation of the NOD-like receptor protein 3 (NLRP3) inflammasome, a known facilitator of pyroptosis. It also is known to bind to a regulator of necroptosis, receptor-interacting protein kinase 3 (RIPK3). A unique binding partner to ZBP1, adenosine deaminase acting on RNA 1 (ADAR1), is involved in RNA editing, stress mechanisms, and disease. In murine bone marrow-derived macrophages (BMDMs) treatment with nuclear export inhibitors (NEIs) has allowed for sequestering of ADAR1 to the nucleus, and increased incidence of cell death. Additionally, the use of interferons (IFNs) to induce ZBP1 has increased the incidence of cell death. Emerging therapies are looking at the efficacy of using a combination of NEI and IFN treatment to rapidly reduce tumor size and growth by inducing PANoptosis. KPT-330 and KPT-8602 are two different NEIs, both of which have shown efficacy in the reduction of tumor size and inhibition of Exportin 1 (XPO1), a transport protein. However, this article posits KPT-8602 as the better of the two. KPT-8602 is more tolerable for the patient and should be pushed to human trials., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Methyltransferase-like 3 (METTL3), a key component of the m6A methyltransferase complex, regulates the splicing, nuclear transport, stability, and translation of its target genes. However, the mechanism underlying the regulation of METTL3 expression by alternative splicing (AS) remains unknown. We analyzed the expression pattern of METTL3 after AS in human tissues and confirmed the expression of an isoform retaining introns 8 and 9 (METTL3-IR). We confirmed the different intracellular localizations of METTL3-IR and METTL3 proteins using immunofluorescence microscopy. Furthermore, the endogenous expression of METTL3-IR at the protein level was different from that at the mRNA level. We found that 3'-UTR generation by intron retention (IR) inhibited the export of METTL3-IR mRNA to the cytoplasm, which in turn suppressed protein expression. To the best of our knowledge, this is the first study to confirm the regulation of METTL3 gene expression by AS, providing evidence that the suppression of METTL3 protein expression by IR is an integral part of the mechanism by which 3'-UTR generation regulates protein expression via inhibition of RNA export to the cytoplasm. [BMB Reports 2023; 56(9): 514-519].

Flowering locus C (FLC) is a central transcriptional repressor that controls flowering time. However, how FLC is imported into the nucleus is unknown. Here, we report that Arabidopsis nucleoporins 62 (NUP62), NUP58, and NUP54 composed NUP62-subcomplex modulates FLC nuclear import during floral transition in an importin α-independent manner, via direct interaction. NUP62 recruits FLC to the cytoplasmic filaments and imports it into the nucleus through the NUP62-subcomplex composed central channel. Importin β supersensitive to ABA and drought 2 (SAD2), a carrier protein, is critical for FLC nuclear import and flower transition, which facilitates FLC import into the nucleus mainly through the NUP62-subcomplex. Proteomics, RNA-seq, and cell biological analyses indicate that the NUP62-subcomplex mainly mediates the nuclear import of cargos with unconventional nuclear localization sequences (NLSs), such as FLC. Our findings illustrate the mechanisms of the NUP62-subcomplex and SAD2 on FLC nuclear import process and floral transition, and provide insights into the role of NUP62-subcomplex and SAD2 in protein nucleocytoplasmic transport in plants., (© 2023 Institute of Botany, Chinese Academy of Sciences.)

The complex between the Rev protein of HIV-1 and the Rev Recognition Element (RRE) within the virus RNA promotes nuclear export of unspliced or incompletely spliced viral transcripts and is required for virus transmission. Here, we have screened a virtual collection of compounds using a pharmacophore based on the chemical similarity of previously characterized inhibitors to identify new chemical scaffolds blocking the RRE-Rev interaction. The best molecules discovered with this strategy inhibited the complex by binding to the RRE and exhibited substantial antiretroviral activity (between 0.582 and 11.3 μM EC 50 values) likely associated to inhibitory actions on viral transcription and Rev function. These results have allowed us to identify structural features required for RRE-Rev inhibition as well as to add new compounds to the pool of possible candidates for developing antiretroviral agents based on blockage of HIV-1 RNA biogenesis., Competing Interests: Declaration of Competing Interest A.S.-L. and J.G. participate as inventors in a patent application involving the antiviral activity of clomiphene citrate, trim and trpi., (Copyright © 2023. Published by Elsevier Inc.)