WW domains form a folded type of nuclear localization signal to guide YAP1 nuclear import.

The nuclear translocation of YAP1 is significantly implicated in the proliferation, stemness, and metastasis of cancer cells. Although the molecular basis underlying YAP1 subcellular distribution has been extensively explored, it remains to be elucidated how the nuclear localization signal guides YAP1 to pass through the nuclear pore complex. Here, we define a globular type of nuclear localization signal composed of folded WW domains, named as WW-NLS. It directs YAP1 nuclear import through the heterodimeric nuclear transport receptors KPNA-KPNB1, bypassing the canonical nuclear localization signal that has been well documented in KPNA/KPNB1-mediated nuclear import. Strikingly, competitive interference with the function of the WW-NLS significantly attenuates YAP1 nuclear translocation and damages stemness gene activation and sphere formation in malignant breast cancer cells. Our findings elucidate a novel globular type of nuclear localization signal to facilitate nuclear entry of WW-containing proteins including YAP1.

Proteomic and yeast 2-hybrid screens to identify PTEN binding partners.

PTEN is a phosphoinositide lipid phosphatase and an important tumour suppressor protein. PTEN function is reduced or lost in around a third of all human cancers through diverse mechanisms, from gene deletion to changes in the function of proteins which regulate PTEN through direct protein binding. Here we present data from SILAC (Stable Isotope Labelling by Amino acids in Cell culture) proteomic screens to identify proteins which bind to PTEN. These experiments using untransformed epithelial cells and glioma cells identified several novel candidate proteins in addition to many previously identified PTEN binding partners and many proteins which are recognised as common false positives using these methods. From subsequent co-expression pull-down experiments we provide further evidence supporting the physical interaction of PTEN with MMP1, Myosin 18A and SHROOM3. We also performed yeast two-hybrid screens which identify the previously recognised PTEN binding partner MSP58 in addition to the nuclear import export receptor TNPO3. These experiments identify several novel candidate binding partners of PTEN and provide further data addressing the set of proteins that interact with this important tumour suppressor.

Human RTEL1 Interacts with KPNB1 (Importin ß) and NUP153 and Connects Nuclear Import to Nuclear Envelope Stability in S-Phase.

Regulator of TElomere Length Helicase 1 (RTEL1) is a helicase required for telomere maintenance and genome replication and repair. RTEL1 has been previously shown to participate in the nuclear export of small nuclear RNAs. Here we show that RTEL1 deficiency leads to a nuclear envelope destabilization exclusively in cells entering S-phase and in direct connection to origin firing. We discovered that inhibiting protein import also leads to similar, albeit non-cell cycle-related, nuclear envelope disruptions. Remarkably, overexpression of wild-type RTEL1, or of its C-terminal part lacking the helicase domain, protects cells against nuclear envelope anomalies mediated by protein import inhibition. We identified distinct domains in the C-terminus of RTEL1 essential for the interaction with KPNB1 (importin ß) and NUP153, respectively, and we demonstrated that, on its own, the latter domain can promote the dynamic nuclear internalization of peptides that freely diffuse through the nuclear pore. Consistent with putative functions exerted in protein import, RTEL1 can be visualized on both sides of the nuclear pore using high-resolution microscopy. In all, our work points to an unanticipated, helicase-independent, role of RTEL1 in connecting both nucleocytoplasmic trafficking and nuclear envelope integrity to genome replication initiation in S-phase.

Defective recognition of a nonclassical nuclear localization signal in neurodevelopmental disorders.

In this issue of Structure, Gonzalez et al. present the cryo-EM structure of Karyopherin-ß2 bound to the proline-tyrosine nuclear localization signal (PY-NLS) of heterogeneous nuclear ribonucleoprotein H2 (HNRNPH2). The structure advances our understanding of not only the diversity of PY-NLSs but also the pathogenic mechanisms arising from HNRNPH2 variants.

KPNB1-mediated nuclear import in cancer.

Dysregulation of nucleocytoplasmic shuttling impairs cellular homeostasis and promotes cancer development. KPNB1 is a member of karyopherin ß 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.

Regulation of FLC nuclear import by coordinated action of the NUP62-subcomplex and importin ß SAD2.

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 ß 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.

Toxoplasma Gondii Importin α Shows Weak Auto-Inhibition.

Importin α is a nuclear transporter that binds to nuclear localization signals (NLSs), consisting of 7-20 positively charged amino acids found within cargo proteins. In addition to cargo binding, intramolecular interactions also occur within the importin α protein due to binding between the importin ß-binding (IBB) domain and the NLS-binding sites, a phenomenon called auto-inhibition. The interactions causing auto-inhibition are driven by a stretch of basic residues, similar to an NLS, in the IBB domain. Consistent with this, importin α proteins that do not have some of these basic residues lack auto-inhibition; a naturally occurring example of such a protein is found in the apicomplexan parasite Plasmodium falciparum. In this report, we show that importin α from another apicomplexan parasite, Toxoplasma gondii, harbors basic residues (KKR) in the IBB domain and exhibits auto-inhibition. This protein has a long, unstructured hinge motif (between the IBB domain and the NLS-binding sites) that does not contribute to auto-inhibition. However, the IBB domain may have a higher propensity to form an α-helical structure, positioning the wild-type KKR motif in an orientation that results in weaker interactions with the NLS-binding site than a KRR mutant. We conclude that the importin α protein from T. gondii shows auto-inhibition, exhibiting a different phenotype from that of P. falciparum importin α. However, our data indicate that T. gondii importin α may have a low strength of auto-inhibition. We hypothesize that low levels of auto-inhibition may confer an advantage to these important human pathogens.

A new Karyopherin-ß2 binding PY-NLS epitope of HNRNPH2 linked to neurodevelopmental disorders.

The HNRNPH2 proline-tyrosine nuclear localization signal (PY-NLS) is mutated in HNRNPH2-related X-linked neurodevelopmental disorder, causing the normally nuclear HNRNPH2 to accumulate in the cytoplasm. We solved the cryoelectron microscopy (cryo-EM) structure of Karyopherin-ß2/Transportin-1 bound to the HNRNPH2 PY-NLS to understand importin-NLS recognition and disruption in disease. HNRNPH2 206RPGPY210 is a typical R-X2-4-P-Y motif comprising PY-NLS epitopes 2 and 3, followed by an additional Karyopherin-ß2-binding epitope, we term epitope 4, at residues 211DRP213; no density is present for PY-NLS epitope 1. Disease variant mutations at epitopes 2-4 impair Karyopherin-ß2 binding and cause aberrant cytoplasmic accumulation in cells, emphasizing the role of nuclear import defect in disease. Sequence/structure analysis suggests that strong PY-NLS epitopes 4 are rare and thus far limited to close paralogs of HNRNPH2, HNRNPH1, and HNRNPF. Epitope 4-binidng hotspot Karyopherin-ß2 W373 corresponds to close paralog Karyopherin-ß2b/Transportin-2 W370, a pathological variant site in neurodevelopmental abnormalities, suggesting that Karyopherin-ß2b/Transportin-2-HNRNPH2/H1/F interactions may be compromised in the abnormalities.

MicroRNA-130a-3p impedes the progression of papillary thyroid carcinoma through downregulation of KPNB1 by targeting PSME3.

BACKGROUND: Papillary thyroid carcinoma (PTC) is the main type of thyroid cancer (THCA). Despite the good prognosis, some PTC patients may deteriorate into more aggressive disease, leading to poor survival. Our study aimed to explore the role of microRNA (miR)-130a-3p in regulating PTC. METHODS: After transfection with miR-130a-3p-mimic, OE-PSME3, or miR-130a-3p-mimic + OE-KPNB1 in PTC cells (TPC-1), CCK-8, Transwell, scratch, and flow cytometry experiments were performed to analyze TPC-1 cell proliferation, invasion, migration, and apoptosis. Western blotting was used to detect proliferation or invasion-related protein markers (PCNA, E-cadherin, and N-cadherin). The RNA22 database, dual-luciferase reporter assay, and RNA pull-down assay were applied for the prediction and verification of the binding site between miR-130a-3p and PSME3. Pan-cancer software identified a positive correlation between PSME3 and KPNB1 in THCA. Co-immunoprecipitation was utilized to verify the interaction of PSME3 with KPNB1. Nude mice were transplanted with TPC-1 cells overexpressing miR-130a-3p. The tumors were isolated for detection of the expression of miR-130a-3p, PSME3, KPNB1, Ki-67, and CD31. RESULTS: miR-130a-3p was lowly expressed in PTC cell lines. Upregulation of miR-130a-3p repressed the expression of PSME3 and KPNB1 and reduced the malignancy of TPC-1 cells in vitro, shown by inhibited cell proliferation, invasion, migration, and the expression of PCNA and N-cadherin. Also, overexpressed miR-130a-3p inhibited the growth of xenograft tumors in nude mice. miR-130a-3p bound to PSME3 which interacted with KPNB1. CONCLUSION: miR-130a-3p impedes the progression of PTC by downregulating PSME3/KPNB1.

Progress in the study of parvovirus entry pathway.

A group of DNA viruses called parvoviruses that have significant effects on cancer therapy and genetic engineering applications. After passing through the cell membrane to reach the cytosol, it moves along the microtubule toward the nuclear membrane. The nuclear localization signal (NLS) is recognized by importin-beta (impß) and other proteins from the complex outside the nuclear membrane and binds to enter the nucleus via the nuclear pore complex (NPC). There are two main pathways for viruses to enter the nucleus. The classical pathway is through the interaction of imp α and impß with NLS via NPC. The other is the NPC mediated by the combination of impß and it. While the capsid is introduced into the nucleus through classical nuclear transduction, there is also a transient nuclear membrane dissolution leading to passive transport into the nucleus, which has been proposed in recent years. This article mainly discusses several nuclear entry pathways and related proteins, providing a reference for subsequent research on viral entry pathways.

Activin and BMP Signalling in Human Testicular Cancer Cell Lines, and a Role for the Nucleocytoplasmic Transport Protein Importin-5 in Their Crosstalk.

Testicular germ cell tumours (TGCTs) are the most common malignancy in young men. Originating from foetal testicular germ cells that fail to differentiate correctly, TGCTs appear after puberty as germ cell neoplasia in situ cells that transform through unknown mechanisms into distinct seminoma and non-seminoma tumour types. A balance between activin and BMP signalling may influence TGCT emergence and progression, and we investigated this using human cell line models of seminoma (TCam-2) and non-seminoma (NT2/D1). Activin A- and BMP4-regulated transcripts measured at 6 h post-treatment by RNA-sequencing revealed fewer altered transcripts in TCam-2 cells but a greater responsiveness to activin A, while BMP4 altered more transcripts in NT2/D1 cells. Activin significantly elevated transcripts linked to pluripotency, cancer, TGF-ß, Notch, p53, and Hippo signalling in both lines, whereas BMP4 altered TGF-ß, pluripotency, Hippo and Wnt signalling components. Dose-dependent antagonism of BMP4 signalling by activin A in TCam-2 cells demonstrated signalling crosstalk between these two TGF-ß superfamily arms. Levels of the nuclear transport protein, IPO5, implicated in BMP4 and WNT signalling, are highly regulated in the foetal mouse germline. IPO5 knockdown in TCam-2 cells using siRNA blunted BMP4-induced transcript changes, indicating that IPO5 levels could determine TGF-ß signalling pathway outcomes in TGCTs.

Importin α/ß-dependent nuclear transport of human parvovirus B19 nonstructural protein 1 is essential for viral replication.

Human parvovirus B19 (B19V) is a major human pathogen causing a variety of diseases, characterized by a selective tropism to human progenitor cells in bone marrow. In similar fashion to all Parvoviridae members, the B19V ssDNA genome is replicated within the nucleus of infected cells through a process which involves both cellular and viral proteins. Among the latter, a crucial role is played by non-structural protein (NS)1, a multifunctional protein involved in genome replication and transcription, as well as modulation of host gene expression and function. Despite the localization of NS1 within the host cell nucleus during infection, little is known regarding the mechanism of its nuclear transport pathway. In this study we undertake structural, biophysical, and cellular approaches to characterize this process. Quantitative confocal laser scanning microscopy (CLSM), gel mobility shift, fluorescence polarization and crystallographic analysis identified a short sequence of amino acids (GACHAKKPRIT-182) as the classical nuclear localization signal (cNLS) responsible for nuclear import, mediated in an energy and importin (IMP) α/ß-dependent fashion. Structure-guided mutagenesis of key residue K177 strongly impaired IMPα binding, nuclear import, and viral gene expression in a minigenome system. Further, treatment with ivermectin, an antiparasitic drug interfering with the IMPα/ß dependent nuclear import pathway, inhibited NS1 nuclear accumulation and viral replication in infected UT7/Epo-S1 cells. Thus, NS1 nuclear transport is a potential target of therapeutic intervention against B19V induced disease.

Importin α/ß promote Kif18B microtubule association and enhance microtubule destabilization activity.

Tight regulation of microtubule (MT) dynamics is necessary for proper spindle assembly and chromosome segregation. The MT destabilizing Kinesin-8, Kif18B, controls astral MT dynamics and spindle positioning. Kif18B interacts with importin α/ß as well as with the plus-tip tracking protein EB1, but how these associations modulate Kif18B is not known. We mapped the key binding sites on Kif18B, made residue-specific mutations, and assessed their impact on Kif18B function. Blocking EB1 interaction disrupted Kif18B MT plus-end accumulation and inhibited its ability to control MT length on monopolar spindles in cells. Blocking importin α/ß interaction disrupted Kif18B localization without affecting aster size. In vitro, importin α/ß increased Kif18B MT association by increasing the on-rate and decreasing the off-rate from MTs, which stimulated MT destabilization. In contrast, EB1 promoted MT destabilization without increasing lattice binding in vitro, which suggests that EB1 and importin α/ß have distinct roles in the regulation of Kif18B-mediated MT destabilization. We propose that importin α/ß spatially modulate Kif18B association with MTs to facilitate its MT destabilization activity. Our results suggest that Ran regulation is important not only to control molecular motor function near chromatin but also to provide a spatial control mechanism to modulate MT binding of nuclear localization signal-containing spindle assembly factors.

Loss of C9orf72 perturbs the Ran-GTPase gradient and nucleocytoplasmic transport, generating compositionally diverse Importin ß-1 granules.

A hexanucleotide (GGGGCC)n repeat expansion in C9orf72 causes amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), eliciting toxic effects through generation of RNA foci, dipeptide repeat proteins, and/or loss of C9orf72 protein. Defects in nucleocytoplasmic transport (NCT) have been implicated as a pathogenic mechanism underlying repeat expansion toxicity. Here, we show that loss of C9orf72 disrupts the Ran-GTPase gradient and NCT in vitro and in vivo. NCT disruption in vivo is enhanced by the presence of compositionally different types of cytoplasmic Importin ß-1 granule that exhibit neuronal subtype-specific properties. We show that the abundance of Importin ß-1 granules is increased in the context of C9orf72 deficiency, disrupting interactions with nuclear pore complex proteins. These granules appear to associate with the nuclear envelope and are co-immunoreactive for G3BP1 and K63-ubiquitin. These findings link loss of C9orf72 protein to gain-of-function mechanisms and defects in NCT.

Transportin 1 is a major nuclear import receptor of the nitric oxide synthase interacting protein.

The nitric oxide synthase interacting protein (NOSIP), an E3-ubiquitin ligase, is involved in various processes like neuronal development, craniofacial development, granulopoiesis, mitogenic signaling, apoptosis, and cell proliferation. The best-characterized function of NOSIP is the regulation of endothelial nitric oxide synthase activity by translocating the membrane-bound enzyme to the cytoskeleton, specifically in the G2 phase of the cell cycle. For this, NOSIP itself has to be translocated from its prominent localization, the nucleus, to the cytoplasm. Nuclear import of NOSIP was suggested to be mediated by the canonical transport receptors importin α/ß. Recently, we found NOSIP in a proteomic screen as a potential importin 13 cargo. Here, we describe the nuclear shuttling characteristics of NOSIP in living cells and in vitro and show that it does not interact directly with importin α. Instead, it formed stable complexes with several importins (-ß, -7, -ß/7, -13, and transportin 1) and was also imported into the nucleus in digitonin-permeabilized cells by these factors. In living HeLa cells, transportin 1 seems to be the major nuclear import receptor for NOSIP. A detailed analysis of the NOSIP-transportin 1 interaction revealed a high affinity and an unusual binding mode, involving the N-terminal half of transportin 1. In contrast to nuclear import, nuclear export of NOSIP seems to occur mostly by passive diffusion. Thus, our results uncover additional layers in the larger process of endothelial nitric oxide synthase regulation.

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.

A minimal construct of nuclear-import receptor Karyopherin-ß2 defines the regions critical for chaperone and disaggregation activity.

Karyopherin-ß2 (Kapß2) is a nuclear-import receptor that recognizes proline-tyrosine nuclear localization signals of diverse cytoplasmic cargo for transport to the nucleus. Kapß2 cargo includes several disease-linked RNA-binding proteins with prion-like domains, such as FUS, TAF15, EWSR1, hnRNPA1, and hnRNPA2. These RNA-binding proteins with prion-like domains are linked via pathology and genetics to debilitating degenerative disorders, including amyotrophic lateral sclerosis, frontotemporal dementia, and multisystem proteinopathy. Remarkably, Kapß2 prevents and reverses aberrant phase transitions of these cargoes, which is cytoprotective. However, the molecular determinants of Kapß2 that enable these activities remain poorly understood, particularly from the standpoint of nuclear-import receptor architecture. Kapß2 is a super-helical protein comprised of 20 HEAT repeats. Here, we design truncated variants of Kapß2 and assess their ability to antagonize FUS aggregation and toxicity in yeast and FUS condensation at the pure protein level and in human cells. We find that HEAT repeats 8 to 20 of Kapß2 recapitulate all salient features of Kapß2 activity. By contrast, Kapß2 truncations lacking even a single cargo-binding HEAT repeat display reduced activity. Thus, we define a minimal Kapß2 construct for delivery in adeno-associated viruses as a potential therapeutic for amyotrophic lateral sclerosis/frontotemporal dementia, multisystem proteinopathy, and related disorders.

Nucleo-Cytoplasmic Transport of ZIKV Non-Structural 3 Protein Is Mediated by Importin-α/ß and Exportin CRM-1.

Flaviviruses have a cytoplasmic replicative cycle, and crucial events, such as genome translation and replication, occur in the endoplasmic reticulum. However, some viral proteins, such as C, NS1, and NS5 from Zika virus (ZIKV) containing nuclear localization signals (NLSs) and nuclear export signals (NESs), are also located in the nucleus of Vero cells. The NS2A, NS3, and NS4A proteins from dengue virus (DENV) have also been reported to be in the nucleus of A549 cells, and our group recently reported that the NS3 protein is also located in the nucleus of Huh7 and C636 cells during DENV infection. However, the NS3 protease-helicase from ZIKV locates in the perinuclear region of infected cells and alters the morphology of the nuclear lamina, a component of the nuclear envelope. Furthermore, ZIKV NS3 has been reported to accumulate on the concave face of altered kidney-shaped nuclei and may be responsible for modifying other elements of the nuclear envelope. However, nuclear localization of NS3 from ZIKV has not been substantially investigated in human host cells. Our group has recently reported that DENV and ZIKV NS3 alter the nuclear pore complex (NPC) by cleaving some nucleoporins. Here, we demonstrate the presence of ZIKV NS3 in the nucleus of Huh7 cells early in infection and in the cytoplasm at later times postinfection. In addition, we found that ZIKV NS3 contains an NLS and a putative NES and uses the classic import (importin-α/ß) and export pathway via CRM-1 to be transported between the cytoplasm and the nucleus. IMPORTANCE Flaviviruses have a cytoplasmic replication cycle, but recent evidence indicates that nuclear elements play a role in their viral replication. Viral proteins, such as NS5 and C, are imported into the nucleus, and blocking their import prevents replication. Because of the importance of the nucleus in viral replication and the role of NS3 in the modification of nuclear components, we investigated whether NS3 can be localized in the nucleus during ZIKV infection. We found that NS3 is imported into the nucleus via the importin pathway and exported to the cytoplasm via CRM-1. The significance of viral protein nuclear import and export and its relationship with infection establishment is highlighted, emphasizing the development of new host-directed antiviral therapeutic strategies.

Identification of the Karyopherin Superfamily in Maize and Its Functional Cues in Plant Development.

Appropriate nucleo-cytoplasmic partitioning of proteins is a vital regulatory mechanism in phytohormone signaling and plant development. However, how this is achieved remains incompletely understood. The Karyopherin (KAP) superfamily is critical for separating the biological processes in the nucleus from those in the cytoplasm. The KAP superfamily is divided into Importin α (IMPα) and Importin ß (IMPß) families and includes the core components in mediating nucleocytoplasmic transport. Recent reports suggest the KAPs play crucial regulatory roles in Arabidopsis development and stress response by regulating the nucleo-cytoplasmic transport of members in hormone signaling. However, the KAP members and their associated molecular mechanisms are still poorly understood in maize. Therefore, we first identified seven IMPα and twenty-seven IMPß genes in the maize genome and described their evolution traits and the recognition rules for substrates with nuclear localization signals (NLSs) or nuclear export signals (NESs) in plants. Next, we searched for the protein interaction partners of the ZmKAPs and selected the ones with Arabidopsis orthologs functioning in auxin biosynthesis, transport, and signaling to predict their potential function. Finally, we found that several ZmKAPs share similar expression patterns with their interacting proteins, implying their function in root development. Overall, this article focuses on the Karyopherin superfamily in maize and starts with this entry point by systematically comprehending the KAP-mediated nucleo-cytoplasmic transport process in plants, and then predicts the function of the ZmKAPs during maize development, with a perspective on a closely associated regulatory mechanism between the nucleo-cytoplasmic transport and the phytohormone network.

Nuclear transport and subcellular localization of the dystrophin Dp71 and Dp40 isoforms in the PC12 cell line.

The shortest dystrophins, Dp71 and Dp40, are transcribed from the DMD gene through an internal promoter located in intron 62. These proteins are the main product of the DMD gene in the nervous system and have been involved in various functions related to cellular differentiation and proliferation as well as other cellular processes. Dp71 mRNA undergoes alternative splicing that results in different Dp71 protein isoforms. The subcellular localization of some of these isoforms in the PC12 cell line has been previously reported, and a differential subcellular distribution was observed, which suggests a particular role for each isoform. With the aim of obtaining information on their function, this study identified factors involved in the nuclear transport of Dp71 and Dp40 isoforms in the PC12 cell line. Cell cultures were treated with specific nuclear import/export inhibitors to determine the Dp71 isoform transport routes. The results showed that all isoforms of Dp71 and Dp40 included in the analysis have the ability to enter the cell nucleus through α/ß importin, and the main route of nuclear export for Dp71 isoforms is through the exportin CRM1, which is not the case for Dp40.