Nuclear Pores Assemble from Nucleoporin Condensates During Oogenesis.

The molecular events that direct nuclear pore complex (NPC) assembly toward nuclear envelopes have been conceptualized in two pathways that occur during mitosis or interphase, respectively. In gametes and embryonic cells, NPCs also occur within stacked cytoplasmic membrane sheets, termed annulate lamellae (AL), which serve as NPC storage for early development. The mechanism of NPC biogenesis at cytoplasmic membranes remains unknown. Here, we show that during Drosophila oogenesis, Nucleoporins condense into different precursor granules that interact and progress into NPCs. Nup358 is a key player that condenses into NPC assembly platforms while its mRNA localizes to their surface in a translation-dependent manner. In concert, Microtubule-dependent transport, the small GTPase Ran and nuclear transport receptors regulate NPC biogenesis in oocytes. We delineate a non-canonical NPC assembly mechanism that relies on Nucleoporin condensates and occurs away from the nucleus under conditions of cell cycle arrest.

Stress Granule Assembly Disrupts Nucleocytoplasmic Transport.

Defects in nucleocytoplasmic transport have been identified as a key pathogenic event in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) mediated by a GGGGCC hexanucleotide repeat expansion in C9ORF72, the most common genetic cause of ALS/FTD. Furthermore, nucleocytoplasmic transport disruption has also been implicated in other neurodegenerative diseases with protein aggregation, suggesting a shared mechanism by which protein stress disrupts nucleocytoplasmic transport. Here, we show that cellular stress disrupts nucleocytoplasmic transport by localizing critical nucleocytoplasmic transport factors into stress granules, RNA/protein complexes that play a crucial role in ALS pathogenesis. Importantly, inhibiting stress granule assembly, such as by knocking down Ataxin-2, suppresses nucleocytoplasmic transport defects as well as neurodegeneration in C9ORF72-mediated ALS/FTD. Our findings identify a link between stress granule assembly and nucleocytoplasmic transport, two fundamental cellular processes implicated in the pathogenesis of C9ORF72-mediated ALS/FTD and other neurodegenerative diseases.

XPO7 is a tumor suppressor regulating p21CIP1-dependent senescence.

Senescence is a key barrier to neoplastic transformation. To identify senescence regulators relevant to cancer, we screened a genome-wide shRNA library. Here, we describe exportin 7 (XPO7) as a novel regulator of senescence and validate its function in telomere-induced, replicative, and oncogene-induced senescence (OIS). XPO7 is a bidirectional transporter that regulates the nuclear-cytoplasmic shuttling of a broad range of substrates. Depletion of XPO7 results in reduced levels of TCF3 and an impaired induction of the cyclin-dependent kinase inhibitor p21CIP1 during OIS. Deletion of XPO7 correlates with poorer overall survival in several cancer types. Moreover, depletion of XPO7 alleviated OIS and increased tumor formation in a mouse model of liver cancer. Our results suggest that XPO7 is a novel tumor suppressor that regulates p21CIP1 expression to control senescence and tumorigenesis.

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

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

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

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

Impeding Transcription of Expanded Microsatellite Repeats by Deactivated Cas9.

Transcription of expanded microsatellite repeats is associated with multiple human diseases, including myotonic dystrophy, Fuchs endothelial corneal dystrophy, and C9orf72-ALS/FTD. Reducing production of RNA and proteins arising from these expanded loci holds therapeutic benefit. Here, we tested the hypothesis that deactivated Cas9 enzyme impedes transcription across expanded microsatellites. We observed a repeat length-, PAM-, and strand-dependent reduction of repeat-containing RNAs upon targeting dCas9 directly to repeat sequences; targeting the non-template strand was more effective. Aberrant splicing patterns were rescued in DM1 cells, and production of RAN peptides characteristic of DM1, DM2, and C9orf72-ALS/FTD cells was drastically decreased. Systemic delivery of dCas9/gRNA by adeno-associated virus led to reductions in pathological RNA foci, rescue of chloride channel 1 protein expression, and decreased myotonia. These observations suggest that transcription of microsatellite repeat-containing RNAs is more sensitive to perturbation than transcription of other RNAs, indicating potentially viable strategies for therapeutic intervention.

Augmin is a Ran-regulated spindle assembly factor.

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

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

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

RanGTP and importin ß regulate meiosis I spindle assembly and function in mouse oocytes.

Homologous chromosome segregation during meiosis I (MI) in mammalian oocytes is carried out by the acentrosomal MI spindles. Whereas studies in human oocytes identified Ran GTPase as a crucial regulator of the MI spindle function, experiments in mouse oocytes questioned the generality of this notion. Here, we use live-cell imaging with fluorescent probes and Förster resonance energy transfer (FRET) biosensors to monitor the changes in Ran and importin ß signaling induced by perturbations of Ran in mouse oocytes while examining the MI spindle dynamics. We show that unlike RanT24N employed in previous studies, a RanT24N, T42A double mutant inhibits RanGEF without perturbing cargo binding to importin ß and disrupts MI spindle function in chromosome segregation. Roles of Ran and importin ß in the coalescence of microtubule organizing centers (MTOCs) and MI spindle assembly are further supported by the use of the chemical inhibitor importazole, whose effects are partially rescued by the GTP hydrolysis-resistant RanQ69L mutant. These results indicate that RanGTP is essential for MI spindle assembly and function both in humans and mice.

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

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

Nuclear lipid droplets and nuclear damage in Caenorhabditis elegans.

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

On the asymmetric partitioning of nucleocytoplasmic transport - recent insights and open questions.

Macromolecular cargoes are asymmetrically partitioned in the nucleus or cytoplasm by nucleocytoplasmic transport (NCT). At the center of this activity lies the nuclear pore complex (NPC), through which soluble factors circulate to orchestrate NCT. These include cargo-carrying importin and exportin receptors from the ß-karyopherin (Kapß) family and the small GTPase Ran, which switches between guanosine triphosphate (GTP)- and guanosine diphosphate (GDP)-bound forms to regulate cargo delivery and compartmentalization. Ongoing efforts have shed considerable light on how these soluble factors traverse the NPC permeability barrier to sustain NCT. However, this does not explain how importins and exportins are partitioned in the cytoplasm and nucleus, respectively, nor how a steep RanGTP-RanGDP gradient is maintained across the nuclear envelope. In this Review, we peel away the multiple layers of control that regulate NCT and juxtapose unresolved features against known aspects of NPC function. Finally, we discuss how NPCs might function synergistically with Kapßs, cargoes and Ran to establish the asymmetry of NCT.

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

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

Separable roles for RanGTP in nuclear and ciliary trafficking of a kinesin-2 subunit.

Kinesin is part of the microtubule-binding motor protein superfamily, which serves important roles in cell division and intraorganellar transport. The heterotrimeric kinesin-2, consisting of the heterodimeric motor subunits, kinesin family member 3A/3B (KIF3A/3B), and kinesin-associated protein 3 (KAP3), is highly conserved across species from the unicellular eukaryote Chlamydomonas to humans. It plays diverse roles in cargo transport including anterograde (base to tip) trafficking in cilia. However, the molecular determinants mediating trafficking of heterotrimeric kinesin-2 itself are poorly understood. It has been previously suggested that ciliary transport is analogous to nuclear transport mechanisms. Using Chlamydomonas and human telomerase reverse transcriptase-retinal pigment epithelial cell line, we show that RanGTP, a small GTPase that dictates nuclear transport, regulates ciliary trafficking of KAP3, a key component for functional kinesin-2. We found that the armadillo-repeat region 6 to 9 (ARM6-9) of KAP3, required for its nuclear translocation, is also necessary and sufficient for its targeting to the ciliary base. Given that KAP3 is essential for cilium formation and there are the emerging roles for RanGTP/importin ß in ciliary protein targeting, we further investigated the effect of RanGTP in cilium formation and maintenance. We found that precise control of RanGTP levels, revealed by different Ran mutants, is crucial for cilium formation and maintenance. Most importantly, we were able to provide orthogonal support in an algal model system that segregates RanGTP regulation of ciliary protein trafficking from its nuclear roles. Our work provides important support for the model that nuclear import mechanisms have been co-opted for independent roles in ciliary import.

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

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

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

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

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

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

Artesunate inhibits intestinal tumorigenesis through inhibiting wnt signaling.

Artesunate (ART) is a clinically approved antimalarial drug and was revealed as a candidate of colorectal cancer chemopreventive agents in our drug screening system. Here, we aimed to understand the suppressive effects of ART on intestinal tumorigenesis. In vitro, ART reduced T-cell factor/lymphoid enhancer factor (TCF/LEF) promoter transcriptional activity. In vivo, ART inhibited intestinal polyp development. We found that ART reduces TCF1/TCF7 nuclear translocation by binding the Ras-related nuclear protein (RAN), suggesting that ART inhibits TCF/LEF transcriptional factor nuclear translocation by binding to RAN, thereby inhibiting Wnt signaling. Our results provide a novel mechanism through which artesunate inhibits intestinal tumorigenesis.

Initiation of DNA replication requires actin dynamics and formin activity.

Nuclear actin regulates transcriptional programmes in a manner dependent on its levels and polymerisation state. This dynamics is determined by the balance of nucleocytoplasmic shuttling, formin- and redox-dependent filament polymerisation. Here, using Xenopus egg extracts and human somatic cells, we show that actin dynamics and formins are essential for DNA replication. In proliferating cells, formin inhibition abolishes nuclear transport and initiation of DNA replication, as well as general transcription. In replicating nuclei from transcriptionally silent Xenopus egg extracts, we identified numerous actin regulators, and disruption of actin dynamics abrogates nuclear transport, preventing NLS (nuclear localisation signal)-cargo release from RanGTP-importin complexes. Nuclear formin activity is further required to promote loading of cyclin-dependent kinase (CDK) and proliferating cell nuclear antigen (PCNA) onto chromatin, as well as initiation and elongation of DNA replication. Therefore, actin dynamics and formins control DNA replication by multiple direct and indirect mechanisms.