SUMOylation modulates the function of DDX19 in mRNA export.

Nuclear export of mRNAs is a critical regulatory step in eukaryotic gene expression. The mRNA transcript undergoes extensive processing, and is loaded with a set of RNA-binding proteins (RBPs) to form export-competent messenger ribonucleoprotein particles (mRNPs) in the nucleus. During the transit of mRNPs through the nuclear pore complex (NPC), the DEAD-box ATPase - DDX19 (herein referring to DDX19A and DDX19B) - remodels mRNPs at the cytoplasmic side of the NPC, by removing a subset of RNA-binding proteins to terminate mRNP export. This requires the RNA-dependent ATPase activity of DDX19 and its dynamic interactions with Gle1 and Nup214. However, the regulatory mechanisms underlying these interactions are unclear. We find that DDX19 gets covalently attached with a small ubiquitin-like modifier (SUMO) at lysine 26, which enhances its interaction with Gle1. Furthermore, a SUMOylation-defective mutant of human DDX19B, K26R, failed to provide a complete rescue of the mRNA export defect caused by DDX19 depletion. Collectively, our results suggest that SUMOylation fine-tunes the function of DDX19 in mRNA export by regulating its interaction with Gle1. This study identifies SUMOylation of DDX19 as a modulatory mechanism during the mRNA export process. This article has an associated First Person interview with the first author of the paper.

RanGTPase links nucleo-cytoplasmic transport to the recruitment of cargoes into small extracellular vesicles.

Small extracellular vesicle (sEV)-mediated intercellular communication regulates multiple aspects of growth and development in multicellular organisms. However, the mechanism underlying cargo recruitment into sEVs is currently unclear. We show that the key nucleo-cytoplasmic transport (NCT) protein-RanGTPase, in its GTP-bound form (RanGTP), is enriched in sEVs secreted by mammalian cells. This recruitment of RanGTP into sEVs depends on the export receptor CRM1 (also called XPO1). The recruitment of GAPDH, a candidate cargo protein, into sEVs is regulated by the RanGTP-CRM1axis in a nuclear export signal (NES)-dependent manner. Perturbation of NCT through overexpression or depletion of nuclear transport components affected the recruitment of Ran, CRM1 and GAPDH into sEVs. Our studies, thus, suggest a link between NCT, particularly the Ran-CRM1 axis, and recruitment of NES-containing cargoes into the sEVs. Collectively, these findings implicate RanGTPase as a link between NCT and sEV mediated intercellular communication.

Nup358 regulates microridge length by controlling SUMOylation-dependent activity of aPKC in zebrafish epidermis.

The Par polarity complex, consisting of Par3, Par6 and atypical protein kinase C (aPKC), plays a crucial role in the establishment and maintenance of cell polarity. Although activation of aPKC is critical for polarity, how this is achieved is unclear. The developing zebrafish epidermis, along with its apical actin-based projections, called microridges, offers a genetically tractable system for unraveling the mechanisms of the cell polarity control. The zebrafish aPKC regulates elongation of microridges by controlling levels of apical Lgl, which acts as a pro-elongation factor. Here, we show that the nucleoporin Nup358 (also known as RanBP2) - a component of the nuclear pore complex and a part of cytoplasmic annulate lamellae (AL) - SUMOylates zebrafish aPKC. Nup358-mediated SUMOylation controls aPKC activity to regulate Lgl-dependent microridge elongation. Our data further suggest that cytoplasmic AL structures are the possible site for Nup358-mediated aPKC SUMOylation. We have unraveled a hitherto unappreciated contribution of Nup358-mediated aPKC SUMOylation in cell polarity regulation.This article has an associated First Person interview with the first author of the paper.

Acute necrotizing encephalopathy-linked mutations in Nup358 impair interaction of Nup358 with TNRC6/GW182 and miRNA function.

MicroRNA (miRNA)-mediated translational suppression of mRNAs is involved in the regulation of multiple cellular processes. A recent study showed that Nup358, a protein mutated in a neurological disorder called acute necrotizing encephalopathy 1 (ANE1), helps in the coupling of miRNA-induced silencing complex (miRISC) - consisting of miRNA, AGO and GW182/TNRC6 proteins - with the target mRNA. Here we provide a detailed characterization of the interaction between Nup358 and GW182. We identified that the N-terminal region of Nup358 directly interacts with the C-terminal silencing domain of GW182. Interestingly, ANE1-associated Nup358 mutants display reduced interaction with GW182. Consistent with this, one of the prevalent ANE1 mutations, 585th threonine (T) residue changed to methionine (M) [T585M] compromised Nup358's ability to function in the miRNA pathway. Collectively, these results suggest that the ANE1-associated mutations in Nup358 might affect the miRNA pathway and contribute to the development of ANE1.

The miRISC component AGO2 has multiple binding sites for Nup358 SUMO-interacting motif.

Micro-RNA mediated suppression of mRNA translation represents a major regulatory mode of post-transcriptional gene expression. Recently, the nucleoporin Nup358 was shown to interact with AGO protein, a key component of miRNA-induced silencing complex (miRISC), and facilitate the coupling of miRISC with target mRNA. Previous results suggested that SUMO-interacting motifs (SIMs) present on Nup358 mediate interaction with AGO protein. Here we show that Nup358-SIM has multiple interacting regions on AGO2, specifically within the N, PAZ and MID domains, with an affinity comparable to SIM-SUMO1 interaction. The study also unraveled specific residues involved in the interaction of AGO2 with miRNA-loading components such as Dicer and HSP90. Collectively, the results support the conclusion that multiple SIMs contribute to the association of Nup358 with AGO2.

Nup358 binds to AGO proteins through its SUMO-interacting motifs and promotes the association of target mRNA with miRISC.

MicroRNA (miRNA)-guided mRNA repression, mediated by the miRNA-induced silencing complex (miRISC), is an important component of post-transcriptional gene silencing. However, how miRISC identifies the target mRNA in vivo is not well understood. Here, we show that the nucleoporin Nup358 plays an important role in this process. Nup358 localizes to the nuclear pore complex and to the cytoplasmic annulate lamellae (AL), and these structures dynamically associate with two mRNP granules: processing bodies (P bodies) and stress granules (SGs). Nup358 depletion disrupts P bodies and concomitantly impairs the miRNA pathway. Furthermore, Nup358 interacts with AGO and GW182 proteins and promotes the association of target mRNA with miRISC A well-characterized SUMO-interacting motif (SIM) in Nup358 is sufficient for Nup358 to directly bind to AGO proteins. Moreover, AGO and PIWI proteins interact with SIMs derived from other SUMO-binding proteins. Our study indicates that Nup358-AGO interaction is important for miRNA-mediated gene silencing and identifies SIM as a new interacting motif for the AGO family of proteins. The findings also support a model wherein the coupling of miRISC with the target mRNA could occur at AL, specialized domains within the ER, and at the nuclear envelope.

Global regulator SATB1 recruits beta-catenin and regulates T(H)2 differentiation in Wnt-dependent manner.

In vertebrates, the conserved Wnt signalling cascade promotes the stabilization and nuclear accumulation of beta-catenin, which then associates with the lymphoid enhancer factor/T cell factor proteins (LEF/TCFs) to activate target genes. Wnt/beta -catenin signalling is essential for T cell development and differentiation. Here we show that special AT-rich binding protein 1 (SATB1), the T lineage-enriched chromatin organizer and global regulator, interacts with beta-catenin and recruits it to SATB1's genomic binding sites. Gene expression profiling revealed that the genes repressed by SATB1 are upregulated upon Wnt signalling. Competition between SATB1 and TCF affects the transcription of TCF-regulated genes upon beta-catenin signalling. GATA-3 is a T helper type 2 (T(H)2) specific transcription factor that regulates production of T(H)2 cytokines and functions as T(H)2 lineage determinant. SATB1 positively regulated GATA-3 and siRNA-mediated knockdown of SATB1 downregulated GATA-3 expression in differentiating human CD4(+) T cells, suggesting that SATB1 influences T(H)2 lineage commitment by reprogramming gene expression. In the presence of Dickkopf 1 (Dkk1), an inhibitor of Wnt signalling, GATA-3 is downregulated and the expression of signature T(H)2 cytokines such as IL-4, IL-10, and IL-13 is reduced, indicating that Wnt signalling is essential for T(H)2 differentiation. Knockdown of beta-catenin also produced similar results, confirming the role of Wnt/beta-catenin signalling in T(H)2 differentiation. Furthermore, chromatin immunoprecipitation analysis revealed that SATB1 recruits beta-catenin and p300 acetyltransferase on GATA-3 promoter in differentiating T(H)2 cells in a Wnt-dependent manner. SATB1 coordinates T(H)2 lineage commitment by reprogramming gene expression. The SATB1:beta-catenin complex activates a number of SATB1 regulated genes, and hence this study has potential to find novel Wnt responsive genes. These results demonstrate that SATB1 orchestrates T(H)2 lineage commitment by mediating Wnt/beta-catenin signalling. This report identifies a new global transcription factor involved in beta-catenin signalling that may play a major role in dictating the functional outcomes of this signalling pathway during development, differentiation, and tumorigenesis.

Crm1 is a mitotic effector of Ran-GTP in somatic cells.

The Ran GTPase controls multiple cellular processes, including nuclear transport, mitotic checkpoints, spindle assembly and post-mitotic nuclear envelope reassembly. Here we examine the mitotic function of Crm1, the Ran-GTP-binding nuclear export receptor for leucine-rich cargo (bearing nuclear export sequence) and Snurportin-1 (ref. 3). We find that Crm1 localizes to kinetochores, and that Crm1 ternary complex assembly is essential for Ran-GTP-dependent recruitment of Ran GTPase-activating protein 1 (Ran-GAP1) and Ran-binding protein 2 (Ran-BP2) to kinetochores. We further show that Crm1 inhibition by leptomycin B disrupts mitotic progression and chromosome segregation. Analysis of spindles within leptomycin B-treated cells shows that their centromeres were under increased tension. In leptomycin B-treated cells, centromeres frequently associated with continuous microtubule bundles that spanned the centromeres, indicating that their kinetochores do not maintain discrete end-on attachments to single kinetochore fibres. Similar spindle defects were observed in temperature-sensitive Ran pathway mutants (tsBN2 cells). Taken together, our findings demonstrate that Crm1 and Ran-GTP are essential for Ran-BP2/Ran-GAP1 recruitment to kinetochores, for definition of kinetochore fibres and for chromosome segregation at anaphase. Thus, Crm1 is a critical Ran-GTP effector for mitotic spindle assembly and function in somatic cells.

Workshop on RanBP2/Nup358 and acute necrotizing encephalopathy.

Dominant missense mutations in RanBP2/Nup358 cause Acute Necrotizing Encephalopathy (ANE), a pediatric disease where seemingly healthy individuals develop a cytokine storm that is restricted to the central nervous system in response to viral infection. Untreated, this condition leads to seizures, coma, long-term neurological damage and a high rate of mortality. The exact mechanism by which RanBP2 mutations contribute to the development of ANE remains elusive. In November 2021, a number of clinicians and basic scientists presented their work on this disease and on the interactions between RanBP2/Nup358, viral infections, the innate immune response and other cellular processes.

Nup358 interacts with APC and plays a role in cell polarization.

Asymmetric localization of adenomatous polyposis coli (APC) to the ends of a subset of microtubules located in the leading edges is essential for the establishment of front-rear polarity during cell migration. APC is known to associate with microtubules in three ways: through interaction with the plus-end tracking protein EB1, direct binding through a C-terminal basic region, and through interaction with the plus-end motor kinesin-2. Here we report that the middle region of APC has a previously unidentified microtubule plus-end-targeting function, suggesting an additional microtubule-binding mode for APC. Through the same region, APC interacts with Nup358 (also called RanBP2), a microtubule-binding nucleoporin. Ectopic expression of the middle region of APC is sufficient to recruit endogenous Nup358 to the plus ends of microtubules. Furthermore, our results indicate that Nup358 cooperates with kinesin-2 to regulate the localization of APC to the cell cortex through a nuclear-transport-independent mechanism. Using RNA interference and a scratch-induced wound-healing assay we demonstrate that Nup358 functions in polarized cell migration. These results reveal a more active role for structural nucleoporins in regulating fundamental cellular processes than previously anticipated.

AMPK: a key regulator of energy stress and calcium-induced autophagy.

Autophagy is a well-known cell-survival strategy orchestrated by a conserved set of proteins. It equips the cells with mechanisms to attain homeostasis during unfavorable conditions such as stress by breaking down the cellular components and reusing them for energy as well as for building new components required for survival. A basal level of autophagy is required for achieving homeostasis under normal conditions through regular turnover of macromolecules and organelles. Initiation of autophagy is regulated by two key components of the nutrient/energy sensor pathways; mammalian target of rapamycin 1 (mTORC1) and AMP-activated kinase (AMPK). Under energy-deprived conditions, AMPK is activated triggering autophagy, whereas, in nutrient-rich conditions, the growth-promoting kinase mTORC1 is activated inhibiting autophagy. Thus, the reciprocal regulation of autophagy by AMPK and mTORC1 defines a fundamental mechanism by which cells respond to nutrient availability. Interestingly, cytoplasmic calcium is also found to be an activator of AMPK and autophagy through a calmodulin/CaMKKß pathway. However, the physiological significance of the regulation of autophagy by cytoplasmic calcium is currently unclear. This review focuses on the current understanding of the mechanism of autophagy and its regulation by AMPK.

SUMO-1 targets RanGAP1 to kinetochores and mitotic spindles.

RanGAP1 was the first documented substrate for conjugation with the ubiquitin-like protein SUMO-1. However, the functional significance of this conjugation has not been fully clarified. We sought to examine RanGAP1 behavior during mitosis. We found that RanGAP1 associates with mitotic spindles and that it is particularly concentrated at foci near kinetochores. Association with kinetochores appeared soon after nuclear envelope breakdown and persisted until late anaphase, but it was lost coincident with nuclear envelope assembly in telophase. A mutant RanGAP1 protein lacking the capacity to be conjugated to SUMO-1 no longer associated with spindles, indicating that conjugation was essential for RanGAP1's mitotic localization. RanBP2, a nuclear pore protein that binds SUMO-1-conjugated RanGAP1 during interphase, colocalized with RanGAP1 on spindles, suggesting that a complex between these two proteins may be involved in mitotic targeting of RanGAP1. This report shows for the first time that SUMO-1 conjugation is required for mitotic localization of RanGAP1, and suggests that a major role of SUMO-1 conjugation to RanGAP1 may be the spatial regulation of the Ran pathway during mitosis.

Nuclear envelope breakdown is coordinated by both Nup358/RanBP2 and Nup153, two nucleoporins with zinc finger modules.

When higher eukaryotic cells transition into mitosis, the nuclear envelope, nuclear pore complexes, and nuclear lamina are coordinately disassembled. The COPI coatomer complex, which plays a major role in membrane remodeling at the Golgi, has been implicated in the process of nuclear envelope breakdown and requires interactions at the nuclear pore complex for recruitment to this new site of action at mitosis. Nup153, a resident of the nuclear pore basket, was found to be involved in COPI recruitment, but the molecular nature of the interface between COPI and the nuclear pore has not been fully elucidated. To better understand what occurs at the nuclear pore at this juncture, we have probed the role of the nucleoporin Nup358/RanBP2. Nup358 contains a repetitive zinc finger domain with overall organization similar to a region within Nup153 that is critical to COPI association, yet inspection of these two zinc finger domains reveals features that also clearly distinguish them. Here, we found that the Nup358 zinc finger domain, but not a zinc finger domain from an unrelated protein, binds to COPI and dominantly inhibits progression of nuclear envelope breakdown in an assay that robustly recapitulates this process in vitro. Moreover, the Nup358 zinc finger domain interferes with COPI recruitment to the nuclear rim. Consistent with a role for this pore protein in coordinating nuclear envelope breakdown, Nup358-specific antibodies impair nuclear disassembly. Significantly, targeting either Nup153 or Nup358 for inhibition perturbs nuclear envelope breakdown, supporting a model in which these nucleoporins play nonredundant roles, perhaps contributing to COPI recruitment platforms on both the nuclear and cytoplasmic faces of the pore. We found that an individual zinc finger is the minimal interface for COPI association, although tandem zinc fingers are optimal. These results provide new information about the critical components of nuclear membrane remodeling and lay the foundation for a better understanding of how this process is regulated.

The nucleoporin Nup358 associates with and regulates interphase microtubules.

The nucleoporin Nup358 resides on the cytoplasmic face of the interphase nuclear pore complex (NPC). During metaphase, its recruitment to kinetochores is important for correct microtubule-kinetochore attachment. Here, we report that a fraction of endogenous Nup358 interacts with interphase microtubules through its N-terminal region (BPN). Cells overexpressing the microtubule targeting domain of Nup358 displayed dramatic alteration in the microtubule organization including increased microtubule bundling and stability. Ectopic expression of BPN and full-length Nup358 exhibited significantly higher levels of acetylated microtubules that were resistant to nocodazole, a microtubule depolymerizing agent. Furthermore, RNAi mediated depletion of Nup358 affected polarized stabilization of microtubules during directed cell migration, confirming the in vivo role of Nup358 in regulating interphase microtubules.

The RanGAP1-RanBP2 complex is essential for microtubule-kinetochore interactions in vivo.

RanGAP1 is the activating protein for the Ran GTPase. Vertebrate RanGAP1 is conjugated to a small ubiquitin-like protein, SUMO-1. This modification promotes association of RanGAP1 with the interphase nuclear pore complex (NPC) through binding to the nucleoporin RanBP2, also known as Nup358. During mitosis, RanGAP1 is concentrated at kinetochores in a microtubule- (MT) and SUMO-1-dependent fashion. RanBP2 is also abundantly found on kinetochores in mitosis. Here we show that ablation of proteins required for MT-kinetochore attachment (Hec1/Ndc80, Nuf2 ) disrupts RanGAP1 and RanBP2 targeting to kinetochores. No similar disruption was observed after ablation of proteins nonessential for MT-kinetochore interactions (CENP-I, Bub1, CENP-E ). Acquisition of RanGAP1 and RanBP2 by kinetochores is temporally correlated in untreated cells with MT attachment. These patterns of accumulation suggest a loading mechanism wherein the RanGAP1-RanBP2 complex may be transferred along the MT onto the kinetochore. Depletion of RanBP2 caused mislocalization of RanGAP1, Mad1, Mad2, CENP-E, and CENP-F, as well as loss of cold-stable kinetochore-MT interactions and accumulation of mitotic cells with multipolar spindles and unaligned chromosomes. Taken together, our observations indicate that RanBP2 and RanGAP1 are targeted as a single complex that is both regulated by and essential for stable kinetochore-MT association.

Selective recruitment of nucleoporins on vaccinia virus factories and the role of Nup358 in viral infection.

Vaccinia virus (VACV), a member of the Poxviridae family, uses cytoplasmic factories for its replication. Recent studies indicated that VACV infection requires a set of nucleoporins. However, how the nucleoporins contribute to viral life cycle remains unclear. Here, we report that the nucleoporins Nup62 and Nup358 localize to the cytoplasmic viral factories (VFs). Nup358 was targeted to the VFs at 6h post-infection (hpi), whereas Nup62, along with the previously reported translation factors such as eIF4E, eIF3η and G3BP1, was recruited to the VFs at 8 hpi. Nup358 depletion led to a decrease in the size and number of viral factories and reduction in viral yield. Further studies showed that Nup358 is involved in recruiting Nup62 and eIF4E to the VFs. Collectively, our results reveal spatio-temporal regulation in the recruitment of nucleoporins and translation factors to VFs, and particularly the importance of Nup358 in VACV infection.

Regulation of aPKC activity by Nup358 dependent SUMO modification.

Atypical PKC (aPKC) family members are involved in regulation of diverse cellular processes, including cell polarization. aPKCs are known to be activated by phosphorylation of specific threonine residues in the activation loop and turn motif. They can also be stimulated by interaction with Cdc42~GTP-Par6 complex. Here we report that PKCζ, a member of the aPKC family, is activated by SUMOylation. We show that aPKC is endogenously modified by SUMO1 and the nucleoporin Nup358 acts as its SUMO E3 ligase. Results from in vitro SUMOylation and kinase assays showed that the modification enhances the kinase activity of PKCζ by ~10-fold. By monitoring the phosphorylation of Lethal giant larvae (Lgl), a downstream target of aPKC, we confirmed these findings in vivo. Consistent with the function of Nup358 as a SUMO E3 ligase for aPKC, depletion of Nup358 attenuated the extent of SUMOylation and the activity of aPKC. Moreover, overexpression of the C-terminal fragment of Nup358 that possesses the E3 ligase activity enhanced SUMOylation of endogenous aPKC and its kinase activity. Collectively, our studies reveal a role for Nup358-dependent SUMOylation in the regulation of aPKC activity and provide a framework for understanding the role of Nup358 in cell polarity.

Glucose induced activation of canonical Wnt signaling pathway in hepatocellular carcinoma is regulated by DKK4.

Elevated glycemic index, an important feature of diabetes is implicated in an increased risk of hepatocellular carcinoma (HCC). However, the underlying molecular mechanisms of this association are relatively less explored. Present study investigates the effect of hyperglycemia over HCC proliferation. We observed that high glucose culture condition (HG) specifically activates canonical Wnt signaling in HCC cells, which is mediated by suppression of DKK4 (a Wnt antagonist) expression and enhanced ß-catenin level. Functional assays demonstrated that a normoglycemic culture condition (NG) maintains constitutive expression of DKK4, which controls HCC proliferation rate by suppressing canonical Wnt signaling pathway. HG diminishes DKK4 expression leading to loss of check at G0/G1/S phases of the cell cycle thereby enhancing HCC proliferation, in a ß-catenin dependent manner. Interestingly, in NOD/SCID mice supplemented with high glucose, HepG2 xenografted tumors grew rapidly in which elevated levels of ß-catenin, c-Myc and decreased levels of DKK4 were detected. Knockdown of DKK4 by shRNA promotes proliferation of HCC cells in NG, which is suppressed by treating cells exogenously with recombinant DKK4 protein. Our in vitro and in vivo results indicate an important functional role of DKK4 in glucose facilitated HCC proliferation.

Inter-cellular transport of ran GTPase.

Ran, a member of the Ras-GTPase superfamily, has a well-established role in regulating the transport of macromolecules across the nuclear envelope (NE). Ran has also been implicated in mitosis, cell cycle progression, and NE formation. Over-expression of Ran is associated with various cancers, although the molecular mechanism underlying this phenomenon is unclear. Serendipitously, we found that Ran possesses the ability to move from cell-to-cell when transiently expressed in mammalian cells. Moreover, we show that the inter-cellular transport of Ran is GTP-dependent. Importantly, Ran displays a similar distribution pattern in the recipient cells as that in the donor cell and co-localizes with the Ran binding protein Nup358 (also called RanBP2). Interestingly, leptomycin B, an inhibitor of CRM1-mediated export, or siRNA mediated depletion of CRM1, significantly impaired the inter-cellular transport of Ran, suggesting a function for CRM1 in this process. These novel findings indicate a possible role for Ran beyond nucleo-cytoplasmic transport, with potential implications in inter-cellular communication and cancers.