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.

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.

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.

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.

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 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.

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.

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.

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.

miR-196b-mediated translation regulation of mouse insulin2 via the 5'UTR.

The 5' and the 3' untranslated regions (UTR) of the insulin genes are very well conserved across species. Although microRNAs (miRNAs) are known to regulate insulin secretion process, direct regulation of insulin biosynthesis by miRNA has not been reported. Here, we show that mouse microRNA miR-196b can specifically target the 5'UTR of the long insulin2 splice isoform. Using reporter assays we show that miR-196b specifically increases the translation of the reporter protein luciferase. We further show that this translation activation is abolished when Argonaute 2 levels are knocked down after transfection with an Argonaute 2-directed siRNA. Binding of miR-196b to the target sequence in insulin 5'UTR causes the removal of HuD (a 5'UTR-associated translation inhibitor), suggesting that both miR-196b and HuD bind to the same RNA element. We present data suggesting that the RNA-binding protein HuD, which represses insulin translation, is displaced by miR-196b. Together, our findings identify a mechanism of post-transcriptional regulation of insulin biosynthesis.

Nup358 interacts with Dishevelled and aPKC to regulate neuronal polarity.

Par polarity complex, consisting of Par3, Par6, and aPKC, plays a conserved role in the establishment and maintenance of polarization in diverse cellular contexts. Recent reports suggest that Dishevelled (Dvl), a cytoplasmic mediator of Wnt signalling, interacts with atypical protein kinase C and regulates its activity during neuronal differentiation and directed cell migration. Here we show that Nup358 (also called RanBP2), a nucleoporin previously implicated in polarity during directed cell migration, interacts with Dishevelled and aPKC through its N-terminal region (BPN) and regulates axon-dendrite differentiation of cultured hippocampal neurons. Depletion of endogenous Nup358 leads to generation of multiple axons, whereas overexpression of BPN abrogates the process of axon formation. Moreover, siRNA-mediated knockdown of Dvl or inhibition of aPKC by a pseudosubstrate inhibitor significantly reverses the multiple axon phenotype produced by Nup358 depletion. Collectively, these data suggest that Nup358 plays an important role in regulating neuronal polarization upstream to Dvl and aPKC.

Serological and molecular studies of a novel virus isolate causing yellow mosaic of Patchouli [Pogostemon cablin (Blanco) Benth].

Here we have identified and characterized a devastating virus capable of inducing yellow mosaic on the leaves of Patchouli [Pogostemon cablin (Blanco) Benth]. The diagnostic tools used were host range, transmission studies, cytopathology, electron microscopy, serology and partial coat protein (CP) gene sequencing. Evidence from biological, serological and sequence data suggested that the causal virus belonged to genus Potyvirus, family Potyviridae. The isolate, designated as Patchouli Yellow Mosaic Virus (PaYMV), was transmitted through grafting, sap and the insect Myzus persicae (Sulz.). Flexuous rod shaped particles with a mean length of 800 nm were consistently observed in leaf-dip preparations from natural as well as alternate hosts, and in purified preparation. Cytoplasmic cylindrical inclusions, pinwheels and laminar aggregates were observed in ultra-thin sections of infected patchouli leaves. The purified capsid protein has a relative mass of 43 kDa. Polyclonal antibodies were raised in rabbits against the coat protein separated on SDS - PAGE; which were used in ELISA and western blotting. Using specific antibodies in ELISA, PaYMV was frequently detected at patchouli plantations at Lucknow and Bengaluru. Potyvirus-specific degenerate primer pair (U335 and D335) had consistently amplified partial CP gene from crude preparations of infected tissues by reverse transcription polymerase chain reaction (RT-PCR). Comparison of the PCR product sequence (290 bp) with the corresponding regions of established potyviruses showed 78-82% and 91-95% sequence similarity at the nucleotide and amino acid levels, respectively. The results clearly established that the virus under study has close homology with watermelon mosaic virus (WMV) in the coat protein region and therefore could share a common ancestor family. Further studies are required to authenticate the identity of PaYMV as a distinct virus or as an isolate of WMV.

Wnt signalling antagonizes stress granule assembly through a Dishevelled-dependent mechanism.

Cells often respond to diverse environmental stresses by inducing stress granules (SGs) as an adaptive mechanism. SGs are generally assembled as a result of aggregation of mRNAs stalled in a translational pre-initiation complex, mediated by a set of RNA-binding proteins such as G3BP and TIA-1. SGs may serve as triage centres for storage, translation re-initiation or degradation of specific mRNAs. However, the mechanism involved in the modulation of their assembly/disassembly is unclear. Here we report that Wnt signalling negatively regulates SG assembly through Dishevelled (Dvl), a cytoplasmic Wnt effector. Overexpression of Dvl2, an isoform of Dvl, leads to impairment of SG assembly through a DEP domain dependent mechanism. Intriguingly, the Dvl2 mutant K446M, which corresponds to an analogous mutation in Drosophila Dishevelled DEP domain (dsh(1)) that results in defective PCP pathway, fails to antagonize SG assembly. Furthermore, we show that Dvl2 exerts the antagonistic effect on SG assembly through a mechanism involving Rac1-mediated inhibition of RhoA. Dvl2 interacts with G3BP, a downstream component of Ras signalling involved in SG assembly, and functional analysis suggests a model wherein the Dvl-Rac1-RhoA axis regulates G3BP's SG-nucleating activity. Collectively, these results define an antagonistic effect of Wnt signalling on SG assembly, and reveal a novel role for Wnt/Dvl pathway in the modulation of mRNA functions.

Ran-dependent docking of importin-beta to RanBP2/Nup358 filaments is essential for protein import and cell viability.

RanBP2/Nup358, the major component of the cytoplasmic filaments of the nuclear pore complex (NPC), is essential for mouse embryogenesis and is implicated in both macromolecular transport and mitosis, but its specific molecular functions are unknown. Using RanBP2 conditional knockout mouse embryonic fibroblasts and a series of mutant constructs, we show that transport, rather than mitotic, functions of RanBP2 are required for cell viability. Cre-mediated RanBP2 inactivation caused cell death with defects in M9- and classical nuclear localization signal (cNLS)-mediated protein import, nuclear export signal-mediated protein export, and messenger ribonucleic acid export but no apparent mitotic failure. A short N-terminal RanBP2 fragment harboring the NPC-binding domain, three phenylalanine-glycine motifs, and one Ran-binding domain (RBD) corrected all transport defects and restored viability. Mutation of the RBD within this fragment caused lethality and perturbed binding to Ran guanosine triphosphate (GTP)-importin-ß, accumulation of importin-ß at nuclear pores, and cNLS-mediated protein import. These data suggest that a critical function of RanBP2 is to capture recycling RanGTP-importin-ß complexes at cytoplasmic fibrils to allow for adequate cNLS-mediated cargo import.

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.

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.