Functions of Gle1 are governed by two distinct modes of self-association.

Gle1 is a conserved, essential regulator of DEAD-box RNA helicases, with critical roles defined in mRNA export, translation initiation, translation termination, and stress granule formation. Mechanisms that specify which, where, and when DDXs are targeted by Gle1 are critical to understand. In addition to roles for stress-induced phosphorylation and inositol hexakisphosphate binding in specifying Gle1 function, Gle1 oligomerizes via its N-terminal domain in a phosphorylation-dependent manner. However, a thorough analysis of the role for Gle1 self-association is lacking. Here, we find that Gle1 self-association is driven by two distinct regions: a coiled-coil domain and a novel 10-amino acid aggregation-prone region, both of which are necessary for proper Gle1 oligomerization. By exogenous expression in HeLa cells, we tested the function of a series of mutations that impact the oligomerization domains of the Gle1A and Gle1B isoforms. Gle1 oligomerization is necessary for many, but not all aspects of Gle1A and Gle1B function, and the requirements for each interaction domain differ. Whereas the coiled-coil domain and aggregation-prone region additively contribute to competent mRNA export and stress granule formation, both self-association domains are independently required for regulation of translation under cellular stress. In contrast, Gle1 self-association is dispensable for phosphorylation and nonstressed translation initiation. Collectively, we reveal self-association functions as an additional mode of Gle1 regulation to ensure proper mRNA export and translation. This work also provides further insight into the mechanisms underlying human gle1 disease mutants found in prenatally lethal forms of arthrogryposis.

The general mRNA exporters Mex67 and Mtr2 play distinct roles in nuclear export of tRNAs in Trypanosoma brucei.

Transfer RNAs (tRNAs) are central players in protein synthesis, which in Eukarya need to be delivered from the nucleus to the cytoplasm by specific transport receptors, most of which belong to the evolutionarily conserved beta-importin family. Based on the available literature, we identified two candidates, Xpo-t and Xpo-5 for tRNA export in Trypanosoma brucei. However, down-regulation of expression of these genes did not disrupt the export of tRNAs to the cytoplasm. In search of alternative pathways, we tested the mRNA export complex Mex67-Mtr2, for a role in tRNA nuclear export, as described previously in yeast. Down-regulation of either exporter affected the subcellular distribution of tRNAs. However, contrary to yeast, TbMex67 and TbMtr2 accumulated different subsets of tRNAs in the nucleus. While TbMtr2 perturbed the export of all the tRNAs tested, silencing of TbMex67, led to the nuclear accumulation of tRNAs that are typically modified with queuosine. In turn, inhibition of tRNA nuclear export also affected the levels of queuosine modification in tRNAs. Taken together, the results presented demonstrate the dynamic nature of tRNA trafficking in T. brucei and its potential impact not only on the availability of tRNAs for protein synthesis but also on their modification status.

Targeting Mybbp1a suppresses HCC progression via inhibiting IGF1/AKT pathway by CpG islands hypo-methylation dependent promotion of IGFBP5.

BACKGROUND: Myb-binding protein 1A (Mybbp1a) is a nucleolar protein that can regulate rRNA metabolism, the stress response and carcinogenesis. However, the function of Mybbp1a in the progression of hepatocellular carcinoma (HCC) is unclear. We aimed to determine the role of Mybbp1a in HCC and the underlying mechanism. METHODS: We investigated the function of Mybbp1a in HCC cell models and the xenograft mouse model. The relationship between Mybbp1a and IGFBP5 was found through expression profile chip. The molecular mechanism of Mybbp1a regulating IGFBP5 was proved through CO-IP, CHIP, Bisulfite Sequencing and Pyrosequencing. FINDINGS: In this study, we observed that Mybbp1a was overexpressed in HCC tissues and associated with the poor prognosis of HCC patients. Suppression of Mybbp1a led to a reduction in the proliferation and migration ability of HCC cells through inhibiting the IGF1/AKT signaling pathway. Further study found that Mybbp1a could form a complex with DNMT1 and induce aberrant hyper-methylation of CpG islands of IGFBP5, which inhibits secretion of IGFBP5 and then activates IGF1/AKT signaling pathway. INTERPRETATION: These findings extend our understanding of the function of Mybbp1a in the progression of HCC. The newly identified Mybbp1a may provide a novel biomarker for developing potential therapeutic targets of HCC. FUND: Science Technology Department of Zhejiang Province (No. 2015C03034), National Health and Family Planning Commission of China (No. 2016138643), Innovative Research Groups of National Natural Science Foundation of China (No. 81721091), Major program of National Natural Science Foundation of China (No. 91542205).

Mechanism-based inhibition of human persulfide dioxygenase by γ-glutamyl-homocysteinyl-glycine.

Hydrogen sulfide (H2S) is a signaling molecule with many beneficial effects. However, its cellular concentration is strictly regulated to avoid toxicity. Persulfide dioxygenase (PDO or ETHE1) is a mononuclear non-heme iron-containing protein in the sulfide oxidation pathway catalyzing the conversion of GSH persulfide (GSSH) to sulfite and GSH. PDO mutations result in the autosomal-recessive disorder ethylmalonic encephalopathy (EE). Here, we developed γ-glutamyl-homocysteinyl-glycine (GHcySH), in which the cysteinyl moiety in GSH is substituted with homocysteine, as a mechanism-based PDO inhibitor. Human PDO used GHcySH as an alternative substrate and converted it to GHcy-SO2H, mimicking GS-SO2H, the putative oxygenated intermediate formed with the natural substrate. Because GHcy-SO2H contains a C-S bond rather than an S-S bond in GS-SO2H, it failed to undergo the final hydrolysis step in the catalytic cycle, leading to PDO inhibition. We also characterized the biochemical penalties incurred by the L55P, T136A, C161Y, and R163W mutations reported in EE patients. The variants displayed lower iron content (1.4-11-fold) and lower thermal stability (1.2-1.7-fold) than WT PDO. They also exhibited varying degrees of catalytic impairment; the kcat/Km values for R163W, L55P, and C161Y PDOs were 18-, 42-, and 65-fold lower, respectively, and the T136A variant was most affected, with a 200-fold lower kcat/Km Like WT enzyme, these variants were inhibited by GHcySH. This study provides the first characterization of an intermediate in the PDO-catalyzed reaction and reports on deficits associated with EE-linked mutations that are distal from the active site.

Inhibition of mTOR/eIF4E by anti-viral drug ribavirin effectively enhances the effects of paclitaxel in oral tongue squamous cell carcinoma.

Upregulation of eIF4E is associated with poor clinical outcome in many human cancers and represents a potential therapeutic target. However, the function of eIF4E remains unknown in oral tongue squamous cell carcinoma (OTSCC). In this work, we show that ribavirin, an anti-viral drug, effectively augments sensitivity of OTSCC cells to paclitaxel via inhibiting mTOR/eIF4E signaling pathway. Ribavirin dose-dependently inhibits proliferation and induces apoptosis in SCC-9 and CAL27 cells. Combination of ribavirin and paclitaxel are more effective in inhibiting proliferation and inducing apoptosis in OTSCC cells. Importantly, the in vivo efficacy of ribavirin and its synergism with paclitaxel is confirmed by two independent OTSCC xenograft mouse models. Mechanistically, ribavirin significantly decreases mTOR/eIF4E signaling pathway in OTSCC cells via suppressing phosphorylation of Akt, mTOR, 4EBP1 and eIF4E. Overexpression of the phosphor-mimetic form of eIF4E (eIF4E S209D) but not the nonphosphorylatable form (eIF4E S209A) reverses the effects of ribavirin, confirming that eIF4E inhibition is the mechanism of action of ribavirin in OTSCC cells. In addition, eIF4E depletion significantly enhances the anti-proliferative and pro-apoptotic effects of paclitaxel, demonstrating the critical role of eIF4E in OTSCC cell response to paclitaxel. Our work is the first to demonstrate the efficacy of ribavirin as a single agent and synergism as combination with paclitaxel in OTSCC in vitro and in vivo. Our findings also demonstrate the therapeutic value of inhibiting eIF4E in OTSCC treatment.

Cotargeting MNK and MEK kinases induces the regression of NF1-mutant cancers.

Neurofibromin 1-mutant (NF1-mutant) cancers are driven by excessive Ras signaling; however, there are currently no effective therapies for these or other Ras-dependent tumors. While combined MEK and mTORC1 suppression causes regression of NF1-deficient malignancies in animal models, the potential toxicity of cotargeting these 2 major signaling pathways in humans may necessitate the identification of more refined, cancer-specific signaling nodes. Here, we have provided evidence that MAPK-interacting kinases (MNKs), which converge on the mTORC1 effector eIF4E, are therapeutic targets in NF1-deficient malignancies. Specifically, we evaluated primary human NF1-deficient peripheral nervous system tumors and found that MNKs are activated in the majority of tumors tested. Genetic and chemical suppression of MNKs in NF1-deficient murine tumor models and human cell lines potently cooperated with MEK inhibitors to kill these cancers through effects on eIF4E. We also demonstrated that MNK kinases are important and direct targets of cabozantinib. Accordingly, coadministration of cabozantinib and MEK inhibitors triggered dramatic regression in an aggressive genetically engineered tumor model. The cytotoxicity of this combination required the suppression of MNK-induced eIF4E phosphorylation and was not recapitulated by suppressing other cabozantinib targets. Collectively, these studies demonstrate that combined MNK and MEK suppression represents a promising therapeutic strategy for these incurable Ras-driven tumors and highlight the utility of developing selective MNK inhibitors for these and possibly other malignancies.

ORC interacts with THSC/TREX-2 and its subunits promote Nxf1 association with mRNP and mRNA export in Drosophila.

The origin recognition complex (ORC) of eukaryotes associates with the replication origins and initiates the pre-replication complex assembly. In the literature, there are several reports of interaction of ORC with different RNAs. Here, we demonstrate for the first time a direct interaction of ORC with the THSC/TREX-2 mRNA nuclear export complex. The THSC/TREX-2 was purified from the Drosophila embryonic extract and found to bind with a fraction of the ORC. This interaction occurred via several subunits and was essential for Drosophila viability. Also, ORC was associated with mRNP, which was facilitated by TREX-2. ORC subunits interacted with the Nxf1 receptor mediating the bulk mRNA export. The knockdown of Orc5 led to a drop in the Nxf1 association with mRNP, while Orc3 knockdown increased the level of mRNP-bound Nxf1. The knockdown of Orc5, Orc3 and several other ORC subunits led to an accumulation of mRNA in the nucleus, suggesting that ORC participates in the regulation of the mRNP export.

An amyotrophic lateral sclerosis-linked mutation in GLE1 alters the cellular pool of human Gle1 functional isoforms.

Amyotrophic lateral sclerosis (ALS) is a lethal late onset motor neuron disease with underlying cellular defects in RNA metabolism. In prior studies, two deleterious heterozygous mutations in the gene encoding human (h)Gle1 were identified in ALS patients. hGle1 is an mRNA processing modulator that requires inositol hexakisphosphate (IP6) binding for function. Interestingly, one hGLE1 mutation (c.1965-2A>C) results in a novel 88 amino acid C-terminal insertion, generating an altered protein. Like hGle1A, at steady state, the altered protein termed hGle1-IVS14-2A>C is absent from the nuclear envelope rim and localizes to the cytoplasm. hGle1A performs essential cytoplasmic functions in translation and stress granule regulation. Therefore, we speculated that the ALS disease pathology results from altered cellular pools of hGle1 and increased cytoplasmic hGle1 activity. GFP-hGle1-IVS14-2A>C localized to stress granules comparably to GFP-hGle1A, and rescued stress granule defects following siRNA-mediated hGle1 depletion. As described for hGle1A, overexpression of the hGle1-IVS14-2A>C protein also induced formation of larger SGs. Interestingly, hGle1A and the disease associated hGle1-IVS14-2A>C overexpression induced the formation of distinct cytoplasmic protein aggregates that appear similar to those found in neurodegenerative diseases. Strikingly, the ALS-linked hGle1-IVS14-2A>C protein also rescued mRNA export defects upon depletion of endogenous hGle1, acting in a potentially novel bi-functional manner. We conclude that the ALS-linked hGle1-c.1965-2A>C mutation generates a protein isoform capable of both hGle1A- and hGle1B-ascribed functions, and thereby uncoupled from normal mechanisms of hGle1 regulation.

Phosphorylation of eIF4E Confers Resistance to Cellular Stress and DNA-Damaging Agents through an Interaction with 4E-T: A Rationale for Novel Therapeutic Approaches.

Phosphorylation of the eukaryotic translation initiation factor eIF4E is associated with malignant progression and poor cancer prognosis. Accordingly, here we have analyzed the association between eIF4E phosphorylation and cellular resistance to oxidative stress, starvation, and DNA-damaging agents in vitro. Using immortalized and cancer cell lines, retroviral expression of a phosphomimetic (S209D) form of eIF4E, but not phospho-dead (S209A) eIF4E or GFP control, significantly increased cellular resistance to stress induced by DNA-damaging agents (cisplatin), starvation (glucose+glutamine withdrawal), and oxidative stress (arsenite). De novo accumulation of eIF4E-containing cytoplasmic bodies colocalizing with the eIF4E-binding protein 4E-T was observed after expression of phosphomimetic S209D, but not S209A or wild-type eIF4E. Increased resistance to cellular stress induced by eIF4E-S209D was lost upon knockdown of endogenous 4E-T or use of an eIF4E-W73A-S209D mutant unable to bind 4E-T. Cancer cells treated with the Mnk1/2 inhibitor CGP57380 to prevent eIF4E phosphorylation and mouse embryonic fibroblasts derived from Mnk1/2 knockout mice were also more sensitive to arsenite and cisplatin treatment. Polysome analysis revealed an 80S peak 2 hours after arsenite treatment in cells overexpressing phosphomimetic eIF4E, indicating translational stalling. Nonetheless, a selective increase was observed in the synthesis of some proteins (cyclin D1, HuR, and Mcl-1). We conclude that phosphorylation of eIF4E confers resistance to various cell stressors and that a direct interaction or regulation of 4E-T by eIF4E is required. Further delineation of this process may identify novel therapeutic avenues for cancer treatment, and these results support the use of modern Mnk1/2 inhibitors in conjunction with standard therapy.

NMD3 regulates both mRNA and rRNA nuclear export in African trypanosomes via an XPOI-linked pathway.

Trypanosomes mostly regulate gene expression through post-transcriptional mechanisms, particularly mRNA stability. However, much mRNA degradation is cytoplasmic such that mRNA nuclear export must represent an important level of regulation. Ribosomal RNAs must also be exported from the nucleus and the trypanosome orthologue of NMD3 has been confirmed to be involved in rRNA processing and export, matching its function in other organisms. Surprisingly, we found that TbNMD3 depletion also generates mRNA accumulation of procyclin-associated genes (PAGs), these being co-transcribed by RNA polymerase I with the procyclin surface antigen genes expressed on trypanosome insect forms. By whole transcriptome RNA-seq analysis of TbNMD3-depleted cells we confirm the regulation of the PAG transcripts by TbNMD3 and using reporter constructs reveal that PAG1 regulation is mediated by its 5'UTR. Dissection of the mechanism of regulation demonstrates that it is not dependent upon translational inhibition mediated by TbNMD3 depletion nor enhanced transcription. However, depletion of the nuclear export factors XPO1 or MEX67 recapitulates the effects of TbNMD3 depletion on PAG mRNAs and mRNAs accumulated in the nucleus of TbNMD3-depleted cells. These results invoke a novel RNA regulatory mechanism involving the NMD3-dependent nuclear export of mRNA cargos, suggesting a shared platform for mRNA and rRNA export.

Influenza polymerase encoding mRNAs utilize atypical mRNA nuclear export.

BACKGROUND: Influenza is a segmented negative strand RNA virus. Each RNA segment is encapsulated by influenza nucleoprotein and bound by the viral RNA dependent RNA polymerase (RdRP) to form viral ribonucleoproteins responsible for RNA synthesis in the nucleus of the host cell. Influenza transcription results in spliced mRNAs (M2 and NS2), intron-containing mRNAs (M1 and NS1), and intron-less mRNAs (HA, NA, NP, PB1, PB2, and PA), all of which undergo nuclear export into the cytoplasm for translation. Most cellular mRNA nuclear export is Nxf1-mediated, while select mRNAs utilize Crm1. METHODS: Here we inhibited Nxf1 and Crm1 nuclear export prior to infection with influenza A/Udorn/307/1972(H3N2) virus and analyzed influenza intron-less mRNAs using cellular fractionation and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). We examined direct interaction between Nxf1 and influenza intron-less mRNAs using immuno purification of Nxf1 and RT-PCR of associated RNA. RESULTS: Inhibition of Nxf1 resulted in less influenza intron-less mRNA export into the cytoplasm for HA and NA influenza mRNAs in both human embryonic kidney cell line (293 T) and human lung adenocarcinoma epithelial cell line (A549). However, in 293 T cells no change was observed for mRNAs encoding the components of the viral ribonucleoproteins; NP, PA, PB1, and PB2, while in A549 cells, only PA, PB1, and PB2 mRNAs, encoding the RdRP, remained unaffected; NP mRNA was reduced in the cytoplasm. In A549 cells NP, NA, HA, mRNAs were found associated with Nxf1 but PA, PB1, and PB2 mRNAs were not. Crm1 inhibition also resulted in no significant difference in PA, PB1, and PB2 mRNA nuclear export. CONCLUSIONS: These results further confirm Nxf1-mediated nuclear export is functional during the influenza life cycle and hijacked for select influenza mRNA nuclear export. We reveal a cell type difference for Nxf1-mediated nuclear export of influenza NP mRNA, a reminder that cell type can influence molecular mechanisms. Importantly, we conclude that in both A549 and 293 T cells, PA, PB1, and PB2 mRNA nuclear export is Nxf1 and Crm1 independent. Our data support the hypothesis that PA, PB1, and PB2 mRNAs, encoding the influenza RdRP, utilize atypical mRNA nuclear export.

HIV-1 Rev protein specifies the viral RNA export pathway by suppressing TAP/NXF1 recruitment.

Nuclear RNA export pathways in eukaryotes are often linked to the fate of a given RNA. Therefore, the choice of export pathway should be well-controlled to avoid an unfavorable effect on gene expression. Although some RNAs could be exported by more than one pathway, little is known about how the choice is regulated. This issue is highlighted when the human immunodeficiency virus type 1 (HIV-1) Rev protein induces the export of singly spliced and unspliced HIV-1 transcripts. How these RNAs are exported is not well understood because such transcripts should have the possibility of utilizing CRM1-dependent export via Rev or cellular TAP/NXF1-dependent export via the transcription/export (TREX) complex, or both. Here we found that Rev suppressed TAP/NXF1-dependent export of model RNA substrates that recapitulated viral transcripts. In this effect, Rev interacted with the cap-binding complex and inhibited the recruitment of the TREX complex. Thus, Rev controls the identity of the factor occupying the cap-proximal region that determines the RNA export pathway. This ribonucleoprotein remodeling activity of Rev may favor viral gene expression.

Structure-activity relationship study of 4EGI-1, small molecule eIF4E/eIF4G protein-protein interaction inhibitors.

Protein-protein interactions are critical for regulating the activity of translation initiation factors and multitude of other cellular process, and form the largest block of untapped albeit most challenging targets for drug development. 4EGI-1, (E/Z)-2-(2-(4-(3,4-dichlorophenyl)thiazol-2-yl)hydrazono)-3-(2-nitrophenyl)propanoic acid, is a hit compound discovered in a screening campaign of small molecule libraries as an inhibitor of translation initiation factors eIF4E and eIF4G protein-protein interaction; it inhibits translation initiation in vitro and in vivo. A series of 4EGI-1-derived thiazol-2-yl hydrazones have been designed and synthesized in order to delineate the structural latitude and improve its binding affinity to eIF4E, and increase its potency in inhibiting the eIF4E/eIF4G interaction. Probing a wide range of substituents on both phenyl rings comprising the 3-phenylpropionic acid and 4-phenylthiazolidine moieties in the context of both E- and Z-isomers of 4EGI-1 led to analogs with enhanced binding affinity and translation initiation inhibitory activities.

Inhibition of ER stress-mediated apoptosis in macrophages by nuclear-cytoplasmic relocalization of eEF1A by the HIV-1 Nef protein.

HIV-1 Nef protein has key roles at almost all stages of the viral life cycle. We assessed the role of the Nef/eEF1A (eukaryotic translation elongation factor 1-alpha) complex in nucleocytoplasmic shuttling in primary human macrophages. Nuclear retention experiments and inhibition of the exportin-t (Exp-t) pathway suggested that cytoplasmic relocalization of eEF1A, mediated by Exp-t, occurs in Nef-treated monocyte-derived macrophages (MDMs). We observed the presence of tRNA in the Nef/eEF1A complexes. Nucleocytoplasmic relocalization of the Nef/eEF1A complexes prevented stress-induced apoptosis of MDMs treated with brefeldin-A. Blockade of stress-induced apoptosis of MDMs treated with HIV-1 Nef resulted from enhanced nucleocytoplasmic transport of eEF1A with decreased release of mitochondrial cytochrome c, and from increased tRNA binding to cytochrome c, ultimately leading to an inhibition of caspase activation. Our results indicate that HIV-1 Nef, through the nucleocytoplasmic relocalization of eEF1A and tRNAs, enhances resistance to stress-induced apoptosis in primary human macrophages.

Pegylated interferon alpha targets Wnt signaling by inducing nuclear export of ß-catenin.

BACKGROUND & AIMS: Pegylated-Interferon-α2a (peg-IFN), a first line therapy for Hepatitis C virus (HCV) patients, also impacts the recurrence of hepatocellular carcinoma (HCC). The activation of the Wnt pathway due to ß-catenin gene mutations contributes to the development of a significant subset of HCC. Herein, we explored the effect of peg-IFN on Wnt/ß-catenin signaling in vitro and in vivo. METHODS: Multiple human hepatoma cell lines were treated with Peg-IFN to assess its effect on the Wnt pathway and the mechanisms involved. Transgenic (TG) mice expressing stable ß-catenin mutant in the liver were exposed to diethylnitrosamine (DEN) and treated with peg-IFN. RESULTS: In vitro, peg-IFN decreased the transcriptional activity of ß-catenin/Tcf and did so independently of JAK/Stat signaling. Peg-IFN treatment led to increased mRNA and protein expression of RanBP3, a known ß-catenin nuclear export factor, in all hepatoma cells. Co-precipitation studies showed an increased association between RanBP3 and ß-catenin after peg-IFN treatment. The siRNA-mediated RanBP3 knockdown abrogated Peg-IFN-induced decrease in TOPFlash reporter activity. In vivo, Peg-IFN treatment led to increased nuclear RanBP3, decreased nuclear ß-catenin and cyclin D1, and decreased cytoplasmic glutamine synthetase. Increased association of RanBP3 and ß-catenin was also observed in vivo in response to Peg-IFN that led to decreased hepatocyte proliferation. CONCLUSIONS: Peg-IFN inhibits ß-catenin signaling through the up-regulation of RanBP3, which may be a contributory mechanism for the delayed HCC and improved survival in treated HCV patients. This observation might have chemo-preventive or chemo-therapeutic implications in tumor with aberrant Wnt pathway activation.

Identification of Myb-binding protein 1A (MYBBP1A) as a novel substrate for aurora B kinase.

Aurora kinases are mitotic enzymes involved in centrosome maturation and separation, spindle assembly and stability, and chromosome condensation, segregation, and cytokinesis and represent well known targets for cancer therapy because their deregulation has been linked to tumorigenesis. The availability of suitable markers is of crucial importance to investigate the functions of Auroras and monitor kinase inhibition in in vivo models and in clinical trials. Extending the knowledge on Aurora substrates could help to better understand their biology and could be a source for clinical biomarkers. Using biochemical, mass spectrometric, and cellular approaches, we identified MYBBP1A as a novel Aurora B substrate and serine 1303 as the major phosphorylation site. MYBBP1A is phosphorylated in nocodazole-arrested cells and is dephosphorylated upon Aurora B silencing or by treatment with Danusertib, a small molecule inhibitor of Aurora kinases. Furthermore, we show that MYBBP1A depletion by RNA interference causes mitotic progression delay and spindle assembly defects. MYBBP1A has until now been described as a nucleolar protein, mainly involved in transcriptional regulation. The results presented herein show MYBBP1A as a novel Aurora B kinase substrate and reveal a not yet recognized link of this nucleolar protein to mitosis.

Individual influenza A virus mRNAs show differential dependence on cellular NXF1/TAP for their nuclear export.

The influenza A virus RNA-dependent RNA polymerase produces capped and polyadenylated mRNAs in the nucleus of infected cells that resemble mature cellular mRNAs, but are made by very different mechanisms. Furthermore, only two of the 10 viral protein-coding mRNAs are spliced: most are intronless, while two contain unremoved introns. The mechanism(s) by which any of these mRNAs are exported from the nucleus is uncertain. To probe the involvement of the primary cellular mRNA export pathway, we treated cells with siRNAs against NXF1, Aly or UAP56, or with the drug 5,6-dichloro-1-beta-d-ribofuranosyl-benzimidazole (DRB), an inhibitor of RNA polymerase II phosphorylation previously shown to inhibit nuclear export of cellular mRNA as well as influenza virus segment 7 mRNAs. Depletion of NXF1 or DRB treatment had similar effects, inhibiting the nuclear export of several of the viral mRNAs. However, differing degrees of sensitivity were seen, depending on the particular segment examined. Intronless HA mRNA and spliced M2 or unspliced M1 transcripts (all encoding late proteins) showed a strong requirement for NXF1, while intronless early gene mRNAs, especially NP mRNA, showed the least dependency. Depletion of Aly had little effect on viral mRNA export, but reduction of UAP56 levels strongly inhibited trafficking and/or translation of the M1, M2 and NS1 mRNAs. Synthesis of NS2 from the spliced segment 8 transcript was, however, resistant. We conclude that influenza A virus co-opts the main cellular mRNA export pathway for a subset of its mRNAs, including most but not all late gene transcripts.

mRNA export from mammalian cell nuclei is dependent on GANP.

Bulk nuclear export of messenger ribonucleoproteins (mRNPs) through nuclear pore complexes (NPCs) is mediated by NXF1. It binds mRNPs through adaptor proteins such as ALY and SR splicing factors and mediates translocation through the central NPC transport channel via transient interactions with FG nucleoporins. Here, we show that mammalian cells require GANP (germinal center-associated nuclear protein) for efficient mRNP nuclear export and for efficient recruitment of NXF1 to NPCs. Separate regions of GANP show local homology to FG nucleoporins, the yeast mRNA export factor Sac3p, and the mammalian MCM3 acetyltransferase. GANP interacts with both NXF1 and NPCs and partitions between NPCs and the nuclear interior. GANP depletion inhibits mRNA export, with retention of mRNPs and NXF1 in punctate foci within the nucleus. The GANP N-terminal region that contains FG motifs interacts with the NXF1 FG-binding domain. Overexpression of this GANP fragment leads to nuclear accumulation of both poly(A)(+)RNA and NXF1. Treatment with transcription inhibitors redistributes GANP from NPCs into foci throughout the nucleus. These results establish GANP as an integral component of the mammalian mRNA export machinery and suggest a model whereby GANP facilitates the transfer of NXF1-containing mRNPs to NPCs.

Nuclear export of Smad2 and Smad3 by RanBP3 facilitates termination of TGF-beta signaling.

Smad2 and Smad3 (Smad2/3) are key intracellular signal transducers for TGF-beta signaling, and their transcriptional activities are controlled through reversible phosphorylation and nucleocytoplasmic shuttling. However, the precise mechanism underlying nuclear export of Smad2/3 remains elusive. Here we report the essential function of RanBP3 in selective nuclear export of Smad2/3 in the TGF-beta pathway. RanBP3 directly recognizes dephosphorylated Smad2/3, which results from the activity of nuclear Smad phosphatases, and mediates nuclear export of Smad2/3 in a Ran-dependent manner. As a result, increased expression of RanBP3 inhibits TGF-beta signaling in mammalian cells and Xenopus embryos. Conversely, depletion of RanBP3 expression or dominant-negative inhibition of RanBP3 enhances TGFbeta-induced antiproliferative and transcriptional responses. In conclusion, our study supports a definitive role for RanBP3 in mediating Smad2/3 nuclear export and terminating TGF-beta signaling.

Venezuelan equine encephalitis virus capsid protein inhibits nuclear import in Mammalian but not in mosquito cells.

Venezuelan equine encephalitis virus (VEEV) represents a continuous public health threat in the United States. It has the ability to cause fatal disease in humans and in horses and other domestic animals. We recently demonstrated that replicating VEEV interferes with cellular transcription and uses this phenomenon as a means of downregulating a cellular antiviral response. VEEV capsid protein was found to play a critical role in this process, and its approximately 35-amino-acid-long peptide, fused with green fluorescent protein, functioned as efficiently as did the entire capsid. We detected a significant fraction of VEEV capsid associated with nuclear envelope, which suggested that this protein might regulate nucleocytoplasmic trafficking. In this study, we demonstrate that VEEV capsid and its N-terminal sequence efficiently inhibit multiple receptor-mediated nuclear import pathways but have no effect on the passive diffusion of small proteins. The capsid protein of the Old World alphavirus Sindbis virus and the VEEV capsid, with a previously defined frameshift mutation, were found to have no detectable effect on nuclear import. Importantly, the VEEV capsid did not noticeably interfere with nuclear import in mosquito cells, and this might play a critical role in the ability of the virus to develop a persistent, life-long infection in mosquito vectors. These findings demonstrate a new aspect of VEEV-host cell interactions, and the results of this study are likely applicable to other New World alphaviruses, such as eastern and western equine encephalitis viruses.