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Sample records for nuclear export signals

  1. Mechanisms of progesterone receptor export from nuclei: role of nuclear localization signal, nuclear export signal, and ran guanosine triphosphate.

    PubMed

    Tyagi, R K; Amazit, L; Lescop, P; Milgrom, E; Guiochon-Mantel, A

    1998-11-01

    Steroid hormone receptors are, in most cases, mainly nuclear proteins that undergo a continuous nucleocytoplasmic shuttling. The mechanism of the nuclear export of these proteins remains largely unknown. To approach this problem experimentally in vivo, we have prepared cell lines permanently coexpressing the wild-type nuclear progesterone receptor (PR) and a cytoplasmic receptor mutant deleted of its nuclear localization signal (NLS) [(deltaNLS)PR]. Each receptor species was deleted from the epitope recognized by a specific monoclonal antibody, thus allowing separated observation of the two receptor forms in the same cells. Administration of hormone provoked formation of heterodimers during nucleocytoplasmic shuttling and import of (deltaNLS)PR into the nucleus. Washing out of the hormone allowed us to follow the export of (deltaNLS)PR into the cytoplasm. Microinjection of BSA coupled to a NLS inhibited the export of (deltaNLS)PR. On the contrary, microinjection of BSA coupled to a nuclear export signal (NES) was without effect. Moreover, leptomycin B, which inhibits NES-mediated export, was also without effect. tsBN2 cells contain a thermosensitive RCC1 protein (Ran GTP exchange protein). At the nonpermissive temperature, the nuclear export of (deltaNLS)PR could be observed, whereas the export of NES-BSA was suppressed. Microinjection of GTPgammaS confirmed that the export of (deltaNLS)PR was not dependent on GTP hydrolysis. These experiments show that the nuclear export of PR is not NES mediated but probably involves the NLS. It does not involve Ran GTP, and it is not dependent on the hydrolysis of GTP. The nucleocytoplasmic shuttling of steroid hormone receptors thus appears to utilize mechanisms different from those previously described for some viral, regulatory, and heterogeneous ribonuclear proteins. PMID:9817595

  2. Morbillivirus nucleoprotein possesses a novel nuclear localization signal and a CRM1-independent nuclear export signal

    SciTech Connect

    Sato, Hiroki; Masuda, Munemitsu; Miura, Ryuichi; Yoneda, Misako; Kai, Chieko . E-mail: ckai@ims.u-tokyo.ac.jp

    2006-08-15

    Morbilliviruses, which belong to the Mononegavirales, replicate its RNA genome in the cytoplasm of the host cell. However, they also form characteristic intranuclear inclusion bodies, consisting of nucleoprotein (N), in infected cells. To analyze the mechanisms of nucleocytoplasmic transport of N protein, we characterized the nuclear localization (NLS) and nuclear export (NES) signals of canine distemper virus (CDV) N protein by deletion mutation and alanine substitution of the protein. The NLS has a novel leucine/isoleucine-rich motif (TGILISIL) at positions 70-77, whereas the NES is composed of a leucine-rich motif (LLRSLTLF) at positions 4-11. The NLS and NES of the N proteins of other morbilliviruses, that is, measles virus (MV) and rinderpest virus (RPV), were also analyzed. The NLS of CDV-N protein is conserved at the same position in MV-N protein, whereas the NES of MV-N protein is located in the C-terminal region. The NES of RPV-N protein is also located at the same position as CDV-N protein, whereas the NLS motif is present not only at the same locus as CDV-N protein but also at other sites. Interestingly, the nuclear export of all these N proteins appears to proceed via a CRM1-independent pathway.

  3. Identification of a putative nuclear export signal motif in human NANOG homeobox domain

    SciTech Connect

    Park, Sung-Won; Do, Hyun-Jin; Huh, Sun-Hyung; Sung, Boreum; Uhm, Sang-Jun; Song, Hyuk; Kim, Nam-Hyung; Kim, Jae-Hwan

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer We found the putative nuclear export signal motif within human NANOG homeodomain. Black-Right-Pointing-Pointer Leucine-rich residues are important for human NANOG homeodomain nuclear export. Black-Right-Pointing-Pointer CRM1-specific inhibitor LMB blocked the potent human NANOG NES-mediated nuclear export. -- Abstract: NANOG is a homeobox-containing transcription factor that plays an important role in pluripotent stem cells and tumorigenic cells. To understand how nuclear localization of human NANOG is regulated, the NANOG sequence was examined and a leucine-rich nuclear export signal (NES) motif ({sup 125}MQELSNILNL{sup 134}) was found in the homeodomain (HD). To functionally validate the putative NES motif, deletion and site-directed mutants were fused to an EGFP expression vector and transfected into COS-7 cells, and the localization of the proteins was examined. While hNANOG HD exclusively localized to the nucleus, a mutant with both NLSs deleted and only the putative NES motif contained (hNANOG HD-{Delta}NLSs) was predominantly cytoplasmic, as observed by nucleo/cytoplasmic fractionation and Western blot analysis as well as confocal microscopy. Furthermore, site-directed mutagenesis of the putative NES motif in a partial hNANOG HD only containing either one of the two NLS motifs led to localization in the nucleus, suggesting that the NES motif may play a functional role in nuclear export. Furthermore, CRM1-specific nuclear export inhibitor LMB blocked the hNANOG potent NES-mediated export, suggesting that the leucine-rich motif may function in CRM1-mediated nuclear export of hNANOG. Collectively, a NES motif is present in the hNANOG HD and may be functionally involved in CRM1-mediated nuclear export pathway.

  4. A novel nuclear export signal in Smad1 is essential for its signaling activity.

    PubMed

    Xiao, Zhan; Brownawell, Amy M; Macara, Ian G; Lodish, Harvey F

    2003-09-01

    To investigate the subcellular distributions of Smad proteins, the intracellular mediators of transforming growth factor-beta family cytokines, we examined their sequences for nuclear export signals (NES). We found a leucine-rich NES-like motif (termed NES2) in the central linker region of the receptor-regulated Smads that is absent from the other two classes of Smads (Co-Smads and I-Smads). In microinjection assays, NES2 peptide caused nuclear export of a fused glutathione S-transferase protein. Mutations in NES2 converted Smad1 from an even distribution throughout the cells into an exclusive nuclear localization in both transiently and stably expressing cell lines, and this nuclear enrichment was more pronounced than that induced by mutations in NES1. Furthermore, overexpression of CRM1, the cellular export receptor, transforms Smad1 into a mostly cytoplasmic profile by enhancing its nuclear export. The Smad1 NES2 mutant but not the Smad1 NES1 mutant is mostly resistant to this cytoplasmic targeting, indicating that NES2, not NES1, is the major target for CRM1 in Smad1. We further confirmed the functionality of NES2 by a heterokaryon assay. The Smad1 NES1 mutant displays good ligand responsiveness and moderately lowered transcriptional activity compared with wild type Smad1. In contrast, the Smad1 NES2 mutant shows a severe disruption in reporter gene activation, minimal response to bone morphogenetic protein stimulation, and significantly lowered bone morphogenetic protein-induced phosphorylation, which may be the reason for its deficient transcription activity. Thus, we have defined a major NES in Smad1 that is essential for its ligand-induced coupling with cell surface receptors and hence, transcriptional activity. Our study, along with recent studies of the nucleocytoplasmic shuttling of Smad2 and Smad3 proteins, demonstrate that continued nucleocytoplasmic shuttling is a common requisite for the active signaling of R-Smads. Although conserved in other R

  5. Nuclear export of cutaneous HPV8 E7 oncoprotein is mediated by a leucine-rich nuclear export signal via a CRM1 pathway

    SciTech Connect

    Onder, Zeynep; Chang, Vivian; Moroianu, Junona

    2015-01-01

    We recently determined that the nuclear import of cutaneous beta genus HPV8 E7 oncoprotein it is mediated by its zinc-binding domain via direct hydrophobic interactions with the FG nucleoporins Nup62 and Nup153 (Onder and Moroianu, 2014). Here we investigated the nuclear export of HPV8 E7 oncoprotein using confocal microscopy after transfections of HeLa cells with EGFP–8cE7 and mutant plasmids and treatment with Ratjadone A nuclear export inhibitor. We determined that HPV8 E7 contains a leucine-rich nuclear export signal (NES), {sub 76}IRTFQELLF{sub 84}, within its zinc-binding domain that mediates its nuclear export via a CRM1 pathway. We found that HPV8 E7 interacts with CRM1 and that the hydrophobic amino acid residues I76, F79 and L82 of the NES are essential for this interaction and for nuclear export of HPV8 E7 oncoprotein. - Highlights: • HPV8 E7 has a leucine-rich NES within its zinc-binding domain that mediates its nuclear export. • CRM1 nuclear export receptor interacts with HPV8 E7 and mediates its export. • Identification of the critical hydrophobic amino acids of the NES of HPV8 E7.

  6. Structural basis for leucine-rich nuclear export signal recognition by CRM1

    SciTech Connect

    Dong, Xiuhua; Biswas, Anindita; Süel, Katherine E.; Jackson, Laurie K.; Martinez, Rita; Gu, Hongmei; Chook, Yuh Min

    2009-07-10

    CRM1 (also known as XPO1 and exportin 1) mediates nuclear export of hundreds of proteins through the recognition of the leucine-rich nuclear export signal (LR-NES). Here we present the 2.9 {angstrom} structure of CRM1 bound to snurportin 1 (SNUPN). Snurportin 1 binds CRM1 in a bipartite manner by means of an amino-terminal LR-NES and its nucleotide-binding domain. The LR-NES is a combined {alpha}-helical-extended structure that occupies a hydrophobic groove between two CRM1 outer helices. The LR-NES interface explains the consensus hydrophobic pattern, preference for intervening electronegative residues and inhibition by leptomycin B. The second nuclear export signal epitope is a basic surface on the snurportin 1 nucleotide-binding domain, which binds an acidic patch on CRM1 adjacent to the LR-NES site. Multipartite recognition of individually weak nuclear export signal epitopes may be common to CRM1 substrates, enhancing CRM1 binding beyond the generally low affinity LR-NES. Similar energetic construction is also used in multipartite nuclear localization signals to provide broad substrate specificity and rapid evolution in nuclear transport.

  7. Identification of a nuclear export signal in the KSHV latent protein LANA2 mediating its export from the nucleus

    SciTech Connect

    Munoz-Fontela, C.; Collado, M.; Rodriguez, E.; Garcia, M.A.; Alvarez-Barrientos, A.; Arroyo, J.; Nombela, C.; Rivas, C. . E-mail: mdcrivas@farm.ucm.es

    2005-11-15

    LANA2 is a latent protein detected in Kaposi's sarcoma-associated herpesvirus (KSHV)-infected B cells that inhibits p53-dependent transcriptional transactivation and apoptosis and PKR-dependent apoptosis, suggesting an important role in the transforming activity of the virus. It has been reported that LANA2 localizes into the nucleus of both KSHV-infected B cells and transiently transfected HeLa cells. In this study, we show that LANA2 is a nucleocytoplasmic shuttling protein that requires a Rev-type nuclear export signal located in the C-terminus to direct the protein to the cytoplasm, through an association with the export receptor CRM1. In addition, a functional protein kinase B (PKB)/Akt phosphorylation motif partially overlapping with the nuclear export signal was identified. Nuclear exclusion of LANA2 was negatively regulated by the phosphorylation of threonine 564 by Akt. The ability of LANA2 to shuttle between nucleus and cytoplasm has implications for the function of this viral protein.

  8. A high-throughput screening system targeting the nuclear export pathway via the third nuclear export signal of influenza A virus nucleoprotein.

    PubMed

    Kakisaka, Michinori; Mano, Takafumi; Aida, Yoko

    2016-06-01

    Two classes of antiviral drugs, M2 channel inhibitors and neuraminidase (NA) inhibitors, are currently approved for the treatment of influenza; however, the development of resistance against these agents limits their efficacy. Therefore, the identification of new targets and the development of new antiviral drugs against influenza are urgently needed. The third nuclear export signal (NES3) of nucleoprotein (NP) is the most important for viral replication among seven NESs encoded by four viral proteins, NP, M1, NS1, and NS2. NP-NES3 is critical for the nuclear export of NP, and targeting NP-NES3 is therefore a promising strategy that may lead to the development of antiviral drugs. However, a high-throughput screening (HTS) system to identify inhibitors of NP nuclear export has not been established. Here, we developed a novel HTS system to evaluate the inhibitory effects of compounds on the nuclear export pathway mediated by NP-NES3 using a MDCK cell line stably expressing NP-NES3 fused to a green fluorescent protein from aequorea coerulescens (AcGFP-NP-NES3) and a cell imaging analyzer. This HTS system was used to screen a 9600-compound library, leading to the identification of several hit compounds with inhibitory activity against the nuclear export of AcGFP-NP-NES3. The present HTS system provides a useful strategy for the identification of inhibitors targeting the nuclear export of NP via its NES3 sequence. PMID:26948263

  9. CRM1/Ran-mediated nuclear export of p27(Kip1) involves a nuclear export signal and links p27 export and proteolysis.

    PubMed

    Connor, Michael K; Kotchetkov, Rouslan; Cariou, Sandrine; Resch, Ansgar; Lupetti, Rafaella; Beniston, Richard G; Melchior, Frauke; Hengst, Ludger; Slingerland, Joyce M

    2003-01-01

    We show that p27 localization is cell cycle regulated and we suggest that active CRM1/RanGTP-mediated nuclear export of p27 may be linked to cytoplasmic p27 proteolysis in early G1. p27 is nuclear in G0 and early G1 and appears transiently in the cytoplasm at the G1/S transition. Association of p27 with the exportin CRM1 was minimal in G0 and increased markedly during G1-to-S phase progression. Proteasome inhibition in mid-G1 did not impair nuclear import of p27, but led to accumulation of p27 in the cytoplasm, suggesting that export precedes degradation for at least part of the cellular p27 pool. p27-CRM1 binding and nuclear export were inhibited by S10A mutation but not by T187A mutation. A putative nuclear export sequence in p27 is identified whose mutation reduced p27-CRM1 interaction, nuclear export, and p27 degradation. Leptomycin B (LMB) did not inhibit p27-CRM1 binding, nor did it prevent p27 export in vitro or in heterokaryon assays. Prebinding of CRM1 to the HIV-1 Rev nuclear export sequence did not inhibit p27-CRM1 interaction, suggesting that p27 binds CRM1 at a non-LMB-sensitive motif. LMB increased total cellular p27 and may do so indirectly, through effects on other p27 regulatory proteins. These data suggest a model in which p27 undergoes active, CRM1-dependent nuclear export and cytoplasmic degradation in early G1. This would permit the incremental activation of cyclin E-Cdk2 leading to cyclin E-Cdk2-mediated T187 phosphorylation and p27 proteolysis in late G1 and S phase. PMID:12529437

  10. Identification of a Functional, CRM-1-Dependent Nuclear Export Signal in Hepatitis C Virus Core Protein

    PubMed Central

    Cerutti, Andrea; Maillard, Patrick; Minisini, Rosalba; Vidalain, Pierre-Olivier; Roohvand, Farzin; Pecheur, Eve-Isabelle; Pirisi, Mario; Budkowska, Agata

    2011-01-01

    Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. HCV core protein is involved in nucleocapsid formation, but it also interacts with multiple cytoplasmic and nuclear molecules and plays a crucial role in the development of liver disease and hepatocarcinogenesis. The core protein is found mostly in the cytoplasm during HCV infection, but also in the nucleus in patients with hepatocarcinoma and in core-transgenic mice. HCV core contains nuclear localization signals (NLS), but no nuclear export signal (NES) has yet been identified. We show here that the aa(109–133) region directs the translocation of core from the nucleus to the cytoplasm by the CRM-1-mediated nuclear export pathway. Mutagenesis of the three hydrophobic residues (L119, I123 and L126) in the identified NES or in the sequence encoding the mature core aa(1–173) significantly enhanced the nuclear localisation of the corresponding proteins in transfected Huh7 cells. Both the NES and the adjacent hydrophobic sequence in domain II of core were required to maintain the core protein or its fragments in the cytoplasmic compartment. Electron microscopy studies of the JFH1 replication model demonstrated that core was translocated into the nucleus a few minutes after the virus entered the cell. The blockade of nucleocytoplasmic export by leptomycin B treatment early in infection led to the detection of core protein in the nucleus by confocal microscopy and coincided with a decrease in virus replication. Our data suggest that the functional NLS and NES direct HCV core protein shuttling between the cytoplasmic and nuclear compartments, with at least some core protein transported to the nucleus. These new properties of HCV core may be essential for virus multiplication and interaction with nuclear molecules, influence cell signaling and the pathogenesis of HCV infection. PMID:22039426

  11. Structural determinants of nuclear export signal orientation in binding to exportin CRM1

    PubMed Central

    Fung, Ho Yee Joyce; Fu, Szu-Chin; Brautigam, Chad A; Chook, Yuh Min

    2015-01-01

    The Chromosome Region of Maintenance 1 (CRM1) protein mediates nuclear export of hundreds of proteins through recognition of their nuclear export signals (NESs), which are highly variable in sequence and structure. The plasticity of the CRM1-NES interaction is not well understood, as there are many NES sequences that seem incompatible with structures of the NES-bound CRM1 groove. Crystal structures of CRM1 bound to two different NESs with unusual sequences showed the NES peptides binding the CRM1 groove in the opposite orientation (minus) to that of previously studied NESs (plus). Comparison of minus and plus NESs identified structural and sequence determinants for NES orientation. The binding of NESs to CRM1 in both orientations results in a large expansion in NES consensus patterns and therefore a corresponding expansion of potential NESs in the proteome. DOI: http://dx.doi.org/10.7554/eLife.10034.001 PMID:26349033

  12. Structural determinants of nuclear export signal orientation in binding to exportin CRM1

    DOE PAGESBeta

    Fung, Ho Yee Joyce; Fu, Szu -Chin; Brautigam, Chad A.; Chook, Yuh Min

    2015-09-08

    The Chromosome Region of Maintenance 1 (CRM1) protein mediates nuclear export of hundreds of proteins through recognition of their nuclear export signals (NESs), which are highly variable in sequence and structure. The plasticity of the CRM1-NES interaction is not well understood, as there are many NES sequences that seem incompatible with structures of the NES-bound CRM1 groove. Crystal structures of CRM1 bound to two different NESs with unusual sequences showed the NES peptides binding the CRM1 groove in the opposite orientation (minus) to that of previously studied NESs (plus). A comparison of minus and plus NESs identified structural and sequencemore » determinants for NES orientation. The binding of NESs to CRM1 in both orientations results in a large expansion in NES consensus patterns and therefore a corresponding expansion of potential NESs in the proteome.« less

  13. Structural determinants of nuclear export signal orientation in binding to exportin CRM1

    SciTech Connect

    Fung, Ho Yee Joyce; Fu, Szu -Chin; Brautigam, Chad A.; Chook, Yuh Min

    2015-09-08

    The Chromosome Region of Maintenance 1 (CRM1) protein mediates nuclear export of hundreds of proteins through recognition of their nuclear export signals (NESs), which are highly variable in sequence and structure. The plasticity of the CRM1-NES interaction is not well understood, as there are many NES sequences that seem incompatible with structures of the NES-bound CRM1 groove. Crystal structures of CRM1 bound to two different NESs with unusual sequences showed the NES peptides binding the CRM1 groove in the opposite orientation (minus) to that of previously studied NESs (plus). A comparison of minus and plus NESs identified structural and sequence determinants for NES orientation. The binding of NESs to CRM1 in both orientations results in a large expansion in NES consensus patterns and therefore a corresponding expansion of potential NESs in the proteome.

  14. Identification of the nuclear localization and export signals of high risk HPV16 E7 oncoprotein

    SciTech Connect

    Knapp, Alixandra A.; McManus, Patrick M.; Bockstall, Katy; Moroianu, Junona

    2009-01-05

    The E7 oncoprotein of high risk human papillomavirus type 16 (HPV16) binds and inactivates the retinoblastoma (RB) family of proteins. Our previous studies suggested that HPV16 E7 enters the nucleus via a novel Ran-dependent pathway independent of the nuclear import receptors (Angeline, M., Merle, E., and Moroianu, J. (2003). The E7 oncoprotein of high-risk human papillomavirus type 16 enters the nucleus via a nonclassical Ran-dependent pathway. Virology 317(1), 13-23.). Here, analysis of the localization of specific E7 mutants revealed that the nuclear localization of E7 is independent of its interaction with pRB or of its phosphorylation by CKII. Fluorescence microscopy analysis of enhanced green fluorescent protein (EGFP) and 2xEGFP fusions with E7 and E7 domains in HeLa cells revealed that E7 contains a novel nuclear localization signal (NLS) in the N-terminal domain (aa 1-37). Interestingly, treatment of transfected HeLa cells with two specific nuclear export inhibitors, Leptomycin B and ratjadone, changed the localization of 2xEGFP-E7{sub 38-98} from cytoplasmic to mostly nuclear. These data suggest the presence of a leucine-rich nuclear export signal (NES) and a second NLS in the C-terminal domain of E7 (aa 38-98). Mutagenesis of critical amino acids in the putative NES sequence ({sub 76}IRTLEDLLM{sub 84}) changed the localization of 2xEGFP-E7{sub 38-98} from cytoplasmic to mostly nuclear suggesting that this is a functional NES. The presence of both NLSs and an NES suggests that HPV16 E7 shuttles between the cytoplasm and nucleus which is consistent with E7 having functions in both of these cell compartments.

  15. NES-REBS: A novel nuclear export signal prediction method using regular expressions and biochemical properties.

    PubMed

    Wu, Tingfang; Wang, Xun; Zhang, Zheng; Gong, Faming; Song, Tao; Chen, Zhihua; Zhang, Pan; Zhao, Yang

    2016-06-01

    A nuclear export signal (NES) is a protein localization signal, which is involved in binding of cargo proteins to nuclear export receptor, thus contributes to regulate localization of cellular proteins. Consensus sequences of NES have been used to detect NES from protein sequences, but suffer from poor predictive power. Some recent peering works were proposed to use biochemical properties of experimental verified NES to refine NES candidates. Those methods can achieve high prediction rates, but their execution time will become unacceptable for large-scale NES searching if too much properties are involved. In this work, we developed a novel computational approach, named NES-REBS, to search NES from protein sequences, where biochemical properties of experimental verified NES, including secondary structure and surface accessibility, are utilized to refine NES candidates obtained by matching popular consensus sequences. We test our method by searching 262 experimental verified NES from 221 NES-containing protein sequences. It is obtained that NES-REBS runs in 2-3[Formula: see text]mins and performs well by achieving precision rate 47.2% and sensitivity 54.6%. PMID:27225342

  16. Coordinate regulation/localization of the carbohydrate responsive binding protein (ChREBP) by two nuclear export signal sites: Discovery of a new leucine-rich nuclear export signal site

    SciTech Connect

    Fukasawa, Masashi; Ge, Qing; Wynn, R. Max; Ishii, Seiji; Uyeda, Kosaku

    2010-01-08

    Carbohydrate response element binding protein (ChREBP) is responsible for conversion of dietary carbohydrate to storage fat in liver by coordinating expression of the enzymes that channel glycolytic pyruvate into lipogenesis. The activation of ChREBP in response to high glucose is nuclear localization and transcription, and the inactivation of ChREBP under low glucose involves export from the nucleus to the cytosol. Here we report a new nuclear export signal site ('NES1') of ChREBP. Together these signals provide ChREBP with two NES sequences, both the previously reported NES2 and now the new NES1 coordinate to interact together with CRM1 (exportin) for nuclear export of the carbohydrate response element binding protein.

  17. Identification of a functional nuclear export signal in the green fluorescent protein asFP499

    SciTech Connect

    Mustafa, Huseyin . E-mail: huseyinm@hotmail.com; Strasser, Bernd; Rauth, Sabine; Irving, Robert A.; Wark, Kim L.

    2006-04-21

    The green fluorescent protein (GFP) asFP499 from Anemonia sulcata is a distant homologue of the GFP from Aequorea victoria. We cloned the asFP499 gene into a mammalian expression vector and showed that this protein was expressed in the human lymphoblast cell line Ramos RA1 and in the embryonic kidney 293T cell line (HEK 293T). In HEK 293T cells, asFP499 was localized mainly in the cytoplasm, suggesting that the protein was excluded from the nucleus. We identified {sub 194}LRMEKLNI{sub 201} as a candidate nuclear export signal in asFP499 and mutated the isoleucine at position 201 to an alanine. Unlike the wildtype form, the mutant protein was distributed throughout the cytoplasm and nucleus. This is First report of a GFP that contains a functional NES.

  18. Nucleocytoplasmic Recycling of the Nuclear Localization Signal Receptor α Subunit In Vivo Is Dependent on a Nuclear Export Signal, Energy, and RCC1

    PubMed Central

    Boche, Irene; Fanning, Ellen

    1997-01-01

    Nuclear protein import requires a nuclear localization signal (NLS) receptor and at least three other cytoplasmic factors. The α subunit of the NLS receptor, Rag cohort 1 (Rch1), enters the nucleus, probably in a complex with the β subunit of the receptor, as well as other import factors and the import substrate. To learn more about which factors and/or events end the import reaction and how the import factors return to the cytoplasm, we have studied nucleocytoplasmic shuttling of Rch1 in vivo. Recombinant Rch1 microinjected into Vero or tsBN2 cells was found primarily in the cytoplasm. Rch1 injected into the nucleus was rapidly exported in a temperature-dependent manner. In contrast, a mutant of Rch1 lacking the first 243 residues accumulated in the nuclei of Vero cells after cytoplasmic injection. After nuclear injection, the truncated Rch1 was retained in the nucleus, but either Rch1 residues 207–217 or a heterologous nuclear export signal, but not a mutant form of residues 207–217, restored nuclear export. Loss of the nuclear transport factor RCC1 (regulator of chromosome condensation) at the nonpermissive temperature in the thermosensitive mutant cell line tsBN2 caused nuclear accumulation of wild-type Rch1 injected into the cytoplasm. However, free Rch1 injected into nuclei of tsBN2 cells at the nonpermissive temperature was exported. These results suggested that RCC1 acts at an earlier step in Rch1 recycling, possibly the disassembly of an import complex that contains Rch1 and the import substrate. Consistent with this possibility, incubation of purified RanGTP and RCC1 with NLS receptor and import substrate prevented assembly of receptor/substrate complexes or stimulated their disassembly. PMID:9334337

  19. Supraphysiological nuclear export signals bind CRM1 independently of RanGTP and arrest at Nup358

    PubMed Central

    Engelsma, Dieuwke; Bernad, Rafael; Calafat, Jero; Fornerod, Maarten

    2004-01-01

    Leucine-rich nuclear export signals (NESs) mediate rapid nuclear export of proteins via interaction with CRM1. This interaction is stimulated by RanGTP but remains of a relatively low affinity. In order to identify strong signals, we screened a 15-mer random peptide library for CRM1 binding, both in the presence and absence of RanGTP. Under each condition, strikingly similar signals were enriched, conforming to the NES consensus sequence. A derivative of an NES selected in the absence of RanGTP exhibits very high affinity for CRM1 in vitro and stably binds without the requirement of RanGTP. Localisation studies and RNA interference demonstrate inefficient CRM1-mediated export and accumulation of CRM1 complexed with the high-affinity NES at nucleoporin Nup358. These results provide in vivo evidence for a nuclear export reaction intermediate. They suggest that NESs have evolved to maintain low affinity for CRM1 to allow efficient export complex disassembly and release from Nup358. PMID:15329671

  20. Identification of a nuclear export signal sequence for bovine papillomavirus E1 protein

    SciTech Connect

    Rosas-Acosta, German; Wilson, Van G.

    2008-03-30

    Recent studies have demonstrated nuclear export by papillomavirus E1 proteins, but the requisite export sequence(s) for bovine papillomavirus (BPV) E1 were not defined. In this report we identify three functional nuclear export sequences (NES) present in BPV E1, with NES2 being the strongest in reporter assays. Nuclear localization of BPV1 E1 was modulated by over- or under-expression of CRM1, the major cellular exportin, and export was strongly reduced by the CRM1 inhibitor, Leptomycin B, indicating that E1 export occurs primarily through a CRM1-dependent process. Consistent with the in vivo functional results, E1 bound CRM1 in an in vitro pull-down assay. In addition, sumoylated E1 bound CRM1 more effectively than unmodified E1, suggesting that E1 export may be regulated by SUMO modification. Lastly, an E1 NES2 mutant accumulated in the nucleus to a greater extent than wild-type E1, yet was defective for viral origin replication in vivo. However, NES2 exhibited no intrinsic replication defect in an in vitro replication assay, implying that nucleocytoplasmic shuttling may be required to maintain E1 in a replication competent state.

  1. ICP27 mediates HSV RNA export by shuttling through a leucine-rich nuclear export signal and binding viral intronless RNAs through an RGG motif

    PubMed Central

    Sandri-Goldin, Rozanne M.

    1998-01-01

    Infection of metazoan cells with some viruses alters the balance of cellular mRNA export to favor viral RNA export and to retain cellular transcripts in the nucleus. Here, evidence is presented to show that the herpes simplex virus 1 (HSV-1) essential regulatory protein ICP27, which inhibits host cell-splicing, resulting in the accumulation of unspliced transcripts in the nucleus, mediates RNA export of viral intronless mRNAs. ICP27 was shown to shuttle between the nucleus and cytoplasm through a leucine-rich nuclear export signal, which alone was able to direct the export of the heterologous green fluorescent protein. In vivo UV irradiation studies demonstrated that ICP27 could be crosslinked to poly(A)+ RNA in the nucleus and the cytoplasm, supporting a role in export. Furthermore, the amount of hnRNP A1, which has been implicated in the export of cellular spliced mRNAs, that was bound to poly(A)+ RNA in HSV-1-infected cells was reduced compared with uninfected cells. In addition, it was demonstrated that ICP27 bound seven intronless HSV-1 transcripts in both the nucleus and the cytoplasm, and export of these transcripts was diminished substantially during infection with an ICP27 null mutant virus. In contrast, ICP27 did not bind to two HSV-1 mRNAs that undergo splicing. Finally, binding of ICP27 to RNA in vivo required an arginine-glycine region that resembles an RGG box. These results indicate that ICP27 is an important viral export factor that promotes the transport of HSV-1 intronless RNAs. PMID:9512520

  2. Nuclear Export of Smads by RanBP3L Regulates Bone Morphogenetic Protein Signaling and Mesenchymal Stem Cell Differentiation

    PubMed Central

    Chen, Fenfang; Lin, Xia; Xu, Pinglong; Zhang, Zhengmao; Chen, Yanzhen; Wang, Chao; Han, Jiahuai; Zhao, Bin; Xiao, Mu

    2015-01-01

    Bone morphogenetic proteins (BMPs) play vital roles in regulating stem cell maintenance and differentiation. BMPs can induce osteogenesis and inhibit myogenesis of mesenchymal stem cells. Canonical BMP signaling is stringently controlled through reversible phosphorylation and nucleocytoplasmic shuttling of Smad1, Smad5, and Smad8 (Smad1/5/8). However, how the nuclear export of Smad1/5/8 is regulated remains unclear. Here we report that the Ran-binding protein RanBP3L acts as a nuclear export factor for Smad1/5/8. RanBP3L directly recognizes dephosphorylated Smad1/5/8 and mediates their nuclear export in a Ran-dependent manner. Increased expression of RanBP3L blocks BMP-induced osteogenesis of mouse bone marrow-derived mesenchymal stem cells and promotes myogenic induction of C2C12 mouse myoblasts, whereas depletion of RanBP3L expression enhances BMP-dependent stem cell differentiation activity and transcriptional responses. In conclusion, our results demonstrate that RanBP3L, as a nuclear exporter for BMP-specific Smads, plays a critical role in terminating BMP signaling and regulating mesenchymal stem cell differentiation. PMID:25755279

  3. Sequence and structural analyses of nuclear export signals in the NESdb database

    PubMed Central

    Xu, Darui; Farmer, Alicia; Collett, Garen; Grishin, Nick V.; Chook, Yuh Min

    2012-01-01

    We compiled >200 nuclear export signal (NES)–containing CRM1 cargoes in a database named NESdb. We analyzed the sequences and three-dimensional structures of natural, experimentally identified NESs and of false-positive NESs that were generated from the database in order to identify properties that might distinguish the two groups of sequences. Analyses of amino acid frequencies, sequence logos, and agreement with existing NES consensus sequences revealed strong preferences for the Φ1-X3-Φ2-X2-Φ3-X-Φ4 pattern and for negatively charged amino acids in the nonhydrophobic positions of experimentally identified NESs but not of false positives. Strong preferences against certain hydrophobic amino acids in the hydrophobic positions were also revealed. These findings led to a new and more precise NES consensus. More important, three-dimensional structures are now available for 68 NESs within 56 different cargo proteins. Analyses of these structures showed that experimentally identified NESs are more likely than the false positives to adopt α-helical conformations that transition to loops at their C-termini and more likely to be surface accessible within their protein domains or be present in disordered or unobserved parts of the structures. Such distinguishing features for real NESs might be useful in future NES prediction efforts. Finally, we also tested CRM1-binding of 40 NESs that were found in the 56 structures. We found that 16 of the NES peptides did not bind CRM1, hence illustrating how NESs are easily misidentified. PMID:22833565

  4. Nuclear Export of African Swine Fever Virus p37 Protein Occurs through Two Distinct Pathways and Is Mediated by Three Independent Signals

    PubMed Central

    Eulálio, Ana; Nunes-Correia, Isabel; Carvalho, Ana Luísa; Faro, Carlos; Citovsky, Vitaly; Salas, José; Salas, Maria L.; Simões, Sérgio; de Lima, Maria C. Pedroso

    2006-01-01

    Nucleocytoplasmic shuttling activity of the African swine fever virus p37 protein, a major structural protein of this highly complex virus, has been recently reported. The systematic characterization of the nuclear export ability of this protein constituted the major purpose of the present study. We report that both the N- and C-terminal regions of p37 protein are actively exported from the nucleus to the cytoplasm of yeast and mammalian cells. Moreover, experiments using leptomycin B and small interfering RNAs targeting the CRM1 receptor have demonstrated that the export of p37 protein is mediated by both the CRM1-dependent and CRM1-independent nuclear export pathways. Two signals responsible for the CRM1-mediated nuclear export of p37 protein were identified at the N terminus of the protein, and an additional signal was identified at the C-terminal region, which mediates the CRM1-independent nuclear export. Interestingly, site-directed mutagenesis revealed that hydrophobic amino acids are critical to the function of these three nuclear export signals. Overall, our results demonstrate that two distinct pathways contribute to the strong nuclear export of full-length p37 protein, which is mediated by three independent nuclear export signals. The existence of overlapping nuclear export mechanisms, together with our observation that p37 protein is localized in the nucleus at early stages of infection and exclusively in the cytoplasm at later stages, suggests that the nuclear transport ability of this protein may be critical to the African swine fever virus replication cycle. PMID:16415017

  5. A CALM-derived nuclear export signal is essential for CALM-AF10–mediated leukemogenesis

    PubMed Central

    Conway, Amanda E.; Scotland, Paula B.; Lavau, Catherine P.

    2013-01-01

    The t(10;11) chromosomal translocation gives rise to the CALM-AF10 fusion gene and is found in patients with aggressive and difficult-to-treat hematopoietic malignancies. CALM-AF10–driven leukemias are characterized by HOXA gene up-regulation and a global reduction in H3K79 methylation. DOT1L, the H3K79 methyltransferase, interacts with the octapeptide/leucine zipper domain of AF10, and this region has been shown to be necessary and sufficient for CALM-AF10–mediated transformation. However, the precise role of CALM in leukemogenesis remains unclear. Here, we show that CALM contains a nuclear export signal (NES) that mediates cytoplasmic localization of CALM-AF10 and is necessary for CALM-AF10–dependent transformation. Fusions of the CALM NES (NESCALM-AF10) or NES motifs from heterologous proteins (ABL1, Rev, PKIA, APC) in-frame with AF10 are sufficient to immortalize murine hematopoietic progenitors in vitro. The CALM NES is essential for CALM-AF10–dependent Hoxa gene up-regulation and aberrant H3K79 methylation, possibly by mislocalization of DOT1L. Finally, we observed that CALM-AF10 leukemia cells are selectively sensitive to inhibition of nuclear export by Leptomycin B. These findings uncover a novel mechanism of leukemogenesis mediated by the nuclear export pathway and support further investigation of the utility of nuclear export inhibitors as therapeutic agents for patients with CALM-AF10 leukemias. PMID:23487024

  6. Sumoylation of Smad3 stimulates its nuclear export during PIASy-mediated suppression of TGF-{beta} signaling

    SciTech Connect

    Imoto, Seiyu; Ohbayashi, Norihiko; Ikeda, Osamu; Kamitani, Shinya; Muromoto, Ryuta; Sekine, Yuichi; Matsuda, Tadashi

    2008-05-30

    Sma- and MAD-related protein 3 (Smad3) plays crucial roles in the transforming growth factor-{beta} (TGF-{beta})-mediated signaling pathway, which produce a variety of cellular responses, including cell proliferation and differentiation. In our previous study, we demonstrated that protein inhibitor of activated STATy (PIASy) suppresses TGF-{beta} signaling by interacting with and sumoylating Smad3. In the present study, we examined the molecular mechanisms of Smad3 sumoylation during PIASy-mediated suppression of TGF-{beta} signaling. We found that small-interfering RNA-mediated reduction of endogenous PIASy expression enhanced TGF-{beta}-induced gene expression. Importantly, coexpression of Smad3 with PIASy and SUMO1 affected the DNA-binding activity of Smad3. Furthermore, coexpression of Smad3 with PIASy and SUMO1 stimulated the nuclear export of Smad3. Finally, fluorescence resonance energy transfer analyses revealed that Smad3 interacted with SUMO1 in the cytoplasm. These results suggest that PIASy regulates TGF-{beta}/Smad3-mediated signaling by stimulating sumoylation and nuclear export of Smad3.

  7. Nucleocytoplasmic shuttling of the rabies virus P protein requires a nuclear localization signal and a CRM1-dependent nuclear export signal

    SciTech Connect

    Pasdeloup, David; Poisson, Nicolas; Raux, Helene; Gaudin, Yves; Ruigrok, Rob W.H. . E-mail: danielle.blondel@vms.cnrs-gif.fr

    2005-04-10

    Rabies virus P protein is a co-factor of the viral RNA polymerase. It has been shown previously that P mRNA directs the synthesis of four N-terminally truncated P products P2, P3, P4, and P5 due to translational initiation by a leaky scanning mechanism at internal Met codons. Whereas P and P2 are located in the cytoplasm, P3, P4, and P5 are found in the nucleus. Here, we have analyzed the molecular basis of the subcellular localization of these proteins. Using deletion mutants fused to GFP protein, we show the presence of a nuclear localization signal (NLS) in the C-terminal part of P (172-297). This domain contains a short lysine-rich stretch ({sup 211}KKYK{sup 214}) located in close proximity with arginine 260 as revealed by the crystal structure of P. We demonstrate the critical role of lysine 214 and arginine 260 in NLS activity. In the presence of Leptomycin B, P is retained in the nucleus indicating that it contains a CRM1-dependent nuclear export signal (NES). The subcellular distribution of P deletion mutants indicates that the domain responsible for export is the amino-terminal part of the protein. The use of fusion proteins that have amino terminal fragments of P fused to {beta}-galactosidase containing the NLS of SV40 T antigen allows us to identify a NES between residues 49 and 58. The localization of NLS and NES determines the cellular distribution of the P gene products.

  8. The Bovine Immunodeficiency Virus Rev Protein: Identification of a Novel Nuclear Import Pathway and Nuclear Export Signal among Retroviral Rev/Rev-Like Proteins

    PubMed Central

    Gomez Corredor, Andrea

    2012-01-01

    The Rev protein is essential for the replication of lentiviruses. Rev is a shuttling protein that transports unspliced and partially spliced lentiviral RNAs from the nucleus to the cytoplasm via the nucleopore. To transport these RNAs, the human immunodeficiency virus type 1 (HIV-1) Rev uses the karyopherin β family importin β and CRM1 proteins that interact with the Rev nuclear localization signal (NLS) and nuclear exportation signal (NES), respectively. Recently, we reported the presence of new types of bipartite NLS and nucleolar localization signal (NoLS) in the bovine immunodeficiency virus (BIV) Rev protein. Here we report the characterization of the nuclear import and export pathways of BIV Rev. By using an in vitro nuclear import assay, we showed that BIV Rev is transported into the nucleus by a cytosolic and energy-dependent importin α/β classical pathway. Results from glutathione S-transferase (GST) pulldown assays that showed the binding of BIV Rev with importins α3 and α5 were in agreement with those from the nuclear import assay. We also identified a leptomycin B-sensitive NES in BIV Rev, which indicates that the protein is exported via CRM1 like HIV-1 Rev. Mutagenesis experiments showed that the BIV Rev NES maps between amino acids 109 to 121 of the protein. Remarkably, the BIV Rev NES was found to be of the cyclic AMP (cAMP)-dependent protein kinase inhibitor (PKI) type instead of the HIV-1 Rev type. In summary, our data showed that the nuclear import mechanism of BIV Rev is novel among Rev proteins characterized so far in lentiviruses. PMID:22379104

  9. Calreticulin Is a Receptor for Nuclear Export

    PubMed Central

    Holaska, James M.; Black, Ben E.; Love, Dona C.; Hanover, John A.; Leszyk, John; Paschal, Bryce M.

    2001-01-01

    In previous work, we used a permeabilized cell assay that reconstitutes nuclear export of protein kinase inhibitor (PKI) to show that cytosol contains an export activity that is distinct from Crm1 (Holaska, J.M., and B.M. Paschal. 1995. Proc. Natl. Acad. Sci. USA. 95: 14739–14744). Here, we describe the purification and characterization of the activity as calreticulin (CRT), a protein previously ascribed to functions in the lumen of the ER. We show that cells contain both ER and cytosolic pools of CRT. The mechanism of CRT-dependent export of PKI requires a functional nuclear export signal (NES) in PKI and involves formation of an export complex that contains RanGTP. Previous studies linking CRT to downregulation of steroid hormone receptor function led us to examine its potential role in nuclear export of the glucocorticoid receptor (GR). We found that CRT mediates nuclear export of GR in permeabilized cell, microinjection, and transfection assays. GR export is insensitive to the Crm1 inhibitor leptomycin B in vivo, and it does not rely on a leucine-rich NES. Rather, GR export is facilitated by its DNA-binding domain, which is shown to function as an NES when transplanted to a green fluorescent protein reporter. CRT defines a new export pathway that may regulate the transcriptional activity of steroid hormone receptors. PMID:11149926

  10. Calreticulin Is a receptor for nuclear export.

    PubMed

    Holaska, J M; Black, B E; Love, D C; Hanover, J A; Leszyk, J; Paschal, B M

    2001-01-01

    In previous work, we used a permeabilized cell assay that reconstitutes nuclear export of protein kinase inhibitor (PKI) to show that cytosol contains an export activity that is distinct from Crm1 (Holaska, J.M., and B.M. Paschal. 1995. Proc. Natl. Acad. Sci. USA. 95: 14739-14744). Here, we describe the purification and characterization of the activity as calreticulin (CRT), a protein previously ascribed to functions in the lumen of the ER. We show that cells contain both ER and cytosolic pools of CRT. The mechanism of CRT-dependent export of PKI requires a functional nuclear export signal (NES) in PKI and involves formation of an export complex that contains RanGTP. Previous studies linking CRT to downregulation of steroid hormone receptor function led us to examine its potential role in nuclear export of the glucocorticoid receptor (GR). We found that CRT mediates nuclear export of GR in permeabilized cell, microinjection, and transfection assays. GR export is insensitive to the Crm1 inhibitor leptomycin B in vivo, and it does not rely on a leucine-rich NES. Rather, GR export is facilitated by its DNA-binding domain, which is shown to function as an NES when transplanted to a green fluorescent protein reporter. CRT defines a new export pathway that may regulate the transcriptional activity of steroid hormone receptors. PMID:11149926

  11. An N-terminal nuclear export signal regulates trafficking and aggregation of Huntingtin (Htt) protein exon 1.

    PubMed

    Zheng, Zhiqiang; Li, Aimin; Holmes, Brandon B; Marasa, Jayne C; Diamond, Marc I

    2013-03-01

    Huntington disease is a dominantly inherited neurodegenerative condition caused by polyglutamine expansion in the N terminus of the huntingtin protein (Htt). The first 17 amino acids (N17) of Htt play a key role in regulating its toxicity and aggregation. Both nuclear export and cytoplasm retention functions have been ascribed to N17. We have determined that N17 acts as a nuclear export sequence (NES) within Htt exon and when fused to yellow fluorescent protein. We have defined amino acids within N17 that constitute the nuclear export sequence (NES). Mutation of any of the conserved residues increases nuclear accumulation of Htt exon 1. Nuclear export of Htt is sensitive to leptomycin B and is reduced by knockdown of exportin 1. In HEK293 cells, NES mutations decrease overall Htt aggregation but increase the fraction of cells with nuclear inclusions. In primary cultured neurons, NES mutations increase nuclear accumulation and increase overall aggregation. This work defines a bona fide nuclear export sequence within N17 and links it to effects on protein aggregation. This may help explain the important role of N17 in controlling Htt toxicity. PMID:23319588

  12. Identification of the nuclear export signals that regulate the intracellular localization of the mouse CMP-sialic acid synthetase

    SciTech Connect

    Fujita, Akiko; Sato, Chihiro; Kitajima, Ken. E-mail: kitajima@agr.nagoya-u.ac.jp

    2007-03-30

    The CMP-sialic acid synthetase (CSS) catalyzes the activation of sialic acid (Sia) to CMP-Sia which is a donor substrate of sialyltransferases. The vertebrate CSSs are usually localized in nucleus due to the nuclear localization signal (NLS) on the molecule. In this study, we first point out that a small, but significant population of the mouse CMP-sialic acid synthetase (mCSS) is also present in cytoplasm, though mostly in nucleus. As a mechanism for the localization in cytoplasm, we first identified two nuclear export signals (NESs) in mCSS, based on the localization studies of the potential NES-deleted mCSS mutants as well as the potential NES-tagged eGFP proteins. These two NESs are conserved among mammalian and fish CSSs, but not present in the bacterial or insect CSS. These results suggest that the intracellular localization of vertebrate CSSs is regulated by not only the NLS, but also the NES sequences.

  13. Functional characterization of nuclear localization and export signals in hepatitis C virus proteins and their role in the membranous web.

    PubMed

    Levin, Aviad; Neufeldt, Christopher J; Pang, Daniel; Wilson, Kristen; Loewen-Dobler, Darci; Joyce, Michael A; Wozniak, Richard W; Tyrrell, D Lorne J

    2014-01-01

    The hepatitis C virus (HCV) is a positive strand RNA virus of the Flavivirus family that replicates in the cytoplasm of infected hepatocytes. Previously, several nuclear localization signals (NLS) and nuclear export signals (NES) have been identified in HCV proteins, however, there is little evidence that these proteins travel into the nucleus during infection. We have recently shown that nuclear pore complex (NPC) proteins (termed nucleoporins or Nups) are present in the membranous web and are required during HCV infection. In this study, we identify a total of 11 NLS and NES sequences in various HCV proteins. We show direct interactions between HCV proteins and importin α5 (IPOA5/kapα1), importin β3 (IPO5/kap β3), and exportin 1 (XPO1/CRM1) both in-vitro and in cell culture. These interactions can be disrupted using peptides containing the specific NLS or NES sequences of HCV proteins. Moreover, using a synchronized infection system, we show that these peptides inhibit HCV infection during distinct phases of the HCV life cycle. The inhibitory effects of these peptides place them in two groups. The first group binds IPOA5 and inhibits infection during the replication stage of HCV life cycle. The second group binds IPO5 and is active during both early replication and early assembly. This work delineates the entire life cycle of HCV and the active involvement of NLS sequences during HCV replication and assembly. Given the abundance of NLS sequences within HCV proteins, our previous finding that Nups play a role in HCV infection, and the relocation of the NLS double-GFP reporter in HCV infected cells, this work supports our previous hypothesis that NPC-like structures and nuclear transport factors function in the membranous web to create an environment conducive to viral replication. PMID:25485706

  14. Functional Characterization of Nuclear Localization and Export Signals in Hepatitis C Virus Proteins and Their Role in the Membranous Web

    PubMed Central

    Levin, Aviad; Neufeldt, Christopher J.; Pang, Daniel; Wilson, Kristen; Loewen-Dobler, Darci; Joyce, Michael A.; Wozniak, Richard W.; Tyrrell, D. Lorne J

    2014-01-01

    The hepatitis C virus (HCV) is a positive strand RNA virus of the Flavivirus family that replicates in the cytoplasm of infected hepatocytes. Previously, several nuclear localization signals (NLS) and nuclear export signals (NES) have been identified in HCV proteins, however, there is little evidence that these proteins travel into the nucleus during infection. We have recently shown that nuclear pore complex (NPC) proteins (termed nucleoporins or Nups) are present in the membranous web and are required during HCV infection. In this study, we identify a total of 11 NLS and NES sequences in various HCV proteins. We show direct interactions between HCV proteins and importin α5 (IPOA5/kapα1), importin β3 (IPO5/kap β3), and exportin 1 (XPO1/CRM1) both in-vitro and in cell culture. These interactions can be disrupted using peptides containing the specific NLS or NES sequences of HCV proteins. Moreover, using a synchronized infection system, we show that these peptides inhibit HCV infection during distinct phases of the HCV life cycle. The inhibitory effects of these peptides place them in two groups. The first group binds IPOA5 and inhibits infection during the replication stage of HCV life cycle. The second group binds IPO5 and is active during both early replication and early assembly. This work delineates the entire life cycle of HCV and the active involvement of NLS sequences during HCV replication and assembly. Given the abundance of NLS sequences within HCV proteins, our previous finding that Nups play a role in HCV infection, and the relocation of the NLS double-GFP reporter in HCV infected cells, this work supports our previous hypothesis that NPC-like structures and nuclear transport factors function in the membranous web to create an environment conducive to viral replication. PMID:25485706

  15. Subcellular localization of CrmA: identification of a novel leucine-rich nuclear export signal conserved in anti-apoptotic serpins.

    PubMed Central

    Rodriguez, Jose A; Span, Simone W; Kruyt, Frank A E; Giaccone, Giuseppe

    2003-01-01

    The cowpox virus-encoded anti-apoptotic protein cytokine response modifier A (CrmA) is a member of the serpin family that specifically inhibits the cellular proteins caspase 1, caspase 8 and granzyme B. In this study, we have used Flag- and yellow fluorescent protein (YFP)-tagged versions of CrmA to investigate the mechanisms that regulate its subcellular localization. We show that CrmA can actively enter and exit the nucleus and we demonstrate the role of the nuclear export receptor CRM1 in this shuttling process. CrmA contains a novel leucine-rich nuclear export signal (NES) that is functionally conserved in the anti-apoptotic cellular serpin PI-9. Besides this leucine-rich export signal, additional sequences mapping to a 103-amino-acid region flanking the NES contribute to the CRM1-dependent nuclear export of CrmA. Although YFP-tagged CrmA is primarily located in the cytoplasm, shifting its localization to be predominantly nuclear by fusion of a heterologous nuclear localization signal did not impair its ability to prevent Fas-induced apoptosis. We propose that nucleocytoplasmic shuttling would allow CrmA to efficiently target cellular pro-apoptotic proteins not only in the cytoplasm, but also in the nucleus, and thus to carry out its anti-apoptotic function in both compartments. PMID:12667137

  16. In Vitro Comparison of Adipokine Export Signals.

    PubMed

    Sharafi, Parisa; Kocaefe, Y Çetin

    2016-01-01

    Mammalian cells are widely used for recombinant protein production in research and biotechnology. Utilization of export signals significantly facilitates production and purification processes. 35 years after the discovery of the mammalian export machinery, there still are obscurities regarding the efficiency of the export signals. The aim of this study was the comparative evaluation of the efficiency of selected export signals using adipocytes as a cell model. Adipocytes have a large capacity for protein secretion including several enzymes, adipokines, and other signaling molecules, providing a valid system for a quantitative evaluation. Constructs that expressed N-terminal fusion export signals were generated to express Enhanced Green Fluorescence Protein (EGFP) as a reporter for quantitative and qualitative evaluation. Furthermore, fluorescent microscopy was used to trace the intracellular traffic of the reporter. The export efficiency of six selected proteins secreted from adipocytes was evaluated. Quantitative comparison of intracellular and exported fractions of the recombinant constructs demonstrated a similar efficiency among the studied sequences with minor variations. The export signal of Retinol Binding Protein (RBP4) exhibited the highest efficiency. This study presents the first quantitative data showing variations among export signals, in adipocytes which will help optimization of recombinant protein distribution. PMID:27064098

  17. Proteome-wide search for functional motifs altered in tumors: Prediction of nuclear export signals inactivated by cancer-related mutations.

    PubMed

    Prieto, Gorka; Fullaondo, Asier; Rodríguez, Jose A

    2016-01-01

    Large-scale sequencing projects are uncovering a growing number of missense mutations in human tumors. Understanding the phenotypic consequences of these alterations represents a formidable challenge. In silico prediction of functionally relevant amino acid motifs disrupted by cancer mutations could provide insight into the potential impact of a mutation, and guide functional tests. We have previously described Wregex, a tool for the identification of potential functional motifs, such as nuclear export signals (NESs), in proteins. Here, we present an improved version that allows motif prediction to be combined with data from large repositories, such as the Catalogue of Somatic Mutations in Cancer (COSMIC), and to be applied to a whole proteome scale. As an example, we have searched the human proteome for candidate NES motifs that could be altered by cancer-related mutations included in the COSMIC database. A subset of the candidate NESs identified was experimentally tested using an in vivo nuclear export assay. A significant proportion of the selected motifs exhibited nuclear export activity, which was abrogated by the COSMIC mutations. In addition, our search identified a cancer mutation that inactivates the NES of the human deubiquitinase USP21, and leads to the aberrant accumulation of this protein in the nucleus. PMID:27174732

  18. Proteome-wide search for functional motifs altered in tumors: Prediction of nuclear export signals inactivated by cancer-related mutations

    PubMed Central

    Prieto, Gorka; Fullaondo, Asier; Rodríguez, Jose A.

    2016-01-01

    Large-scale sequencing projects are uncovering a growing number of missense mutations in human tumors. Understanding the phenotypic consequences of these alterations represents a formidable challenge. In silico prediction of functionally relevant amino acid motifs disrupted by cancer mutations could provide insight into the potential impact of a mutation, and guide functional tests. We have previously described Wregex, a tool for the identification of potential functional motifs, such as nuclear export signals (NESs), in proteins. Here, we present an improved version that allows motif prediction to be combined with data from large repositories, such as the Catalogue of Somatic Mutations in Cancer (COSMIC), and to be applied to a whole proteome scale. As an example, we have searched the human proteome for candidate NES motifs that could be altered by cancer-related mutations included in the COSMIC database. A subset of the candidate NESs identified was experimentally tested using an in vivo nuclear export assay. A significant proportion of the selected motifs exhibited nuclear export activity, which was abrogated by the COSMIC mutations. In addition, our search identified a cancer mutation that inactivates the NES of the human deubiquitinase USP21, and leads to the aberrant accumulation of this protein in the nucleus. PMID:27174732

  19. Characterization of specific antigenic epitopes and the nuclear export signal of the Porcine circovirus 2 ORF3 protein.

    PubMed

    Gu, Jinyan; Wang, Lun; Jin, Yulan; Lin, Cui; Wang, Huijuan; Zhou, Niu; Xing, Gang; Liao, Min; Zhou, Jiyong

    2016-02-29

    Porcine circovirus 2 (PCV2) is the etiological agent of postweaning multisystemic wasting syndrome. PCV2 ORF3 protein is a nonstructural protein known to induce apoptosis, but little is known about the biological function of ORF3 protein. Therefore, we undertook this study to map ORF3 protein epitopes recognized by a panel of monoclonal antibodies (mAbs) and to characterize putative nuclear localization (NLS) and nuclear export (NES) sequences in ORF3. The linear epitopes targeted by two previously published mAbs 3B1 and 1H3 and a novel mouse mAb 3C3 were defined using overlapping pools of peptides. Here, we find that ORF3 in PCV2 infected cells contains a conformational epitope targeted by the antibody 3C3, which is distinct from linear epitopes recognized by the antibodies 3B1 and 1H3 in recombinant ORF3 protein. These results suggest that the linear epitope recognized by 3B1 and 1H3 is masked in PCV2 infected cells, and that the conformational epitope is unique to PCV2 infection. Furthermore, we find that ORF3 protein expressed in cytoplasm in early stages of PCV2 infection and then accumulated in nucleus over time. Moreover, we localize a NES at the N-terminus (residues 1-35aa) of ORF3 which plays critical role in nuclear export activity. These findings provide a novel insight that deepens our understanding of the biological function of PCV2 ORF3. PMID:26854343

  20. Identification of the Nuclear Export Signal and STAT-Binding Domains of the Nipah Virus V Protein Reveals Mechanisms Underlying Interferon Evasion

    PubMed Central

    Rodriguez, Jason J.; Cruz, Cristian D.; Horvath, Curt M.

    2004-01-01

    The V proteins of Nipah virus and Hendra virus have been demonstrated to bind to cellular STAT1 and STAT2 proteins to form high-molecular-weight complexes that inhibit interferon (IFN)-induced antiviral transcription by preventing STAT nuclear accumulation. Analysis of the Nipah virus V protein has revealed a region between amino acids 174 and 192 that functions as a CRM1-dependent nuclear export signal (NES). This peptide is sufficient to complement an export-defective human immunodeficiency virus Rev protein, and deletion and substitution mutagenesis revealed that this peptide is necessary for both V protein shuttling and cytoplasmic retention of STAT1 and STAT2 proteins. However, the NES is not required for V-dependent IFN signaling inhibition. IFN signaling is blocked primarily by interaction between Nipah virus V residues 100 to 160 and STAT1 residues 509 to 712. Interaction with STAT2 requires a larger Nipah virus V segment between amino acids 100 and 300, but deletion of residues 230 to 237 greatly reduced STAT2 coprecipitation. Further, V protein interactions with cellular STAT1 is a prerequisite for STAT2 binding, and sequential immunoprecipitations demonstrate that V, STAT1, and STAT2 can form a tripartite complex. These findings characterize essential regions for Henipavirus V proteins that represent potential targets for therapeutic intervention. PMID:15113915

  1. Mechanisms of Nuclear Export in Cancer and Resistance to Chemotherapy

    PubMed Central

    El-Tanani, Mohamed; Dakir, El-Habib; Raynor, Bethany; Morgan, Richard

    2016-01-01

    Tumour suppressor proteins, such as p53, BRCA1, and ABC, play key roles in preventing the development of a malignant phenotype, but those that function as transcriptional regulators need to enter the nucleus in order to function. The export of proteins between the nucleus and cytoplasm is complex. It occurs through nuclear pores and exported proteins need a nuclear export signal (NES) to bind to nuclear exportin proteins, including CRM1 (Chromosomal Region Maintenance protein 1), and the energy for this process is provided by the RanGTP/RanGDP gradient. Due to the loss of DNA repair and cell cycle checkpoints, drug resistance is a major problem in cancer treatment, and often an initially successful treatment will fail due to the development of resistance. An important mechanism underlying resistance is nuclear export, and a number of strategies that can prevent nuclear export may reverse resistance. Examples include inhibitors of CRM1, antibodies to the nuclear export signal, and alteration of nuclear pore structure. Each of these are considered in this review. PMID:26985906

  2. The subcellular localization of PBX1 and EXD proteins depends on nuclear import and export signals and is modulated by association with PREP1 and HTH

    PubMed Central

    Berthelsen, Jens; Kilstrup-Nielsen, Charlotte; Blasi, Francesco; Mavilio, Fulvio; Zappavigna, Vincenzo

    1999-01-01

    Nuclear localization of the Extradenticle (EXD) and PBX1 proteins is regionally restricted during Drosophila and mammalian development. We studied the subcellular localization of EXD, PBX, and their partners Homothorax (HTH) and PREP1, in different cell contexts. HTH and PREP1 are cytoplasmic and require association with EXD/PBX for nuclear localization. EXD and PBX1 are nuclear in murine fibroblasts but not in Drosophila Schneider cells, in which they are actively exported to the cytoplasm. Coexpression of EXD/PBX with HTH/PREP1 causes nuclear localization of their heterodimers in both cell contexts. We propose that heterodimerization with HTH/PREP induces nuclear translocation of EXD and PBX1 in specific cell contexts by blocking their nuclear export. PMID:10215622

  3. The subcellular localization of PBX1 and EXD proteins depends on nuclear import and export signals and is modulated by association with PREP1 and HTH.

    PubMed

    Berthelsen, J; Kilstrup-Nielsen, C; Blasi, F; Mavilio, F; Zappavigna, V

    1999-04-15

    Nuclear localization of the Extradenticle (EXD) and PBX1 proteins is regionally restricted during Drosophila and mammalian development. We studied the subcellular localization of EXD, PBX, and their partners Homothorax (HTH) and PREP1, in different cell contexts. HTH and PREP1 are cytoplasmic and require association with EXD/PBX for nuclear localization. EXD and PBX1 are nuclear in murine fibroblasts but not in Drosophila Schneider cells, in which they are actively exported to the cytoplasm. Coexpression of EXD/PBX with HTH/PREP1 causes nuclear localization of their heterodimers in both cell contexts. We propose that heterodimerization with HTH/PREP induces nuclear translocation of EXD and PBX1 in specific cell contexts by blocking their nuclear export. PMID:10215622

  4. Biogenesis and nuclear export of ribosomal subunits in higher eukaryotes depend on the CRM1 export pathway.

    PubMed

    Thomas, Franziska; Kutay, Ulrike

    2003-06-15

    The production of ribosomes constitutes a major biosynthetic task for cells. Eukaryotic small and large ribosomal subunits are assembled in the nucleolus and independently exported to the cytoplasm. Most nuclear export pathways require RanGTP-binding export receptors. We analyzed the role of CRM1, the export receptor for leucine-rich nuclear export signals (NES), in the biogenesis of ribosomal subunits in vertebrate cells. Inhibition of the CRM1 export pathway led to a defect in nuclear export of both 40S and 60S subunits in HeLa cells. Moreover, the export of newly made ribosomal subunits in Xenopus oocytes was efficiently and specifically competed by BSA-NES conjugates. The CRM1 dependence of 60S subunit export suggested a conserved function for NMD3, a factor proposed to be a 60S subunit export adaptor in yeast. Indeed, we observed that nuclear export of human NMD3 (hNMD3) is sensitive to leptomycin B (LMB), which inactivates CRM1. It had, however, not yet been demonstrated that Nmd3 can interact with CRM1. Using purified recombinant proteins we have shown here that hNMD3 binds to CRM1 directly, in a RanGTP-dependent manner, by way of a C-terminal NES sequence. Our results suggest that the functions of CRM1 and NMD3 in ribosomal subunit export are conserved from yeast to higher eukaryotes. PMID:12724356

  5. Nuclear export of proteins and drug resistance in cancer

    PubMed Central

    Turner, Joel G.; Dawson, Jana; Sullivan, Daniel M.

    2015-01-01

    The intracellular location of a protein is crucial to its normal functioning in a cell. Cancer cells utilize the normal processes of nuclear-cytoplasmic transport through the nuclear pore complex of a cell to effectively evade anti-neoplastic mechanisms. CRM1-mediated export is increased in various cancers. Proteins that are exported in cancer include tumor-suppressive proteins such as retinoblastoma, APC, p53, BRAC1, FOXO proteins, INI1/hSNF5, galectin-3, Bok, nucleophosmin, RASSF2, Merlin, p21CIP, p27KIP1, N-WASP/FAK, estradiol receptor and Tob, drug targets topoisomerase I and IIα and BCR-ABL, and the molecular chaperone protein Hsp90. Here, we review in detail the current processes and known structures involved in the export of a protein through the nuclear pore complex. We also discuss the export receptor molecule CRM1 and its binding to the leucine-rich nuclear export signal of the cargo protein and the formation of a nuclear export trimer with RanGTP. The therapeutic potential of various CRM1 inhibitors will be addressed, including leptomycin B, ratjadone, KOS-2464, and specific small molecule inhibitors of CRM1, N-azolylacrylate analogs, FOXO export inhibitors, valtrate, acetoxychavicol acetate, CBS9106, and SINE inhibitors. We will also discuss examples of how drug resistance may be reversed by targeting the exported proteins topoisomerase IIα, BCR-ABL, and galectin-3. As effective and less toxic CRM1 export inhibitors become available, they may be used as both single agents and in combination with current chemotherapeutic drugs. We believe that the future development of low-toxicity, small-molecule CRM1 inhibitors may provide a new approach to treating cancer. PMID:22209898

  6. Optogenetic control of nuclear protein export

    PubMed Central

    Niopek, Dominik; Wehler, Pierre; Roensch, Julia; Eils, Roland; Di Ventura, Barbara

    2016-01-01

    Active nucleocytoplasmic transport is a key mechanism underlying protein regulation in eukaryotes. While nuclear protein import can be controlled in space and time with a portfolio of optogenetic tools, protein export has not been tackled so far. Here we present a light-inducible nuclear export system (LEXY) based on a single, genetically encoded tag, which enables precise spatiotemporal control over the export of tagged proteins. A constitutively nuclear, chromatin-anchored LEXY variant expands the method towards light inhibition of endogenous protein export by sequestering cellular CRM1 receptors. We showcase the utility of LEXY for cell biology applications by regulating a synthetic repressor as well as human p53 transcriptional activity with light. LEXY is a powerful addition to the optogenetic toolbox, allowing various novel applications in synthetic and cell biology. PMID:26853913

  7. Nucleocytoplasmic transfer of the NF2 tumor suppressor protein merlin is regulated by exon 2 and a CRM1-dependent nuclear export signal in exon 15.

    PubMed

    Kressel, Michael; Schmucker, Beatrice

    2002-09-15

    The neurofibromatosis 2 protein merlin is a classical tumor suppressor protein. Germline mutations predispose to the development of schwannomas, meningiomas and ependymomas. Merlin has been implicated in cellular migration and adhesion. This function is reflected in its subcellular localization at the plasma membrane and known interacting partners. Merlin has been regarded as an exception in not exerting a functional role within the nucleus as other tumor suppressors do. Here, we show that detection of wild-type protein in the nucleus is a rare event. However, splicing out of exon 2 leads to unrestricted entry into the nucleus. Skipping of adjacent exon 3 has no comparable effect ruling out an unspecific effect due to misfolding of the 4.1/JEF domain. Exon 2 functions as a cytoplasmic retention factor as it is able to confer sole cytoplasmic localization to a GFP fusion protein. Nuclear entry of merlin is thus regulated by alternative splicing within the 4.1/JEF domain and analogous to band 4.1 protein. Merlin's ability to enter the nucleus is complemented by a full nuclear-cytoplasmic shuttle protein with a functional Rev-type nuclear export sequence (NES) within exon 15 that facilitates export via the CRM1/exportin pathway. Deletion of this NES or treatment with the CRM1-specific inhibitor leptomycin B leads to overall nuclear accumulation of merlin isoforms missing exon 2. A cellular function different to the wild-type protein is implied for naturally occurring splice variants lacking exon 2. A putative effect of merlin as a transcriptional regulator and identification of nuclear binding partners remains to be elucidated. PMID:12217955

  8. Intracellular trafficking of LET-756, a fibroblast growth factor of C. elegans, is controlled by a balance of export and nuclear signals

    SciTech Connect

    Popovici, Cornel; Fallet, Mathieu; Marguet, Didier; Birnbaum, Daniel; Roubin, Regine . E-mail: roubin@marseille.inserm.fr

    2006-05-15

    The superfamily of fibroblast growth factors (FGF), which counts 22 members in humans, exerts many functions during animal development and adult life. LET-756 is one of the two FGFs of the nematode C. elegans. Re-introduction of LET-756 in a null mutant strain restores viability, allowing the study of structural requirements for LET-756 trafficking and function. LET-756 protein has several regions and motifs, including a non-classical internal motif required for secretion. We show here that a main difference in the wild-type LET-756 molecule and a truncated molecule that mimics a partial loss-of-function mutant lies on subnuclear expression. Using Cos-1 cells and rescue activity we show that: (i) nuclear localization is due to various redundant NLS, one of them acting as a nucleolar localization signal; (ii) nuclear LET-756 is addressed to the speckles by a stretch of glutamine residues; (iii) nuclear LET-756 is trafficking between speckles and nucleoli; (iv) in the nucleolus, LET-756 is associated with proteins of the rRNA splicing compartment; (v) changing LET-756 secretion signal prevents its nuclear localization. We propose that LET-756 exerts its functions through a balance between secreted and nuclear forms due to two opposite addressing signals (i) synergy of several NLS and (ii) attenuated secretion signal.

  9. Nuclear export modulates the cytoplasmic Sir2 homologue Hst2

    PubMed Central

    Wilson, Jeanne M; Le, Viet Q; Zimmerman, Collin; Marmorstein, Ronen; Pillus, Lorraine

    2006-01-01

    Modulating transcription factors is crucial to executing sophisticated gene expression programs. The silent information regulator 2 (Sir2) family of NAD-dependent protein deacetylases influences transcription by targeting proteins such as histones, p53 and forkhead-box family transcription factors. Although apparently cytoplasmic, both mammalian SIRT2 and its yeast orthologue Hst2 have been implicated in transcriptional regulation. Here, we show that Hst2 moves between the nucleus and cytoplasm, but is largely cytoplasmic owing to efficient nuclear export. This nuclear exclusion is mediated by the exportin chromosomal region maintenance 1 (Crm1) and a putative leucine-rich nuclear export sequence in Hst2, which overlaps a unique autoregulatory helix. Disruption of Hst2 export shows that nuclear exclusion inhibits the activity of Hst2 as a transcriptional repressor. Our identification of putative nuclear export sequences in numerous vertebrate SIRT2 proteins shows that active nuclear export can be a conserved mechanism for regulating Sir2 homologues. PMID:17110954

  10. Enzymatically Driven Transport: A Kinetic Theory for Nuclear Export

    PubMed Central

    Kim, Sanghyun; Elbaum, M.

    2013-01-01

    Nuclear import and export are often considered inverse processes whereby transport receptors ferry protein cargo through the nuclear pore. In contrast to import, where the reversible binding of receptor to nuclear RanGTP leads to a balanced bidirectional exchange, termination of export by physiologically irreversible hydrolysis of the Ran-bound GTP leads to unidirectional transport. We present a concise mathematical model that predicts protein distributions and kinetic rates for receptor-mediated nuclear export, which further exhibit an unexpected pseudolinear relation one to the other. Predictions of the model are verified with permeabilized and live cell measurements. PMID:24209844

  11. Insights into the nuclear export of murine leukemia virus intron-containing RNA

    PubMed Central

    Pessel-Vivares, Lucie; Houzet, Laurent; Lainé, Sébastien; Mougel, Marylène

    2015-01-01

    The retroviral genome consists of an intron-containing transcript that has essential cytoplasmic functions in the infected cell. This viral transcript can escape splicing, circumvent the nuclear checkpoint mechanisms and be transported to the cytoplasm by hijacking the host machinery. Once in the cytoplasm, viral unspliced RNA acts as mRNA to be translated and as genomic RNA to be packaged into nascent viruses. The murine leukemia virus (MLV) is among the first retroviruses discovered and is classified as simple Retroviridae due to its minimal encoding capacity. The oncogenic and transduction abilities of MLV are extensively studied, whereas surprisingly the crucial step of its nuclear export has remained unsolved until 2014. Recent work has revealed the recruitment by MLV of the cellular NXF1/Tap-dependent pathway for export. Unconventionally, MLV uses of Tap to export both spliced and unspliced viral RNAs. Unlike other retroviruses, MLV does not harbor a unique RNA signal for export. Indeed, multiple sequences throughout the MLV genome appear to promote export of the unspliced MLV RNA. We review here the current understanding of the export mechanism and highlight the determinants that influence MLV export. As the molecular mechanism of MLV export is elucidated, we will gain insight into the contribution of the export pathway to the cytoplasmic fate of the viral RNA. PMID:26158194

  12. Light-induced nuclear export reveals rapid dynamics of epigenetic modifications.

    PubMed

    Yumerefendi, Hayretin; Lerner, Andrew Michael; Zimmerman, Seth Parker; Hahn, Klaus; Bear, James E; Strahl, Brian D; Kuhlman, Brian

    2016-06-01

    We engineered a photoactivatable system for rapidly and reversibly exporting proteins from the nucleus by embedding a nuclear export signal in the LOV2 domain from phototropin 1. Fusing the chromatin modifier Bre1 to the photoswitch, we achieved light-dependent control of histone H2B monoubiquitylation in yeast, revealing fast turnover of the ubiquitin mark. Moreover, this inducible system allowed us to dynamically monitor the status of epigenetic modifications dependent on H2B ubiquitylation. PMID:27089030

  13. Multiple cis-acting signals for export of pre-U1 snRNA from the nucleus.

    PubMed

    Terns, M P; Dahlberg, J E; Lund, E

    1993-10-01

    We have identified cis-acting sequences that promote nuclear export of pre-U1 RNA injected into Xenopus oocyte nuclei. At least three elements, the 5' m7G cap, the 3'-terminal stem-loop structure, and sequences in the 5'-terminal 124 nucleotides, contribute to efficient export of this RNA. Both the 5' and 3' export signals can function separately and do so independently of the cap structure. Experiments using hybrid RNAs indicate that the 5' and 3' export sequences of U1 RNA are sufficient to direct export of the heterologous, otherwise nonexportable, U6 RNA. The absence of comparable export signals in U6 RNA appears to be responsible for its retention in the nucleus. Stability of the pre-snRNAs in the nucleus depends on the presence of both a 5' cap structure and a 3' base-paired stem. The 5' m7G cap is neither sufficient nor necessary for nuclear export. The m7G cap by itself did not promote export of U6 RNA or nonspecific small RNAs. Moreover, substitution of this cap with either an AppG cap or gamma-mppG cap did not eliminate export of either full-length or a "minimal" U1 RNA (lacking most of the internal U1 RNA sequences), but it reduced the rate of export by about two to threefold. However, in the absence of the 3' stem-loop, substitution of the m7G cap led to a greater decrease in export rate, underscoring the cooperative action of the three different export elements of pre-U1 RNA. The m7G cap analog, m7GpppG, selectively destabilized pre-U1 RNA within the nucleus. Thus, nuclear components that recognize the 5' m7G cap may be important for both the stability and the export of pre-U1 RNA. PMID:8405997

  14. The export receptor Crm1 forms a dimer to promote nuclear export of HIV RNA

    PubMed Central

    Booth, David S; Cheng, Yifan; Frankel, Alan D

    2014-01-01

    The HIV Rev protein routes viral RNAs containing the Rev Response Element (RRE) through the Crm1 nuclear export pathway to the cytoplasm where viral proteins are expressed and genomic RNA is delivered to assembling virions. The RRE assembles a Rev oligomer that displays nuclear export sequences (NESs) for recognition by the Crm1-RanGTP nuclear receptor complex. Here we provide the first view of an assembled HIV-host nuclear export complex using single-particle electron microscopy. Unexpectedly, Crm1 forms a dimer with an extensive interface that enhances association with Rev-RRE and poises NES binding sites to interact with a Rev oligomer. The interface between Crm1 monomers explains differences between Crm1 orthologs that alter nuclear export and determine cellular tropism for viral replication. The arrangement of the export complex identifies a novel binding surface to possibly target an HIV inhibitor and may point to a broader role for Crm1 dimerization in regulating host gene expression. DOI: http://dx.doi.org/10.7554/eLife.04121.001 PMID:25486595

  15. RNA export factor Ddx19 is required for nuclear import of the SRF coactivator MKL1

    PubMed Central

    Rajakylä, Eeva Kaisa; Viita, Tiina; Kyheröinen, Salla; Huet, Guillaume; Treisman, Richard; Vartiainen, Maria K.

    2015-01-01

    Controlled transport of macromolecules between the cytoplasm and nucleus is essential for homeostatic regulation of cellular functions. For instance, gene expression entails coordinated nuclear import of transcriptional regulators to activate transcription and nuclear export of the resulting messenger RNAs for cytoplasmic translation. Here we link these two processes by reporting a novel role for the mRNA export factor Ddx19/Dbp5 in nuclear import of MKL1, the signal-responsive transcriptional activator of SRF. We show that Ddx19 is not a general nuclear import factor, and that its specific effect on MKL1 nuclear import is separate from its role in mRNA export. Both helicase and nuclear pore-binding activities of Ddx19 are dispensable for MKL1 nuclear import, but RNA binding is required. Mechanistically, Ddx19 operates by modulating the conformation of MKL1, which affects its interaction with Importin-β for efficient nuclear import. Thus, Ddx19 participates in mRNA export, translation and nuclear import of a key transcriptional regulator. PMID:25585691

  16. RNA export factor Ddx19 is required for nuclear import of the SRF coactivator MKL1.

    PubMed

    Rajakylä, Eeva Kaisa; Viita, Tiina; Kyheröinen, Salla; Huet, Guillaume; Treisman, Richard; Vartiainen, Maria K

    2015-01-01

    Controlled transport of macromolecules between the cytoplasm and nucleus is essential for homeostatic regulation of cellular functions. For instance, gene expression entails coordinated nuclear import of transcriptional regulators to activate transcription and nuclear export of the resulting messenger RNAs for cytoplasmic translation. Here we link these two processes by reporting a novel role for the mRNA export factor Ddx19/Dbp5 in nuclear import of MKL1, the signal-responsive transcriptional activator of SRF. We show that Ddx19 is not a general nuclear import factor, and that its specific effect on MKL1 nuclear import is separate from its role in mRNA export. Both helicase and nuclear pore-binding activities of Ddx19 are dispensable for MKL1 nuclear import, but RNA binding is required. Mechanistically, Ddx19 operates by modulating the conformation of MKL1, which affects its interaction with Importin-β for efficient nuclear import. Thus, Ddx19 participates in mRNA export, translation and nuclear import of a key transcriptional regulator. PMID:25585691

  17. 10 CFR 110.26 - General license for the export of nuclear reactor components.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 10 Energy 2 2014-01-01 2014-01-01 false General license for the export of nuclear reactor components. 110.26 Section 110.26 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) EXPORT AND IMPORT OF NUCLEAR EQUIPMENT AND MATERIAL Licenses § 110.26 General license for the export of nuclear...

  18. 10 CFR 110.9 - List of Nuclear Material under NRC export licensing authority.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 10 Energy 2 2014-01-01 2014-01-01 false List of Nuclear Material under NRC export licensing authority. 110.9 Section 110.9 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) EXPORT AND IMPORT OF NUCLEAR EQUIPMENT AND MATERIAL General Provisions § 110.9 List of Nuclear Material under NRC export...

  19. 10 CFR 110.21 - General license for the export of special nuclear material.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 10 Energy 2 2013-01-01 2013-01-01 false General license for the export of special nuclear material. 110.21 Section 110.21 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) EXPORT AND IMPORT OF NUCLEAR EQUIPMENT AND MATERIAL Licenses § 110.21 General license for the export of special nuclear material....

  20. 10 CFR 110.21 - General license for the export of special nuclear material.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 10 Energy 2 2014-01-01 2014-01-01 false General license for the export of special nuclear material. 110.21 Section 110.21 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) EXPORT AND IMPORT OF NUCLEAR EQUIPMENT AND MATERIAL Licenses § 110.21 General license for the export of special nuclear material....

  1. 10 CFR 110.9 - List of Nuclear Material under NRC export licensing authority.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 10 Energy 2 2013-01-01 2013-01-01 false List of Nuclear Material under NRC export licensing authority. 110.9 Section 110.9 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) EXPORT AND IMPORT OF NUCLEAR EQUIPMENT AND MATERIAL General Provisions § 110.9 List of Nuclear Material under NRC export...

  2. 10 CFR 110.26 - General license for the export of nuclear reactor components.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 10 Energy 2 2013-01-01 2013-01-01 false General license for the export of nuclear reactor components. 110.26 Section 110.26 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) EXPORT AND IMPORT OF NUCLEAR EQUIPMENT AND MATERIAL Licenses § 110.26 General license for the export of nuclear...

  3. 10 CFR 110.21 - General license for the export of special nuclear material.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 2 2010-01-01 2010-01-01 false General license for the export of special nuclear material. 110.21 Section 110.21 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) EXPORT AND IMPORT OF NUCLEAR EQUIPMENT AND MATERIAL Licenses § 110.21 General license for the export of special nuclear material....

  4. 10 CFR 110.26 - General license for the export of nuclear reactor components.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 2 2010-01-01 2010-01-01 false General license for the export of nuclear reactor components. 110.26 Section 110.26 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) EXPORT AND IMPORT OF NUCLEAR EQUIPMENT AND MATERIAL Licenses § 110.26 General license for the export of nuclear...

  5. 10 CFR 110.9 - List of Nuclear Material under NRC export licensing authority.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 2 2010-01-01 2010-01-01 false List of Nuclear Material under NRC export licensing authority. 110.9 Section 110.9 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) EXPORT AND IMPORT OF NUCLEAR EQUIPMENT AND MATERIAL General Provisions § 110.9 List of Nuclear Material under NRC export...

  6. 10 CFR 110.26 - General license for the export of nuclear reactor components.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 10 Energy 2 2011-01-01 2011-01-01 false General license for the export of nuclear reactor components. 110.26 Section 110.26 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) EXPORT AND IMPORT OF NUCLEAR EQUIPMENT AND MATERIAL Licenses § 110.26 General license for the export of nuclear...

  7. 10 CFR 110.21 - General license for the export of special nuclear material.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 10 Energy 2 2011-01-01 2011-01-01 false General license for the export of special nuclear material. 110.21 Section 110.21 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) EXPORT AND IMPORT OF NUCLEAR EQUIPMENT AND MATERIAL Licenses § 110.21 General license for the export of special nuclear material....

  8. 10 CFR 110.9 - List of Nuclear Material under NRC export licensing authority.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 10 Energy 2 2011-01-01 2011-01-01 false List of Nuclear Material under NRC export licensing authority. 110.9 Section 110.9 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) EXPORT AND IMPORT OF NUCLEAR EQUIPMENT AND MATERIAL General Provisions § 110.9 List of Nuclear Material under NRC export...

  9. 10 CFR 110.26 - General license for the export of nuclear reactor components.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 10 Energy 2 2012-01-01 2012-01-01 false General license for the export of nuclear reactor components. 110.26 Section 110.26 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) EXPORT AND IMPORT OF NUCLEAR EQUIPMENT AND MATERIAL Licenses § 110.26 General license for the export of nuclear...

  10. 10 CFR 110.21 - General license for the export of special nuclear material.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 10 Energy 2 2012-01-01 2012-01-01 false General license for the export of special nuclear material. 110.21 Section 110.21 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) EXPORT AND IMPORT OF NUCLEAR EQUIPMENT AND MATERIAL Licenses § 110.21 General license for the export of special nuclear material....

  11. 10 CFR 110.9 - List of Nuclear Material under NRC export licensing authority.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 10 Energy 2 2012-01-01 2012-01-01 false List of Nuclear Material under NRC export licensing authority. 110.9 Section 110.9 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) EXPORT AND IMPORT OF NUCLEAR EQUIPMENT AND MATERIAL General Provisions § 110.9 List of Nuclear Material under NRC export...

  12. Splicing promotes the nuclear export of β-globin mRNA by overcoming nuclear retention elements

    PubMed Central

    Akef, Abdalla; Lee, Eliza S.; Palazzo, Alexander F.

    2015-01-01

    Most current models of mRNA nuclear export in vertebrate cells assume that an mRNA must have specialized signals in order to be exported from the nucleus. Under such a scenario, mRNAs that lack these specialized signals would be shunted into a default pathway where they are retained in the nucleus and eventually degraded. These ideas were based on the selective use of model mRNA reporters. For example, it has been shown that splicing promotes the nuclear export of certain model mRNAs, such as human β-globin, and that in the absence of splicing, the cDNA-derived mRNA is retained in the nucleus and degraded. Here we provide evidence that β-globin mRNA contains an element that actively retains it in the nucleus and degrades it. Interestingly, this nuclear retention activity can be overcome by increasing the length of the mRNA or by splicing. Our results suggest that contrary to many current models, the default pathway for most intronless RNAs is to be exported from the nucleus, unless the RNA contains elements that actively promote its nuclear retention. PMID:26362019

  13. Cofactor Requirements for Nuclear Export of Rev Response Element (Rre)–And Constitutive Transport Element (Cte)–Containing Retroviral Rnas

    PubMed Central

    Hofmann, Wilma; Reichart, Beate; Ewald, Andrea; Müller, Eleonora; Schmitt, Iris; Stauber, Roland H.; Lottspeich, Friedrich; Jockusch, Brigitte M.; Scheer, Ulrich; Hauber, Joachim; Dabauvalle, Marie-Christine

    2001-01-01

    Nuclear export of proteins containing leucine-rich nuclear export signals (NESs) is mediated by the export receptor CRM1/exportin1. However, additional protein factors interacting with leucine-rich NESs have been described. Here, we investigate human immunodeficiency virus type 1 (HIV-1) Rev-mediated nuclear export and Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE)–mediated nuclear export in microinjected Xenopus laevis oocytes. We show that eukaryotic initiation factor 5A (eIF-5A) is essential for Rev and Rev-mediated viral RNA export, but not for nuclear export of CTE RNA. In vitro binding studies demonstrate that eIF-5A is required for efficient interaction of Rev–NES with CRM1/exportin1 and that eIF-5A interacts with the nucleoporins CAN/nup214, nup153, nup98, and nup62. Quite unexpectedly, nuclear actin was also identified as an eIF-5A binding protein. We show that actin is associated with the nucleoplasmic filaments of nuclear pore complexes and is critically involved in export processes. Finally, actin- and energy-dependent nuclear export of HIV-1 Rev is reconstituted by using a novel in vitro egg extract system. In summary, our data provide evidence that actin plays an important functional role in nuclear export not only of retroviral RNAs but also of host proteins such as protein kinase inhibitor (PKI). PMID:11238447

  14. Calcium-dependent regulation of NEMO nuclear export in response to genotoxic stimuli.

    PubMed

    Berchtold, Craig M; Wu, Zhao-Hui; Huang, Tony T; Miyamoto, Shigeki

    2007-01-01

    The mechanisms involved in activation of the transcription factor NF-kappaB by genotoxic agents are not well understood. Previously, we provided evidence that a regulatory subunit of the IkappaB kinase (IKK) complex, NF-kappaB essential modulator (NEMO)/IKKgamma, is a component of a nuclear signal that is generated after DNA damage to mediate NF-kappaB activation. Here, we found that etoposide (VP16) and camptothecin induced increases in intracellular free calcium levels at 60 min after stimulation of CEM T leukemic cells. Inhibition of calcium increases by calcium chelators, BAPTA-AM and EGTA-AM, abrogated NF-kappaB activation by these agents in several cell types examined. Conversely, thapsigargin and ionomycin attenuated the BAPTA-AM effects and promoted NF-kappaB activation by the genotoxic stimuli. Analyses of nuclear NEMO levels in VP16-treated cells suggested that calcium was required for nuclear export of NEMO. Inhibition of the nuclear exporter CRM1 by leptomycin B did not interfere with NEMO nuclear export. Similarly, deficiency of a plausible calcium-dependent nuclear export receptor, calreticulin, failed to prevent NF-kappaB activation by VP16. However, temperature inactivation of the Ran guanine nucleotide exchange factor RCC1 in the tsBN2 cell line harboring a temperature-sensitive mutant of RCC1 blocked NF-kappaB activation induced by genotoxic stimuli. Overexpression of Ran in this cell model showed that DNA damage stimuli induced formation of a complex between Ran and NEMO, suggesting that RCC1 regulated NF-kappaB activation through the modulation of RanGTP. Indeed, evidence for VP16-inducible interaction between Ran-GTP and NEMO could be obtained by means of glutathione S-transferase (GST) pull-down assays using GST fused to the Ran binding domain of RanBP2, which specifically interacts with the GTP-bound form of Ran. BAPTA-AM did not alter these interactions, suggesting that calcium is a necessary step beyond the formation of a Ran

  15. 77 FR 27113 - Export and Import of Nuclear Equipment and Material; Export of International Atomic Energy Agency...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-05-09

    ...) part 110, ``Export and Import of Nuclear Equipment and Material,'' to current nuclear non-proliferation... and import of nuclear materials and equipment. This rulemaking is necessary to reflect the nuclear non... OMB control number. Regulatory Analysis This rulemaking is necessary to reflect the nuclear...

  16. The BRO proteins of Bombyx mori nucleopolyhedrovirus are nucleocytoplasmic shuttling proteins that utilize the CRM1-mediated nuclear export pathway

    SciTech Connect

    Kang, Won Kyung . E-mail: wkkang@riken.jp; Kurihara, Masaaki . E-mail: mkuri@riken.jp; Matsumoto, Shogo . E-mail: smatsu@riken.jp

    2006-06-20

    The BRO proteins of Bombyx mori nucleopolyhedrovirus (BmNPV) display a biphasic pattern of intracellular localization during infection. At early times, they reside in the nucleus but then show both cytoplasmic and nuclear localization as the infection proceeds. Therefore, we examined the possibility of nuclear export. Using inhibitors, we reveal that BmNPV BRO proteins shuttle between the nucleus and cytoplasm. Mutations on the leucine-rich region of BRO proteins resulted in nuclear accumulation of transiently expressed proteins, suggesting that this region functions as a CRM1-dependent nuclear export signal (NES). On the contrary, mutant BRO-D with an altered NES did not show nuclear accumulation in infected cells, although protein production seemed to be reduced. RT-PCR analysis showed that the lower level of protein production was due to a reduction in RNA synthesis. Taken together, our results suggest that BRO proteins are nucleocytoplasmic shuttling proteins that utilize the CRM1-mediated nuclear export pathway.

  17. The net repressor is regulated by nuclear export in response to anisomycin, UV, and heat shock.

    PubMed

    Ducret, C; Maira, S M; Dierich, A; Wasylyk, B

    1999-10-01

    The ternary complex factors (TCFs) are targets for Ras/mitogen-activated protein kinase signalling pathways. They integrate the transcriptional response at the level of serum response elements in early-response genes, such as the c-fos proto-oncogene. An important aim is to understand the individual roles played by the three TCFs, Net, Elk1, and Sap1a. Net, in contrast to Elk1 and Sap1a, is a strong repressor of transcription. We now show that Net is regulated by nuclear-cytoplasmic shuttling in response to specific signalling pathways. Net is mainly nuclear under both normal and basal serum conditions. Net contains two nuclear localization signals (NLSs); one is located in the Ets domain, and the other corresponds to the D box. Net also has a nuclear export signal (NES) in the conserved Ets DNA binding domain. Net is apparently unique among Ets proteins in that a particular leucine in helix 1, a structural element, generates a NES. Anisomycin, UV, and heat shock induce active nuclear exclusion of Net through a pathway that involves c-Jun N-terminal kinase kinase and is inhibited by leptomycin B. Nuclear exclusion relieves transcriptional repression by Net. The specific induction of nuclear exclusion of Net by particular signalling pathways shows that nuclear-cytoplasmic transport of transcription factors can add to the specificity of the response to signalling cascades. PMID:10490644

  18. Novel Nuclear Localization Signal Regulated by Ambient Tonicity in Vertebrates*

    PubMed Central

    Kwon, Min Seong; Lee, Sang Do; Kim, Jeong-Ah; Colla, Emanuela; Choi, Yu Jeong; Suh, Pann-Ghil; Kwon, H. Moo

    2008-01-01

    TonEBP is a Rel domain-containing transcription factor implicated in adaptive immunity, viral replication, and cancer. In the mammalian kidney, TonEBP is a central regulator of water homeostasis. Animals deficient in TonEBP suffer from life-threatening dehydration due to renal water loss. Ambient tonicity (effective osmolality) is the prominent signal for TonEBP in a bidirectional manner; TonEBP activity decreases in hypotonicity, whereas it increases in hypertonicity. Here we found that TonEBP displayed nuclear export in response to hypotonicity and nuclear import in response to hypertonicity. The nuclear export of TonEBP was not mediated by the nuclear export receptor CRM1 or discrete nuclear export signal. In contrast, a dominant nuclear localization signal (NLS) was found in a small region of 16 amino acid residues. When short peptides containing the NLS were fused to constitutively cytoplasmic proteins, the fusion proteins displayed tonicity-dependent nucleocytoplasmic trafficking like TonEBP. Thus, tonicity-dependent activation of the NLS is crucial in the nucleocytoplasmic trafficking of TonEBP. The novel NLS is present only in the vertebrates, indicating that it developed late in evolution. PMID:18579527

  19. Identification of CRM1-dependent Nuclear Export Cargos Using Quantitative Mass Spectrometry*

    PubMed Central

    Thakar, Ketan; Karaca, Samir; Port, Sarah A.; Urlaub, Henning; Kehlenbach, Ralph H.

    2013-01-01

    Chromosome region maintenance 1/exportin1/Exp1/Xpo1 (CRM1) is the major transport receptor for the export of proteins from the nucleus. It binds to nuclear export signals (NESs) that are rich in leucines and other hydrophobic amino acids. The prediction of NESs is difficult because of the extreme recognition flexibility of CRM1. Furthermore, proteins can be exported upon binding to an NES-containing adaptor protein. Here we present an approach for identifying targets of the CRM1-export pathway via quantitative mass spectrometry using stable isotope labeling with amino acids in cell culture. With this approach, we identified >100 proteins from HeLa cells that were depleted from cytosolic fractions and/or enriched in nuclear fractions in the presence of the selective CRM1-inhibitor leptomycin B. Novel and validated substrates are the polyubiquitin-binding protein sequestosome 1, the cancerous inhibitor of protein phosphatase 2A (PP2A), the guanine nucleotide-binding protein-like 3-like protein, the programmed cell death protein 2-like protein, and the cytosolic carboxypeptidase 1 (CCP1). We identified a functional NES in CCP1 that mediates direct binding to the export receptor CRM1. The method will be applicable to other nucleocytoplasmic transport pathways, as well as to the analysis of nucleocytoplasmic shuttling proteins under different growth conditions. PMID:23242554

  20. Arabidopsis NMD3 Is Required for Nuclear Export of 60S Ribosomal Subunits and Affects Secondary Cell Wall Thickening

    PubMed Central

    Chen, Mei-Qin; Zhang, Ai-Hong; Zhang, Quan; Zhang, Bao-Cai; Nan, Jie; Li, Xia; Liu, Na; Qu, Hong; Lu, Cong-Ming; Sudmorgen; Zhou, Yi-Hua; Xu, Zhi-Hong; Bai, Shu-Nong

    2012-01-01

    NMD3 is required for nuclear export of the 60S ribosomal subunit in yeast and vertebrate cells, but no corresponding function of NMD3 has been reported in plants. Here we report that Arabidopsis thaliana NMD3 (AtNMD3) showed a similar function in the nuclear export of the 60S ribosomal subunit. Interference with AtNMD3 function by overexpressing a truncated dominant negative form of the protein lacking the nuclear export signal sequence caused retainment of the 60S ribosomal subunits in the nuclei. More interestingly, the transgenic Arabidopsis with dominant negative interference of AtNMD3 function showed a striking failure of secondary cell wall thickening, consistent with the altered expression of related genes and composition of cell wall components. Observation of a significant decrease of rough endoplasmic reticulum (RER) in the differentiating interfascicular fiber cells of the transgenic plant stems suggested a link between the defective nuclear export of 60S ribosomal subunits and the abnormal formation of the secondary cell wall. These findings not only clarified the evolutionary conservation of NMD3 functions in the nuclear export of 60S ribosomal subunits in yeast, animals and plants, but also revealed a new facet of the regulatory mechanism underlying secondary cell wall thickening in Arabidopsis. This new facet is that the nuclear export of 60S ribosomal subunits and the formation of RER may play regulatory roles in coordinating protein synthesis in cytoplasm and transcription in nuclei. PMID:22558264

  1. Nuclear Export and Centrosome Targeting of the Protein Phosphatase 2A Subunit B56α

    PubMed Central

    Flegg, Cameron P.; Sharma, Manisha; Medina-Palazon, Cahora; Jamieson, Cara; Galea, Melanie; Brocardo, Mariana G.; Mills, Kate; Henderson, Beric R.

    2010-01-01

    Protein phosphatase (PP) 2A is a heterotrimeric enzyme regulated by specific subunits. The B56 (or B′/PR61/PPP2R5) class of B-subunits direct PP2A or its substrates to different cellular locations, and the B56α, -β, and -ϵ isoforms are known to localize primarily in the cytoplasm. Here we studied the pathways that regulate B56α subcellular localization. We detected B56α in the cytoplasm and nucleus, and at the nuclear envelope and centrosomes, and show that cytoplasmic localization is dependent on CRM1-mediated nuclear export. The inactivation of CRM1 by leptomycin B or by siRNA knockdown caused nuclear accumulation of ectopic and endogenous B56α. Conversely, CRM1 overexpression shifted B56α to the cytoplasm. We identified a functional nuclear export signal at the C terminus (NES; amino acids 451–469), and site-directed mutagenesis of the NES (L461A) caused nuclear retention of full-length B56α. Active NESs were identified at similar positions in the cytoplasmic B56-β and ϵ isoforms, but not in the nuclear-localized B56-δ or γ isoforms. The transient expression of B56α induced nuclear export of the PP2A catalytic (C) subunit, and this was blocked by the L461A NES mutation. In addition, B56α co-located with the PP2A active (A) subunit at centrosomes, and its centrosome targeting involved sequences that bind to the A-subunit. Fluorescence Recovery after Photobleaching (FRAP) assays revealed dynamic and immobile pools of B56α-GFP, which was rapidly exported from the nucleus and subject to retention at centrosomes. We propose that B56α can act as a PP2A C-subunit chaperone and regulates PP2A activity at diverse subcellular locations. PMID:20378546

  2. Clinical translation of nuclear export inhibitors in cancer.

    PubMed

    Senapedis, William T; Baloglu, Erkan; Landesman, Yosef

    2014-08-01

    Clinical targeting of multi-dimensional proteins such as the proteasome has been efficacious in recent years. Inhibitors such as bortezomib and carfilzomib have been used successfully to treat multiple myeloma despite early skepticism surrounding unsubstantiated toxic side effects. Another target of this magnitude is ready to emerge as a clinically viable option for targeting various neoplasias. This target, XPO1 (exportin-1 also known as Chromosome Region Maintenance 1 (CRM1)), is the transport protein responsible for nuclear export of many of the major tumor suppressor proteins and cell growth regulators. Up-regulation of XPO1 protein, a common occurrence in a variety of cancers, can lead to aberrant cytoplasmic localization and degradation of tumor suppressors such as p53 and FOXO. Therefore, inhibition of XPO1 using specific small molecules collectively called Selective Inhibitors of Nuclear Export (SINE) could potentially restore normal tumor suppressor function and have universal application for the treatment of cancer. This review will discuss the current pre-clinical data on SINE compounds in both hematological and solid malignancies. Cancer treatment through direct inhibition of the proteasome and the nuclear export machinery should instill optimism for further targeting of critical cellular pathways. PMID:24755012

  3. Life Extension of a Nuclear Facility: Export Control Implications

    SciTech Connect

    Kerschner, Harrison F.; Cunningham, Julia A.; Sportelli, James M.; Yarbro, Steve; Bedell, Jeffrey J.

    2010-04-11

    This paper discusses life extension upgrades to an operational nuclear research facility and identifies export control implications. The Department of Energy (DOE) is engaged in a multi-year program of deactivating and decommissioning (D&D) the majority of the Hanford Site 300 Area facilities. In 2006, the DOE decided to retain the Pacific Northwest National Laboratory’s Radiochemical Processing Laboratory (RPL), which was on the D&D list. As part of the facility life-extension to ensure continued mission capability, the DOE decided to consolidate replacement hot cell capability into the RPL. Physical limitations within the facility dictated that new hot cell design and construction would be modularized—a process that allows for ease of fabrication and introduction into existing space. A review of the fabrication and installation techniques has identified potential export control issues.

  4. Mechanistic insights from the recent structures of the CRM1 nuclear export complex and its disassembly intermediate

    PubMed Central

    Koyama, Masako; Matsuura, Yoshiyuki

    2012-01-01

    CRM1 (also known as exportin 1 or Xpo1) is the most versatile nuclear export receptor (exportin) that carries a broad range of proteins and ribonucleoproteins from the nucleus to the cytoplasm through the nuclear pore complex. The majority of the export substrates of CRM1 contain a short peptide sequence, so-called leucine-rich nuclear export signal (NES), which typically harbor four or five characteristically spaced hydrophobic residues. The transport directionality is determined by the small GTPase Ran and Ran-binding proteins that control the binding and dissociation of cargo. Here we review recent structural studies that advanced understanding of how NES is specifically recognized by CRM1 in the nucleus, and how NES is rapidly dissociated from CRM1 in the cytoplasm.

  5. 10 CFR 110.46 - Conduct resulting in termination of nuclear exports.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 10 Energy 2 2014-01-01 2014-01-01 false Conduct resulting in termination of nuclear exports. 110.46 Section 110.46 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) EXPORT AND IMPORT OF NUCLEAR... agreement or understanding to which the United States subscribes. (c) Under section 129 of the Atomic...

  6. 10 CFR 110.46 - Conduct resulting in termination of nuclear exports.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 10 Energy 2 2013-01-01 2013-01-01 false Conduct resulting in termination of nuclear exports. 110.46 Section 110.46 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) EXPORT AND IMPORT OF NUCLEAR... agreement or understanding to which the United States subscribes. (c) Under section 129 of the Atomic...

  7. 10 CFR 110.46 - Conduct resulting in termination of nuclear exports.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 2 2010-01-01 2010-01-01 false Conduct resulting in termination of nuclear exports. 110.46 Section 110.46 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) EXPORT AND IMPORT OF NUCLEAR EQUIPMENT AND MATERIAL Review of License Applications § 110.46 Conduct resulting in termination of...

  8. 10 CFR 110.46 - Conduct resulting in termination of nuclear exports.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 10 Energy 2 2011-01-01 2011-01-01 false Conduct resulting in termination of nuclear exports. 110.46 Section 110.46 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) EXPORT AND IMPORT OF NUCLEAR EQUIPMENT AND MATERIAL Review of License Applications § 110.46 Conduct resulting in termination of...

  9. 10 CFR 110.46 - Conduct resulting in termination of nuclear exports.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 10 Energy 2 2012-01-01 2012-01-01 false Conduct resulting in termination of nuclear exports. 110.46 Section 110.46 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) EXPORT AND IMPORT OF NUCLEAR... agreement or understanding to which the United States subscribes. (c) Under section 129 of the Atomic...

  10. Inhibition of CRM1-dependent nuclear export sensitizes malignant cells to cytotoxic and targeted agents.

    PubMed

    Turner, Joel G; Dawson, Jana; Cubitt, Christopher L; Baz, Rachid; Sullivan, Daniel M

    2014-08-01

    Nuclear-cytoplasmic trafficking of proteins is a significant factor in the development of cancer and drug resistance. Subcellular localization of exported proteins linked to cancer development include those involved in cell growth and proliferation, apoptosis, cell cycle regulation, transformation, angiogenesis, cell adhesion, invasion, and metastasis. Here, we examined the basic mechanisms involved in the export of proteins from the nucleus to the cytoplasm. All proteins over 40kDa use the nuclear pore complex to gain entry or exit from the nucleus, with the primary nuclear export molecule involved in these processes being chromosome region maintenance 1 (CRM1, exportin 1 or XPO1). Proteins exported from the nucleus must possess a hydrophobic nuclear export signal (NES) peptide that binds to a hydrophobic groove containing an active-site Cys528 in the CRM1 protein. CRM1 inhibitors function largely by covalent modification of the active site Cys528 and prevent binding to the cargo protein NES. In the absence of a CRM1 inhibitor, CRM1 binds cooperatively to the NES of the cargo protein and RanGTP, forming a trimer that is actively transported out of the nucleus by facilitated diffusion. Nuclear export can be blocked by CRM1 inhibitors, NES peptide inhibitors or by preventing post-translational modification of cargo proteins. Clinical trials using the classic CRM1 inhibitor leptomycin B proved too toxic for patients; however, a new generation of less toxic small molecule inhibitors is being used in clinical trials in patients with both hematological malignancies and solid tumors. Additional trials are being initiated using small-molecule CRM1 inhibitors in combination with chemotherapeutics such as pegylated liposomal doxorubicin. In this review, we present evidence that combining the new CRM1 inhibitors with other classes of therapeutics may prove effective in the treatment of cancer. Potential combinatorial therapies discussed include the use of CRM1 inhibitors and

  11. Mechanistic Insights from Structural Analyses of Ran-GTPase-Driven Nuclear Export of Proteins and RNAs.

    PubMed

    Matsuura, Yoshiyuki

    2016-05-22

    Understanding how macromolecules are rapidly exchanged between the nucleus and the cytoplasm through nuclear pore complexes is a fundamental problem in biology. Exportins are Ran-GTPase-dependent nuclear transport factors that belong to the karyopherin-β family and mediate nuclear export of a plethora of proteins and RNAs, except for bulk mRNA nuclear export. Exportins bind cargo macromolecules in a Ran-GTP-dependent manner in the nucleus, forming exportin-cargo-Ran-GTP complexes (nuclear export complexes). Transient weak interactions between exportins and nucleoporins containing characteristic FG (phenylalanine-glycine) repeat motifs facilitate nuclear pore complex passage of nuclear export complexes. In the cytoplasm, nuclear export complexes are disassembled, thereby releasing the cargo. GTP hydrolysis by Ran promoted in the cytoplasm makes the disassembly reaction virtually irreversible and provides thermodynamic driving force for the overall export reaction. In the past decade, X-ray crystallography of some of the exportins in various functional states coupled with functional analyses, single-particle electron microscopy, molecular dynamics simulations, and small-angle solution X-ray scattering has provided rich insights into the mechanism of cargo binding and release and also begins to elucidate how exportins interact with the FG repeat motifs. The knowledge gained from structural analyses of nuclear export is being translated into development of clinically useful inhibitors of nuclear export to treat human diseases such as cancer and influenza. PMID:26519791

  12. Nuclear sensor signal processing circuit

    DOEpatents

    Kallenbach, Gene A.; Noda, Frank T.; Mitchell, Dean J.; Etzkin, Joshua L.

    2007-02-20

    An apparatus and method are disclosed for a compact and temperature-insensitive nuclear sensor that can be calibrated with a non-hazardous radioactive sample. The nuclear sensor includes a gamma ray sensor that generates tail pulses from radioactive samples. An analog conditioning circuit conditions the tail-pulse signals from the gamma ray sensor, and a tail-pulse simulator circuit generates a plurality of simulated tail-pulse signals. A computer system processes the tail pulses from the gamma ray sensor and the simulated tail pulses from the tail-pulse simulator circuit. The nuclear sensor is calibrated under the control of the computer. The offset is adjusted using the simulated tail pulses. Since the offset is set to zero or near zero, the sensor gain can be adjusted with a non-hazardous radioactive source such as, for example, naturally occurring radiation and potassium chloride.

  13. Substrate-induced Nuclear Export and Peripheral Compartmentalization of Hepatic Glucokinase Correlates with Glycogen Deposition

    PubMed Central

    Shiota, Masa; Knobel, Susan M.; Piston, David W.; Cherrington, Alan D.; Magnuson, Mark A.

    2001-01-01

    Hepatic glucokinase (GK) is acutely regulated by binding to its nuclear-anchored regulatory protein (GKRP). Although GK release by GKRP is tightly coupled to the rate of glycogen synthesis, the nature of this association is obscure. To gain insight into this coupling mechanism under physiological stimulating conditions in primary rat hepatocytes, we analyzed the subcellular distribution of GK and GKRP with immunofluorescence, and glycogen deposition with glycogen cytochemical fluorescence, using confocal microscopyand quantitative image analysis. Following stimulation, a fraction of the GK signal translocated from the nucleus to the cytoplasm. The reduction in the nuclear to cytoplasmic ratio of GK, an index of nuclear export, correlated with a >50% increase in glycogen cytochemical fluorescence over a 60min stimulation period. Furthermore, glycogen accumulation was initially deposited in a peripheral pattern in hepatocytes similar to that of GK. These data suggest that a compartmentalization exists of both active GK and the initial sites of glycogen deposition at the hepatocyte surface. PMID:12369705

  14. The metalloid arsenite induces nuclear export of Id3 possibly via binding to the N-terminal cysteine residues

    SciTech Connect

    Kurooka, Hisanori; Sugai, Manabu; Mori, Kentaro; Yokota, Yoshifumi

    2013-04-19

    Highlights: •Sodium arsenite induces cytoplasmic accumulation of Id3. •Arsenite binds to closely spaced N-terminal cysteine residues of Id3. •N-terminal cysteines are essential for arsenite-induced nuclear export of Id3. •Nuclear export of Id3 counteracts its transcriptional repression activity. -- Abstract: Ids are versatile transcriptional repressors that regulate cell proliferation and differentiation, and appropriate subcellular localization of the Id proteins is important for their functions. We previously identified distinct functional nuclear export signals (NESs) in Id1 and Id2, but no active NES has been reported in Id3. In this study, we found that treatment with the stress-inducing metalloid arsenite led to the accumulation of GFP-tagged Id3 in the cytoplasm. Cytoplasmic accumulation was impaired by a mutation in the Id3 NES-like sequence resembling the Id1 NES, located at the end of the HLH domain. It was also blocked by co-treatment with the CRM1-specific nuclear export inhibitor leptomycin B (LMB), but not with the inhibitors for mitogen-activated protein kinases (MAPKs). Importantly, we showed that the closely spaced N-terminal cysteine residues of Id3 interacted with the arsenic derivative phenylarsine oxide (PAO) and were essential for the arsenite-induced cytoplasmic accumulation, suggesting that arsenite induces the CRM1-dependent nuclear export of Id3 via binding to the N-terminal cysteines. Finally, we demonstrated that Id3 significantly repressed arsenite-stimulated transcription of the immediate-early gene Egr-1 and that this repression activity was inversely correlated with the arsenite-induced nuclear export. Our results imply that Id3 may be involved in the biological action of arsenite.

  15. 37 CFR 5.20 - Export of technical data relating to sensitive nuclear technology.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ....20 Export of technical data relating to sensitive nuclear technology. Under regulations (10 CFR 810.7... 37 Patents, Trademarks, and Copyrights 1 2011-07-01 2011-07-01 false Export of technical data relating to sensitive nuclear technology. 5.20 Section 5.20 Patents, Trademarks, and Copyrights...

  16. 37 CFR 5.20 - Export of technical data relating to sensitive nuclear technology.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ....20 Export of technical data relating to sensitive nuclear technology. Under regulations (10 CFR 810.7... 37 Patents, Trademarks, and Copyrights 1 2014-07-01 2014-07-01 false Export of technical data relating to sensitive nuclear technology. 5.20 Section 5.20 Patents, Trademarks, and Copyrights...

  17. 37 CFR 5.20 - Export of technical data relating to sensitive nuclear technology.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ....20 Export of technical data relating to sensitive nuclear technology. Under regulations (10 CFR 810.7... 37 Patents, Trademarks, and Copyrights 1 2013-07-01 2013-07-01 false Export of technical data relating to sensitive nuclear technology. 5.20 Section 5.20 Patents, Trademarks, and Copyrights...

  18. A signal sequence is not required for protein export in prlA mutants of Escherichia coli.

    PubMed Central

    Derman, A I; Puziss, J W; Bassford, P J; Beckwith, J

    1993-01-01

    The prlA/secY gene, which codes for an integral membrane protein component of the Escherichia coli protein export machinery, is the locus of the strongest suppressors of signal sequence mutations. We demonstrate that two exported proteins of E.coli, maltose-binding protein and alkaline phosphatase, each lacking its entire signal sequence, are exported to the periplasm in several prlA mutants. The export efficiency can be substantial; in a strain carrying the prlA4 allele, 30% of signal-sequenceless alkaline phosphatase is exported to the periplasm. Other components of the E.coli export machinery, including SecA, are required for this export. SecB is required for the export of signal-sequenceless alkaline phosphatase even though the normal export of alkaline phosphatase does not require this chaperonin. Our findings indicate that signal sequences confer speed and efficiency upon the export process, but that they are not always essential for export. Entry into the export pathway may involve components that so overlap in function that the absence of a signal sequence can be compensated for, or there may exist one or more means of entry that do not require signal sequences at all. Images PMID:8458344

  19. Olig1 Acetylation and Nuclear Export Mediate Oligodendrocyte Development

    PubMed Central

    Dai, Jinxiang; Bercury, Kathryn K.; Jin, Weilin

    2015-01-01

    The oligodendrocyte transcription factor Olig1 is critical for both oligodendrocyte development and remyelination in mice. Nuclear to cytoplasmic translocation of Olig1 protein occurs during brain development and in multiple sclerosis, but the detailed molecular mechanism of this translocation remains elusive. Here, we report that Olig1 acetylation and deacetylation drive its active translocation between the nucleus and the cytoplasm in both mouse and rat oligodendrocytes. We identified three functional nuclear export sequences (NES) localized in the basic helix-loop-helix domain and one specific acetylation site at Lys 150 (human Olig1) in NES1. Olig1 acetylation and deacetylation are regulated by the acetyltransferase CREB-binding protein and the histone deacetylases HDAC1, HDAC3, and HDAC10. Acetylation of Olig1 decreased its chromatin association, increased its interaction with inhibitor of DNA binding 2 and facilitated its retention in the cytoplasm of mature oligodendrocytes. These studies establish that acetylation of Olig1 regulates its chromatin dissociation and subsequent translocation to the cytoplasm and is required for its function in oligodendrocyte maturation. SIGNIFICANCE STATEMENT The nuclear to cytoplasmic translocation of Olig1 protein has been observed during mouse and human brain development and in multiple sclerosis in several studies, but the detailed molecular mechanism of this translocation remains elusive. Here, we provide insight into the mechanism by which acetylation of Olig1 regulates its unique nuclear-cytoplasmic shuttling during oligodendrocyte development and how the acetylation status of Olig1 modulates its distinct function in the nucleus versus the cytoplasm. The current study provides a unique example of a lineage-specific transcription factor that is actively translocated from the nucleus to the cytoplasm as the cell differentiates. Importantly, we demonstrate that this process is tightly controlled by acetylation at a single

  20. Olig1 Acetylation and Nuclear Export Mediate Oligodendrocyte Development.

    PubMed

    Dai, Jinxiang; Bercury, Kathryn K; Jin, Weilin; Macklin, Wendy B

    2015-12-01

    The oligodendrocyte transcription factor Olig1 is critical for both oligodendrocyte development and remyelination in mice. Nuclear to cytoplasmic translocation of Olig1 protein occurs during brain development and in multiple sclerosis, but the detailed molecular mechanism of this translocation remains elusive. Here, we report that Olig1 acetylation and deacetylation drive its active translocation between the nucleus and the cytoplasm in both mouse and rat oligodendrocytes. We identified three functional nuclear export sequences (NES) localized in the basic helix-loop-helix domain and one specific acetylation site at Lys 150 (human Olig1) in NES1. Olig1 acetylation and deacetylation are regulated by the acetyltransferase CREB-binding protein and the histone deacetylases HDAC1, HDAC3, and HDAC10. Acetylation of Olig1 decreased its chromatin association, increased its interaction with inhibitor of DNA binding 2 and facilitated its retention in the cytoplasm of mature oligodendrocytes. These studies establish that acetylation of Olig1 regulates its chromatin dissociation and subsequent translocation to the cytoplasm and is required for its function in oligodendrocyte maturation. PMID:26631469

  1. The Cellular Distribution of RanGAP1 Is Regulated by CRM1-Mediated Nuclear Export in Mammalian Cells

    PubMed Central

    Raghunayakula, Sarita; Zhang, Xiang-Dong

    2015-01-01

    The Ran GTPase activating protein RanGAP1 plays an essential role in nuclear transport by stimulating RanGTP hydrolysis in the cytoplasmic compartment. In mammalian cells, unmodified RanGAP1 is predominantly cytoplasmic, whereas modification by small ubiquitin-related modifier protein (SUMO) targets RanGAP1 to the cytoplasmic filaments of nuclear pore complex (NPC). Although RanGAP1 contains nine putative nuclear export signals and a nuclear localization signal, little is known if RanGAP1 shuttles between the nuclear and cytoplasmic compartments and how its primary localization in the cytoplasm and at the NPC is regulated. Here we show that inhibition of CRM1-mediated nuclear export using RNAi-knockdown of CRM1 and inactivation of CRM1 by leptomycin B (LMB) results in nuclear accumulation of RanGAP1. LMB treatment induced a more robust redistribution of RanGAP1 from the cytoplasm to the nucleoplasm compared to CRM1 RNAi and also uniquely triggered a decrease or loss of RanGAP1 localization at the NPC, suggesting that LMB treatment is more effective in inhibiting CRM1-mediated nuclear export of RanGAP1. Our time-course analysis of LMB treatment reveals that the NPC-associated RanGAP1 is much more slowly redistributed to the nucleoplasm than the cytoplasmic RanGAP1. Furthermore, LMB-induced nuclear accumulation of RanGAP1 is positively correlated with an increase in levels of SUMO-modified RanGAP1, suggesting that SUMOylation of RanGAP1 may mainly take place in the nucleoplasm. Lastly, we demonstrate that the nuclear localization signal at the C-terminus of RanGAP1 is required for its nuclear accumulation in cells treated with LMB. Taken together, our results elucidate that RanGAP1 is actively transported between the nuclear and cytoplasmic compartments, and that the cytoplasmic and NPC localization of RanGAP1 is dependent on CRM1-mediated nuclear export. PMID:26506250

  2. The Cellular Distribution of RanGAP1 Is Regulated by CRM1-Mediated Nuclear Export in Mammalian Cells.

    PubMed

    Cha, Keith; Sen, Progga; Raghunayakula, Sarita; Zhang, Xiang-Dong

    2015-01-01

    The Ran GTPase activating protein RanGAP1 plays an essential role in nuclear transport by stimulating RanGTP hydrolysis in the cytoplasmic compartment. In mammalian cells, unmodified RanGAP1 is predominantly cytoplasmic, whereas modification by small ubiquitin-related modifier protein (SUMO) targets RanGAP1 to the cytoplasmic filaments of nuclear pore complex (NPC). Although RanGAP1 contains nine putative nuclear export signals and a nuclear localization signal, little is known if RanGAP1 shuttles between the nuclear and cytoplasmic compartments and how its primary localization in the cytoplasm and at the NPC is regulated. Here we show that inhibition of CRM1-mediated nuclear export using RNAi-knockdown of CRM1 and inactivation of CRM1 by leptomycin B (LMB) results in nuclear accumulation of RanGAP1. LMB treatment induced a more robust redistribution of RanGAP1 from the cytoplasm to the nucleoplasm compared to CRM1 RNAi and also uniquely triggered a decrease or loss of RanGAP1 localization at the NPC, suggesting that LMB treatment is more effective in inhibiting CRM1-mediated nuclear export of RanGAP1. Our time-course analysis of LMB treatment reveals that the NPC-associated RanGAP1 is much more slowly redistributed to the nucleoplasm than the cytoplasmic RanGAP1. Furthermore, LMB-induced nuclear accumulation of RanGAP1 is positively correlated with an increase in levels of SUMO-modified RanGAP1, suggesting that SUMOylation of RanGAP1 may mainly take place in the nucleoplasm. Lastly, we demonstrate that the nuclear localization signal at the C-terminus of RanGAP1 is required for its nuclear accumulation in cells treated with LMB. Taken together, our results elucidate that RanGAP1 is actively transported between the nuclear and cytoplasmic compartments, and that the cytoplasmic and NPC localization of RanGAP1 is dependent on CRM1-mediated nuclear export. PMID:26506250

  3. Inner nuclear envelope protein SUN1 plays a prominent role in mammalian mRNA export

    PubMed Central

    Li, Ping; Noegel, Angelika A.

    2015-01-01

    Nuclear export of messenger ribonucleoproteins (mRNPs) through the nuclear pore complex (NPC) can be roughly classified into two forms: bulk and specific export, involving an nuclear RNA export factor 1 (NXF1)-dependent pathway and chromosome region maintenance 1 (CRM1)-dependent pathway, respectively. SUN proteins constitute the inner nuclear envelope component of the linker of nucleoskeleton and cytoskeleton (LINC) complex. Here, we show that mammalian cells require SUN1 for efficient nuclear mRNP export. The results indicate that both SUN1 and SUN2 interact with heterogeneous nuclear ribonucleoprotein (hnRNP) F/H and hnRNP K/J. SUN1 depletion inhibits the mRNP export, with accumulations of both hnRNPs and poly(A)+RNA in the nucleus. Leptomycin B treatment indicates that SUN1 functions in mammalian mRNA export involving the NXF1-dependent pathway. SUN1 mediates mRNA export through its association with mRNP complexes via a direct interaction with NXF1. Additionally, SUN1 associates with the NPC through a direct interaction with Nup153, a nuclear pore component involved in mRNA export. Taken together, our results reveal that the inner nuclear envelope protein SUN1 has additional functions aside from being a central component of the LINC complex and that it is an integral component of the mammalian mRNA export pathway suggesting a model whereby SUN1 recruits NXF1-containing mRNP onto the nuclear envelope and hands it over to Nup153. PMID:26476453

  4. Steady-state nuclear actin levels are determined by export competent actin pool.

    PubMed

    Skarp, Kari-Pekka; Huet, Guillaume; Vartiainen, Maria K

    2013-10-01

    A number of studies in the last decade have irrevocably promoted actin into a fully fledged member of the nuclear compartment, where it, among other crucial tasks, facilitates transcription and chromatin remodeling. Changes in nuclear actin levels have been linked to different cellular processes: decreased nuclear actin to quiescence and increased nuclear actin to differentiation. Importin 9 and exportin 6 transport factors are responsible for the continuous nucleocytoplasmic shuttling of actin, but the mechanisms, which result in modulated actin levels, have not been characterized. We find that in cells growing under normal growth conditions, the levels of nuclear actin vary considerably from cell to cell. To understand the basis for this, we have extensively quantified several cellular parameters while at the same time recording the import and export rates of green fluorescent protein (GFP)-tagged actin. Surprisingly, our dataset shows that the ratio of nuclear to cytoplasmic fluorescence intensity, but not nuclear shape, size, cytoplasm size, or their ratio, correlates negatively with both import and export rate of actin. This suggests that high-nuclear actin content is maintained by both diminished import and export. The high nuclear actin containing cells still show high mobility of actin, but it is not export competent, suggesting increased binding of actin to nuclear complexes. Creation of such export incompetent actin pool would ensure enough actin is retained in the nucleus and make it available for the various nuclear functions described for actin. PMID:23749625

  5. GLE2, a Saccharomyces cerevisiae homologue of the Schizosaccharomyces pombe export factor RAE1, is required for nuclear pore complex structure and function.

    PubMed Central

    Murphy, R; Watkins, J L; Wente, S R

    1996-01-01

    To identify and characterize novel factors required for nuclear transport, a genetic screen was conducted in the yeast Saccharomyces cerevisiae. Mutations that were lethal in combination with a null allele of the gene encoding the nucleoporin Nup100p were isolated using a colony-sectoring assay. Three complementation groups of gle (for GLFG lethal) mutants were identified. In this report, the characterization of GLE2 is detailed. GLE2 encodes a 40.5-kDa polypeptide with striking similarity to that of Schizosaccharomyces pombe RAE1. In indirect immunofluorescence and nuclear pore complex fractionation experiments, Gle2p was associated with nuclear pore complexes. Mutated alleles of GLE2 displayed blockage of polyadenylated RNA export; however, nuclear protein import was not apparently diminished. Immunofluorescence and thin-section electron microscopic analysis revealed that the nuclear pore complex and nuclear envelope structure was grossly perturbed in gle2 mutants. Because the clusters of herniated pore complexes appeared subsequent to the export block, the structural perturbations were likely indirect consequences of the export phenotype. Interestingly, a two-hybrid interaction was detected between Gle2p and Srp1p, the nuclear localization signal receptor, as well as Rip1p, a nuclear export signal-interacting protein. We propose that Gle2p has a novel role in mediating nuclear transport. Images PMID:8970155

  6. Visualizing nuclear export of different classes of RNA by electron microscopy.

    PubMed

    Panté, N; Jarmolowski, A; Izaurralde, E; Sauder, U; Baschong, W; Mattaj, I W

    1997-05-01

    Export of RNA from the cell nucleus to the cytoplasm occurs through nuclear pore complexes (NPCs). To examine nuclear export of RNA, we have gold-labeled different types of RNA (i.e., mRNA, tRNA, U snRNAs), and followed their export by electron microscopy (EM) after their microinjection into Xenopus oocyte nuclei. By changing the polarity of the negatively charged colloidal gold, complexes with mRNA, tRNA, and U1 snRNA can be formed efficiently, and gold-tagged RNAs are exported to the cytoplasm with kinetics and specific saturation behavior similar to that of unlabeled RNAs. U6 snRNA conjugates, in contrast, remain in the nucleus, as does naked U6 snRNA. During export, RNA-gold was found distributed along the central axis of the NPC, within the nuclear basket, or accumulated at the nuclear and cytoplasmic periphery of the central gated channel, but not associated with the cytoplasmic fibrils. In an attempt to identify the initial NPC docking site(s) for RNA, we have explored various conditions that either yield docking of import ligands to the NPC or inhibit the export of nuclear RNAs. Surprisingly, we failed to observe docking of RNA destined for export at the nuclear periphery of the NPC under any of these conditions. Instead, each condition in which export of any of the RNA-gold conjugates was inhibited caused accumulation of gold particles scattered uniformly throughout the nucleoplasm. These results point to the existence of steps in export involving mobilization of the export substrate from the nucleoplasm to the NPC. PMID:9149231

  7. TNF{alpha} release from peripheral blood leukocytes depends on a CRM1-mediated nuclear export

    SciTech Connect

    Miskolci, Veronika; Ghosh, Chandra C.; Rollins, Janet; Romero, Carlos; Vu, Hai-Yen; Robinson, Staci; Davidson, Dennis; Vancurova, Ivana . E-mail: vancuroi@stjohns.edu

    2006-12-15

    Tumor necrosis factor-{alpha} (TNF{alpha}) is a potent pro-inflammatory cytokine that plays a major role in the pathogenesis of acute and chronic inflammatory disorders such as septic shock and arthritis, respectively. Leukocytes stimulated with inflammatory signals such as lipopolysaccharide (LPS) are the predominant producers of TNF{alpha}, and thus control of TNF{alpha} release from stimulated leukocytes represents a potential therapeutic target. Here, we report that leptomycin B (LMB), a specific inhibitor of CRM1-dependent nuclear protein export, inhibits TNF{alpha} release from LPS-stimulated human peripheral blood neutrophils and mononuclear cells. In addition, immunofluorescence confocal microscopy and immunoblotting analysis indicate that TNF{alpha} is localized in the nucleus of human neutrophils and mononuclear cells. This study demonstrates that the cellular release of TNF{alpha} from stimulated leukocytes is mediated by the CRM1-dependent nuclear export mechanism. Inhibition of CRM1-dependent cellular release of TNF{alpha} could thus provide a novel therapeutic approach for disorders involving excessive TNF{alpha} release.

  8. Expression, function, and targeting of the nuclear exporter chromosome region maintenance 1 (CRM1) protein

    PubMed Central

    Ishizawa, Jo; Kojima, Kensuke; Hail, Numsen; Tabe, Yoko; Andreeff, Michael

    2015-01-01

    Nucleocytoplasmic trafficking of proteins/RNAs is essential to normal cellular function. Indeed, accumulating evidence suggests that cancer cells escape anti-neoplastic mechanisms and benefit from pro-survival signals via the dysregulation of this system. The nuclear exporter chromosome region maintenance 1 (CRM1) protein is the only protein in the karyopherin-β protein family that contributes to the trafficking of numerous proteins and RNAs from the nucleus. It is considered to be an oncogenic, anti-apoptotic protein in transformed cells, since it reportedly functions as a gatekeeper for cell survival, including affecting p53 function, and ribosomal biogenesis. Furthermore, abnormally high expression of CRM1 is correlated with poor patient prognosis in various malignancies. Therapeutic targeting of CRM1 has emerged as a novel cancer treatment strategy, starting with a clinical trial with leptomycin B, the original specific inhibitor of CRM1, followed by development of several next-generation small molecules. KPT-330, a novel member of the CRM1-selective inhibitors of nuclear export (SINE) class of compounds, is currently undergoing clinical evaluation for the therapy of various malignancies. Results from these trials suggest that SINE compounds may be particularly useful against hematological malignancies, which often become refractory to standard chemotherapeutic agents. PMID:26048327

  9. Design principles of nuclear receptor signaling: how complex networking improves signal transduction

    PubMed Central

    Kolodkin, Alexey N; Bruggeman, Frank J; Plant, Nick; Moné, Martijn J; Bakker, Barbara M; Campbell, Moray J; van Leeuwen, Johannes P T M; Carlberg, Carsten; Snoep, Jacky L; Westerhoff, Hans V

    2010-01-01

    The topology of nuclear receptor (NR) signaling is captured in a systems biological graphical notation. This enables us to identify a number of ‘design' aspects of the topology of these networks that might appear unnecessarily complex or even functionally paradoxical. In realistic kinetic models of increasing complexity, calculations show how these features correspond to potentially important design principles, e.g.: (i) cytosolic ‘nuclear' receptor may shuttle signal molecules to the nucleus, (ii) the active export of NRs may ensure that there is sufficient receptor protein to capture ligand at the cytoplasmic membrane, (iii) a three conveyor belts design dissipating GTP-free energy, greatly aids response, (iv) the active export of importins may prevent sequestration of NRs by importins in the nucleus and (v) the unspecific nature of the nuclear pore may ensure signal-flux robustness. In addition, the models developed are suitable for implementation in specific cases of NR-mediated signaling, to predict individual receptor functions and differential sensitivity toward physiological and pharmacological ligands. PMID:21179018

  10. Promising SINEs for embargoing nuclear-cytoplasmic export as an anticancer strategy.

    PubMed

    Tan, David S P; Bedard, Philippe L; Kuruvilla, John; Siu, Lillian L; Razak, Albiruni R Abdul

    2014-05-01

    In cancer cells, the nuclear-cytoplasmic transport machinery is frequently disrupted, resulting in mislocalization and loss of function for many key regulatory proteins. In this review, the mechanisms by which tumor cells co-opt the nuclear transport machinery to facilitate carcinogenesis, cell survival, drug resistance, and tumor progression will be elucidated, with a particular focus on the role of the nuclear-cytoplasmic export protein. The recent development of a new generation of selective inhibitors of nuclear export (XPO1 antagonists) and how these novel anticancer drugs may bring us closer to the implementation of this therapeutic strategy in the clinic will be discussed. PMID:24743138

  11. Cytoplasmic Sequestration of Rel Proteins by IκBα Requires CRM1-Dependent Nuclear Export

    PubMed Central

    Tam, Winnie F.; Lee, Linda H.; Davis, Laura; Sen, Ranjan

    2000-01-01

    Rel and IκB protein families form a complex cellular regulatory network. A major regulatory function of IκB proteins is to retain Rel proteins in the cell cytoplasm. In addition, IκB proteins have also been postulated to serve nuclear functions. These include the maintenance of inducible NF-κB-dependent gene transcription, as well as termination of inducible transcription. We show that IκBα shuttles between the nucleus and the cytoplasm, utilizing the nuclear export receptor CRM1. A CRM1-binding export sequence was identified in the N-terminal domain of IκBα but not in that of IκBβ or IκBɛ. By reconstituting major aspects of NF-κB–IκB sequestration in yeast, we demonstrate that cytoplasmic retention of p65 (also called RelA) by IκBα requires Crm1p-dependent nuclear export. In mammalian cells, inhibition of CRM1 by leptomycin B resulted in nuclear localization of cotransfected p65 and IκBα in COS cells and enhanced nuclear relocation of endogenous p65 in T cells. These observations suggest that the main function of IκBα is that of a nuclear export chaperone rather than a cytoplasmic tether. We propose that the nucleus is the major site of p65-IκBα association, from where these complexes must be exported in order to create the cytoplasmic pool. PMID:10688673

  12. Piperlongumine is a novel nuclear export inhibitor with potent anticancer activity.

    PubMed

    Niu, Mingshan; Xu, Xiaoyu; Shen, Yangling; Yao, Yao; Qiao, Jianlin; Zhu, Feng; Zeng, Lingyu; Liu, Xuejiao; Xu, Kailin

    2015-07-25

    Piperlongumine is a natural compound recently identified to be toxic selectively to tumor cells in vitro and in vivo. However, the molecular mechanism underlying its anti-tumor action still remains unclear. In this report, we describe another novel mechanism by which piperlongumine mediates its anti-tumor effects. We found that piperlongumine is a novel nuclear export inhibitor. Piperlongumine could induce nuclear retention of tumor suppressor proteins and inhibit the interactions between CRM1 and these proteins. Piperlongumine could directly bind to the conserved Cys528 of CRM1 but not to a Cys528 mutant peptide. More importantly, cancer cells expressing mutant CRM1 (C528S) are resistant to piperlongumine, demonstrating the nuclear export inhibition via direct interaction with Cys528 of CRM1. The inhibition of nuclear export by piperlongumine may account for its therapeutic properties in cancer diseases. Our findings provide a good starting point for development of novel CRM1 inhibitors. PMID:26026911

  13. 77 FR 51581 - Request for a License To Export Nuclear Grade Graphite

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-08-24

    ... August 2007, 72 FR 49139 (Aug. 28, 2007). Information about filing electronically is available on the NRC... From the Federal Register Online via the Government Publishing Office NUCLEAR REGULATORY COMMISSION Request for a License To Export Nuclear Grade Graphite Pursuant to 10 CFR 110.70 (b)...

  14. 37 CFR 5.20 - Export of technical data relating to sensitive nuclear technology.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ....20 Export of technical data relating to sensitive nuclear technology. Under regulations (10 CFR 810.7... relating to sensitive nuclear technology. 5.20 Section 5.20 Patents, Trademarks, and Copyrights UNITED.... 184, is considered to be information available to the public in published form and a...

  15. 77 FR 39521 - Application for a License To Export Nuclear Reactor Major Components and Equipment

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-07-03

    ... COMMISSION Application for a License To Export Nuclear Reactor Major Components and Equipment Pursuant to 10... Reactor internals, Components and For use in Braka nuclear power Company LLC reactor coolant equipment for... plant in Braka. 110060011 control equipment, auxiliary equipment and emergency cooling systems....

  16. Coupling pre-mRNA splicing and 3' end formation to mRNA export: alternative ways to punch the nuclear export clock.

    PubMed

    Elbarbary, Reyad A; Maquat, Lynne E

    2016-03-01

    How does a mammalian cell determine when newly synthesized mRNAs are fully processed and appropriate for nuclear export? Müller-McNicoll and colleagues (pp. 553-566) expand on mechanisms known to be mediated by nuclear export factor 1 (NXF1) by describing SR proteins as NXF1 adaptors that flag alternatively spliced and polyadenylated mRNA isoforms as cargo ready for the cytoplasm. PMID:26944675

  17. Dcas is required for importin-alpha3 nuclear export and mechano-sensory organ cell fate specification in Drosophila.

    PubMed

    Tekotte, Hildegard; Berdnik, Daniela; Török, Tibor; Buszczak, Michael; Jones, Lynn M; Cooley, Lynn; Knoblich, Jürgen A; Davis, Ilan

    2002-04-15

    We have studied the in vivo function and tissue specificity of Dcas, the Drosophila ortholog of CAS, the importin beta-like export receptor for importin alpha. While dcas mRNA is specifically expressed in the embryonic central nervous system, Dcas protein is maternally supplied to all embryonic cells and its nuclear/cytoplasmic distribution varies in different tissues and times in development. Unexpectedly, hypomorphic alleles of dcas show specific transformations in mechano-sensory organ cell identity, characteristic of mutations that increase Notch signaling. Dcas is essential for efficient importin-alpha3 nuclear export in mechano-sensory cells and the surrounding epidermal cells and is indirectly required for the import of one component of the Notch pathway, but not others tested. We interpret the specificity of the dcas phenotype as indicating that one or more Notch signaling components are particularly sensitive to a disruption in nuclear protein import. We propose that mutations in house keeping genes often cause specific developmental phenotypes, such as those observed in many human genetic disorders. PMID:11944946

  18. Selective inhibitors of nuclear export (SINE)--a novel class of anti-cancer agents.

    PubMed

    Parikh, Kaushal; Cang, Shundong; Sekhri, Arunabh; Liu, Delong

    2014-01-01

    Dysregulation of the nucleo-cytoplasmic transport of proteins plays an important role in carcinogenesis. The nuclear export of proteins depends on the activity of transport proteins, exportins. Exportins belong to the karyopherin β superfamily. Exportin-1 (XPO1), also known as chromosomal region maintenance 1 (CRM1), mediates transport of around 220 proteins. In this review, we summarized the development of a new class of antitumor drugs, collectively known as selective inhibitors of nuclear export (SINE). KPT-330 (selinexor) as an oral agent is showing activities in early clinical trials in both solid tumors and hematological malignancies. PMID:25316614

  19. Nuclear Export of Pre-Ribosomal Subunits Requires Dbp5, but Not as an RNA-Helicase as for mRNA Export

    PubMed Central

    Neumann, Bettina; Wu, Haijia; Hackmann, Alexandra; Krebber, Heike

    2016-01-01

    The DEAD-box RNA-helicase Dbp5/Rat8 is known for its function in nuclear mRNA export, where it displaces the export receptor Mex67 from the mRNA at the cytoplasmic side of the nuclear pore complex (NPC). Here we show that Dbp5 is also required for the nuclear export of both pre-ribosomal subunits. Yeast temperature-sensitive dbp5 mutants accumulate both ribosomal particles in their nuclei. Furthermore, Dbp5 genetically and physically interacts with known ribosomal transport factors such as Nmd3. Similar to mRNA export we show that also for ribosomal transport Dbp5 is required at the cytoplasmic side of the NPC. However, unlike its role in mRNA export, Dbp5 does not seem to undergo its ATPase cycle for this function, as ATPase-deficient dbp5 mutants that selectively inhibit mRNA export do not affect ribosomal transport. Furthermore, mutants of GLE1, the ATPase stimulating factor of Dbp5, show no major ribosomal export defects. Consequently, while Dbp5 uses its ATPase cycle to displace the export receptor Mex67 from the translocated mRNAs, Mex67 remains bound to ribosomal subunits upon transit to the cytoplasm, where it is detectable on translating ribosomes. Therefore, we propose a model, in which Dbp5 supports ribosomal transport by capturing ribosomal subunits upon their cytoplasmic appearance at the NPC, possibly by binding export factors such as Mex67. Thus, our findings reveal that although different ribonucleoparticles, mRNAs and pre-ribosomal subunits, use shared export factors, they utilize different transport mechanisms. PMID:26872259

  20. Nuclear export controls and the CTBT: Where we`ve been and challenges ahead -- Views of an engineer

    SciTech Connect

    Lundy, A.S.

    1998-09-01

    The paper discusses the following topics: the importance of export controls; the uniqueness of nuclear weapons and their export control requirements; ``dual-use`` controls; and recent developments in nonproliferation beyond export control. Also discussed are some non-obvious challenges which include computer modeling and visualization, and fissile material availability and instant nukes. The author concludes by asking the Nuclear Suppliers Group to consider whether there are ways to make its controls more effective.

  1. Nuclear PI3K signaling in cell growth and tumorigenesis

    PubMed Central

    Davis, William J.; Lehmann, Peter Z.; Li, Weimin

    2015-01-01

    The PI3K/Akt signaling pathway is a major driving force in a variety of cellular functions. Dysregulation of this pathway has been implicated in many human diseases including cancer. While the activity of the cytoplasmic PI3K/Akt pathway has been extensively studied, the functions of these molecules and their effector proteins within the nucleus are poorly understood. Harboring key cellular processes such as DNA replication and repair as well as nascent messenger RNA transcription, the nucleus provides a unique compartmental environment for protein–protein and protein–DNA/RNA interactions required for cell survival, growth, and proliferation. Here we summarize recent advances made toward elucidating the nuclear PI3K/Akt signaling cascade and its key components within the nucleus as they pertain to cell growth and tumorigenesis. This review covers the spatial and temporal localization of the major nuclear kinases having PI3K activities and the counteracting phosphatases as well as the role of nuclear PI3K/Akt signaling in mRNA processing and exportation, DNA replication and repair, ribosome biogenesis, cell survival, and tumorigenesis. PMID:25918701

  2. Dss1 associating with the proteasome functions in selective nuclear mRNA export in yeast

    SciTech Connect

    Mannen, Taro; Andoh, Tomoko; Tani, Tokio

    2008-01-25

    Dss1p is an evolutionarily conserved small protein that interacts with BRCA2, a tumor suppressor protein, in humans. The Schizosaccharomyces pombe strain lacking the dss1{sup +} gene ({delta}dss1) shows a temperature-sensitive growth defect and accumulation of bulk poly(A){sup +} RNA in the nucleus at a nonpermissive temperature. In situ hybridization using probes for several specific mRNAs, however, revealed that the analyzed mRNAs were exported normally to the cytoplasm in {delta}dss1, suggesting that Dss1p is required for export of some subsets of mRNAs. We identified the pad1{sup +} gene, which encodes a component of the 26S proteasome, as a suppressor for the ts{sup -} phenotype of {delta}dss1. Unexpectedly, overexpression of Pad1p could suppress neither the defect in nuclear mRNA export nor a defect in proteasome function. In addition, loss of proteasome functions does not cause defective nuclear mRNA export. Dss1p seems to be a multifunctional protein involved in nuclear export of specific sets of mRNAs and the ubiquitin-proteasome pathway in fission yeast.

  3. High-resolution imaging reveals new features of nuclear export of mRNA through the nuclear pore complexes.

    PubMed

    Kelich, Joseph M; Yang, Weidong

    2014-01-01

    The nuclear envelope (NE) of eukaryotic cells provides a physical barrier for messenger RNA (mRNA) and the associated proteins (mRNPs) traveling from sites of transcription in the nucleus to locations of translation processing in the cytoplasm. Nuclear pore complexes (NPCs) embedded in the NE serve as a dominant gateway for nuclear export of mRNA. However, the fundamental characterization of export dynamics of mRNPs through the NPC has been hindered by several technical limits. First, the size of NPC that is barely below the diffraction limit of conventional light microscopy requires a super-resolution microscopy imaging approach. Next, the fast transit of mRNPs through the NPC further demands a high temporal resolution by the imaging approach. Finally, the inherent three-dimensional (3D) movements of mRNPs through the NPC demand the method to provide a 3D mapping of both transport kinetics and transport pathways of mRNPs. This review will highlight the recently developed super-resolution imaging techniques advanced from 1D to 3D for nuclear export of mRNPs and summarize the new features in the dynamic nuclear export process of mRNPs revealed from these technical advances. PMID:25141104

  4. Intragenic reversion mutations that improve export of maltose-binding protein in Escherichia coli malE signal sequence mutants.

    PubMed

    Ryan, J P; Duncan, M C; Bankaitis, V A; Bassford, P J

    1986-03-01

    Escherichia coli strains harboring malE signal sequence point mutations accumulate export-defective precursor maltose-binding protein (MBP) in the cytoplasm. Beginning with these mutants, a number of spontaneous intragenic revertants have been obtained in which export of the MBP to the periplasm is either partially or totally restored. With a single exception, each of the reversion mutations resulted in an increase in the overall hydrophobicity of the signal peptide hydrophobic core by one of five different mechanisms. In some revertants, MBP export was achieved at a rate comparable to the wild type MBP; in other cases, the rate of MBP export was significantly slower than wild type. The results indicate that the overall hydrophobicity of the signal peptide, rather than the absolute length of its uninterrupted hydrophobic core, is a major determinant of MBP export competency. An alteration at residue 19 of the mature MBP also has been identified that provides fairly efficient suppression of the export defect in the adjacent signal peptide, further suggesting that important export information may reside in this region of the precursor protein. PMID:3512555

  5. Sec24 is a coincidence detector that simultaneously binds two signals to drive ER export

    PubMed Central

    Pagant, Silvere; Wu, Alexander; Edwards, Samuel; Diehl, Frances; Miller, Elizabeth A.

    2014-01-01

    Summary Background Incorporation of secretory proteins into ER-derived vesicles involves recognition of cytosolic signals by the COPII coat protein, Sec24. Additional cargo diversity is achieved through cargo receptors, which include the Erv14/Cornichon family that mediate export of transmembrane proteins despite the potential for such clients to directly interact with Sec24. The molecular function of Erv14 thus remains unclear, with possible roles in COPII-binding, membrane domain chaperoning and lipid organization. Results Using a targeted mutagenesis approach to define the mechanism of Erv14 function, we identify conserved residues in the second transmembrane domain of Erv14 that mediate interaction with a subset of Erv14 clients. We further show that interaction of Erv14 with a novel cargo-binding surface on Sec24 is necessary for efficient trafficking of all of its clients. However, we also determine that some Erv14 clients also engage directly an adjacent cargo-binding domain of Sec24, suggesting a novel mode of dual interaction between cargo and coat. Conclusions We conclude that Erv14 functions as a canonical cargo receptor that couples membrane proteins to the COPII coat, but that maximal export requires a bivalent signal that derives from motifs on both the cargo protein and Erv14. Sec24 can thus be considered a coincidence detector that binds simultaneously to multiple signals to drive packaging of polytopic membrane proteins. This mode of dual signal binding to a single coat protein might serve as a general mechanism to trigger efficient capture, or may be specifically employed in ER export to control deployment of nascent proteins. PMID:25619760

  6. 37 CFR 5.20 - Export of technical data relating to sensitive nuclear technology.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ....20 Export of technical data relating to sensitive nuclear technology. Under regulations (10 CFR 810.7) established by the United States Department of Energy, an application filed in accordance with the regulations... authorized activity for the purposes of the Department of Energy regulations....

  7. Export Control Guide: Loose Parts Monitoring Systems for Nuclear Power Plants

    SciTech Connect

    Langenberg, Donald W.

    2012-12-01

    This report describes a typical LPMS, emphasizing its application to the RCS of a modern NPP. The report also examines the versatility of AE monitoring technology by describing several nuclear applications other than loose parts monitoring, as well as some non-nuclear applications. In addition, LPMS implementation requirements are outlined, and LPMS suppliers are identified. Finally, U.S. export controls applicable to LPMSs are discussed.

  8. Thyroid hormone receptor alpha1 follows a cooperative CRM1/calreticulin-mediated nuclear export pathway.

    PubMed

    Grespin, Matthew E; Bonamy, Ghislain M C; Roggero, Vincent R; Cameron, Nicole G; Adam, Lindsay E; Atchison, Andrew P; Fratto, Victoria M; Allison, Lizabeth A

    2008-09-12

    The thyroid hormone receptor alpha1 (TRalpha) exhibits a dual role as an activator or repressor of its target genes in response to thyroid hormone (T(3)). Previously, we have shown that TRalpha, formerly thought to reside solely in the nucleus bound to DNA, actually shuttles rapidly between the nucleus and cytoplasm. An important aspect of the shuttling activity of TRalpha is its ability to exit the nucleus through the nuclear pore complex. TRalpha export is not sensitive to treatment with the CRM1-specific inhibitor leptomycin B (LMB) in heterokaryon assays, suggesting a role for an export receptor other than CRM1. Here, we have used a combined approach of in vivo fluorescence recovery after photobleaching experiments, in vitro permeabilized cell nuclear export assays, and glutathione S-transferase pull-down assays to investigate the export pathway used by TRalpha. We show that, in addition to shuttling in heterokaryons, TRalpha shuttles rapidly in an unfused monokaryon system as well. Furthermore, our data show that TRalpha directly interacts with calreticulin, and point to the intriguing possibility that TRalpha follows a cooperative export pathway in which both calreticulin and CRM1 play a role in facilitating efficient translocation of TRalpha from the nucleus to cytoplasm. PMID:18641393

  9. Assembly and nuclear export of pre-ribosomal particles in budding yeast.

    PubMed

    Gerhardy, Stefan; Menet, Anna Maria; Peña, Cohue; Petkowski, Janusz Jurand; Panse, Vikram Govind

    2014-08-01

    The ribosome is responsible for the final step of decoding genetic information into proteins. Therefore, correct assembly of ribosomes is a fundamental task for all living cells. In eukaryotes, the construction of the ribosome which begins in the nucleolus requires coordinated efforts of >350 specialized factors that associate with pre-ribosomal particles at distinct stages to perform specific assembly steps. On their way through the nucleus, diverse energy-consuming enzymes are thought to release assembly factors from maturing pre-ribosomal particles after accomplishing their task(s). Subsequently, recruitment of export factors prepares pre-ribosomal particles for transport through nuclear pore complexes. Pre-ribosomes are exported into the cytoplasm in a functionally inactive state, where they undergo final maturation before initiating translation. Accumulating evidence indicates a tight coupling between nuclear export, cytoplasmic maturation, and final proofreading of the ribosome. In this review, we summarize our current understanding of nuclear export of pre-ribosomal subunits and cytoplasmic maturation steps that render pre-ribosomal subunits translation-competent. PMID:24817020

  10. Nuclear export of the human immunodeficiency virus type 1 nucleocytoplasmic shuttle protein Rev is mediated by its activation domain and is blocked by transdominant negative mutants.

    PubMed Central

    Szilvay, A M; Brokstad, K A; Kopperud, R; Haukenes, G; Kalland, K H

    1995-01-01

    The human immunodeficiency virus type 1 nucleocytoplasmic shuttle protein Rev moves repeatedly between the cytoplasm, a perinuclear zone, the nucleoli, and nucleoplasmic speckles. In this study, we demonstrated by both indirect immunofluorescence and Western immunoblot analysis that nuclear exit of Rev transdominant negative mutants was defective compared with that of wild-type Rev. The basic and activation domains of Rev signal import and export, respectively, of Rev across the nuclear membrane. In cotransfection experiments, mutants containing mutations of Rev inhibited the nuclear egress of wild-type Rev, thus revealing a novel transdominant negative phenotype. PMID:7745679

  11. Extracellular signaling cues for nuclear actin polymerization.

    PubMed

    Plessner, Matthias; Grosse, Robert

    2015-01-01

    Contrary to cytoplasmic actin structures, the biological functions of nuclear actin filaments remain largely enigmatic. Recent progress in the field, however, has determined nuclear actin structures in somatic cells either under steady state conditions or in response to extracellular signaling cues. These actin structures differ in size and shape as well as in their temporal appearance and dynamics. Thus, a picture emerges that suggests that mammalian cells may have different pathways and mechanisms to assemble nuclear actin filaments. Apart from serum- or LPA-triggered nuclear actin polymerization, integrin activation by extracellular matrix interaction was recently implicated in nuclear actin polymerization through the linker of nucleoskeleton and cytoskeleton (LINC) complex. Some of these extracellular cues known so far appear to converge at the level of nuclear formin activity and subsequent regulation of myocardin-related transcription factors. Nevertheless, as the precise signaling events are as yet unknown, the regulation of nuclear actin polymerization may be of significant importance for different cellular functions as well as disease conditions caused by altered nuclear dynamics and architecture. PMID:26059398

  12. Combinatorial targeting of nuclear export and translation of RNA inhibits aggressive B-cell lymphomas

    PubMed Central

    Culjkovic-Kraljacic, Biljana; Fernando, Tharu M.; Marullo, Rossella; Calvo-Vidal, Nieves; Verma, Akanksha; Yang, ShaoNing; Tabbò, Fabrizio; Gaudiano, Marcello; Zahreddine, Hiba; Goldstein, Rebecca L.; Patel, Jayeshkumar; Taldone, Tony; Chiosis, Gabriela; Ladetto, Marco; Ghione, Paola; Machiorlatti, Rodolfo; Elemento, Olivier; Inghirami, Giorgio; Melnick, Ari; Borden, Katherine L. B.

    2016-01-01

    Aggressive double- and triple-hit (DH/TH) diffuse large B-cell lymphomas (DLBCLs) feature activation of Hsp90 stress pathways. Herein, we show that Hsp90 controls posttranscriptional dynamics of key messenger RNA (mRNA) species including those encoding BCL6, MYC, and BCL2. Using a proteomics approach, we found that Hsp90 binds to and maintains activity of eIF4E. eIF4E drives nuclear export and translation of BCL6, MYC, and BCL2 mRNA. eIF4E RNA-immunoprecipitation sequencing in DLBCL suggests that nuclear eIF4E controls an extended program that includes B-cell receptor signaling, cellular metabolism, and epigenetic regulation. Accordingly, eIF4E was required for survival of DLBCL including the most aggressive subtypes, DH/TH lymphomas. Indeed, eIF4E inhibition induces tumor regression in cell line and patient-derived tumorgrafts of TH-DLBCL, even in the presence of elevated Hsp90 activity. Targeting Hsp90 is typically limited by counterregulatory elevation of Hsp70B, which induces resistance to Hsp90 inhibitors. Surprisingly, we identify Hsp70 mRNA as an eIF4E target. In this way, eIF4E inhibition can overcome drug resistance to Hsp90 inhibitors. Accordingly, rational combinatorial inhibition of eIF4E and Hsp90 inhibitors resulted in cooperative antilymphoma activity in DH/TH DLBCL in vitro and in vivo. PMID:26603836

  13. Dual functions for the Schizosaccharomyces pombe inositol kinase Ipk1 in nuclear mRNA export and polarized cell growth.

    PubMed

    Sarmah, Bhaskarjyoti; Wente, Susan R

    2009-02-01

    The inositol 1,3,4,5,6-pentakisphosphate (IP(5)) 2-kinase (Ipk1) catalyzes the production of inositol hexakisphosphate (IP(6)) in eukaryotic cells. Previous studies have shown that IP(6) is required for efficient nuclear mRNA export in the budding yeast Saccharomyces cerevisiae. Here, we report the first functional analysis of ipk1(+) in Schizosaccharomyces pombe. S. pombe Ipk1 (SpIpk1) is unique among Ipk1 orthologues in that it harbors a novel amino (N)-terminal domain with coiled-coil structural motifs similar to those of BAR (Bin-amphiphysin-Rvs) domain proteins. Mutants with ipk1(+) deleted (ipk1Delta) had mRNA export defects as well as pleiotropic defects in polarized growth, cell morphology, endocytosis, and cell separation. The SpIpk1 catalytic carboxy-terminal domain was required to rescue these defects, and the mRNA export block was genetically linked to SpDbp5 function and, likely, IP(6) production. However, the overexpression of the N-terminal domain alone also inhibited these functions in wild-type cells. This revealed a distinct noncatalytic function for the N-terminal domain. To test for connections with other inositol polyphosphates, we also analyzed whether the loss of asp1(+) function, encoding an IP(6) kinase downstream of Ipk1, had an effect on ipk1Delta cells. The asp1Delta mutant alone did not block mRNA export, and its cell morphology, polarized growth, and endocytosis defects were less severe than those of ipk1Delta cells. Moreover, ipk1Delta asp1Delta double mutants had altered inositol polyphosphate levels distinct from those of the ipk1Delta mutant. This suggested novel roles for asp1(+) upstream of ipk1(+). We propose that IP(6) production is a key signaling linchpin for regulating multiple essential cellular processes. PMID:19047361

  14. Nucleolin regulates phosphorylation and nuclear export of fibroblast growth factor 1 (FGF1).

    PubMed

    Sletten, Torunn; Kostas, Michal; Bober, Joanna; Sorensen, Vigdis; Yadollahi, Mandana; Olsnes, Sjur; Tomala, Justyna; Otlewski, Jacek; Zakrzewska, Malgorzata; Wiedlocha, Antoni

    2014-01-01

    Extracellular fibroblast growth factor 1 (FGF1) acts through cell surface tyrosine kinase receptors, but FGF1 can also act directly in the cell nucleus, as a result of nuclear import of endogenously produced, non-secreted FGF1 or by transport of extracellular FGF1 via endosomes and cytosol into the nucleus. In the nucleus, FGF1 can be phosphorylated by protein kinase C δ (PKCδ), and this event induces nuclear export of FGF1. To identify intracellular targets of FGF1 we performed affinity pull-down assays and identified nucleolin, a nuclear multifunctional protein, as an interaction partner of FGF1. We confirmed a direct nucleolin-FGF1 interaction by surface plasmon resonance and identified residues of FGF1 involved in the binding to be located within the heparin binding site. To assess the biological role of the nucleolin-FGF1 interaction, we studied the intracellular trafficking of FGF1. In nucleolin depleted cells, exogenous FGF1 was endocytosed and translocated to the cytosol and nucleus, but FGF1 was not phosphorylated by PKCδ or exported from the nucleus. Using FGF1 mutants with reduced binding to nucleolin and a FGF1-phosphomimetic mutant, we showed that the nucleolin-FGF1 interaction is critical for the intranuclear phosphorylation of FGF1 by PKCδ and thereby the regulation of nuclear export of FGF1. PMID:24595027

  15. The Consensus 5' Splice Site Motif Inhibits mRNA Nuclear Export

    PubMed Central

    Lee, Eliza S.; Akef, Abdalla; Mahadevan, Kohila; Palazzo, Alexander F.

    2015-01-01

    In eukaryotes, mRNAs are synthesized in the nucleus and then exported to the cytoplasm where they are translated into proteins. We have mapped an element, which when present in the 3’terminal exon or in an unspliced mRNA, inhibits mRNA nuclear export. This element has the same sequence as the consensus 5’splice site motif that is used to define the start of introns. Previously it was shown that when this motif is retained in the mRNA, it causes defects in 3’cleavage and polyadenylation and promotes mRNA decay. Our new data indicates that this motif also inhibits nuclear export and promotes the targeting of transcripts to nuclear speckles, foci within the nucleus which have been linked to splicing. The motif, however, does not disrupt splicing or the recruitment of UAP56 or TAP/Nxf1 to the RNA, which are normally required for nuclear export. Genome wide analysis of human mRNAs, lncRNA and eRNAs indicates that this motif is depleted from naturally intronless mRNAs and eRNAs, but less so in lncRNAs. This motif is also depleted from the beginning and ends of the 3’terminal exons of spliced mRNAs, but less so for lncRNAs. Our data suggests that the presence of the 5’splice site motif in mature RNAs promotes their nuclear retention and may help to distinguish mRNAs from misprocessed transcripts and transcriptional noise. PMID:25826302

  16. Nuclear Signaling from Cadherin Adhesion Complexes

    PubMed Central

    McCrea, Pierre D.; Maher, Meghan T.; Gottardi, Cara J.

    2015-01-01

    The arrival of multicellularity in evolution facilitated cell–cell signaling in conjunction with adhesion. As the ectodomains of cadherins interact with each other directly in trans (as well as in cis), spanning the plasma membrane and associating with multiple other entities, cadherins enable the transduction of “outside-in” or “inside-out” signals. We focus this review on signals that originate from the larger family of cadherins that are inwardly directed to the nucleus, and thus have roles in gene control or nuclear structure–function. The nature of cadherin complexes varies considerably depending on the type of cadherin and its context, and we will address some of these variables for classical cadherins versus other family members. Substantial but still fragmentary progress has been made in understanding the signaling mediators used by varied cadherin complexes to coordinate the state of cell–cell adhesion with gene expression. Evidence that cadherin intracellular binding partners also localize to the nucleus is a major point of interest. In some models, catenins show reduced binding to cadherin cytoplasmic tails favoring their engagement in gene control. When bound, cadherins may serve as stoichiometric competitors of nuclear signals. Cadherins also directly or indirectly affect numerous signaling pathways (e.g., Wnt, receptor tyrosine kinase, Hippo, NFκB, and JAK/STAT), enabling cell–cell contacts to touch upon multiple biological outcomes in embryonic development and tissue homeostasis. PMID:25733140

  17. Nuclear signaling from cadherin adhesion complexes.

    PubMed

    McCrea, Pierre D; Maher, Meghan T; Gottardi, Cara J

    2015-01-01

    The arrival of multicellularity in evolution facilitated cell-cell signaling in conjunction with adhesion. As the ectodomains of cadherins interact with each other directly in trans (as well as in cis), spanning the plasma membrane and associating with multiple other entities, cadherins enable the transduction of "outside-in" or "inside-out" signals. We focus this review on signals that originate from the larger family of cadherins that are inwardly directed to the nucleus, and thus have roles in gene control or nuclear structure-function. The nature of cadherin complexes varies considerably depending on the type of cadherin and its context, and we will address some of these variables for classical cadherins versus other family members. Substantial but still fragmentary progress has been made in understanding the signaling mediators used by varied cadherin complexes to coordinate the state of cell-cell adhesion with gene expression. Evidence that cadherin intracellular binding partners also localize to the nucleus is a major point of interest. In some models, catenins show reduced binding to cadherin cytoplasmic tails favoring their engagement in gene control. When bound, cadherins may serve as stoichiometric competitors of nuclear signals. Cadherins also directly or indirectly affect numerous signaling pathways (e.g., Wnt, receptor tyrosine kinase, Hippo, NFκB, and JAK/STAT), enabling cell-cell contacts to touch upon multiple biological outcomes in embryonic development and tissue homeostasis. PMID:25733140

  18. Structural basis for assembly and disassembly of the CRM1 nuclear export complex

    SciTech Connect

    Dong, Xiuhua; Biswas, Anindita; Chook, Yuh Min

    2009-09-15

    CRM1 (or exportin 1, Xpo1) transports proteins out of the cell nucleus through the nuclear pore complex. In the cytoplasm, GTP hydrolysis and consequent dissociation of Ran from CRM1 releases low-affinity substrates, while additional factors facilitate release of high-affinity substrates. Here we provide a model for human CRM1 export complex assembly and disassembly through structural and biochemical analyses of CRM1 bound to the substrate snurportin 1 (SNUPN, also called snuportin 1).

  19. Nuclear inositide signaling in myelodysplastic syndromes.

    PubMed

    Follo, Matilde Y; Mongiorgi, Sara; Finelli, Carlo; Clissa, Cristina; Ramazzotti, Giulia; Fiume, Roberta; Faenza, Irene; Manzoli, Lucia; Martelli, Alberto M; Cocco, Lucio

    2010-04-15

    Myelodysplastic syndromes (MDS) are defined as clonal hematopoietic stem-cell disorders characterized by ineffective hematopoiesis in one or more of the lineages of the bone marrow. Although distinct morphologic subgroups exist, the natural history of MDS is progression to acute myeloid leukemia (AML). However, the molecular the mechanisms the underlying MDS evolution to AML are not completely understood. Inositides are key cellular second messengers with well-established roles in signal transduction pathways, and nuclear metabolism elicited by phosphoinositide-specific phospholipase C (PI-PLC) beta1 and Akt plays an important role in the control of the balance between cell cycle progression and apoptosis in both normal and pathologic conditions. Recent findings evidenced the role played by nuclear lipid signaling pathways, which could become promising therapeutic targets in MDS. This review will provide a concise and updated revision of the state of art on this topic. PMID:20058233

  20. Endogenous IGFBP-3 Mediates Intrinsic Apoptosis Through Modulation of Nur77 Phosphorylation and Nuclear Export.

    PubMed

    Agostini-Dreyer, Allyson; Jetzt, Amanda E; Stires, Hillary; Cohick, Wendie S

    2015-11-01

    In nontransformed bovine mammary epithelial cells, the intrinsic apoptosis inducer anisomycin (ANS) induces IGFBP-3 expression and nuclear localization and knockdown of IGFBP-3 attenuates ANS-induced apoptosis. Others have shown in prostate cancer cells that exogenous IGFBP-3 induces apoptosis by facilitating nuclear export of the orphan nuclear receptor Nur77 and its binding partner, retinoid X receptor-α (RXRα). The goal of the present work was to determine whether endogenous IGFBP-3 plays a role in ANS-induced apoptosis by facilitating nuclear transport of Nur77 and/or RXRα in nontransformed cells. Knockdown of Nur77 with siRNA decreased ANS-induced cleavage of caspase-3 and -7 and their downstream target, PARP, indicating a role for Nur77 in ANS-induced apoptosis. In cells transfected with IGFBP-3, IGFBP-3 associated with RXRα but not Nur77 under basal conditions, however, IGFBP-3 co-precipitated with phosphorylated forms of both proteins in ANS-treated cells. Indirect immunofluorescence and cell fractionation techniques showed that ANS induced phosphorylation and transport of Nur77 from the nucleus to the cytoplasm and these effects were attenuated by knockdown of IGFBP-3. These data suggest that endogenous IGFBP-3 plays a role in intrinsic apoptosis by facilitating phosphorylation and nuclear export of Nur77 to the cytoplasm where it exerts its apoptotic effect. Whether this mechanism involves a physical association between endogenous IGFBP-3 and Nur77 or RXRα remains to be determined. PMID:26340041

  1. Nup358/RanBP2 Attaches to the Nuclear Pore Complex via Association with Nup88 and Nup214/CAN and Plays a Supporting Role in CRM1-Mediated Nuclear Protein Export

    PubMed Central

    Bernad, Rafael; van der Velde, Hella; Fornerod, Maarten; Pickersgill, Helen

    2004-01-01

    Nuclear pore complexes (NPCs) traverse the nuclear envelope (NE), providing a channel through which nucleocytoplasmic transport occurs. Nup358/RanBP2, Nup214/CAN, and Nup88 are components of the cytoplasmic face of the NPC. Here we show that Nup88 localizes midway between Nup358 and Nup214 and physically interacts with them. RNA interference of either Nup88 or Nup214 in human cells caused a strong reduction of Nup358 at the NE. Nup88 and Nup214 showed an interdependence at the NPC and were not affected by the absence of Nup358. These data indicate that Nup88 and Nup214 mediate the attachment of Nup358 to the NPC. We show that localization of the export receptor CRM1 at the cytoplasmic face of the NE is Nup358 dependent and represents its empty state. Also, removal of Nup358 causes a distinct reduction in nuclear export signal-dependent nuclear export. We propose that Nup358 provides both a platform for rapid disassembly of CRM1 export complexes and a binding site for empty CRM1 recycling into the nucleus. PMID:14993277

  2. HIV-1 and M-PMV RNA Nuclear Export Elements Program Viral Genomes for Distinct Cytoplasmic Trafficking Behaviors.

    PubMed

    Pocock, Ginger M; Becker, Jordan T; Swanson, Chad M; Ahlquist, Paul; Sherer, Nathan M

    2016-04-01

    Retroviruses encode cis-acting RNA nuclear export elements that override nuclear retention of intron-containing viral mRNAs including the full-length, unspliced genomic RNAs (gRNAs) packaged into assembling virions. The HIV-1 Rev-response element (RRE) recruits the cellular nuclear export receptor CRM1 (also known as exportin-1/XPO1) using the viral protein Rev, while simple retroviruses encode constitutive transport elements (CTEs) that directly recruit components of the NXF1(Tap)/NXT1(p15) mRNA nuclear export machinery. How gRNA nuclear export is linked to trafficking machineries in the cytoplasm upstream of virus particle assembly is unknown. Here we used long-term (>24 h), multicolor live cell imaging to directly visualize HIV-1 gRNA nuclear export, translation, cytoplasmic trafficking, and virus particle production in single cells. We show that the HIV-1 RRE regulates unique, en masse, Rev- and CRM1-dependent "burst-like" transitions of mRNAs from the nucleus to flood the cytoplasm in a non-localized fashion. By contrast, the CTE derived from Mason-Pfizer monkey virus (M-PMV) links gRNAs to microtubules in the cytoplasm, driving them to cluster markedly to the centrosome that forms the pericentriolar core of the microtubule-organizing center (MTOC). Adding each export element to selected heterologous mRNAs was sufficient to confer each distinct export behavior, as was directing Rev/CRM1 or NXF1/NXT1 transport modules to mRNAs using a site-specific RNA tethering strategy. Moreover, multiple CTEs per transcript enhanced MTOC targeting, suggesting that a cooperative mechanism links NXF1/NXT1 to microtubules. Combined, these results reveal striking, unexpected features of retroviral gRNA nucleocytoplasmic transport and demonstrate roles for mRNA export elements that extend beyond nuclear pores to impact gRNA distribution in the cytoplasm. PMID:27070420

  3. HIV-1 and M-PMV RNA Nuclear Export Elements Program Viral Genomes for Distinct Cytoplasmic Trafficking Behaviors

    PubMed Central

    Pocock, Ginger M.; Becker, Jordan T.; Swanson, Chad M.; Ahlquist, Paul; Sherer, Nathan M.

    2016-01-01

    Retroviruses encode cis-acting RNA nuclear export elements that override nuclear retention of intron-containing viral mRNAs including the full-length, unspliced genomic RNAs (gRNAs) packaged into assembling virions. The HIV-1 Rev-response element (RRE) recruits the cellular nuclear export receptor CRM1 (also known as exportin-1/XPO1) using the viral protein Rev, while simple retroviruses encode constitutive transport elements (CTEs) that directly recruit components of the NXF1(Tap)/NXT1(p15) mRNA nuclear export machinery. How gRNA nuclear export is linked to trafficking machineries in the cytoplasm upstream of virus particle assembly is unknown. Here we used long-term (>24 h), multicolor live cell imaging to directly visualize HIV-1 gRNA nuclear export, translation, cytoplasmic trafficking, and virus particle production in single cells. We show that the HIV-1 RRE regulates unique, en masse, Rev- and CRM1-dependent “burst-like” transitions of mRNAs from the nucleus to flood the cytoplasm in a non-localized fashion. By contrast, the CTE derived from Mason-Pfizer monkey virus (M-PMV) links gRNAs to microtubules in the cytoplasm, driving them to cluster markedly to the centrosome that forms the pericentriolar core of the microtubule-organizing center (MTOC). Adding each export element to selected heterologous mRNAs was sufficient to confer each distinct export behavior, as was directing Rev/CRM1 or NXF1/NXT1 transport modules to mRNAs using a site-specific RNA tethering strategy. Moreover, multiple CTEs per transcript enhanced MTOC targeting, suggesting that a cooperative mechanism links NXF1/NXT1 to microtubules. Combined, these results reveal striking, unexpected features of retroviral gRNA nucleocytoplasmic transport and demonstrate roles for mRNA export elements that extend beyond nuclear pores to impact gRNA distribution in the cytoplasm. PMID:27070420

  4. Nuclear retention of Fbw7 by specific inhibitors of nuclear export leads to Notch1 degradation in pancreatic cancer.

    PubMed

    Gao, Jiankun; Azmi, Asfar S; Aboukameel, Amro; Kauffman, Michael; Shacham, Sharon; Abou-Samra, Abdul-Badi; Mohammad, Ramzi M

    2014-06-15

    Chromosome maintenance region 1 (CRM1) also called Exportin 1 (Xpo1), a protein found elevated in pancreatic ductal adenocarcinoma (PDAC), blocks tumor suppressor protein (TSP) function through constant nuclear export. Earlier we had shown that targeting CRM1 by our newly developed specific inhibitors of nuclear export (SINE) leads to inhibition of pancreatic cancer cell proliferation and tumor growth arrest. In this paper we define the mechanism of SINE action. Our lead SINE KPT-185 inhibits PDAC cell growth, cell migration, tumor invasion and induces apoptosis and G2-M cell cycle arrest in low nano molar range (IC50s~150 nM). Mechanistically we demonstrate that the activity of KPT-185 is associated with nuclear retention of Fbw7; which degrades nuclear Notch-1 leading to decreased tumor promoting markers such as C-Myc, Cyclin-D1, Hes1 and VEGF. The orally bioavailable SINE (KPT-251) showed potent anti-tumor activity in a Colo-357 PDAC xenografts model; residual tumor analysis showed activation of Fbw7 concomitant with attenuation of Notch1 and its downstream genes. These results suggest that the antitumor activity of KPT-185 is in part due to nuclear retention of Fbw7 and consequent Notch1 degradation. The new CRM1 inhibitors, therefore, hold strong potential and warrant further clinical investigations for PDAC. PMID:24899509

  5. A human RNA helicase-like protein, HRH1, facilitates nuclear export of spliced mRNA by releasing the RNA from the spliceosome.

    PubMed

    Ohno, M; Shimura, Y

    1996-04-15

    Because the nuclear export of mRNA occurs only after the splicing reaction is completed, intron-containing pre-mRNA does not normally appear in the cytoplasm. As a mechanism to secure this, intron-containing RNA is retained in the nucleus via formation of the spliceosome. Therefore, the process of releasing spliced mRNA from the spliceosome after completion of splicing is an essential step for triggering the nuclear export of the spliced mRNA. In budding yeast, RNA helicase-like protein Prp22 is implicated in this process. Here we demonstrate the function of HRH1, a human protein homologous to Prp22, in mammalian cells using dominant-negative HRH1++ mutants (dn-HRH1). dn-HRH1 protein stalls on the spliceosome and prevents release of the spliced RNA from the spliceosome in vitro. Expression of dn-HRH1 in mammalian cells leads to inhibition of splicing and to extensive nuclear export of unspliced pre-mRNA, probably because of the incapability of recycling spliceosome components that normally retain the pre-mRNA in the nucleus. The arginine/serine-rich domain (RS domain) of HRH1, which is missing in Prp22, confers a nuclear localization signal, and appears to facilitate the interaction of HRH1 with the spliceosome. This is the first report on a bona fide mammalian homolog of yeast Prp splicing factor, and also on a mammalian RNA helicase-like splicing factor. PMID:8608946

  6. Targeting the Nuclear Export Protein XPO1/CRM1 Reverses Epithelial to Mesenchymal Transition

    PubMed Central

    Azmi, Asfar S.; Muqbil, Irfana; Wu, Jack; Aboukameel, Amro; Senapedis, William; Baloglu, Erkan; Bollig-Fischer, Aliccia; Dyson, Gregory; Kauffman, Michael; Landesman, Yosef; Shacham, Sharon; Philip, Philip A.; Mohammad, Ramzi M.

    2015-01-01

    Here we demonstrate for the first time that targeted inhibition of nuclear exporter protein exportin 1 (XPO1) also known as chromosome maintenance region 1 (CRM1) by Selective Inhibitor of Nuclear Export (SINE) compounds results in reversal of EMT in snail-transduced primary human mammary epithelial cells (HMECs). SINE compounds selinexor (KPT-330) and KPT-185, leptomycin B (LMB as +ve control) but not KPT-301 (–ve control) reverse EMT, suppress mesenchymal markers and consequently induce growth inhibition, apoptosis and prevent spheroid formation. SINE treatment resulted in nuclear retention of snail regulator FBXL5 that was concurrent with suppression of snail and down-regulation of mesenchymal markers. FBXL5 siRNA or transfection with cys528 mut-Xpo1 (lacking SINE binding site) markedly abrogated SINE activity highlighting an XPO1 and FBXL5 mediated mechanism of action. Silencing XPO1 or snail caused re-expression of FBXL5 as well as EMT reversal. Pathway analysis on SINE treated HMECs further verified the involvement of additional F-Box family proteins and confirmed the suppression of snail network. Oral administration of selinexor (15 mg/kg p.o. QoDx3/week for 3weeks) resulted in complete cures (no tumor rebound at 120 days) of HMLER-Snail xenografts. These findings raise the unique possibility of blocking EMT at the nuclear pore. PMID:26536918

  7. Targeting the Nuclear Export Protein XPO1/CRM1 Reverses Epithelial to Mesenchymal Transition.

    PubMed

    Azmi, Asfar S; Muqbil, Irfana; Wu, Jack; Aboukameel, Amro; Senapedis, William; Baloglu, Erkan; Bollig-Fischer, Aliccia; Dyson, Gregory; Kauffman, Michael; Landesman, Yosef; Shacham, Sharon; Philip, Philip A; Mohammad, Ramzi M

    2015-01-01

    Here we demonstrate for the first time that targeted inhibition of nuclear exporter protein exportin 1 (XPO1) also known as chromosome maintenance region 1 (CRM1) by Selective Inhibitor of Nuclear Export (SINE) compounds results in reversal of EMT in snail-transduced primary human mammary epithelial cells (HMECs). SINE compounds selinexor (KPT-330) and KPT-185, leptomycin B (LMB as +ve control) but not KPT-301 (-ve control) reverse EMT, suppress mesenchymal markers and consequently induce growth inhibition, apoptosis and prevent spheroid formation. SINE treatment resulted in nuclear retention of snail regulator FBXL5 that was concurrent with suppression of snail and down-regulation of mesenchymal markers. FBXL5 siRNA or transfection with cys528 mut-Xpo1 (lacking SINE binding site) markedly abrogated SINE activity highlighting an XPO1 and FBXL5 mediated mechanism of action. Silencing XPO1 or snail caused re-expression of FBXL5 as well as EMT reversal. Pathway analysis on SINE treated HMECs further verified the involvement of additional F-Box family proteins and confirmed the suppression of snail network. Oral administration of selinexor (15 mg/kg p.o. QoDx3/week for 3weeks) resulted in complete cures (no tumor rebound at 120 days) of HMLER-Snail xenografts. These findings raise the unique possibility of blocking EMT at the nuclear pore. PMID:26536918

  8. Export control guide: Spent nuclear fuel reprocessing and preparation of plutonium metal

    SciTech Connect

    1993-10-01

    The international Treaty on the Non-Proliferation of Nuclear Weapons, also referred to as the Non-Proliferation Treaty (NPT), states in Article III, paragraph 2(b) that {open_quotes}Each State Party to the Treaty undertakes not to provide . . . equipment or material especially designed or prepared for the processing, use or production of special fissionable material to any non-nuclear-weapon State for peaceful purposes, unless the source or special fissionable material shall be subject to the safeguards required by this Article.{close_quotes} This guide was prepared to assist export control officials in the interpretation, understanding, and implementation of export laws and controls relating to the international Trigger List for irradiated nuclear fuel reprocessing equipment, components, and materials. The guide also contains information related to the production of plutonium metal. Reprocessing and its place in the nuclear fuel cycle are described briefly; the standard procedure to prepare metallic plutonium is discussed; steps used to prepare Trigger List controls are cited; descriptions of controlled items are given; and special materials of construction are noted. This is followed by a comprehensive description of especially designed or prepared equipment, materials, and components of reprocessing and plutonium metal processes and includes photographs and/or pictorial representations. The nomenclature of the Trigger List has been retained in the numbered sections of this document for clarity.

  9. Inhibition of pyrimidine synthesis reverses viral virulence factor-mediated block of mRNA nuclear export

    PubMed Central

    Zhang, Liang; Das, Priyabrata; Schmolke, Mirco; Manicassamy, Balaji; Wang, Yaming; Deng, Xiaoyi; Cai, Ling; Tu, Benjamin P.; Forst, Christian V.; Roth, Michael G.; Levy, David E.; García-Sastre, Adolfo; de Brabander, Jef; Phillips, Margaret A.

    2012-01-01

    The NS1 protein of influenza virus is a major virulence factor essential for virus replication, as it redirects the host cell to promote viral protein expression. NS1 inhibits cellular messenger ribonucleic acid (mRNA) processing and export, down-regulating host gene expression and enhancing viral gene expression. We report in this paper the identification of a nontoxic quinoline carboxylic acid that reverts the inhibition of mRNA nuclear export by NS1, in the absence or presence of the virus. This quinoline carboxylic acid directly inhibited dihydroorotate dehydrogenase (DHODH), a host enzyme required for de novo pyrimidine biosynthesis, and partially reduced pyrimidine levels. This effect induced NXF1 expression, which promoted mRNA nuclear export in the presence of NS1. The release of NS1-mediated mRNA export block by DHODH inhibition also occurred in the presence of vesicular stomatitis virus M (matrix) protein, another viral inhibitor of mRNA export. This reversal of mRNA export block allowed expression of antiviral factors. Thus, pyrimidines play a necessary role in the inhibition of mRNA nuclear export by virulence factors. PMID:22312003

  10. Src Subfamily Kinases Regulate Nuclear Export and Degradation of Transcription Factor Nrf2 to Switch Off Nrf2-mediated Antioxidant Activation of Cytoprotective Gene Expression*

    PubMed Central

    Niture, Suryakant K.; Jain, Abhinav K.; Shelton, Phillip M.; Jaiswal, Anil K.

    2011-01-01

    Nrf2 (NF-E2-related factor 2) is a nuclear transcription factor that in response to chemical and radiation stress regulates coordinated induction of a battery of cytoprotective gene expressions leading to cellular protection. In this study, we investigated the role of Src kinases in the regulation of Nrf2 and downstream signaling. siRNA-mediated inhibition of Fyn, Src, Yes, and Fgr, but not Lyn, in mouse hepatoma Hepa-1 cells, led to nuclear accumulation of Nrf2 and up-regulation of Nrf2 downstream gene expression. Mouse embryonic fibroblasts with combined deficiency of Fyn/Src/Yes/Fgr supported results from siRNA. In addition, steady-state overexpression of Fyn, Src, and Yes phosphorylated Nrf2Tyr568 that triggered nuclear export and degradation of Nrf2 and down-regulation of Nrf2 downstream gene expression. Exposure of cells to antioxidant, oxidant, or UV radiation increased nuclear import of Fyn, Src, and Yes kinases, which phosphorylated Nrf2Tyr568 resulting in nuclear export and degradation of Nrf2. Further analysis revealed that stress-activated GSK3β acted upstream to the Src kinases and phosphorylated the Src kinases, leading to their nuclear localization and Nrf2 phosphorylation. The overexpression of Src kinases in Hepa-1 cells led to decreased Nrf2, increased apoptosis, and decreased cell survival. Mouse embryonic fibroblasts deficient in Src kinases showed nuclear accumulation of Nrf2, induction of Nrf2 and downstream gene expression, reduced apoptosis, and increased cell survival. The studies together demonstrate that Src kinases play a critical role in nuclear export and degradation of Nrf2, thereby providing a negative feedback mechanism to switch off Nrf2 activation and restore normal cellular homeostasis. PMID:21690096

  11. HIV-1 Nef-associated Factor 1 Enhances Viral Production by Interacting with CRM1 to Promote Nuclear Export of Unspliced HIV-1 gag mRNA.

    PubMed

    Ren, Xiao-Xin; Wang, Hai-Bo; Li, Chuan; Jiang, Jin-Feng; Xiong, Si-Dong; Jin, Xia; Wu, Li; Wang, Jian-Hua

    2016-02-26

    HIV-1 depends on host-cell-encoded factors to complete its life cycle. A comprehensive understanding of how HIV-1 manipulates host machineries during viral infection can facilitate the identification of host targets for antiviral drugs or gene therapy. The cellular protein Naf1 (HIV-1 Nef-associated factor 1) is a CRM1-dependent nucleo-cytoplasmic shuttling protein, and has been identified to regulate multiple receptor-mediated signal pathways in inflammation. The cytoplasm-located Naf1 can inhibit NF-κB activation through binding to A20, and the loss of Naf1 controlled NF-κB activation is associated with multiple autoimmune diseases. However, the effect of Naf1 on HIV-1 mRNA expression has not been characterized. In this study we found that the nucleus-located Naf1 could promote nuclear export of unspliced HIV-1 gag mRNA. We demonstrated that the association between Naf1 and CRM1 was required for this function as the inhibition or knockdown of CRM1 expression significantly impaired Naf1-promoted HIV-1 production. The mutation of Naf1 nuclear export signals (NESs) that account for CRM1 recruitment for nuclear export decreased Naf1 function. Additionally, the mutation of the nuclear localization signal (NLS) of Naf1 diminished its ability to promote HIV-1 production, demonstrating that the shuttling property of Naf1 is required for this function. Our results reveal a novel role of Naf1 in enhancing HIV-1 production, and provide a potential therapeutic target for controlling HIV-1 infection. PMID:26733199

  12. An N-Terminal ER Export Signal Facilitates the Plasma Membrane Targeting of HCN1 Channels in Photoreceptors

    PubMed Central

    Pan, Yuan; Laird, Joseph G.; Yamaguchi, David M.; Baker, Sheila A.

    2015-01-01

    Purpose. Hyperpolarization-activated cyclic nucleotide-gated 1 (HCN1) channels are widely expressed in the retina. In photoreceptors, the hyperpolarization-activated current (Ih) carried by HCN1 is important for shaping the light response. It has been shown in multiple systems that trafficking HCN1 channels to specific compartments is key to their function. The localization of HCN1 in photoreceptors is concentrated in the plasma membrane of the inner segment (IS). The mechanisms controlling this localization are not understood. We previously identified a di-arginine endoplasmic reticulum (ER) retention motif that negatively regulates the surface targeting of HCN1. In this study, we sought to identify a forward trafficking signal that could counter the function of the ER retention signal. Methods. We studied trafficking of HCN1 and several mutants by imaging their subcellular localization in transgenic X. laevis photoreceptors. Velocity sedimentation was used to assay the assembly state of HCN1 channels. Results. We found the HCN1 N-terminus can redirect a membrane reporter from outer segments (OS) to the plasma membrane of the IS. The sequence necessary for this behavior was mapped to a 20 amino acid region containing a leucine-based ER export motif. The ER export signal is necessary for forward trafficking but not channel oligomerization. Moreover, this ER export signal alone counteracted the di-arginine ER retention signal. Conclusions. We identified an ER export signal in HCN1 that functions with the ER retention signal to maintain equilibrium of HCN1 between the endomembrane system and the plasma membrane. PMID:26030105

  13. Nuclear export inhibitors avert progression in preclinical models of inflammatory demyelination.

    PubMed

    Haines, Jeffery D; Herbin, Olivier; de la Hera, Belén; Vidaurre, Oscar G; Moy, Gregory A; Sun, Qingxiang; Fung, Ho Yee Joyce; Albrecht, Stefanie; Alexandropoulos, Konstantina; McCauley, Dilara; Chook, Yuh Min; Kuhlmann, Tanja; Kidd, Grahame J; Shacham, Sharon; Casaccia, Patrizia

    2015-04-01

    Axonal damage has been associated with aberrant protein trafficking. We examined a newly characterized class of compounds that target nucleo-cytoplasmic shuttling by binding to the catalytic groove of the nuclear export protein XPO1 (also known as CRM1, chromosome region maintenance protein 1). Oral administration of reversible CRM1 inhibitors in preclinical murine models of demyelination significantly attenuated disease progression, even when started after the onset of paralysis. Clinical efficacy was associated with decreased proliferation of immune cells, characterized by nuclear accumulation of cell cycle inhibitors, and preservation of cytoskeletal integrity even in demyelinated axons. Neuroprotection was not limited to models of demyelination, but was also observed in another mouse model of axonal damage (that is, kainic acid injection) and detected in cultured neurons after knockdown of Xpo1, the gene encoding CRM1. A proteomic screen for target molecules revealed that CRM1 inhibitors in neurons prevented nuclear export of molecules associated with axonal damage while retaining transcription factors modulating neuroprotection. PMID:25706475

  14. Seismic signals from asymmetric underground nuclear explosions

    SciTech Connect

    Davis, C.G.

    1993-09-01

    The methods discussed to estimate the effect on the seismic signals from asymmetric underground nuclear explosions, depends on the use of large-scale numerical codes and high-speed computers. The use of a two-dimensional (2D) radiation diffusion coupled Eulerian hydrodynamic code (SOIL) for the early time phenomenology is discussed. The results from this calculation are then coupled into a 2D Lagrangian code that treats the strength of the materials and the effects of fractures, ground reflections and spells. The final step in the simulation is the use of a seismic code (which uses the representation theory) to develop the actual far field seismic signals. These calculations were run on the CRAY YMP computers at the Los Alamos National Laboratory.

  15. Research Resources for Nuclear Receptor Signaling Pathways.

    PubMed

    McKenna, Neil J

    2016-08-01

    Nuclear receptor (NR) signaling pathways impact cellular function in a broad variety of tissues in both normal physiology and disease states. The complex tissue-specific biology of these pathways is an enduring impediment to the development of clinical NR small-molecule modulators that combine therapeutically desirable effects in specific target tissues with suppression of off-target effects in other tissues. Supporting the important primary research in this area is a variety of web-based resources that assist researchers in gaining an appreciation of the molecular determinants of the pharmacology of a NR pathway in a given tissue. In this study, selected representative examples of these tools are reviewed, along with discussions on how current and future generations of tools might optimally adapt to the future of NR signaling research. PMID:27216565

  16. UAP56- a key player with surprisingly diverse roles in pre-mRNA splicing and nuclear export.

    PubMed

    Shen, Haihong

    2009-04-30

    Transcripts contain introns that are usually removed from premessenger RNA (MRNA) in the process of pre-mRNA splicing. After splicing, the mature RNA is exported from the nucleus to the cytoplasm. The splicing and export processes are coupled. UAP56 protein, which is ubiquitously present in organisms from yeasts to humans, is a DExD/H-box family RNA helicase that is an essential splicing factor with various functions in the prespliceosome assembly and mature spliceosome assembly. Collective evidence indicates that UAP56 has an essential role in mRNA nuclear export. This mini-review summarizes recent evidence for the role of UAP56 in pre-mRNA splicing and nuclear export. PMID:19403039

  17. Heat shock disassembles the nucleolus and inhibits nuclear protein import and poly(A)+ RNA export.

    PubMed Central

    Liu, Y; Liang, S; Tartakoff, A M

    1996-01-01

    Heat shock causes major positive and negative changes in gene expression, drastically alters the appearance of the nucleolus and inhibits rRNA synthesis. We here show that it causes many yeast nucleolar proteins, including the fibrillarin homolog Nop1p, to relocate to the cytoplasm. Relocation depends on several proteins implicated in mRNA transport (Mtrps) and is reversible. Two observations indicate, surprisingly, that disassembly results from a reduction in Ssa protein (Hsp70) levels: (i) selective depletion of Ssa1p leads to disassembly of the nucleolus; (ii) preincubation at 37 degrees C protects the nucleolus against disassembly by heat shock, unless expression of Ssa proteins is specifically inhibited. We observed that heat shock or reduction of Ssa1p levels inhibits protein import into the nucleus and therefore we propose that inhibition of import leads to disassembly of the nucleolus. These observations provide a simple explanation of the effects of heat shock on the anatomy of the nucleolus and rRNA transcription. They also extend understanding of the path of nuclear export. Since a number of nucleoplasmic proteins also relocate upon heat shock, these observations can provide a general mechanism for regulation of gene expression. Relocation of the hnRNP-like protein Mtr13p (= Npl3p, Nop3p), explains the heat shock sensitivity of export of average poly(A)+ RNA. Strikingly, Hsp mRNA export appears not to be affected. Images PMID:8978700

  18. 10 CFR 73.73 - Requirement for advance notice and protection of export shipments of special nuclear material of...

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 2 2010-01-01 2010-01-01 false Requirement for advance notice and protection of export shipments of special nuclear material of low strategic significance. 73.73 Section 73.73 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) PHYSICAL PROTECTION OF PLANTS AND MATERIALS Records and Reports § 73.73 Requirement for advance notice...

  19. 10 CFR 73.73 - Requirement for advance notice and protection of export shipments of special nuclear material of...

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 10 Energy 2 2014-01-01 2014-01-01 false Requirement for advance notice and protection of export shipments of special nuclear material of low strategic significance. 73.73 Section 73.73 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) PHYSICAL PROTECTION OF PLANTS AND MATERIALS Records and Reports §...

  20. Dominant-negative mutants of importin-beta block multiple pathways of import and export through the nuclear pore complex.

    PubMed Central

    Kutay, U; Izaurralde, E; Bischoff, F R; Mattaj, I W; Görlich, D

    1997-01-01

    Nuclear protein import proceeds through the nuclear pore complex (NPC). Importin-beta mediates translocation via direct interaction with NPC components and carries importin-alpha with the NLS substrate from the cytoplasm into the nucleus. The import reaction is terminated by the direct binding of nuclear RanGTP to importin-beta which dissociates the importin heterodimer. Here, we analyse the sites of interaction on importin-beta for its multiple partners. Ran and importin-alpha respectively require residues 1-364 and 331-876 of importin-beta for binding. Thus, RanGTP-mediated release of importin-alpha from importin-beta is likely to be an active displacement rather than due to simple competition between Ran and importin-alpha for a common binding site. Importin-beta has at least two non-overlapping sites of interaction with the NPC, which could potentially be used sequentially during translocation. Our data also suggest that termination of import involves a transient release of importin-beta from the NPC. Importin-beta fragments which bind to the NPC, but not to Ran, resist this release mechanism. As would be predicted from this, these importin-beta mutants are very efficient inhibitors of NLS-dependent protein import. Surprisingly, however, they also inhibit M9 signal-mediated nuclear import as well as nuclear export of mRNA, U snRNA, and the NES-containing Rev protein. This suggests that mediators of these various transport events share binding sites on the NPC and/or that mechanisms exist to coordinate translocation through the NPC via different nucleocytoplasmic transport pathways. PMID:9135132

  1. Characterization of the ptr5{sup +} gene involved in nuclear mRNA export in fission yeast

    SciTech Connect

    Watanabe, Nobuyoshi; Ikeda, Terumasa; Mizuki, Fumitaka; Tani, Tokio

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer We cloned the ptr5{sup +} gene involved in nuclear mRNA export in fission yeast. Black-Right-Pointing-Pointer The ptr5{sup +} gene was found to encode nucleoporin 85 (Nup85). Black-Right-Pointing-Pointer Seh1p and Mlo3p are multi-copy suppressors for the ptr5 mutation. Black-Right-Pointing-Pointer Ptr5p/Nup85p functions in nuclear mRNA export through the mRNA export factor Rae1p. Black-Right-Pointing-Pointer Ptr5p/Nup85p interacts genetically with pre-mRNA splicing factors. -- Abstract: To analyze the mechanisms of mRNA export from the nucleus to the cytoplasm, we have isolated eleven mutants, ptr [poly(A){sup +} RNA transport] 1 to 11, which accumulate poly(A){sup +} RNA in the nucleus at a nonpermissive temperature in Schizosaccharomyces pombe. Of those, the ptr5-1 mutant shows dots- or a ring-like accumulation of poly(A){sup +} RNA at the nuclear periphery after shifting to the nonpermissive temperature. We cloned the ptr5{sup +} gene and found that it encodes a component of the nuclear pore complex (NPC), nucleoporin 85 (Nup85). The ptr5-1 mutant shows no defects in protein transport, suggesting the specific involvement of Ptr5p/Nup85p in nuclear mRNA export in S. pombe. We identified Seh1p, a nucleoporin interacting with Nup85p, an mRNA-binding protein Mlo3p, and Sac3p, a component of the TREX-2 complex involved in coupling of nuclear mRNA export with transcription, as multi-copy suppressors for the ptr5-1 mutation. In addition, we found that the ptr5-1 mutation is synthetically lethal with a mutation of the mRNA export factor Rae1p, and that the double mutant exaggerates defective nuclear mRNA export, suggesting that Ptr5p/Nup85p is involved in nuclear mRNA export through Rae1p. Interestingly, the ptr5-1 mutation also showed synthetic effects with several prp pre-mRNA splicing mutations, suggesting a functional linkage between the NPCs and the splicing apparatus in the yeast nucleus.

  2. Characterization of the nuclear import signal of herpes simplex virus 1 UL31.

    PubMed

    Cai, Mingsheng; Chen, Daixiong; Zeng, Zhancheng; Yang, Hang; Jiang, Si; Li, Xiaowei; Mai, Jingying; Peng, Tao; Li, Meili

    2016-09-01

    The herpes simplex virus 1 (HSV-1) UL31 protein is a multifunctional nucleoprotein that is important for viral infection; however, little is known concerning its subcellular localization signal. Here, by transfection with a series of HSV-1 UL31 deletion mutants fused to enhanced yellow fluorescent protein (EYFP), a bipartite nuclear localization signal (NLS) was identified and mapped to amino acids (aa) 1 to 27 (MYDTDPHRRGSRPGPYHGKERRRSRSS). Additionally, fluorescence results showed that the predicted nuclear export signal (NES) might be nonfunctional, and the functional NES of UL31 might require a specific conformation. Taken together, these results would provide significant information for the study of the biological function of UL31 during HSV-1 infection. PMID:27276975

  3. High-resolution three-dimensional mapping of mRNA export through the nuclear pore

    PubMed Central

    Ma, Jiong; Liu, Zhen; Michelotti, Nicole; Pitchiaya, Sethuramasundaram; Veerapaneni, Ram; Androsavich, John R.; Walter, Nils G.; Yang, Weidong

    2013-01-01

    The flow of genetic information is regulated by selective nucleocytoplasmic transport of messenger RNA:protein complexes (mRNPs) through the nuclear pore complexes (NPCs) of eukaryotic cells. However, the three-dimensional pathway taken by mRNPs as they transit through the NPC, and the kinetics and selectivity of transport, remain obscure. Here we employ single-molecule fluorescence microscopy with an unprecedented spatiotemporal accuracy of 8 nm and 2 ms to provide new insights into the mechanism of nuclear mRNP export in live human cells. We find that mRNPs exiting the nucleus are decelerated and selected at the centre of the NPC, and adopt a fast-slow-fast diffusion pattern during their brief, ~12 ms interaction with the NPC. A 3D reconstruction of the export route indicates that mRNPs primarily interact with the periphery on the nucleoplasmic side and in the center of the NPC, without entering the central axial conduit utilized for passive diffusion of small molecules, and eventually dissociate on the cytoplasmic side. PMID:24008311

  4. Mapping regions in Ste5 that support Msn5-dependent and -independent nuclear export.

    PubMed

    Hu, Zhenhua; Wang, Yunmei; Yu, Lu; Mahanty, Sanjoy K; Mendoza, Natalia; Elion, Elaine A

    2016-04-01

    Careful control of the available pool of the MAPK scaffold Ste5 is important for mating-pathway activation and the prevention of inappropriate mating differentiation in haploid Saccharomyces cerevisiae. Ste5 shuttles constitutively through the nucleus, where it is degraded by a ubiquitin-dependent mechanism triggered by G1 CDK phosphorylation. Here we narrow-down regions of Ste5 that mediate nuclear export. Four regions in Ste5 relocalize SV40-TAgNLS-GFP-GFP from nucleus to cytoplasm. One region is N-terminal, dependent on exportin Msn5/Ste21/Kap142, and interacts with Msn5 in 2 hybrid assays independently of mating pheromone, Fus3, Kss1, Ptc1, the NLS/PM, and RING-H2. A second region overlaps the PH domain and Ste11 binding site and 2 others are on the vWA domain and include residues essential for MAPK activation. We find no evidence for dependence on Crm1/Xpo1, despite numerous potential nuclear export sequences (NESs) detected by LocNES and NetNES1.1 predictors. Thus, Msn5 (homolog of human Exportin-5) and one or more exportins or adaptor molecules besides Crm1/Xpo1 may regulate Ste5 through multiple recognition sites. PMID:26824509

  5. PLTP is present in the nucleus, and its nuclear export is CRM1-dependent

    PubMed Central

    Vuletic, Simona; Dong, Weijiang; Wolfbauer, Gertrud; Day, Joseph R.; Albers, John J.

    2009-01-01

    Phospholipid transfer protein (PLTP), one of the key lipid transfer proteins in plasma and cerebrospinal fluid, is nearly ubiquitously expressed in cells and tissues. Functions of secreted PLTP have been extensively studied. However, very little is known about potential intracellular PLTP functions. In the current study, we provide evidence for PLTP localization in the nucleus of cells that constitutively express PLTP (human neuroblastoma cells, SK-N-SH; and human cortical neurons, HCN2) and in cells transfected with human PLTP (Chinese hamster ovary and baby hamster kidney cells). Furthermore, we have shown that incubation of these cells with leptomycin B (LMB), a specific inhibitor of nuclear export mediated by chromosome region maintenance 1 (CRM1), leads to intranuclear accumulation of PLTP, suggesting that PLTP nuclear export is CRM1-dependent. We also provide evidence for entry of secreted PLTP into the cell and its translocation to the nucleus, and show that intranuclear PLTP is active in phospholipid transfer. These findings suggest that PLTP is involved in novel intracellular functions. PMID:19321130

  6. Ty1 Gag enhances the stability and nuclear export of Ty1 mRNA

    PubMed Central

    Checkley, Mary Ann; Mitchell, Jessica A.; Eizenstat, Linda D.; Lockett, Stephen J.; Garfinkel, David J.

    2012-01-01

    Retrotransposon and retroviral RNA delivery to particle assembly sites is essential for their replication. mRNA and Gag from the Ty1 retrotransposon colocalize in cytoplasmic foci, which are required for transposition and may be sites for virus-like particle (VLP) assembly. To determine which Ty1 components are required to form mRNA/Gag foci, localization studies were performed in a Ty1-less strain expressing galactose-inducible Ty1 plasmids (pGTy1) containing mutations in GAG or POL. Ty1 mRNA/Gag foci remained unaltered in mutants defective in Ty1 protease or deleted for POL. However, Ty1 mRNA containing a frameshift mutation (Ty1fs) that prevents the synthesis of all proteins accumulated in the nucleus. Ty1fs RNA showed a decrease in stability that was mediated by the cytoplasmic exosome, nonsense mediated decay, and the processing-body. Localization of Ty1fs RNA remained unchanged in an nmd2Δ mutant. When Gag and Ty1fs mRNA were expressed independently, Gag provided in trans increased Ty1fs RNA level and restored localization of Ty1fs RNA in cytoplasmic foci. Endogenously expressed Gag also localized to the nuclear periphery independent of RNA export. These results suggest that Gag is required for Ty1 mRNA stability, efficient nuclear export, and localization into cytoplasmic foci. PMID:22998189

  7. Evidence that Armadillo transduces wingless by mediating nuclear export or cytosolic activation of Pangolin.

    PubMed

    Chan, Siu-Kwong; Struhl, Gary

    2002-10-18

    Secreted proteins of the Wnt family have profound organizing roles during animal development and are transduced via the activities of the Frizzled (Fz) class of transmembrane receptors and the TCF/LEF/Pangolin class of transcription factors. beta-catenins, including Drosophila Armadillo (Arm), link activation of Fz at the cell surface to transcriptional regulation by TCF in the nucleus. The consensus view is that Wnt signaling induces beta-catenin to enter the nucleus and combine with TCF to form a transcription factor complex in which TCF binds DNA and the C-terminal domain of beta-catenin activates transcription. Here, we present findings, which challenge this view and suggest instead that beta-catenin may transduce Wnt signals by exporting TCF from the nucleus or activating it in the cytoplasm. PMID:12408870

  8. Acetylation regulates subcellular localization of the Wnt signaling nuclear effector POP-1

    PubMed Central

    Gay, Frédérique; Calvo, Dominica; Lo, Miao-Chia; Ceron, Julian; Maduro, Morris; Lin, Rueyling; Shi, Yang

    2003-01-01

    Lymphoid enhancer factor/T-cell factor (LEF/TCF) are transcription factors that mediate the Wnt signaling pathway, and have crucial roles during embryonic development in various organisms. Here we report that acetylation enhances nuclear retention of POP-1, the Caenorhabditis elegans LEF/TCF homolog, through increasing nuclear import and blocking nuclear export. We identify three lysines that are acetylated in vivo, and demonstrate their essential requirement for proper nuclear localization and biological activity of POP-1 during C. elegans embryogenesis. The conservation of these lysines among other LEF/TCF family members suggests that acetylation may be an important, evolutionarily conserved mechanism regulating subcellular distribution of LEF/TCF factors. PMID:12651889

  9. Nuclear RhoA signaling regulates MRTF-dependent SMC-specific transcription

    PubMed Central

    Staus, Dean P.; Weise-Cross, Laura; Mangum, Kevin D.; Medlin, Matt D.; Mangiante, Lee; Taylor, Joan M.

    2014-01-01

    We have previously shown that RhoA-mediated actin polymerization stimulates smooth muscle cell (SMC)-specific transcription by regulating the nuclear localization of the myocardin-related transcription factors (MRTFs). On the basis of the recent demonstration that nuclear G-actin regulates MRTF nuclear export and observations from our laboratory and others that the RhoA effector, mDia2, shuttles between the nucleus and cytoplasm, we investigated whether nuclear RhoA signaling plays a role in regulating MRTF activity. We identified sequences that control mDia2 nuclear-cytoplasmic shuttling and used mDia2 variants to demonstrate that the ability of mDia2 to fully stimulate MRTF nuclear accumulation and SMC-specific gene transcription was dependent on its localization to the nucleus. To test whether RhoA signaling promotes nuclear actin polymerization, we established a fluorescence recovery after photobleaching (FRAP)-based assay to measure green fluorescent protein-actin diffusion in the nuclear compartment. Nuclear actin FRAP was delayed in cells expressing nuclear-targeted constitutively active mDia1 and mDia2 variants and in cells treated with the polymerization inducer, jasplakinolide. In contrast, FRAP was enhanced in cells expressing a nuclear-targeted variant of mDia that inhibits both mDia1 and mDia2. Treatment of 10T1/2 cells with sphingosine 1-phosphate induced RhoA activity in the nucleus and forced nuclear localization of RhoA or the Rho-specific guanine nucleotide exchange factor (GEF), leukemia-associated RhoGEF, enhanced the ability of these proteins to stimulate MRTF activity. Taken together, these data support the emerging idea that RhoA-dependent nuclear actin polymerization has important effects on transcription and nuclear structure. PMID:24906914

  10. A Common Signal Patch Drives AP-1 Protein-dependent Golgi Export of Inwardly Rectifying Potassium Channels.

    PubMed

    Li, Xiangming; Ortega, Bernardo; Kim, Boyoung; Welling, Paul A

    2016-07-15

    Nearly all members of the inwardly rectifying potassium (Kir) channel family share a cytoplasmic domain structure that serves as an unusual AP-1 clathrin adaptor-dependent Golgi export signal in one Kir channel, Kir2.1 (KCNJ2), raising the question whether Kir channels share a common Golgi export mechanism. Here we explore this idea, focusing on two structurally and functionally divergent Kir family members, Kir2.3 (KCNJ4) and Kir4.1/5.1 (KCNJ10/16), which have ∼50% amino identity. We found that Golgi export of both channels is blocked upon siRNA-mediated knockdown of the AP-1 γ subunit, as predicted for the common AP-1-dependent trafficking process. A comprehensive mutagenic analysis, guided by homology mapping in atomic resolution models of Kir2.1, Kir2.3, and Kir4.1/5.1, identified a common structure that serves as a recognition site for AP-1 binding and governs Golgi export. Larger than realized from previous studies with Kir2.1, the signal is created by a patch of residues distributed at the confluence of cytoplasmic N and C termini. The signal involves a stretch of hydrophobic residues from the C-terminal region that form a hydrophobic cleft, an adjacent cluster of basic residues within the N terminus, and a potential network of salt bridges that join the N- and C-terminal poles together. Because patch formation and AP-1 binding are dependent on proper folding of the cytoplasmic domains, the signal provides a common quality control mechanism at the Golgi for Kir channels. These findings identify a new proteostatic mechanism that couples protein folding of channels to forward trafficking in the secretory pathway. PMID:27226616

  11. Leukemia-Associated Nup214 Fusion Proteins Disturb the XPO1-Mediated Nuclear-Cytoplasmic Transport Pathway and Thereby the NF-κB Signaling Pathway.

    PubMed

    Saito, Shoko; Cigdem, Sadik; Okuwaki, Mitsuru; Nagata, Kyosuke

    2016-07-01

    Nuclear-cytoplasmic transport through nuclear pore complexes is mediated by nuclear transport receptors. Previous reports have suggested that aberrant nuclear-cytoplasmic transport due to mutations or overexpression of nuclear pore complexes and nuclear transport receptors is closely linked to diseases. Nup214, a component of nuclear pore complexes, has been found as chimeric fusion proteins in leukemia. Among various Nup214 fusion proteins, SET-Nup214 and DEK-Nup214 have been shown to be engaged in tumorigenesis, but their oncogenic mechanisms remain unclear. In this study, we examined the functions of the Nup214 fusion proteins by focusing on their effects on nuclear-cytoplasmic transport. We found that SET-Nup214 and DEK-Nup214 interact with exportin-1 (XPO1)/CRM1 and nuclear RNA export factor 1 (NXF1)/TAP, which mediate leucine-rich nuclear export signal (NES)-dependent protein export and mRNA export, respectively. SET-Nup214 and DEK-Nup214 decreased the XPO1-mediated nuclear export of NES proteins such as cyclin B and proteins involved in the NF-κB signaling pathway by tethering XPO1 onto nuclear dots where Nup214 fusion proteins are localized. We also demonstrated that SET-Nup214 and DEK-Nup214 expression inhibited NF-κB-mediated transcription by abnormal tethering of the complex containing p65 and its inhibitor, IκB, in the nucleus. These results suggest that SET-Nup214 and DEK-Nup214 perturb the regulation of gene expression through alteration of the nuclear-cytoplasmic transport system. PMID:27114368

  12. Sorting of influenza A virus RNA genome segments after nuclear export

    SciTech Connect

    Takizawa, Naoki; Kumakura, Michiko; Takeuchi, Kaoru; Kobayashi, Nobuyuki; Nagata, Kyosuke

    2010-06-05

    The genome of the influenza A virus consists of eight different segments. These eight segments are thought to be sorted selectively in infected cells. However, the cellular compartment where segments are sorted is not known. We examined using temperature sensitive (ts) mutant viruses and cell fusion where segments are sorted in infected cells. Different cells were infected with different ts mutant viruses, and these cells were fused. In fused cells, genome segments are mixed only in the cytoplasm, because M1 prevents their re-import into the nucleus. We made a marker ts53 virus, which has silent mutations in given segments and determined the reassortment frequency on all segments using ts1 and marker ts53. In both co-infected and fused cells, all of marker ts53 segments and ts1 segments were incorporated into progeny virions in a random fashion. These results suggest that influenza virus genome segments are sorted after nuclear export.

  13. Lamin Mutations Accelerate Aging via Defective Export of Mitochondrial mRNAs through Nuclear Envelope Budding.

    PubMed

    Li, Yihang; Hassinger, Linda; Thomson, Travis; Ding, Baojin; Ashley, James; Hassinger, William; Budnik, Vivian

    2016-08-01

    Defective RNA metabolism and transport are implicated in aging and degeneration [1, 2], but the underlying mechanisms remain poorly understood. A prevalent feature of aging is mitochondrial deterioration [3]. Here, we link a novel mechanism for RNA export through nuclear envelope (NE) budding [4, 5] that requires A-type lamin, an inner nuclear membrane-associated protein, to accelerated aging observed in Drosophila LaminC (LamC) mutations. These LamC mutations were modeled after A-lamin (LMNA) mutations causing progeroid syndromes (PSs) in humans. We identified mitochondrial assembly regulatory factor (Marf), a mitochondrial fusion factor (mitofusin), as well as other transcripts required for mitochondrial integrity and function, in a screen for RNAs that exit the nucleus through NE budding. PS-modeled LamC mutations induced premature aging in adult flight muscles, including decreased levels of specific mitochondrial protein transcripts (RNA) and progressive mitochondrial degradation. PS-modeled LamC mutations also induced the accelerated appearance of other phenotypes associated with aging, including a progressive accumulation of polyubiquitin aggregates [6, 7] and myofibril disorganization [8, 9]. Consistent with these observations, the mutants had progressive jumping and flight defects. Downregulating marf alone induced the above aging defects. Nevertheless, restoring marf was insufficient for rescuing the aging phenotypes in PS-modeled LamC mutations, as other mitochondrial RNAs are affected by inhibition of NE budding. Analysis of NE budding in dominant and recessive PS-modeled LamC mutations suggests a mechanism by which abnormal lamina organization prevents the egress of these RNAs via NE budding. These studies connect defects in RNA export through NE budding to progressive loss of mitochondrial integrity and premature aging. PMID:27451905

  14. A novel function for the 90 kDa heat-shock protein (Hsp90): facilitating nuclear export of 60 S ribosomal subunits.

    PubMed Central

    Schlatter, Harald; Langer, Thomas; Rosmus, Susann; Onneken, Marie-Luise; Fasold, Hugo

    2002-01-01

    Ribosomal subunits are assembled in the nucleus, and mature 40 S and 60 S subunits are exported stoichiometrically into the cytoplasm. The nuclear export of ribosomal subunits is a unidirectional, saturable and energy-dependent process. An in vitro assay for the nuclear export of 60 S ribosomal subunits involves the use of resealed nuclear envelopes. The export of ribosomal subunits from resealed nuclear envelopes is enhanced by cytoplasmic proteins. Here we present evidence that the export-promoting activity was due to the cytoplasmic 90 kDa heat-shock protein (Hsp90). Isolated, purified Hsp90 vastly enhanced the export of 60 S ribosomal subunits from resealed nuclear envelopes, while inhibition of Hsp90 function, either with the Hsp90-binding drug geldanamycin or with anti-Hsp90 antibodies, resulted in reduced release of 60 S ribosomal subunits. To confirm these findings under in vivo conditions, corresponding experiments were performed with Xenopus oocytes using microinjection techniques; the results obtained confirmed the findings obtained with resealed nuclear envelopes. These findings suggest that Hsp90 facilitates the nuclear export of 60 S ribosomal subunits, probably by chaperoning protein interactions during the export process. PMID:11879195

  15. Competitive Nuclear Export of Cyclin D1 and Hic-5 Regulates Anchorage Dependence of Cell Growth and Survival

    PubMed Central

    Mori, Kazunori; Hirao, Etsuko; Toya, Yosuke; Oshima, Yukiko; Ishikawa, Fumihiro; Nose, Kiyoshi

    2009-01-01

    Anchorage dependence of cell growth and survival is a critical trait that distinguishes nontransformed cells from transformed cells. We demonstrate that anchorage dependence is determined by anchorage-dependent nuclear retention of cyclin D1, which is regulated by the focal adhesion protein, Hic-5, whose CRM1-dependent nuclear export counteracts that of cyclin D1. An adaptor protein, PINCH, interacts with cyclin D1 and Hic-5 and potentially serves as an interface for the competition between cyclin D1 and Hic-5 for CRM1. In nonadherent cells, the nuclear export of Hic-5, which is redox-sensitive, was interrupted due to elevated production of reactive oxygen species, and cyclin D1 was exported from the nucleus. When an Hic-5 mutant that was continuously exported in a reactive oxygen species-insensitive manner was introduced into the cells, cyclin D1 was retained in the nucleus under nonadherent conditions, and a significant population of cells escaped from growth arrest or apoptosis. Interestingly, activated ras achieved predominant cyclin D1 nuclear localization and thus, growth in nonadherent cells. We report a failsafe system for anchorage dependence of cell growth and survival. PMID:18946086

  16. Nuclear Receptor Signaling Atlas: Opening Access to the Biology of Nuclear Receptor Signaling Pathways.

    PubMed

    Becnel, Lauren B; Darlington, Yolanda F; Ochsner, Scott A; Easton-Marks, Jeremy R; Watkins, Christopher M; McOwiti, Apollo; Kankanamge, Wasula H; Wise, Michael W; DeHart, Michael; Margolis, Ronald N; McKenna, Neil J

    2015-01-01

    Signaling pathways involving nuclear receptors (NRs), their ligands and coregulators, regulate tissue-specific transcriptomes in diverse processes, including development, metabolism, reproduction, the immune response and neuronal function, as well as in their associated pathologies. The Nuclear Receptor Signaling Atlas (NURSA) is a Consortium focused around a Hub website (www.nursa.org) that annotates and integrates diverse 'omics datasets originating from the published literature and NURSA-funded Data Source Projects (NDSPs). These datasets are then exposed to the scientific community on an Open Access basis through user-friendly data browsing and search interfaces. Here, we describe the redesign of the Hub, version 3.0, to deploy "Web 2.0" technologies and add richer, more diverse content. The Molecule Pages, which aggregate information relevant to NR signaling pathways from myriad external databases, have been enhanced to include resources for basic scientists, such as post-translational modification sites and targeting miRNAs, and for clinicians, such as clinical trials. A portal to NURSA's Open Access, PubMed-indexed journal Nuclear Receptor Signaling has been added to facilitate manuscript submissions. Datasets and information on reagents generated by NDSPs are available, as is information concerning periodic new NDSP funding solicitations. Finally, the new website integrates the Transcriptomine analysis tool, which allows for mining of millions of richly annotated public transcriptomic data points in the field, providing an environment for dataset re-use and citation, bench data validation and hypothesis generation. We anticipate that this new release of the NURSA database will have tangible, long term benefits for both basic and clinical research in this field. PMID:26325041

  17. Nuclear Receptor Signaling Atlas: Opening Access to the Biology of Nuclear Receptor Signaling Pathways

    PubMed Central

    Becnel, Lauren B.; Darlington, Yolanda F.; Ochsner, Scott A.; Easton-Marks, Jeremy R.; Watkins, Christopher M.; McOwiti, Apollo; Kankanamge, Wasula H.; Wise, Michael W.; DeHart, Michael; Margolis, Ronald N.; McKenna, Neil J.

    2015-01-01

    Signaling pathways involving nuclear receptors (NRs), their ligands and coregulators, regulate tissue-specific transcriptomes in diverse processes, including development, metabolism, reproduction, the immune response and neuronal function, as well as in their associated pathologies. The Nuclear Receptor Signaling Atlas (NURSA) is a Consortium focused around a Hub website (www.nursa.org) that annotates and integrates diverse ‘omics datasets originating from the published literature and NURSA-funded Data Source Projects (NDSPs). These datasets are then exposed to the scientific community on an Open Access basis through user-friendly data browsing and search interfaces. Here, we describe the redesign of the Hub, version 3.0, to deploy “Web 2.0” technologies and add richer, more diverse content. The Molecule Pages, which aggregate information relevant to NR signaling pathways from myriad external databases, have been enhanced to include resources for basic scientists, such as post-translational modification sites and targeting miRNAs, and for clinicians, such as clinical trials. A portal to NURSA’s Open Access, PubMed-indexed journal Nuclear Receptor Signaling has been added to facilitate manuscript submissions. Datasets and information on reagents generated by NDSPs are available, as is information concerning periodic new NDSP funding solicitations. Finally, the new website integrates the Transcriptomine analysis tool, which allows for mining of millions of richly annotated public transcriptomic data points in the field, providing an environment for dataset re-use and citation, bench data validation and hypothesis generation. We anticipate that this new release of the NURSA database will have tangible, long term benefits for both basic and clinical research in this field. PMID:26325041

  18. Disorders of nuclear-mitochondrial intergenomic signaling.

    PubMed

    Spinazzola, Antonella; Zeviani, Massimo

    2005-07-18

    Depletion and multiple deletions of mitochondrial DNA (mtDNA) have been associated with a number of autosomal disorders classified as defects of nuclear-mitochondrial intergenomic signaling. The mendelian forms of progressive external ophthalmoplegia (PEO) are clinically and genetically heterogeneous disorders characterized by the accumulation of multiple deletions of mtDNA in postmitotic patient's tissues. Most of the autosomal dominant PEO (adPEO) families carry heterozygous mutations in either one of three genes: ANT1, Twinkle, and POLG1. Mutations in POLG1 can also cause autosomal recessive PEO (arPEO) and apparently sporadic cases. In addition, recessive POLG1 mutations are responsible for sensory-atactic neuropathy, dysarthria and ophthalmoplegia (SANDO), juvenile spino-cerebellar ataxia-epilepsy syndrome (SCAE) and Alpers-Huttenlocher hepatopathic poliodystrophy. Mutations in thymidine phosphorylase gene (TP) are linked to mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), an autosomal recessive disorder in which PEO is associated with gastrointestinal dysmotility and leukodystrophy. Finally, mitochondrial DNA depletion syndromes (MDS), defined by tissue-reduction in mtDNA copy number, have been linked to mutations in two genes involved in deoxyribonucleotide (dNTP) metabolism: thymidine kinase 2 (TK2) and deoxyguanosine kinase (DGUOK). PMID:15921863

  19. The Karyopherin Kap142p/Msn5p Mediates Nuclear Import and Nuclear Export of Different Cargo Proteins

    PubMed Central

    Yoshida, Kimihisa; Blobel, Günter

    2001-01-01

    We have identified a novel pathway for protein import into the nucleus. Although the product of Saccharomyces cerevisiae gene MSN5 was previously shown to function as a karyopherin (Kap) for nuclear export of various proteins, we discovered a nuclear import pathway mediated by Msn5p (also referred to as Kap142p). We have purified from yeast cytosol a complex containing Kap142p and the trimeric replication protein A (RPA), which is required for multiple aspects of DNA metabolism, including DNA replication, DNA repair, and recombination. In wild-type cells, RPA was localized primarily to the nucleus but, in a KAP142 deletion strain, RPA was mislocalized to the cytoplasm and the strain was highly sensitive to bleomycin (BLM). BLM causes DNA double-strand breaks and, in S. cerevisiae, the DNA damage is repaired predominantly by RPA-dependent homologous recombination. Therefore, our results indicate that in wild-type cells a critical portion of RPA was imported into the nucleus by Kap142p. Like several other import-related Kap–substrate complexes, the endogenous RPA–Kap142p complex was dissociated by RanGTP, but not by RanGDP. All three RPA genes are essential for viability, whereas KAP142 is not. Perhaps explaining this disparity, we observed an interaction between RPA and Kap95p in a strain lacking Kap142p. This interaction could provide a mechanism for import of RPA into the nucleus and cell viability in the absence of Kap142p. Together with published results (Kaffman, A., N.M. Rank, E.M. O'Neill, L.S. Huang, and E.K. O'Shea. 1998. Nature. 396:482–486; Blondel, M., P.M. Alepuz, L.S. Huang, S. Shaham, G. Ammerer, and M. Peter. 1999. Genes Dev. 13:2284–2300; DeVit, M.J., and M. Johnston. 1999. Curr. Biol. 9:1231–1241; Mahanty, S.K., Y. Wang, F.W. Farley, and E.A. Elion. 1999. Cell. 98:501–512) our data indicate that the karyopherin Kap142p is able to mediate nuclear import of one set of proteins and nuclear export of a different set of proteins. PMID

  20. Phosphorylation-dependent regulation of cyclin D1 nuclear export and cyclin D1–dependent cellular transformation

    PubMed Central

    Alt, Jodi R.; Cleveland, John L.; Hannink, Mark; Diehl, J. Alan

    2000-01-01

    GSK-3β-dependent phosphorylation of cyclin D1 at Thr-286 promotes the nuclear-to-cytoplasmic redistribution of cyclin D1 during S phase of the cell cycle, but how phosphorylation regulates redistribution has not been resolved. For example, phosphorylation of nuclear cyclin D1 could increase its rate of nuclear export relative to nuclear import; alternatively, phosphorylation of cytoplasmic cyclin D1 by GSK-3β could inhibit nuclear import. Here, we report that GSK-3β-dependent phosphorylation promotes cyclin D1 nuclear export by facilitating the association of cyclin D1 with the nuclear exportin CRM1. D1-T286A, a cyclin D1 mutant that cannot be phosphorylated by GSK-3β, remains nuclear throughout the cell cycle, a consequence of its reduced binding to CRM1. Constitutive overexpression of the nuclear cyclin D1-T286A in murine fibroblasts results in cellular transformation and promotes tumor growth in immune compromised mice. Thus, removal of cyclin D1 from the nucleus during S phase appears essential for regulated cell division. PMID:11124803

  1. Nuclear DNA damage signalling to mitochondria in ageing.

    PubMed

    Fang, Evandro Fei; Scheibye-Knudsen, Morten; Chua, Katrin F; Mattson, Mark P; Croteau, Deborah L; Bohr, Vilhelm A

    2016-05-01

    Mitochondrial dysfunction is a hallmark of ageing, and mitochondrial maintenance may lead to increased healthspan. Emerging evidence suggests a crucial role for signalling from the nucleus to mitochondria (NM signalling) in regulating mitochondrial function and ageing. An important initiator of NM signalling is nuclear DNA damage, which accumulates with age and may contribute to the development of age-associated diseases. DNA damage-dependent NM signalling constitutes a network that includes nuclear sirtuins and controls genomic stability and mitochondrial integrity. Pharmacological modulation of NM signalling is a promising novel approach for the prevention and treatment of age-associated diseases. PMID:26956196

  2. Nuclear Export as a Novel Therapeutic Target: The CRM1 Connection.

    PubMed

    Lu, Chuanwen; Figueroa, Jose A; Liu, Zhongwei; Konala, Venu; Aulakh, Amardeep; Verma, Rashmi; Cobos, Everardo; Chiriva-Internati, Maurizio; Gao, Weimin

    2015-01-01

    The integrity of eukaryotic cellular function depends on molecular and biochemical compartmentalization. The transport of macromolecules between compartments requires specific and energydriven mechanisms. It occurs through a class of transport proteins known as karyopherins, which are divided in three different groups (exportins, importins, and transportins). The ubiquitous exportin Chromosome Region Maintenance 1 (CRM1) is involved in the transport of many proteins and RNA molecules from nucleus to cytoplasm. We have reviewed the available evidence supporting the relevance of CRM1 in the biology of several human neoplasms, its potential role in drug resistance, and its promise as a therapeutic target. Here we discuss different cancer related proteins (tumor suppressor genes, oncogenes, and enzymatic therapeutic targets), their function, and their association with CRM1, as well as agents that specifically inhibit CRM1, their mechanism of action, and their clinical relevance in certain human neoplasms. The directionality of nuclear transport and the specific molecular cargo in question are of paramount importance when examining the effects that CRM1 inhibition may have on cellular pathophysiology. The available data point out the potential role of CRM1-dependent nuclear export of regulatory proteins in the biology of certain human malignancies. Further studies should expand and clarify the importance of this mechanism in the pathobiology of human neoplasia. PMID:26324128

  3. SINE (selective inhibitor of nuclear export)--translational science in a new class of anti-cancer agents.

    PubMed

    Gerecitano, John

    2014-01-01

    Regulation of protein trafficking between the nucleus and cytoplasm represents a novel control point for antineoplastic intervention. Several proteins involved with cellular growth and survival depend on precise and timely positioning within the cell to fulfill their functions, and the nuclear membrane defines one of the most important compartmental barriers. Chromosome Region Maintenance 1, or exportin-1 (CRM1/XPO1), is involved with the export of more than 200 nuclear proteins, and has intriguingly been shown to have an increased expression in several tumor cell types. Selinexor (KPT-330) is a first-in-class selective inhibitor of nuclear export (SINE) to be developed for clinical use. Preclinical data has demonstrated antineoplastic activity of SINE compounds in many human solid and hematologic malignancies. The clinical development of Selinexor provides an excellent model for translational research. PMID:25281264

  4. Combined effects of the signal sequence and the major chaperone proteins on the export of human cytokines in Escherichia coli.

    PubMed Central

    Bergès, H; Joseph-Liauzun, E; Fayet, O

    1996-01-01

    We have studied the export of two human proteins in the course of their production in Escherichia coli. The coding sequences of the granulocyte-macrophage colony-stimulating factor and of interleukin 13 were fused to those of two synthetic signal sequences to direct the human proteins to the bacterial periplasm. We found that the total amount of protein varies with the signal peptide-cytokine combination, as does the fraction of it that is soluble in a periplasmic extract. The possibility that the major chaperone proteins such as SecB and the GroEL-GroES and DnaK-DnaJ pairs are limiting factors for the export was tested by overexpressing one or the other of these chaperones concomitantly with the heterologous protein. The GroEL-GroES chaperone pair had no effect on protein production. Overproduction of SecB or DnaK plus DnaJ resulted in a marked increase of the quantity of human proteins in the periplasmic fraction, but this increase depends on the signal peptide-heterologous protein-chaperone association involved. PMID:8572712

  5. The signal sequence of exported protein-1 directs the green fluorescent protein to the parasitophorous vacuole of transfected malaria parasites.

    PubMed

    Adisa, Akinola; Rug, Melanie; Klonis, Nectarios; Foley, Michael; Cowman, Alan F; Tilley, Leann

    2003-02-21

    The malaria parasite, Plasmodium falciparum, spends part of its life cycle inside the erythrocytes of its human host. In the mature stages of intraerythrocytic growth, the parasite undertakes extensive remodeling of its adopted cellular home by exporting proteins beyond the confines of its own plasma membrane. To examine the signals involved in export of parasite proteins, we have prepared transfected parasites expressing a chimeric protein comprising the N-terminal region of the Plasmodium falciparum exported protein-1 appended to green fluorescent protein. The majority of the population of the chimeric protein appears to be correctly processed and trafficked to the parasitophorous vacuole, indicating that this is the default destination for protein secretion. Some of the protein is redirected to the parasite food vacuole and further degraded. Photobleaching studies reveal that the parasitophorous vacuole contains subcompartments that are only partially interconnected. Dual labeling with the lipid probe, BODIPY-TR-ceramide, reveals the presence of membrane-bound extensions that can bleb from the parasitophorous vacuole to produce double membrane-bound compartments. We also observed regions and extensions of the parasitophorous vacuole, where there is segregation of the lumenal chimera from the lipid components. These regions may represent sites for the sorting of proteins destined for the trafficking to sites beyond the parasitophorous vacuole membrane. PMID:12456681

  6. Cycling of the signaling protein phospholipase D through cilia requires the BBSome only for the export phase

    PubMed Central

    Brown, Jason M.; Sampaio, Julio L.; Craft, Julie M.; Shevchenko, Andrej; Evans, James E.; Witman, George B.

    2013-01-01

    The BBSome is a complex of seven proteins, including BBS4, that is cycled through cilia by intraflagellar transport (IFT). Previous work has shown that the membrane-associated signaling protein phospholipase D (PLD) accumulates abnormally in cilia of Chlamydomonas reinhardtii bbs mutants. Here we show that PLD is a component of wild-type cilia but is enriched ∼150-fold in bbs4 cilia; this accumulation occurs progressively over time and results in altered ciliary lipid composition. When wild-type BBSomes were introduced into bbs cells, PLD was rapidly removed from the mutant cilia, indicating the presence of an efficient BBSome-dependent mechanism for exporting ciliary PLD. This export requires retrograde IFT. Importantly, entry of PLD into cilia is BBSome and IFT independent. Therefore, the BBSome is required only for the export phase of a process that continuously cycles PLD through cilia. Another protein, carbonic anhydrase 6, is initially imported normally into bbs4 cilia but lost with time, suggesting that its loss is a secondary effect of BBSome deficiency. PMID:23589493

  7. An integrated mechanism of cardiomyocyte nuclear Ca2+ signaling

    PubMed Central

    Ibarra, Cristián; Vicencio, Jose Miguel; Varas-Godoy, Manuel; Jaimovich, Enrique; Rothermel, Beverly A.; Uhlén, Per; Hill, Joseph A.; Lavandero, Sergio

    2015-01-01

    In cardiomyocytes, Ca2+ plays a central role in governing both contraction and signaling events that regulate gene expression. Current evidence indicates that discrimination between these two critical functions is achieved by segregating Ca2+ within subcellular microdomains: transcription is regulated by Ca2+ release within nuclear microdomains, and excitation–contraction coupling is regulated by cytosolic Ca2+. Accordingly, a variety of agonists that control cardiomyocyte gene expression, such as endothelin-1, angiotensin-II or insulin-like growth factor-1, share the feature of triggering nuclear Ca2+ signals. However, signaling pathways coupling surface receptor activation to nuclear Ca2+ release, and the phenotypic responses to such signals, differ between agonists. According to earlier hypotheses, the selective control of nuclear Ca2+ signals by activation of plasma membrane receptors relies on the strategic localization of inositol trisphosphate receptors at the nuclear envelope. There, they mediate Ca2+ release from perinuclear Ca2+ stores upon binding of inositol trisphosphate generated in the cytosol, which diffuses into the nucleus. More recently, identification of such receptors at nuclear membranes or perinuclear sarcolemmal invaginations has uncovered novel mechanisms whereby agonists control nuclear Ca2+ release. In this review, we discuss mechanisms for the selective control of nuclear Ca2+ signals with special focus on emerging models of agonist receptor activation. PMID:24997440

  8. An integrated mechanism of cardiomyocyte nuclear Ca(2+) signaling.

    PubMed

    Ibarra, Cristián; Vicencio, Jose Miguel; Varas-Godoy, Manuel; Jaimovich, Enrique; Rothermel, Beverly A; Uhlén, Per; Hill, Joseph A; Lavandero, Sergio

    2014-10-01

    In cardiomyocytes, Ca(2+) plays a central role in governing both contraction and signaling events that regulate gene expression. Current evidence indicates that discrimination between these two critical functions is achieved by segregating Ca(2+) within subcellular microdomains: transcription is regulated by Ca(2+) release within nuclear microdomains, and excitation-contraction coupling is regulated by cytosolic Ca(2+). Accordingly, a variety of agonists that control cardiomyocyte gene expression, such as endothelin-1, angiotensin-II or insulin-like growth factor-1, share the feature of triggering nuclear Ca(2+) signals. However, signaling pathways coupling surface receptor activation to nuclear Ca(2+) release, and the phenotypic responses to such signals, differ between agonists. According to earlier hypotheses, the selective control of nuclear Ca(2+) signals by activation of plasma membrane receptors relies on the strategic localization of inositol trisphosphate receptors at the nuclear envelope. There, they mediate Ca(2+) release from perinuclear Ca(2+) stores upon binding of inositol trisphosphate generated in the cytosol, which diffuses into the nucleus. More recently, identification of such receptors at nuclear membranes or perinuclear sarcolemmal invaginations has uncovered novel mechanisms whereby agonists control nuclear Ca(2+) release. In this review, we discuss mechanisms for the selective control of nuclear Ca(2+) signals with special focus on emerging models of agonist receptor activation. PMID:24997440

  9. Fast Retrograde Signaling in Response to High Light Involves Metabolite Export, MITOGEN-ACTIVATED PROTEIN KINASE6, and AP2/ERF Transcription Factors in Arabidopsis[C][W

    PubMed Central

    Vogel, Marc Oliver; Moore, Marten; König, Katharina; Pecher, Pascal; Alsharafa, Khalid; Lee, Justin; Dietz, Karl-Josef

    2014-01-01

    Regulation of the expression of nuclear genes encoding chloroplast proteins allows for metabolic adjustment in response to changing environmental conditions. This regulation is linked to retrograde signals that transmit information on the metabolic state of the chloroplast to the nucleus. Transcripts of several APETALA2/ETHYLENE RESPONSE FACTOR transcription factors (AP2/ERF-TFs) were found to respond within 10 min after transfer of low-light-acclimated Arabidopsis thaliana plants to high light. Initiation of this transcriptional response was completed within 1 min after transfer to high light. The fast responses of four AP2/ERF genes, ERF6, RRTF1, ERF104, and ERF105, were entirely deregulated in triose phosphate/phosphate translocator (tpt) mutants. Similarly, activation of MITOGEN-ACTIVATED PROTEIN KINASE6 (MPK6) was upregulated after 1 min in the wild type but not in the tpt mutant. Based on this, together with altered transcript regulation in mpk6 and erf6 mutants, a retrograde signal transmission model is proposed starting with metabolite export through the triose phosphate/phosphate translocator with subsequent MPK6 activation leading to initiation of AP2/ERF-TF gene expression and other downstream gene targets. The results show that operational retrograde signaling in response to high light involves a metabolite-linked pathway in addition to previously described redox and hormonal pathways. PMID:24668746

  10. Dual Functions for the Schizosaccharomyces pombe Inositol Kinase Ipk1 in Nuclear mRNA Export and Polarized Cell Growth▿ †

    PubMed Central

    Sarmah, Bhaskarjyoti; Wente, Susan R.

    2009-01-01

    The inositol 1,3,4,5,6-pentakisphosphate (IP5) 2-kinase (Ipk1) catalyzes the production of inositol hexakisphosphate (IP6) in eukaryotic cells. Previous studies have shown that IP6 is required for efficient nuclear mRNA export in the budding yeast Saccharomyces cerevisiae. Here, we report the first functional analysis of ipk1+ in Schizosaccharomyces pombe. S. pombe Ipk1 (SpIpk1) is unique among Ipk1 orthologues in that it harbors a novel amino (N)-terminal domain with coiled-coil structural motifs similar to those of BAR (Bin-amphiphysin-Rvs) domain proteins. Mutants with ipk1+ deleted (ipk1Δ) had mRNA export defects as well as pleiotropic defects in polarized growth, cell morphology, endocytosis, and cell separation. The SpIpk1 catalytic carboxy-terminal domain was required to rescue these defects, and the mRNA export block was genetically linked to SpDbp5 function and, likely, IP6 production. However, the overexpression of the N-terminal domain alone also inhibited these functions in wild-type cells. This revealed a distinct noncatalytic function for the N-terminal domain. To test for connections with other inositol polyphosphates, we also analyzed whether the loss of asp1+ function, encoding an IP6 kinase downstream of Ipk1, had an effect on ipk1Δ cells. The asp1Δ mutant alone did not block mRNA export, and its cell morphology, polarized growth, and endocytosis defects were less severe than those of ipk1Δ cells. Moreover, ipk1Δ asp1Δ double mutants had altered inositol polyphosphate levels distinct from those of the ipk1Δ mutant. This suggested novel roles for asp1+ upstream of ipk1+. We propose that IP6 production is a key signaling linchpin for regulating multiple essential cellular processes. PMID:19047361

  11. A Novel Antiviral Target Structure Involved in the RNA Binding, Dimerization, and Nuclear Export Functions of the Influenza A Virus Nucleoprotein

    PubMed Central

    Yamada, Kazunori; Kondoh, Yasumitsu; Hikono, Hirokazu; Osada, Hiroyuki; Tomii, Kentaro; Saito, Takehiko; Aida, Yoko

    2015-01-01

    Developing antiviral therapies for influenza A virus (IAV) infection is an ongoing process because of the rapid rate of antigenic mutation and the emergence of drug-resistant viruses. The ideal strategy is to develop drugs that target well-conserved, functionally restricted, and unique surface structures without affecting host cell function. We recently identified the antiviral compound, RK424, by screening a library of 50,000 compounds using cell-based infection assays. RK424 showed potent antiviral activity against many different subtypes of IAV in vitro and partially protected mice from a lethal dose of A/WSN/1933 (H1N1) virus in vivo. Here, we show that RK424 inhibits viral ribonucleoprotein complex (vRNP) activity, causing the viral nucleoprotein (NP) to accumulate in the cell nucleus. In silico docking analysis revealed that RK424 bound to a small pocket in the viral NP. This pocket was surrounded by three functionally important domains: the RNA binding groove, the NP dimer interface, and nuclear export signal (NES) 3, indicating that it may be involved in the RNA binding, oligomerization, and nuclear export functions of NP. The accuracy of this binding model was confirmed in a NP-RK424 binding assay incorporating photo-cross-linked RK424 affinity beads and in a plaque assay evaluating the structure-activity relationship of RK424. Surface plasmon resonance (SPR) and pull-down assays showed that RK424 inhibited both the NP-RNA and NP-NP interactions, whereas size exclusion chromatography showed that RK424 disrupted viral RNA-induced NP oligomerization. In addition, in vitro nuclear export assays confirmed that RK424 inhibited nuclear export of NP. The amino acid residues comprising the NP pocket play a crucial role in viral replication and are highly conserved in more than 7,000 NP sequences from avian, human, and swine influenza viruses. Furthermore, we found that the NP pocket has a surface structure different from that of the pocket in host molecules. Taken

  12. A Novel Antiviral Target Structure Involved in the RNA Binding, Dimerization, and Nuclear Export Functions of the Influenza A Virus Nucleoprotein.

    PubMed

    Kakisaka, Michinori; Sasaki, Yutaka; Yamada, Kazunori; Kondoh, Yasumitsu; Hikono, Hirokazu; Osada, Hiroyuki; Tomii, Kentaro; Saito, Takehiko; Aida, Yoko

    2015-07-01

    Developing antiviral therapies for influenza A virus (IAV) infection is an ongoing process because of the rapid rate of antigenic mutation and the emergence of drug-resistant viruses. The ideal strategy is to develop drugs that target well-conserved, functionally restricted, and unique surface structures without affecting host cell function. We recently identified the antiviral compound, RK424, by screening a library of 50,000 compounds using cell-based infection assays. RK424 showed potent antiviral activity against many different subtypes of IAV in vitro and partially protected mice from a lethal dose of A/WSN/1933 (H1N1) virus in vivo. Here, we show that RK424 inhibits viral ribonucleoprotein complex (vRNP) activity, causing the viral nucleoprotein (NP) to accumulate in the cell nucleus. In silico docking analysis revealed that RK424 bound to a small pocket in the viral NP. This pocket was surrounded by three functionally important domains: the RNA binding groove, the NP dimer interface, and nuclear export signal (NES) 3, indicating that it may be involved in the RNA binding, oligomerization, and nuclear export functions of NP. The accuracy of this binding model was confirmed in a NP-RK424 binding assay incorporating photo-cross-linked RK424 affinity beads and in a plaque assay evaluating the structure-activity relationship of RK424. Surface plasmon resonance (SPR) and pull-down assays showed that RK424 inhibited both the NP-RNA and NP-NP interactions, whereas size exclusion chromatography showed that RK424 disrupted viral RNA-induced NP oligomerization. In addition, in vitro nuclear export assays confirmed that RK424 inhibited nuclear export of NP. The amino acid residues comprising the NP pocket play a crucial role in viral replication and are highly conserved in more than 7,000 NP sequences from avian, human, and swine influenza viruses. Furthermore, we found that the NP pocket has a surface structure different from that of the pocket in host molecules. Taken

  13. Characterisation of Cdc25B localisation and nuclear export during the cell cycle and in response to stress.

    PubMed

    Lindqvist, Arne; Källström, Helena; Karlsson Rosenthal, Christina

    2004-10-01

    Cdc25 phosphatases are essential regulators of the cell cycle. In mammalian cells, the Cdc25B isoform activates cyclin A- and cyclin B1-containing complexes and is necessary for entry into mitosis. In this report, we characterise the subcellular localisation of Cdc25B by immunofluorescence in combination with RNA interference to identify specific antibody staining. We find that endogenous Cdc25B is mainly nuclear, but a fraction resides in the cytoplasm during the G2 phase of the cell cycle. Cdc25B starts to appear in S-phase cells and accumulates until prophase, after which the protein disappears. We characterise a nuclear export sequence in the N-terminus of Cdc25B (amino acids 54-67) that, when mutated, greatly reduces the ability of Cdc25B to shuttle in a fluorescence loss in photobleaching assay. Mutation of the nuclear export sequence makes Cdc25B less efficient in inducing mitosis, suggesting that an important mitotic function of Cdc25B occurs in the cytoplasm. Furthermore, we find that when cells are exposed to cycloheximide or ultraviolet irradiation, Cdc25B partially translocates to the cytoplasm. The dependence of this translocation event on a functional nuclear export sequence, an intact serine 323 residue (a 14-3-3 binding site) and p38 mitogen-activated protein kinase activity indicates that the p38 pathway regulates Cdc25B localisation in different situations of cellular stress. PMID:15456846

  14. Decreased activity and enhanced nuclear export of CCAAT-enhancer-binding protein beta during inhibition of adipogenesis by ceramide.

    PubMed Central

    Sprott, Kam M; Chumley, Michael J; Hanson, Janean M; Dobrowsky, Rick T

    2002-01-01

    To identify novel molecular mechanisms by which ceramide regulates cell differentiation, we examined its effect on adipogenesis of 3T3-L1 preadipocytes. Hormonal stimulation of 3T3-L1 preadipocytes induced formation of triacylglycerol-laden adipocytes over 7 days; in part, via the co-ordinated action of CCAAT-enhancer-binding proteins alpha, beta and delta (C/EBP-alpha, -beta and -delta) and peroxisome-proliferator-activated receptor gamma (PPARgamma). The addition of exogenous N-acetylsphingosine (C2-ceramide) or increasing endogenous ceramide levels inhibited the expression of C/EBPalpha and PPARgamma, and blocked adipocyte development. C2-ceramide did not decrease the cellular expression of C/EBPbeta, which is required for expression of C/EBPalpha and PPARgamma, but significantly blocked its transcriptional activity from a promoter construct after 24 h. The ceramide-induced decrease in the transcriptional activity of C/EBPbeta correlated with a strong decrease in its phosphorylation, DNA-binding ability and nuclear localization at 24 h. However, ceramide did not change the nuclear level of C/EBPbeta after a period of 4 or 16 h, suggesting that it was not affecting nuclear import. CRM1 (more recently named 'exportin-1') is a nuclear membrane protein that regulates protein export from the nucleus by binding to a specific nuclear export sequence. Leptomycin B is an inhibitor of CRM1/exportin-1, and reversed the ceramide-induced decrease in nuclear C/EBPbeta at 24 h. Taken together, these data support the hypothesis that ceramide may inhibit adipogenesis, at least in part, by enhancing dephosphorylation and premature nuclear export of C/EBPbeta at a time when its maximal transcriptional activity is required to drive adipogenesis. PMID:12071851

  15. A deep proteomics perspective on CRM1-mediated nuclear export and nucleocytoplasmic partitioning.

    PubMed

    Kırlı, Koray; Karaca, Samir; Dehne, Heinz Jürgen; Samwer, Matthias; Pan, Kuan Ting; Lenz, Christof; Urlaub, Henning; Görlich, Dirk

    2015-01-01

    CRM1 is a highly conserved, RanGTPase-driven exportin that carries proteins and RNPs from the nucleus to the cytoplasm. We now explored the cargo-spectrum of CRM1 in depth and identified surprisingly large numbers, namely >700 export substrates from the yeast S. cerevisiae, ≈1000 from Xenopus oocytes and >1050 from human cells. In addition, we quantified the partitioning of ≈5000 unique proteins between nucleus and cytoplasm of Xenopus oocytes. The data suggest new CRM1 functions in spatial control of vesicle coat-assembly, centrosomes, autophagy, peroxisome biogenesis, cytoskeleton, ribosome maturation, translation, mRNA degradation, and more generally in precluding a potentially detrimental action of cytoplasmic pathways within the nuclear interior. There are also numerous new instances where CRM1 appears to act in regulatory circuits. Altogether, our dataset allows unprecedented insights into the nucleocytoplasmic organisation of eukaryotic cells, into the contributions of an exceedingly promiscuous exportin and it provides a new basis for NES prediction. PMID:26673895

  16. A deep proteomics perspective on CRM1-mediated nuclear export and nucleocytoplasmic partitioning

    PubMed Central

    Kırlı, Koray; Karaca, Samir; Dehne, Heinz Jürgen; Samwer, Matthias; Pan, Kuan Ting; Lenz, Christof; Urlaub, Henning; Görlich, Dirk

    2015-01-01

    CRM1 is a highly conserved, RanGTPase-driven exportin that carries proteins and RNPs from the nucleus to the cytoplasm. We now explored the cargo-spectrum of CRM1 in depth and identified surprisingly large numbers, namely >700 export substrates from the yeast S. cerevisiae, ≈1000 from Xenopus oocytes and >1050 from human cells. In addition, we quantified the partitioning of ≈5000 unique proteins between nucleus and cytoplasm of Xenopus oocytes. The data suggest new CRM1 functions in spatial control of vesicle coat-assembly, centrosomes, autophagy, peroxisome biogenesis, cytoskeleton, ribosome maturation, translation, mRNA degradation, and more generally in precluding a potentially detrimental action of cytoplasmic pathways within the nuclear interior. There are also numerous new instances where CRM1 appears to act in regulatory circuits. Altogether, our dataset allows unprecedented insights into the nucleocytoplasmic organisation of eukaryotic cells, into the contributions of an exceedingly promiscuous exportin and it provides a new basis for NES prediction. DOI: http://dx.doi.org/10.7554/eLife.11466.001 PMID:26673895

  17. AKT3 controls mitochondrial biogenesis and autophagy via regulation of the major nuclear export protein CRM-1

    PubMed Central

    Corum, Daniel G.; Tsichlis, Philip N.; Muise-Helmericks, Robin C.

    2014-01-01

    Our previous work has shown that Akt3 is required for mitochondrial biogenesis in primary human endothelial cells (ECs) and in Akt3-null mice; Akt3 affects subcellular localization of peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1α), the master regulator of mitochondrial biogenesis. The purpose of this study is to determine the mechanism by which Akt3 controls the subcellular distribution of PGC-1α and to explore the effect on mitochondrial biogenesis and turnover during angiogenesis. Here we use standard biochemical analyses and Akt3-knockdown strategies to show that Akt3 controls the stabilization of chromosome maintenance region-1 (CRM-1), the major nuclear export receptor. Site-directed mutagenesis and association analyses show that PGC-1α nuclear export is CRM-1 dependent. Akt3 knockdown and CRM-1 overexpression cause 3-fold reductions in PGC-1α target gene expression, compared to control levels. Akt3 inhibition causes autophagy, as measured by autophagosome formation, in a CRM-1-dependent, Akt1/mTOR-independent pathway. In vivo, Akt3-null and heterozygous mice show dose-dependent decreases in angiogenesis compared to wild-type littermates (∼5- and 2.5-fold decreases, respectively), as assessed by Matrigel plug assays. This correlates with an ∼1.5-fold decrease in mitochondrial Cox IV expression. Our studies suggest that Akt3 is a regulator of mitochondrial dynamics in the vasculature via regulation of CRM-1-dependent nuclear export.—Corum, D. G., Tsichlis, P. N., Muise-Helmericks, R. C. AKT3 controls mitochondrial biogenesis and autophagy via regulation of the major nuclear export protein CRM-1. PMID:24081905

  18. Friedrich Miescher Prize awardee lecture review. A conserved family of nuclear export receptors mediates the exit of messenger RNA to the cytoplasm.

    PubMed

    Izaurralde, E

    2001-07-01

    The distinguishing feature of eukaryotic cells is the segregation of RNA biogenesis and DNA replication in the nucleus, separate from the cytoplasmic machinery for protein synthesis. As a consequence, messenger RNAs (mRNAs) and all cytoplasmic RNAs from nuclear origin need to be transported from their site of synthesis in the nucleus to their final cytoplasmic destination. Nuclear export occurs through nuclear pore complexes (NPCs) and is mediated by saturable transport receptors, which shuttle between the nucleus and cytoplasm. The past years have seen great progress in the characterization of the mRNA export pathway and the identification of proteins involved in this process. A novel family of nuclear export receptors (the NXF family), distinct from the well-characterized family of importin beta-like proteins, has been implicated in the export of mRNA to the cytoplasm. PMID:11529502

  19. Structural basis for binding the TREX2 complex to nuclear pores, GAL1 localisation and mRNA export.

    PubMed

    Jani, Divyang; Valkov, Eugene; Stewart, Murray

    2014-06-01

    The conserved Sac3:Thp1:Sem1:Sus1:Cdc31 (TREX2) complex binds to nuclear pore complexes (NPCs) and, in addition to integrating mRNA nuclear export with preceding steps in the gene expression pathway, facilitates re-positioning of highly regulated actively transcribing genes (such as GAL1) to NPCs. Although TREX2 is thought to bind NPC protein Nup1, defining the precise role of this interaction has been frustrated by the complex pleiotropic phenotype exhibited by nup1Δ strains. To provide a structural framework for understanding the binding of TREX2 to NPCs and its function in the gene expression pathway, we have determined the structure of the Nup1:TREX2 interaction interface and used this information to engineer a Sac3 variant that impairs NPC binding while not compromising TREX2 assembly. This variant inhibited the NPC association of both de-repressed and activated GAL1 and also produced mRNA export and growth defects. These results indicate that the TREX2:Nup1 interaction facilitates the efficient nuclear export of bulk mRNA together with the re-positioning of GAL1 to NPCs that is required for transcriptional control that is mediated by removal of SUMO from repressors by NPC-bound Ulp1. PMID:24705649

  20. NXT1, a Novel Influenza A NP Binding Protein, Promotes the Nuclear Export of NP via a CRM1-Dependent Pathway

    PubMed Central

    Chutiwitoonchai, Nopporn; Aida, Yoko

    2016-01-01

    Influenza remains a serious worldwide public health problem. After infection, viral genomic RNA is replicated in the nucleus and packed into viral ribonucleoprotein, which will then be exported to the cytoplasm via a cellular chromosome region maintenance 1 (CRM1)-dependent pathway for further assembly and budding. However, the nuclear export mechanism of influenza virus remains controversial. Here, we identify cellular nuclear transport factor 2 (NTF2)-like export protein 1 (NXT1) as a novel binding partner of nucleoprotein (NP) that stimulates NP-mediated nuclear export via the CRM1-dependent pathway. NXT1-knockdown cells exhibit decreased viral replication kinetics and nuclear accumulated viral RNA and NP. By contrast, NXT1 overexpression promotes nuclear export of NP in a CRM1-dependent manner. Pull-down assays suggest the formation of an NXT1, NP, and CRM1 complex, and demonstrate that NXT1 binds to the C-terminal region of NP. These findings reveal a distinct mechanism for nuclear export of the influenza virus and identify the NXT1/NP interaction as a potential target for antiviral drug development. PMID:27483302

  1. Electromagnetic signals from underground nuclear explosions

    SciTech Connect

    Malik, J.; Fitzhugh, R.; Homuth, F.

    1985-10-01

    Electromagnetic fields and ground currents resulting from underground nuclear explosions have been observed since the first such event. A few measurements have been reported, but most have not. There also have been some speculations as to their origin; the two most generally proposed are the magnetic bubble and the seismoelectric effect. The evidence seems to favor the latter mechanism. 15 refs., 36 figs.

  2. Use of synthetic signal sequences to explore the protein export machinery.

    PubMed

    Clérico, Eugenia M; Maki, Jenny L; Gierasch, Lila M

    2008-01-01

    The information for correct localization of newly synthesized proteins in both prokaryotes and eukaryotes resides in self-contained, often transportable targeting sequences. Of these, signal sequences specify that a protein should be secreted from a cell or incorporated into the cytoplasmic membrane. A central puzzle is presented by the lack of primary structural homology among signal sequences, although they share common features in their sequences. Synthetic signal peptides have enabled a wide range of studies of how these "zipcodes" for protein secretion are decoded and used to target proteins to the protein machinery that facilitates their translocation across and integration into membranes. We review research on how the information in signal sequences enables their passenger proteins to be correctly and efficiently localized. Synthetic signal peptides have made possible binding and crosslinking studies to explore how selectivity is achieved in recognition by the signal sequence-binding receptors, signal recognition particle, or SRP, which functions in all organisms, and SecA, which functions in prokaryotes and some organelles of prokaryotic origins. While progress has been made, the absence of atomic resolution structures for complexes of signal peptides and their receptors has definitely left many questions to be answered in the future. PMID:17918185

  3. Interactions between mRNA Export Commitment, 3′-End Quality Control, and Nuclear Degradation

    PubMed Central

    Libri, Domenico; Dower, Ken; Boulay, Jocelyne; Thomsen, Rune; Rosbash, Michael; Jensen, Torben Heick

    2002-01-01

    Several aspects of eukaryotic mRNA processing are linked to transcription. In Saccharomyces cerevisiae, overexpression of the mRNA export factor Sub2p suppresses the growth defect of hpr1 null cells, yet the protein Hpr1p and the associated THO protein complex are implicated in transcriptional elongation. Indeed, we find that a pool of heat shock HSP104 transcripts are 3′-end truncated in THO complex mutant as well as sub2 mutant backgrounds. Surprisingly, however, this defect can be suppressed by deletion of the 3′-5′ exonuclease Rrp6p. This indicates that incomplete RNAs result from nuclear degradation rather than from a failure to efficiently elongate transcription. RNAs that are not degraded are retained at the transcription site in a Rrp6p-dependent manner. Interestingly, the addition of a RRP6 deletion to sub2 or to THO complex mutants shows a strong synthetic growth phenotype, suggesting that the failure to retain and/or degrade defective mRNAs is deleterious. mRNAs produced in the 3′-end processing mutants rna14-3 and rna15-2, as well as an RNA harboring a 3′ end generated by a self-cleaving hammerhead ribozyme, are also retained in Rrp6p-dependent transcription site foci. Taken together, our results show that several classes of defective RNPs are subject to a quality control step that impedes release from transcription site foci and suggest that suboptimal messenger ribonucleoprotein assembly leads to RNA degradation by Rrp6p. PMID:12417728

  4. Structural mechanism for signal transduction in RXR nuclear receptor heterodimers

    PubMed Central

    Kojetin, Douglas J.; Matta-Camacho, Edna; Hughes, Travis S.; Srinivasan, Sathish; Nwachukwu, Jerome C.; Cavett, Valerie; Nowak, Jason; Chalmers, Michael J.; Marciano, David P.; Kamenecka, Theodore M.; Shulman, Andrew I.; Rance, Mark; Griffin, Patrick R.; Bruning, John B.; Nettles, Kendall W.

    2015-01-01

    A subset of nuclear receptors (NRs) function as obligate heterodimers with retinoid X receptor (RXR), allowing integration of ligand-dependent signals across the dimer interface via an unknown structural mechanism. Using nuclear magnetic resonance (NMR) spectroscopy, x-ray crystallography and hydrogen/deuterium exchange (HDX) mass spectrometry, here we show an allosteric mechanism through which RXR co-operates with a permissive dimer partner, peroxisome proliferator-activated receptor (PPAR)-γ, while rendered generally unresponsive by a non-permissive dimer partner, thyroid hormone (TR) receptor. Amino acid residues that mediate this allosteric mechanism comprise an evolutionarily conserved network discovered by statistical coupling analysis (SCA). This SCA network acts as a signalling rheostat to integrate signals between dimer partners, ligands and coregulator-binding sites, thereby affecting signal transmission in RXR heterodimers. These findings define rules guiding how NRs integrate two ligand-dependent signalling pathways into RXR heterodimer-specific responses. PMID:26289479

  5. Signal Recognition Particle and SecA Cooperate during Export of Secretory Proteins with Highly Hydrophobic Signal Sequences

    PubMed Central

    Zhou, Yufan; Ueda, Takuya; Müller, Matthias

    2014-01-01

    The Sec translocon of bacterial plasma membranes mediates the linear translocation of secretory proteins as well as the lateral integration of membrane proteins. Integration of many membrane proteins occurs co-translationally via the signal recognition particle (SRP)-dependent targeting of ribosome-associated nascent chains to the Sec translocon. In contrast, translocation of classical secretory proteins across the Sec translocon is a post-translational event requiring no SRP but the motor protein SecA. Secretory proteins were, however, reported to utilize SRP in addition to SecA, if the hydrophobicity of their signal sequences exceeds a certain threshold value. Here we have analyzed transport of this subgroup of secretory proteins across the Sec translocon employing an entirely defined in vitro system. We thus found SecA to be both necessary and sufficient for translocation of secretory proteins with hydrophobic signal sequences, whereas SRP and its receptor improved translocation efficiency. This SRP-mediated boost of translocation is likely due to the early capture of the hydrophobic signal sequence by SRP as revealed by site-specific photo cross-linking of ribosome nascent chain complexes. PMID:24717922

  6. Structural and Functional Characterization of CRM1-Nup214 Interactions Reveals Multiple FG-Binding Sites Involved in Nuclear Export.

    PubMed

    Port, Sarah A; Monecke, Thomas; Dickmanns, Achim; Spillner, Christiane; Hofele, Romina; Urlaub, Henning; Ficner, Ralf; Kehlenbach, Ralph H

    2015-10-27

    CRM1 is the major nuclear export receptor. During translocation through the nuclear pore, transport complexes transiently interact with phenylalanine-glycine (FG) repeats of multiple nucleoporins. On the cytoplasmic side of the nuclear pore, CRM1 tightly interacts with the nucleoporin Nup214. Here, we present the crystal structure of a 117-amino-acid FG-repeat-containing fragment of Nup214, in complex with CRM1, Snurportin 1, and RanGTP at 2.85 Å resolution. The structure reveals eight binding sites for Nup214 FG motifs on CRM1, with intervening stretches that are loosely attached to the transport receptor. Nup214 binds to N- and C-terminal regions of CRM1, thereby clamping CRM1 in a closed conformation and stabilizing the export complex. The role of conserved hydrophobic pockets for the recognition of FG motifs was analyzed in biochemical and cell-based assays. Comparative studies with RanBP3 and Nup62 shed light on specificities of CRM1-nucleoporin binding, which serves as a paradigm for transport receptor-nucleoporin interactions. PMID:26489467

  7. Lymphoid enhancer factor-1 blocks adenomatous polyposis coli-mediated nuclear export and degradation of beta-catenin. Regulation by histone deacetylase 1.

    PubMed

    Henderson, Beric R; Galea, Melanie; Schuechner, Stefan; Leung, Louie

    2002-07-01

    The oncogenic protein beta-catenin is overexpressed in many cancers, frequently accumulating in nuclei where it forms active complexes with lymphoid enhancer factor-1 (LEF-1)/T-cell transcription factors, inducing genes such as c-myc and cyclin D1. In normal cells, nuclear beta-catenin levels are controlled by the adenomatous polyposis coli (APC) protein through nuclear export and cytoplasmic degradation. Transient expression of LEF-1 is known to increase nuclear beta-catenin levels by an unknown mechanism. Here, we show that APC and LEF-1 compete for nuclear beta-catenin with opposing consequences. APC can export nuclear beta-catenin to the cytoplasm for degradation. In contrast, LEF-1 anchors beta-catenin in the nucleus by blocking APC-mediated nuclear export. LEF-1 also prevented the APC/CRM1-independent nuclear export of beta-catenin as revealed by in vitro assays. Importantly, LEF-1-bound beta-catenin was protected from degradation by APC and axin in SW480 colon cancer cells. The ability of LEF-1 to trap beta-catenin in the nucleus was down-regulated by histone deacetylase 1, and this correlated with a decrease in LEF1 transcription activity. Our findings identify LEF-1 as key regulator of beta-catenin nuclear localization and stability and suggest that overexpression of LEF-1 in colon cancer and melanoma cells may contribute to the accumulation of oncogenic beta-catenin in the nucleus. PMID:11986304

  8. Nuclear proton dynamics and interactions with calcium signaling.

    PubMed

    Hulikova, Alzbeta; Swietach, Pawel

    2016-07-01

    Biochemical signals acting on the nucleus can regulate gene expression. Despite the inherent affinity of nucleic acids and nuclear proteins (e.g. transcription factors) for protons, little is known about the mechanisms that regulate nuclear pH (pHnuc), and how these could be exploited to control gene expression. Here, we show that pHnuc dynamics can be imaged using the DNA-binding dye Hoechst 33342. Nuclear pores allow the passage of medium-sized molecules (calcein), but protons must first bind to mobile buffers in order to gain access to the nucleoplasm. Fixed buffering residing in the nucleus of permeabilized cells was estimated to be very weak on the basis of the large amplitude of pHnuc transients evoked by photolytic H(+)-uncaging or exposure to weak acids/bases. Consequently, the majority of nuclear pH buffering is sourced from the cytoplasm in the form of mobile buffers. Effective proton diffusion was faster in nucleoplasm than in cytoplasm, in agreement with the higher mobile-to-fixed buffering ratio in the nucleus. Cardiac myocyte pHnuc changed in response to maneuvers that alter nuclear Ca(2+) signals. Blocking Ca(2+) release from inositol-1,4,5-trisphosphate receptors stably alkalinized the nucleus. This Ca(2+)-pH interaction may arise from competitive binding to common chemical moieties. Competitive binding to mobile buffers may couple the efflux of Ca(2+)via nuclear pores with a counterflux of protons. This would generate a stable pH gradient between cytoplasm and nucleus that is sensitive to the state of nuclear Ca(2+) signaling. The unusual behavior of protons in the nucleus provides new mechanisms for regulating cardiac nuclear biology. PMID:26183898

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

    PubMed

    Bühlmann, Melanie; Walrad, Pegine; Rico, Eva; Ivens, Alasdair; Capewell, Paul; Naguleswaran, Arunasalam; Roditi, Isabel; Matthews, Keith R

    2015-05-19

    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. PMID:25873624

  10. Nuclear bile acid signaling through the farnesoid X receptor.

    PubMed

    Mazuy, Claire; Helleboid, Audrey; Staels, Bart; Lefebvre, Philippe

    2015-05-01

    Bile acids (BAs) are amphipathic molecules produced from cholesterol by the liver. Expelled from the gallbladder upon meal ingestion, BAs serve as fat solubilizers in the intestine. BAs are reabsorbed in the ileum and return via the portal vein to the liver where, together with nutrients, they provide signals to coordinate metabolic responses. BAs act on energy and metabolic homeostasis through the activation of membrane and nuclear receptors, among which the nuclear receptor farnesoid X receptor (FXR) is an important regulator of several metabolic pathways. Highly expressed in the liver and the small intestine, FXR contributes to BA effects on metabolism, inflammation and cell cycle control. The pharmacological modulation of its activity has emerged as a potential therapeutic strategy for liver and metabolic diseases. This review highlights recent advances regarding the mechanisms by which the BA sensor FXR contributes to global signaling effects of BAs, and how FXR activity may be regulated by nutrient-sensitive signaling pathways. PMID:25511198

  11. Mapping of export signals of Pseudomonas aeruginosa pilin with alkaline phosphatase fusions.

    PubMed Central

    Strom, M S; Lory, S

    1987-01-01

    Pili of Pseudomonas aeruginosa are assembled from monomers of the structural subunit, pilin, after secretion of this protein across the bacterial membrane. These subunits are initally synthesized as precursors (prepilin) with a six-amino-acid leader peptide that is cleaved off during or after membrane traversal, followed by methylation of the amino-terminal phenylalanine residue. This report demonstrates that additional sequences from the N terminus of the mature protein are necessary for membrane translocation. Gene fusions were made between amino-terminal coding sequences of the cloned pilin gene (pilA) and the structural gene for Escherichia coli alkaline phosphatase (phoA) devoid of a signal sequence. Fusions between at least 45 amino acid residues of the mature pilin and alkaline phosphatase resulted in translocation of the fusion proteins across the cytoplasmic membranes of both P. aeruginosa and E. coli strains carrying recombinant plasmids, as measured by alkaline phosphatase activity and Western blotting. Fusion proteins constructed with the first 10 amino acids of prepilin (including the 6-amino-acid leader peptide) were not secreted, although they were detected in the cytoplasm. Therefore, unlike that of the majority of secreted proteins that are synthesized with transient signal sequences, the membrane traversal of pilin across the bacterial membrane requires the transient six-amino-acid leader peptide as well as sequences contained in the N-terminal region of the mature pilin protein. Images PMID:2885309

  12. A New Class of Endoplasmic Reticulum Export Signal ΦXΦXΦ for Transmembrane Proteins and Its Selective Interaction with Sec24C*

    PubMed Central

    Otsu, Wataru; Kurooka, Takao; Otsuka, Yayoi; Sato, Kota; Inaba, Mutsumi

    2013-01-01

    Protein export from the endoplasmic reticulum (ER) depends on the interaction between a signal motif on the cargo and a cargo recognition site on the coatomer protein complex II. A hydrophobic sequence in the N terminus of the bovine anion exchanger 1 (AE1) anion exchanger facilitated the ER export of human AE1Δ11, an ER-retained AE1 mutant, through interaction with a specific Sec24 isoform. The cell surface expression and N-glycan processing of various substitution mutants or chimeras of human and bovine AE1 proteins and their Δ11 mutants in HEK293 cells were examined. The N-terminal sequence (V/L/F)X(I/L)X(M/L), 26VSIPM30 in bovine AE1, which is comparable with ΦXΦXΦ, acted as the ER export signal for AE1 and AE1Δ11 (Φ is a hydrophobic amino acid, and X is any amino acid). The AE1-Ly49E chimeric protein possessing the ΦXΦXΦ motif exhibited effective cell surface expression and N-glycan maturation via the coatomer protein complex II pathway, whereas a chimera lacking this motif was retained in the ER. A synthetic polypeptide containing the N terminus of bovine AE1 bound the Sec23A-Sec24C complex through a selective interaction with Sec24C. Co-transfection of Sec24C-AAA, in which the residues 895LIL897 (the binding site for another ER export signal motif IXM on Sec24C and Sec24D) were mutated to 895AAA897, specifically increased ER retention of the AE1-Ly49E chimera. These findings demonstrate that the ΦXΦXΦ sequence functions as a novel signal motif for the ER export of cargo proteins through an exclusive interaction with Sec24C. PMID:23658022

  13. A method for quantification of exportin-1 (XPO1) occupancy by Selective Inhibitor of Nuclear Export (SINE) compounds

    PubMed Central

    Crochiere, Marsha L.; Baloglu, Erkan; Klebanov, Boris; Donovan, Scott; del Alamo, Diego; Lee, Margaret; Kauffman, Michael; Shacham, Sharon; Landesman, Yosef

    2016-01-01

    Selective Inhibitor of Nuclear Export (SINE) compounds are a family of small-molecules that inhibit nuclear export through covalent binding to cysteine 528 (Cys528) in the cargo-binding pocket of Exportin 1 (XPO1/CRM1) and promote cancer cell death. Selinexor is the lead SINE compound currently in phase I and II clinical trials for advanced solid and hematological malignancies. In an effort to understand selinexor-XPO1 interaction and to establish whether cancer cell response is a function of drug-target engagement, we developed a quantitative XPO1 occupancy assay. Biotinylated leptomycin B (b-LMB) was utilized as a tool compound to measure SINE-free XPO1. Binding to XPO1 was quantitated from SINE compound treated adherent and suspension cells in vitro, dosed ex vivo human peripheral blood mononuclear cells (PBMCs), and PBMCs from mice dosed orally with drug in vivo. Evaluation of a panel of selinexor sensitive and resistant cell lines revealed that resistance was not attributed to XPO1 occupancy by selinexor. Administration of a single dose of selinexor bound XPO1 for minimally 72 hours both in vitro and in vivo. While XPO1 inhibition directly correlates with selinexor pharmacokinetics, the biological outcome of this inhibition depends on modulation of pathways downstream of XPO1, which ultimately determines cancer cell responsiveness. PMID:26654943

  14. A method for quantification of exportin-1 (XPO1) occupancy by Selective Inhibitor of Nuclear Export (SINE) compounds.

    PubMed

    Crochiere, Marsha L; Baloglu, Erkan; Klebanov, Boris; Donovan, Scott; Del Alamo, Diego; Lee, Margaret; Kauffman, Michael; Shacham, Sharon; Landesman, Yosef

    2016-01-12

    Selective Inhibitor of Nuclear Export (SINE) compounds are a family of small-molecules that inhibit nuclear export through covalent binding to cysteine 528 (Cys528) in the cargo-binding pocket of Exportin 1 (XPO1/CRM1) and promote cancer cell death. Selinexor is the lead SINE compound currently in phase I and II clinical trials for advanced solid and hematological malignancies. In an effort to understand selinexor-XPO1 interaction and to establish whether cancer cell response is a function of drug-target engagement, we developed a quantitative XPO1 occupancy assay. Biotinylated leptomycin B (b-LMB) was utilized as a tool compound to measure SINE-free XPO1. Binding to XPO1 was quantitated from SINE compound treated adherent and suspension cells in vitro, dosed ex vivo human peripheral blood mononuclear cells (PBMCs), and PBMCs from mice dosed orally with drug in vivo. Evaluation of a panel of selinexor sensitive and resistant cell lines revealed that resistance was not attributed to XPO1 occupancy by selinexor. Administration of a single dose of selinexor bound XPO1 for minimally 72 hours both in vitro and in vivo. While XPO1 inhibition directly correlates with selinexor pharmacokinetics, the biological outcome of this inhibition depends on modulation of pathways downstream of XPO1, which ultimately determines cancer cell responsiveness. PMID:26654943

  15. Nuclear export and mitochondrial and endoplasmic reticulum localization of IGF-binding protein 3 regulate its apoptotic properties

    PubMed Central

    Paharkova-Vatchkova, Vladislava; Lee, Kuk-Wha

    2011-01-01

    Tumor suppression by IGF-binding protein 3 (IGFBP3) may occur in an IGF-independent manner, in addition to its role as a regulator of IGF bioavailability. After secretion, IGFBP3 is internalized, rapidly localized to the nucleus, and is later detected in the cytoplasm. We identified a putative nuclear export sequence (NES) in IGFBP3 between amino acids 217 and 228, analogous to the leucine-rich NES sequence of p53 and HIV Rev. Mutation of the NES prevents nucleocytoplasmic shuttling of IGFBP3 and blocks its ability to induce apoptosis. Targeting of IGFBP3 to the mitochondria and endoplasmic reticulum (ER) was confirmed by co-localization with organelle markers using fluorescence confocal microscopy and subcellular fractionation. Mitochondrial targeting was also demonstrated in vivo in IGFBP3-treated prostate cancer xenografts. These results show that IGFBP3 shuttles from the nucleus to the mitochondria and ER, and that nuclear export is essential for its effects on prostate cancer apoptosis. PMID:20228135

  16. Ketamine produces antidepressant-like effects through phosphorylation-dependent nuclear export of histone deacetylase 5 (HDAC5) in rats

    PubMed Central

    Choi, Miyeon; Lee, Seung Hoon; Wang, Sung Eun; Ko, Seung Yeon; Song, Mihee; Choi, June-Seek; Duman, Ronald S.; Son, Hyeon

    2015-01-01

    Ketamine produces rapid antidepressant-like effects in animal assays for depression, although the molecular mechanisms underlying these behavioral actions remain incomplete. Here, we demonstrate that ketamine rapidly stimulates histone deacetylase 5 (HDAC5) phosphorylation and nuclear export in rat hippocampal neurons through calcium/calmodulin kinase II- and protein kinase D-dependent pathways. Consequently, ketamine enhanced the transcriptional activity of myocyte enhancer factor 2 (MEF2), which leads to regulation of MEF2 target genes. Transfection of a HDAC5 phosphorylation-defective mutant (Ser259/Ser498 replaced by Ala259/Ala498, HDAC5-S/A), resulted in resistance to ketamine-induced nuclear export, suppression of ketamine-mediated MEF2 transcriptional activity, and decreased expression of MEF2 target genes. Behaviorally, viral-mediated hippocampal knockdown of HDAC5 blocked or occluded the antidepressant effects of ketamine both in unstressed and stressed animals. Taken together, our results reveal a novel role of HDAC5 in the actions of ketamine and suggest that HDAC5 could be a potential mechanism contributing to the therapeutic actions of ketamine. PMID:26647181

  17. Verdinexor, a Novel Selective Inhibitor of Nuclear Export, Reduces Influenza A Virus Replication In Vitro and In Vivo

    PubMed Central

    Perwitasari, Olivia; Johnson, Scott; Yan, Xiuzhen; Howerth, Elizabeth; Shacham, Sharon; Landesman, Yosef; Baloglu, Erkan; McCauley, Dilara; Tamir, Sharon; Tompkins, S. Mark

    2014-01-01

    ABSTRACT Influenza is a global health concern, causing death, morbidity, and economic losses. Chemotherapeutics that target influenza virus are available; however, rapid emergence of drug-resistant strains is common. Therapeutic targeting of host proteins hijacked by influenza virus to facilitate replication is an antiviral strategy to reduce the development of drug resistance. Nuclear export of influenza virus ribonucleoprotein (vRNP) from infected cells has been shown to be mediated by exportin 1 (XPO1) interaction with viral nuclear export protein tethered to vRNP. RNA interference screening has identified XPO1 as a host proinfluenza factor where XPO1 silencing results in reduced influenza virus replication. The Streptomyces metabolite XPO1 inhibitor leptomycin B (LMB) has been shown to limit influenza virus replication in vitro; however, LMB is toxic in vivo, which makes it unsuitable for therapeutic use. In this study, we tested the anti-influenza virus activity of a new class of orally available small-molecule selective inhibitors of nuclear export, specifically, the XPO1 antagonist KPT-335 (verdinexor). Verdinexor was shown to potently and selectively inhibit vRNP export and effectively inhibited the replication of various influenza virus A and B strains in vitro, including pandemic H1N1 virus, highly pathogenic H5N1 avian influenza virus, and the recently emerged H7N9 strain. In vivo, prophylactic and therapeutic administration of verdinexor protected mice against disease pathology following a challenge with influenza virus A/California/04/09 or A/Philippines/2/82-X79, as well as reduced lung viral loads and proinflammatory cytokine expression, while having minimal toxicity. These studies show that verdinexor acts as a novel anti-influenza virus therapeutic agent. IMPORTANCE Antiviral drugs represent important means of influenza virus control. However, substantial resistance to currently approved influenza therapeutic drugs has developed. New antiviral

  18. Infrasound signals from the underground nuclear explosions of North Korea

    NASA Astrophysics Data System (ADS)

    Che, Il-Young; Park, Junghyun; Kim, Inho; Kim, Tae Sung; Lee, Hee-Il

    2014-07-01

    We investigated the infrasound signals from seismic ground motions induced by North Korea's underground nuclear explosions, including the recent third explosion on 2013 February 12. For the third explosion, the epicentral infrasound signals were detected not only by three infrasound network stations (KSGAR, ULDAR and YAGAR) in South Korea but also by two nearby International Monitoring System infrasound stations, IS45 and IS30. The detectability of the signals was limited at stations located on the relatively east side of the epicentre, with large azimuth deviations due to very favourable atmospheric conditions for eastward propagation at stratospheric height in 2013. The stratospheric wind direction was the reverse of that when the second explosion was conducted in 2009 May. The source location of the epicentral infrasound with wave parameters determined at the multiple stations has an offset by about 16.6 km from the reference seismic location. It was possible to determine the infrasonic location with moderate accuracy by the correction of the azimuth deviation due to the eastward winds in the stratosphere. In addition to the epicentral infrasonic signals, diffracted infrasound signals were observed from the second underground nuclear explosion in 2009. The exceptional detectability of the diffracted infrasound was a consequence of the temporal formation of a thin atmospheric inversion layer over the ocean surface when the event occurred.

  19. A chloroplast retrograde signal regulates nuclear alternative splicing

    PubMed Central

    Petrillo, Ezequiel; Herz, Micaela A. Godoy; Fuchs, Armin; Reifer, Dominik; Fuller, John; Yanovsky, Marcelo J.; Simpson, Craig; Brown, John W. S.; Barta, Andrea; Kalyna, Maria; Kornblihtt, Alberto R.

    2015-01-01

    Light is a source of energy and also a regulator of plant physiological adaptations. We show here that light/dark conditions affect alternative splicing of a subset of Arabidopsis genes preferentially encoding proteins involved in RNA processing. The effect requires functional chloroplasts and is also observed in roots when the communication with the photosynthetic tissues is not interrupted, suggesting that a signaling molecule travels through the plant. Using photosynthetic electron transfer inhibitors with different mechanisms of action we deduce that the reduced pool of plastoquinones initiates a chloroplast retrograde signaling that regulates nuclear alternative splicing and is necessary for proper plant responses to varying light conditions. PMID:24763593

  20. Nuclear Gln3 Import Is Regulated by Nitrogen Catabolite Repression Whereas Export Is Specifically Regulated by Glutamine.

    PubMed

    Rai, Rajendra; Tate, Jennifer J; Shanmuganatham, Karthik; Howe, Martha M; Nelson, David; Cooper, Terrance G

    2015-11-01

    Gln3, a transcription activator mediating nitrogen-responsive gene expression in Saccharomyces cerevisiae, is sequestered in the cytoplasm, thereby minimizing nitrogen catabolite repression (NCR)-sensitive transcription when cells are grown in nitrogen-rich environments. In the face of adverse nitrogen supplies, Gln3 relocates to the nucleus and activates transcription of the NCR-sensitive regulon whose products transport and degrade a variety of poorly used nitrogen sources, thus expanding the cell's nitrogen-acquisition capability. Rapamycin also elicits nuclear Gln3 localization, implicating Target-of-rapamycin Complex 1 (TorC1) in nitrogen-responsive Gln3 regulation. However, we long ago established that TorC1 was not the sole regulatory system through which nitrogen-responsive regulation is achieved. Here we demonstrate two different ways in which intracellular Gln3 localization is regulated. Nuclear Gln3 entry is regulated by the cell's overall nitrogen supply, i.e., by NCR, as long accepted. However, once within the nucleus, Gln3 can follow one of two courses depending on the glutamine levels themselves or a metabolite directly related to glutamine. When glutamine levels are high, e.g., glutamine or ammonia as the sole nitrogen source or addition of glutamine analogues, Gln3 can exit from the nucleus without binding to DNA. In contrast, when glutamine levels are lowered, e.g., adding additional nitrogen sources to glutamine-grown cells or providing repressive nonglutamine nitrogen sources, Gln3 export does not occur in the absence of DNA binding. We also demonstrate that Gln3 residues 64-73 are required for nuclear Gln3 export. PMID:26333687

  1. Cotranscriptional Recruitment to the mRNA Export Receptor Mex67p Contributes to Nuclear Pore Anchoring of Activated Genes▿

    PubMed Central

    Dieppois, Guennaelle; Iglesias, Nahid; Stutz, Françoise

    2006-01-01

    Transcription activation of some Saccharomyces cerevisiae genes is paralleled by their repositioning to the nuclear periphery, but the mechanism underlying gene anchoring is poorly defined. We show that the nuclear pore complex-associated Mlp1p and the shuttling mRNA export receptor Mex67p contribute to the stable association of the activated GAL10 and HSP104 genes with the nuclear periphery. However, we find no obligatory link between gene positioning and gene expression. Furthermore, gene anchoring correlates with the cotranscriptional recruitment of Mex67p to transcribing genes. Notably, the association of Mex67p with chromatin is not mediated by RNA. Interestingly, a mutant GAL2 gene lacking the coding region is still able to recruit Mex67p upon transcriptional activation and to relocate to the nuclear periphery. Together these data suggest that, at least for GAL2, nascent messenger ribonucleoprotein does not play a major role in gene anchoring and that the early recruitment of Mex67p contributes to gene repositioning by virtue of an RNA-independent process. PMID:16954382

  2. In vivo single-particle imaging of nuclear mRNA export in budding yeast demonstrates an essential role for Mex67p

    PubMed Central

    Smith, Carlas; Lari, Azra; Derrer, Carina Patrizia; Ouwehand, Anette; Rossouw, Ammeret; Huisman, Maximiliaan; Dange, Thomas; Hopman, Mark; Joseph, Aviva; Zenklusen, Daniel

    2015-01-01

    Many messenger RNA export proteins have been identified; yet the spatial and temporal activities of these proteins and how they determine directionality of messenger ribonucleoprotein (mRNP) complex export from the nucleus remain largely undefined. Here, the bacteriophage PP7 RNA-labeling system was used in Saccharomyces cerevisiae to follow single-particle mRNP export events with high spatial precision and temporal resolution. These data reveal that mRNP export, consisting of nuclear docking, transport, and cytoplasmic release from a nuclear pore complex (NPC), is fast (∼200 ms) and that upon arrival in the cytoplasm, mRNPs are frequently confined near the nuclear envelope. Mex67p functions as the principal mRNP export receptor in budding yeast. In a mex67-5 mutant, delayed cytoplasmic release from NPCs and retrograde transport of mRNPs was observed. This proves an essential role for Mex67p in cytoplasmic mRNP release and directionality of transport. PMID:26694837

  3. CRM1-dependent nuclear export and dimerization with hMSH5 contribute to the regulation of hMSH4 subcellular localization

    SciTech Connect

    Neyton, Sophie; Lespinasse, Francoise; Lahaye, Francois; Staccini, Pascal; Paquis-Flucklinger, Veronique; Santucci-Darmanin, Sabine

    2007-10-15

    MSH4 and MSH5 are members of the MutS homolog family, a conserved group of proteins involved in DNA mismatch correction and homologous recombination. Although several studies have provided compelling evidences suggesting that MSH4 and MSH5 could act together in early and late stages of meiotic recombination, their precise roles are poorly understood and recent findings suggest that the human MSH4 protein may also exert a cytoplasmic function. Here we show that MSH4 is present in the cytoplasm and the nucleus of both testicular cells and transfected somatic cells. Confocal studies on transfected cells provide the first evidence that the subcellular localization of MSH4 is regulated, at least in part, by an active nuclear export pathway dependent on the exportin CRM1. We used deletion mapping and mutagenesis to define two functional nuclear export sequences within the C-terminal part of hMSH4 that mediate nuclear export through the CRM1 pathway. Our results suggest that CRM1 is also involved in MSH5 nuclear export. In addition, we demonstrate that dimerization of MSH4 and MSH5 facilitates their nuclear localization suggesting that dimerization may regulate the intracellular trafficking of these proteins. Our findings suggest that nucleocytoplasmic traffic may constitute a regulatory mechanism for MSH4 and MSH5 functions.

  4. A natural component from Euphorbia humifusa Willd displays novel, broad-spectrum anti-influenza activity by blocking nuclear export of viral ribonucleoprotein.

    PubMed

    Chang, So Young; Park, Ji Hoon; Kim, Young Ho; Kang, Jong Seong; Min, Ji-Young

    2016-03-01

    The need to develop anti-influenza drugs with novel antiviral mechanisms is urgent because of the rapid rate of antigenic mutation and the emergence of drug-resistant viruses. We identified a novel anti-influenza molecule by screening 861 plant-derived natural components using a high-throughput image-based assay that measures inhibition of the influenza virus infection. 1,3,4,6-tetra-O-galloyl-β-D-glucopyranoside (TGBG) from Euphorbia humifusa Willd showed broad-spectrum anti-influenza activity against two seasonal influenza A strains, A/California/07/2009 (H1N1) and A/Perth/16/2009 (H3N2), and seasonal influenza B strain B/Florida/04/2006. We investigated the mode of action of TGBG using neuraminidase activity inhibition and time-of-addition assays, which evaluate the viral release and entry steps, respectively. We found that TGBG exhibits a novel antiviral mechanism that differs from the FDA-approved anti-influenza drugs oseltamivir which inhibits viral release, and amantadine which inhibits viral entry. Immunofluorescence assay demonstrated that TGBG significantly inhibits nuclear export of influenza nucleoproteins (NP) during the early stages of infection causing NP to accumulate in the nucleus. In addition, influenza-induced activation of the Akt signaling pathway was suppressed by TGBG in a dose-dependent manner. These data suggest that a putative mode of action of TGBG involves inhibition of viral ribonucleoprotein (vRNP) export from the nucleus to the cytoplasm consequently disrupting the assembly of progeny virions. In summary, TGBG has potential as novel anti-influenza therapeutic with a novel mechanism of action. PMID:26850850

  5. Herpes simplex virus type 1 protein IE63 affects the nuclear export of virus intron-containing transcripts.

    PubMed Central

    Phelan, A; Dunlop, J; Clements, J B

    1996-01-01

    Using in situ hybridization labelling methods, we have determined that the herpes simplex virus type 1 immediate-early protein IE63 (ICP27) affects the cellular localization of virus transcripts. Intronless transcripts from the IE63, UL38, and UL44 genes are rapidly exported to and accumulate in the cytoplasm throughout infection, in either the presence or absence of IE63 expression. The intron-containing transcripts from the IE110 and UL15 genes, while initially cytoplasmic, are increasingly retained in the nucleus in distinct clumps as infection proceeds, and the clumps colocalize with the redistributed small nuclear ribonucleoprotein particles. Infections with the IE63 mutant virus 27-lacZ demonstrated that in the absence of IE63 expression, nuclear retention of intron-containing transcripts was lost. The nuclear retention of UL15 transcripts, which demonstrated both nuclear and cytoplasmic label, was not as pronounced as that of the IE110 transcripts, and we propose that this is due to the late expression of UL15. Infections with the mutant virus 110C1, in which both introns of IE110 have been precisely removed (R.D. Everett, J. Gen. Virol. 72:651-659, 1991), demonstrated IE110 transcripts in both the nucleus and the cytoplasm; thus, exon definition sequences which regulate viral RNA transport are present in the IE110 transcript. By in situ hybridization a stable population of polyadenylated RNAs was found to accumulate in the nucleus in spots, most of which were separate from the small nuclear ribonucleoprotein particle clumps. The IE63 protein has an involvement, either direct or indirect, in the regulation of nucleocytoplasmic transport of viral transcripts, a function which contrasts with the recently proposed role of herpes simplex virus type 1 Us11 in promoting the nuclear export of partially spliced or unspliced transcripts (J.-J. Diaz, M. Duc Dodon, N. Schaerer-Uthurraly, D. Simonin, K. Kindbeiter, L. Gazzolo, and J.-J. Madjar, Nature [London] 379

  6. Regulation of Nuclear Localization of Signaling Proteins by Cytokinin

    SciTech Connect

    Kieber, J.J.

    2010-05-01

    Cytokinins are a class of mitogenic plant hormones that play an important role in most aspects of plant development, including shoot and root growth, vascular and photomorphogenic development and leaf senescence. A model for cytokinin perception and signaling has emerged that is similar to bacterial two-component phosphorelays. In this model, binding of cytokinin to the extracellular domain of the Arabidopsis histidine kinase (AHKs) receptors induces autophosphorylation within the intracellular histidine-kinase domain. The phosphoryl group is subsequently transferred to cytosolic Arabidopsis histidine phosphotransfer proteins (AHPs), which have been suggested to translocate to the nucleus in response to cytokinin treatment, where they then transfer the phosphoryl group to nuclear-localized response regulators (Type-A and Type-B ARRs). We examined the effects of cytokinin on AHP subcellular localization in Arabidopsis and, contrary to expectations, the AHPs maintained a constant nuclear/cytosolic distribution following cytokinin treatment. Furthermore, mutation of the conserved phosphoacceptor histidine residue of the AHP, as well as disruption of multiple cytokinin signaling elements, did not affect the subcellular localization of the AHP proteins. Finally, we present data indicating that AHPs maintain a nuclear/cytosolic distribution by balancing active transport into and out of the nucleus. Our findings suggest that the current models indicating relocalization of AHP protein into the nucleus in response to cytokinin are incorrect. Rather, AHPs actively maintain a consistent nuclear/cytosolic distribution regardless of the status of the cytokinin response pathway.

  7. A novel role for the nuclear localization signal in regulating hnRNP K protein stability in vivo.

    PubMed

    Hutchins, Erica J; Belrose, Jamie L; Szaro, Ben G

    2016-09-16

    hnRNP K is a highly conserved nucleocytoplasmic shuttling protein, which associates with RNAs through synergistic binding via its three KH domains. hnRNP K is required for proper nuclear export and translational control of its mRNA targets, and these processes are controlled by hnRNP K's movement between subcellular compartments. Whereas the nuclear export and localization of hnRNP K that is associated with mRNP complexes has been well studied, the trafficking of hnRNP K that is unbound to mRNA has yet to be elucidated. To that end, we expressed an EGFP-tagged RNA binding-defective form of hnRNP K in intact Xenopus embryos, and found it was rapidly degraded in vivo. Deleting hnRNP K's nuclear localization signal (NLS), which contains two prospective ubiquitination sites, rescued the protein from degradation. These data demonstrate a novel activity for the NLS of hnRNP K in regulating the protein's stability in vivo when it is unbound to nucleic acids. PMID:27501755

  8. KPT-330 inhibitor of XPO1-mediated nuclear export has anti-proliferative activity in hepatocellular carcinoma

    PubMed Central

    Zheng, Yun; Gery, Sigal; Sun, Haibo; Shacham, Sharon; Kauffman, Michael; Koeffler, H. Phillip

    2014-01-01

    PURPOSE Exportin-1 (XPO1, CRM1) mediates the nuclear export of several key growth regulatory and tumor suppressor proteins. Cancer cells often overexpress XPO1 resulting in cytoplasmic mislocalization and aberrant activity of its target proteins. Orally bioavailable selective inhibitors of nuclear export (SINE) that irreversibly bind to and inhibit the function of XPO1 have been recently developed. The aim of this study was to investigate the efficacy of the clinical staged, orally available, SINE compound, KPT-330 in Hepatocellular carcinoma (HCC). METHODS In silico meta-analysis showed that XPO1 is overexpressed in HCC. Six HCC cell lines were treated with KPT-330 and cell proliferation and expression of cell growth regulators were examined by cell proliferation assays and Western blot analysis, respectively. The in vivo anti-cancer activity of KPT-330 was examined in a HCC xenograft murine model. RESULTS KPT-330 reduced the viability of HCC cell lines in vitro and this anti-proliferative effect was associated with cell cycle arrest and induction of apoptosis. The expression of the pro-apoptotic protein PUMA was markedly up-regulated by KPT-330. In addition, SINE treatment increased the expression of the tumor suppressor proteins p53 and p27, while it reduced the expression of HCC promoting proteins, c-Myc and c-Met. XPO1 levels itself were also down-regulated following KPT-330 treatment. Finally, a HCC xenograft murine model showed that treatment of mice with oral KPT-330 significantly inhibited tumor growth with little evidence of toxicity. CONCLUSION Our results suggest that SINE compounds, such as KPT-330 are promising novel drugs for the targeted therapy of HCC. PMID:25030088

  9. Tap and Dbp5, but not Gag, are involved in DR-mediated nuclear export of unspliced Rous sarcoma virus RNA

    SciTech Connect

    LeBlanc, Jason J.; Uddowla, Sabena; Abraham, Benjamin; Clatterbuck, Sarah; Beemon, Karen L. . E-mail: KLB@jhu.edu

    2007-07-05

    All retroviruses must circumvent cellular restrictions on the export of unspliced RNAs from the nucleus. While the unspliced RNA export pathways for HIV and Mason-Pfizer monkey virus are well characterized, that of Rous sarcoma virus (RSV) is not. We have previously reported that the RSV direct repeat (DR) elements are involved in the cytoplasmic accumulation of unspliced viral RNA. Here, using fluorescent in situ hybridization (FISH), we demonstrate that unspliced viral RNAs bearing a single point mutation (G8863C) in the DR exhibit a restricted cellular localization in and around the nucleus. In contrast, wild type unspliced viral RNA had a diffuse localization throughout the nucleus and cytoplasm. Since the RSV Gag protein has a transient localization in the nucleus, we examined the effect of Gag over-expression on a DR-mediated reporter construct. While Gag did not enhance DR-mediated nuclear export, the dominant-negative expression of two cellular export factors, Tap and Dbp5, inhibited expression of the same reporter construct. Furthermore, FISH studies using the dominant-negative Dbp5 demonstrated that unspliced wild type RSV RNA was retained within the nucleus. Taken together, these results further implicate the DR in nuclear RNA export through interactions with Tap and Dbp5.

  10. Nuclear localization signal receptor importin alpha associates with the cytoskeleton.

    PubMed Central

    Smith, H M; Raikhel, N V

    1998-01-01

    Importin alpha is the nuclear localization signal (NLS) receptor that is involved in the nuclear import of proteins containing basic NLSs. Using importin alpha as a tool, we were interested in determining whether the cytoskeleton could function in the transport of NLS-containing proteins from the cytoplasm to the nucleus. Double-labeling immunofluorescence studies showed that most of the cytoplasmic importin alpha coaligned with microtubules and microfilaments in tobacco protoplasts. Treatment of tobacco protoplasts with microtubule- or microfilament-depolymerizing agents disrupted the strands of importin alpha in the cytoplasm, whereas a microtubule-stabilizing agent had no effect. Biochemical analysis showed that importin alpha associated with microtubules and microfilaments in vitro in an NLS-dependent manner. The interaction of importin alpha with the cytoskeleton could be an essential element of protein transport from the cytoplasm to the nucleus in vivo. PMID:9811789

  11. Nuclear shuttling precedes dimerization in mineralocorticoid receptor signaling.

    PubMed

    Grossmann, Claudia; Ruhs, Stefanie; Langenbruch, Lisa; Mildenberger, Sigrid; Strätz, Nicole; Schumann, Katja; Gekle, Michael

    2012-06-22

    The mineralocorticoid receptor (MR), a member of the steroid receptor superfamily, regulates water-electrolyte balance and mediates pathophysiological effects in the renocardiovascular system. Previously, it was assumed that after binding aldosterone, the MR dissociates from HSP90, forms homodimers, and then translocates into the nucleus where it acts as a transcription factor (Guiochon-Mantel et al., 1989; Robertson et al., 1993; Savory et al., 2001). We found that, during aldosterone-induced nuclear translocation, MR is bound to HSP90 both in the cytosol and the nucleus. Homodimerization measured by eBRET and FRET takes place when the MR is already predominantly nuclear. In vitro binding of MR to DNA was independent of ligand but could be partially inhibited by geldanamycin. Overall, here we provide insights into classical MR signaling necessary for elucidating the mechanisms of pathophysiological MR effects and MR specificity. PMID:22726688

  12. Novel reversible selective inhibitor of nuclear export shows that CRM1 is a target in colorectal cancer cells.

    PubMed

    Niu, Mingshan; Chong, Yulong; Han, Yan; Liu, Xuejiao

    2015-01-01

    Colorectal cancer arises via a multistep carcinogenic process and the deregulation of multiple pathways. Thus, the simultaneous targeting of multiple pathways may be a promising therapeutic approach for colorectal treatment. CRM1 is an attractive cancer drug target, because it can regulate multiple pathways and tumor suppressor proteins. In this study, we investigated the anti-tumor activity of a novel reversible CRM1 inhibitor S109 in colorectal cancer. Our data demonstrate that S109 inhibits proliferation and induces cell cycle arrest in colorectal cancer cells. Mechanistically, we demonstrate that the activity of S109 is associated with the nuclear retention of major tumor suppress proteins. Furthermore, the Cys528 mutation of CRM1 prevented the ability of S109 to block nuclear export and inhibit the proliferation of colorectal cancer cells. Interestingly, S109 decreased the CRM1 protein level via proteasomal pathway. We argue that reversible CRM1 inhibitors but not irreversible inhibitors can induce the degradation of CRM1, because the dissociation of reversible inhibitors of CRM1 changes the conformation of CRM1. Taken together, these findings demonstrate that CRM1 is a valid target for the treatment of colorectal cancer and provide a basis for the development of S109 therapies for colorectal cancer. PMID:25996664

  13. Longitudinal tracking of single live cancer cells to understand cell cycle effects of the nuclear export inhibitor, selinexor

    PubMed Central

    Marcus, Joshua M.; Burke, Russell T.; DeSisto, John A.; Landesman, Yosef; Orth, James D.

    2015-01-01

    Longitudinal tracking is a powerful approach to understand the biology of single cells. In cancer therapy, outcome is determined at the molecular and cellular scale, yet relationships between cellular response and cell fate are often unknown. The selective inhibitor of nuclear export, selinexor, is in development for the treatment of various cancers. Selinexor covalently binds exportin-1, causing nuclear sequestration of cargo proteins, including key regulators of the cell cycle and apoptosis. The cell cycle effects of selinexor and the relationships between cell cycle effects and cell fates, has not been described for individual cells. Using fluorescent cell cycle indicators we report the majority of cell death after selinexor treatment occurs from a protracted G1-phase and early S-phase. G1- or early S-phase treated cells show the strongest response and either die or arrest, while those treated in late S- or G2-phase progress to mitosis and divide. Importantly, the progeny of cell divisions also die or arrest, mostly in the next G1-phase. Cells that survive selinexor are negative for multiple proliferation biomarkers, indicating a penetrant, arrested state. Selinexor acts quickly, shows strong cell cycle selectivity, and is highly effective at arresting cell growth and inducing death in cancer-derived cells. PMID:26399741

  14. Hypo-osmotic shock induces nuclear export and proteasome-dependent decrease of UBL5

    SciTech Connect

    Hatanaka, Ken; Ikegami, Koji; Takagi, Hiroshi; Setou, Mitsutoshi . E-mail: setou@nips.ac.jp

    2006-11-24

    The osmolarity of body fluid is strictly controlled through the action of diuretic hormones, which are secreted in the hypothalamus. In the mammalian brain, ubiquitin-like 5 (UBL5) is expressed in oxytocin- and vasopressin-positive neurons in the hypothalamus, and these neurons play a role in regulating osmolarity. We examined the dynamics of UBL5 levels in response to hyper- or hypo-osmotic conditions. Hypo-osmotic conditions led to significantly reduced levels of UBL5 both in brain slices from the hypothalamus and in NIH-3T3 cells. This decrease in UBL5 was transcription-independent and proteasome-dependent. Time-course immunocytochemical studies using exogenous UBL5 revealed that the protein was exported from the nucleus under hypo-osmotic conditions and decreased in a proteasome-dependent manner. This report is the first to describe changes in the intracellular and subcellular localization of UBL5 in response to hypo-osmotic conditions. Our results imply osmoregulation of UBL5.

  15. Nuclear receptor TLX inhibits TGF-β signaling in glioblastoma.

    PubMed

    Johansson, Erik; Zhai, Qiwei; Zeng, Zhao-Jun; Yoshida, Takeshi; Funa, Keiko

    2016-05-01

    TLX (also called NR2E1) is an orphan nuclear receptor that maintains stemness of neuronal stem cells. TLX is highly expressed in the most malignant form of glioma, glioblastoma multiforme (GBM), and is important for the proliferation and maintenance of the stem/progenitor cells of the tumor. Transforming Growth Factor-β (TGF-β) is a cytokine regulating many different cellular processes such as differentiation, migration, adhesion, cell death and proliferation. TGF-β has an important function in cancer where it can work as either a tumor suppressor or oncogene, depending on the cancer type and stage of tumor development. Since glioblastoma often have dysfunctional TGF-β signaling we wanted to find out if there is any interaction between TLX and TGF-β in glioblastoma cells. We demonstrate that knockdown of TLX enhances the canonical TGF-β signaling response in glioblastoma cell lines. TLX physically interacts with and stabilizes Smurf1, which can ubiquitinate and target TGF-β receptor II for degradation, whereas knockdown of TLX leads to stabilization of TGF-β receptor II, increased nuclear translocation of Smad2/3 and enhanced expression of TGF-β target genes. The interaction between TLX and TGF-β may play an important role in the regulation of proliferation and tumor-initiating properties of glioblastoma cells. PMID:27048878

  16. Identification of two functional nuclear localization signals mediating nuclear import of liver receptor homologue-1.

    PubMed

    Yang, Feng-Ming; Lin, Yu-Chi; Hu, Meng-Chun

    2011-04-01

    Liver receptor homologue-1 (LRH-1) is a member of the nuclear receptor superfamily. We characterized two functional nuclear localization signals (NLSs) in LRH-1. NLS1 (residues 117-168) overlaps the second zinc finger in the DNA binding domain. Mutagenesis showed that the zinc finger structure and two basic clusters on either side of the zinc finger loop are critical for nuclear import of NLS1. NLS2 (residues 169-204) is located in the Ftz-F1 box that contains a bipartite signal. In full-length LRH-1, mutation of either NLS1 or NLS2 had no effect on nuclear localization, but disruption of both NLS1 and NLS2 resulted in the cytoplasmic accumulation of LRH-1. Either NLS1 or NLS2 alone was sufficient to target LRH-1 to the nucleus. Both NLS1 and NLS2 mediate nuclear transport by a mechanism involving importin α/β. Finally, we showed that three crucial basic clusters in the NLSs are involved in the DNA binding and transcriptional activities of LRH-1. PMID:20853131

  17. Spherical tensor analysis of nuclear magnetic resonance signals.

    PubMed

    van Beek, Jacco D; Carravetta, Marina; Antonioli, Gian Carlo; Levitt, Malcolm H

    2005-06-22

    In a nuclear magnetic-resonance (NMR) experiment, the spin density operator may be regarded as a superposition of irreducible spherical tensor operators. Each of these spin operators evolves during the NMR experiment and may give rise to an NMR signal at a later time. The NMR signal at the end of a pulse sequence may, therefore, be regarded as a superposition of spherical components, each derived from a different spherical tensor operator. We describe an experimental method, called spherical tensor analysis (STA), which allows the complete resolution of the NMR signal into its individual spherical components. The method is demonstrated on a powder of a (13)C-labeled amino acid, exposed to a pulse sequence generating a double-quantum effective Hamiltonian. The propagation of spin order through the space of spherical tensor operators is revealed by the STA procedure, both in static and rotating solids. Possible applications of STA to the NMR of liquids, liquid crystals, and solids are discussed. PMID:16035785

  18. A novel tribasic Golgi export signal directs cargo protein interaction with activated Rab11 and AP-1-dependent Golgi-plasma membrane trafficking.

    PubMed

    Parmar, Hirendrasinh B; Duncan, Roy

    2016-04-15

    The reovirus fusion-associated small transmembrane (FAST) proteins comprise a unique family of viral membrane fusion proteins dedicated to inducing cell-cell fusion. We recently reported that a polybasic motif (PBM) in the cytosolic tail of reptilian reovirus p14 FAST protein functions as a novel tribasic Golgi export signal. Using coimmunoprecipitation and fluorescence resonance energy transfer (FRET) assays, we now show the PBM directs interaction of p14 with GTP-Rab11. Overexpression of dominant-negative Rab11 and RNA interference knockdown of endogenous Rab11 inhibited p14 plasma membrane trafficking and resulted in p14 accumulation in the Golgi complex. This is the first example of Golgi export to the plasma membrane that is dependent on the interaction of membrane protein cargo with activated Rab11. RNA interference and immunofluorescence microscopy further revealed that p14 Golgi export is dependent on AP-1 (but not AP-3 or AP-4) and that Rab11 and AP-1 both colocalize with p14 at the TGN. Together these results imply the PBM mediates interactions of p14 with activated Rab11 at the TGN, resulting in p14 sorting into AP1-coated vesicles for anterograde TGN-plasma membrane transport. PMID:26941330

  19. A novel tribasic Golgi export signal directs cargo protein interaction with activated Rab11 and AP-1–dependent Golgi–plasma membrane trafficking

    PubMed Central

    Parmar, Hirendrasinh B.; Duncan, Roy

    2016-01-01

    The reovirus fusion–associated small transmembrane (FAST) proteins comprise a unique family of viral membrane fusion proteins dedicated to inducing cell–cell fusion. We recently reported that a polybasic motif (PBM) in the cytosolic tail of reptilian reovirus p14 FAST protein functions as a novel tribasic Golgi export signal. Using coimmunoprecipitation and fluorescence resonance energy transfer (FRET) assays, we now show the PBM directs interaction of p14 with GTP-Rab11. Overexpression of dominant-negative Rab11 and RNA interference knockdown of endogenous Rab11 inhibited p14 plasma membrane trafficking and resulted in p14 accumulation in the Golgi complex. This is the first example of Golgi export to the plasma membrane that is dependent on the interaction of membrane protein cargo with activated Rab11. RNA interference and immunofluorescence microscopy further revealed that p14 Golgi export is dependent on AP-1 (but not AP-3 or AP-4) and that Rab11 and AP-1 both colocalize with p14 at the TGN. Together these results imply the PBM mediates interactions of p14 with activated Rab11 at the TGN, resulting in p14 sorting into AP1-coated vesicles for anterograde TGN–plasma membrane transport. PMID:26941330

  20. Cell cycle-dependent nuclear export of Cdh1p may contribute to the inactivation of APC/C(Cdh1).

    PubMed

    Jaquenoud, Malika; van Drogen, Frank; Peter, Matthias

    2002-12-01

    Cdh1p is a substrate-specific subunit of the anaphase-promoting complex (APC/C), which functions as an E3 ubiquitin ligase to degrade the mitotic cyclin Clb2p and other substrates during the G(1) phase of the cell cycle. Cdh1p is phosphorylated and thereby inactivated at the G(1)/S transition predominantly by Cdc28p-Clb5p. Here we show that Cdh1p is nuclear during the G(1) phase of the cell cycle, but redistributes to the cytoplasm between S phase and the end of mitosis. Nuclear export of Cdh1p is regulated by phosphorylation and requires active Cdc28p kinase. Cdh1p binds to the importin Pse1p and the exportin Msn5p, which is necessary and sufficient to promote efficient export of Cdh1p in vivo. Although msn5delta cells are viable, they are sensitive to Cdh1p overexpression. Likewise, a mutant form of Cdh1p, which is constitutively nuclear, prevents accumulation of Clb2p and leads to cell cycle arrest when overexpressed in wild-type cells. Taken together, these results suggest that phosphorylation-dependent nuclear export of Cdh1p by Msn5p contributes to efficient inactivation of APC/C(Cdh1). PMID:12456658

  1. Novel roles of nuclear angiotensin receptors and signaling mechanisms.

    PubMed

    Gwathmey, TanYa M; Alzayadneh, Ebaa M; Pendergrass, Karl D; Chappell, Mark C

    2012-03-01

    The renin-angiotensin system (RAS) constitutes an important hormonal system in the physiological regulation of blood pressure. The dysregulation of the RAS is considered a major influence in the development and progression of cardiovascular disease and other pathologies. Indeed, experimental and clinical evidence indicates that blockade of this system with angiotensin-converting enzyme (ACE) inhibitors or angiotensin type 1 receptor (AT1R) antagonists is an effective therapy to attenuate hypertension and diabetic renal injury, and to improve heart failure. Originally defined as a circulating system, multiple tissues express a complete RAS, and compelling evidence now favors an intracellular system involved in cell signaling and function. Within the kidney, intracellular expression of the three predominant ANG receptor subtypes is evident in the nuclear compartment. The ANG type 1 receptor (AT1R) is coupled to the generation of reactive oxygen species (ROS) through the activation of phosphoinositol-3 kinase (PI3K) and PKC. In contrast, both ANG type 2 (AT2R) and ANG-(1-7) (AT7R) receptors stimulate nitric oxide (NO) formation, which may involve nuclear endothelial NO synthase (eNOS). Moreover, blockade of either ACE2-the enzyme that converts ANG II to ANG-(1-7)-or the AT7 receptor exacerbates the ANG II-ROS response on renal nuclei. Finally, in a model of fetal programmed hypertension, the nuclear ROS response to ANG II is enhanced, while both AT2 and AT7 stimulation of NO is attenuated, suggesting that an imbalance in the intracellular RAS may contribute to the development of programming events. We conclude that a functional intracellular or nuclear RAS may have important implications in the therapeutic approaches to cardiovascular disease. PMID:22170620

  2. 10 CFR 110.42 - Export licensing criteria.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 2 2010-01-01 2010-01-01 false Export licensing criteria. 110.42 Section 110.42 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) EXPORT AND IMPORT OF NUCLEAR EQUIPMENT AND MATERIAL Review of License Applications § 110.42 Export licensing criteria. (a) The review of license applications for export for peaceful nuclear uses of...

  3. 10 CFR 110.42 - Export licensing criteria.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 10 Energy 2 2014-01-01 2014-01-01 false Export licensing criteria. 110.42 Section 110.42 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) EXPORT AND IMPORT OF NUCLEAR EQUIPMENT AND MATERIAL Review of License Applications § 110.42 Export licensing criteria. (a) The review of license applications for export for peaceful nuclear uses of...

  4. Definition of a consensus transportin-specific nucleocytoplasmic transport signal.

    PubMed

    Bogerd, H P; Benson, R E; Truant, R; Herold, A; Phingbodhipakkiya, M; Cullen, B R

    1999-04-01

    The low cytoplasmic and high nuclear concentration of the GTP-bound form of Ran provides directionality for both nuclear protein import and export. Both import and export factors bind RanGTP directly, yet this interaction produces opposite effects; in the former case, RanGTP binding induces nuclear cargo release, whereas in the latter, RanGTP binding induces nuclear cargo assembly. Therefore, nuclear import and export receptors and their protein recognition sites are predicted to be distinct. Nevertheless, the approximately 38-amino acid M9 sequence present in heterogeneous nuclear ribonucleoprotein A1 has been reported to serve as both a nuclear localization signal and a nuclear export signal, even though only one protein, the nuclear import factor transportin, has been shown to bind M9 directly. We have used a combination of mutational randomization followed by selection for transportin binding to exhaustively define amino acids in M9 that are critical for transportin binding in vivo. As expected, the resultant approximately 12-amino acid transportin-binding consensus sequence is also predictive of nuclear localization signal activity. Surprisingly, however, this extensive mutational analysis failed to dissect M9 nuclear localization signal and nuclear export signal function. Nevertheless, transportin appears unlikely to be the M9 export receptor, as RanGTP can be shown to block M9 binding by transportin not only in vitro, but also in the nucleus in vivo. This analysis therefore predicts the existence of a nuclear export receptor distinct from transportin that nevertheless shares a common protein-binding site on heterogeneous nuclear ribonucleoprotein A1. PMID:10092666

  5. Intelligent signal analysis methodologies for nuclear detection, identification and attribution

    NASA Astrophysics Data System (ADS)

    Alamaniotis, Miltiadis

    Detection and identification of special nuclear materials can be fully performed with a radiation detector-spectrometer. Due to several physical and computational limitations, development of fast and accurate radioisotope identifier (RIID) algorithms is essential for automated radioactive source detection and characterization. The challenge is to identify individual isotope signatures embedded in spectral signature aggregation. In addition, background and isotope spectra overlap to further complicate the signal analysis. These concerns are addressed, in this thesis, through a set of intelligent methodologies recognizing signature spectra, background spectrum and, subsequently, identifying radionuclides. Initially, a method for detection and extraction of signature patterns is accomplished by means of fuzzy logic. The fuzzy logic methodology is applied on three types of radiation signal processing applications, where it exhibits high positive detection, low false alarm rate and very short execution time, while outperforming the maximum likelihood fitting approach. In addition, an innovative Pareto optimal multiobjective fitting of gamma ray spectra using evolutionary computing is presented. The methodology exhibits perfect identification while performs better than single objective fitting. Lastly, an innovative kernel based machine learning methodology was developed for estimating natural background spectrum in gamma ray spectra. The novelty of the methodology lies in the fact that it implements a data based approach and does not require any explicit physics modeling. Results show that kernel based method adequately estimates the gamma background, but algorithm's performance exhibits a strong dependence on the selected kernel.

  6. Novel role of cortactin in G protein-coupled receptor agonist-induced nuclear export and degradation of p21Cip1

    PubMed Central

    Janjanam, Jagadeesh; Rao, Gadiparthi N.

    2016-01-01

    Monocyte chemotactic protein 1 (MCP1) stimulates phosphorylation of cortactin on Y421 and Y446 residues in a time-dependent manner and phosphorylation at Y446 but not Y421 residue is required for MCP1-induced CDK-interacting protein 1 (p21Cip1) nuclear export and degradation in facilitating human aortic smooth muscle cell (HASMC) proliferation. In addition, MCP1-induced cortactin tyrosine phosphorylation, p21Cip1 degradation and HASMC proliferation are dependent on Fyn activation. Upstream to Fyn, MCP1 stimulated C-C chemokine receptor type 2 (CCR2) and Gi/o and inhibition of either one of these molecules using their specific antagonists or inhibitors attenuated MCP1-induced cortactin tyrosine phosphorylation, p21Cip1 degradation and HASMC proliferation. Cortactin phosphorylation at Y446 residue is also required for another G protein-coupled receptor (GPCR) agonist, thrombin-induced p21Cip1 nuclear export and its degradation in promoting HASMC proliferation. Quite interestingly, the receptor tyrosine kinase (RTK) agonist, platelet-derived growth factor-BB (PDGF-BB)-induced p21Cip1 degradation and HASMC proliferation do not require cortactin tyrosine phosphorylation. Together, these findings demonstrate that tyrosine phosphorylation of cortactin at Y446 residue is selective for only GPCR but not RTK agonist-induced nuclear export and proteolytic degradation of p21Cip1 in HASMC proliferation. PMID:27363897

  7. Novel role of cortactin in G protein-coupled receptor agonist-induced nuclear export and degradation of p21Cip1.

    PubMed

    Janjanam, Jagadeesh; Rao, Gadiparthi N

    2016-01-01

    Monocyte chemotactic protein 1 (MCP1) stimulates phosphorylation of cortactin on Y421 and Y446 residues in a time-dependent manner and phosphorylation at Y446 but not Y421 residue is required for MCP1-induced CDK-interacting protein 1 (p21Cip1) nuclear export and degradation in facilitating human aortic smooth muscle cell (HASMC) proliferation. In addition, MCP1-induced cortactin tyrosine phosphorylation, p21Cip1 degradation and HASMC proliferation are dependent on Fyn activation. Upstream to Fyn, MCP1 stimulated C-C chemokine receptor type 2 (CCR2) and Gi/o and inhibition of either one of these molecules using their specific antagonists or inhibitors attenuated MCP1-induced cortactin tyrosine phosphorylation, p21Cip1 degradation and HASMC proliferation. Cortactin phosphorylation at Y446 residue is also required for another G protein-coupled receptor (GPCR) agonist, thrombin-induced p21Cip1 nuclear export and its degradation in promoting HASMC proliferation. Quite interestingly, the receptor tyrosine kinase (RTK) agonist, platelet-derived growth factor-BB (PDGF-BB)-induced p21Cip1 degradation and HASMC proliferation do not require cortactin tyrosine phosphorylation. Together, these findings demonstrate that tyrosine phosphorylation of cortactin at Y446 residue is selective for only GPCR but not RTK agonist-induced nuclear export and proteolytic degradation of p21Cip1 in HASMC proliferation. PMID:27363897

  8. Nuclear PRP20 protein is required for mRNA export.

    PubMed Central

    Amberg, D C; Fleischmann, M; Stagljar, I; Cole, C N; Aebi, M

    1993-01-01

    The yeast PRP20 protein is highly homologous in structure and function to the RCC1 protein of higher eukaryotes. The RCC1 protein is involved in the regulation of the onset of mitosis, whereas the PRP20 protein was shown to be required for accurate and efficient mRNA metabolism. The first observable phenotype in mutant prp20 cells when shifted from permissive to non-permissive temperature is a loss of nuclear PRP20 protein. Concomitantly, an accumulation of poly(A)+ RNA in the nucleus is observed. The temperature-sensitive RCC1 allele in the mutant hamster cell line tsBN2 leads to a similar accumulation of mRNA in the nucleus. Images PMID:7679070

  9. Transcriptomine, a web resource for nuclear receptor signaling transcriptomes.

    PubMed

    Ochsner, Scott A; Watkins, Christopher M; McOwiti, Apollo; Xu, Xueping; Darlington, Yolanda F; Dehart, Michael D; Cooney, Austin J; Steffen, David L; Becnel, Lauren B; McKenna, Neil J

    2012-09-01

    The nuclear receptor (NR) superfamily of ligand-regulated transcription factors directs ligand- and tissue-specific transcriptomes in myriad developmental, metabolic, immunological, and reproductive processes. The NR signaling field has generated a wealth of genome-wide expression data points, but due to deficits in their accessibility, annotation, and integration, the full potential of these studies has not yet been realized. We searched public gene expression databases and MEDLINE for global transcriptomic datasets relevant to NRs, their ligands, and coregulators. We carried out extensive, deep reannotation of the datasets using controlled vocabularies for RNA Source and regulating molecule and resolved disparate gene identifiers to official gene symbols to facilitate comparison of fold changes and their significance across multiple datasets. We assembled these data points into a database, Transcriptomine (http://www.nursa.org/transcriptomine), that allows for multiple, menu-driven querying strategies of this transcriptomic "superdataset," including single and multiple genes, Gene Ontology terms, disease terms, and uploaded custom gene lists. Experimental variables such as regulating molecule, RNA Source, as well as fold-change and P value cutoff values can be modified, and full data records can be either browsed or downloaded for downstream analysis. We demonstrate the utility of Transcriptomine as a hypothesis generation and validation tool using in silico and experimental use cases. Our resource empowers users to instantly and routinely mine the collective biology of millions of previously disparate transcriptomic data points. By incorporating future transcriptome-wide datasets in the NR signaling field, we anticipate Transcriptomine developing into a powerful resource for the NR- and other signal transduction research communities. PMID:22786849

  10. Nuclear export and expression of human T-cell leukemia virus type 1 tax/rex mRNA are RxRE/Rex dependent.

    PubMed

    Bai, X T; Sinha-Datta, U; Ko, N L; Bellon, M; Nicot, C

    2012-04-01

    Human T-cell leukemia virus type 1 (HTLV-1) is a complex retrovirus associated with the lymphoproliferative disease adult T-cell leukemia/lymphoma (ATL) and the neurodegenerative disorder tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM). Replication of HTLV-1 is under the control of two major trans-acting proteins, Tax and Rex. Previous studies suggested that Tax activates transcription from the viral long terminal repeat (LTR) through recruitment of cellular CREB and transcriptional coactivators. Other studies reported that Rex acts posttranscriptionally and allows the cytoplasmic export of unspliced or incompletely spliced viral mRNAs carrying gag/pol and env only. As opposed to HIV's Rev-responsive element (RRE), the Rex-responsive element (RxRE) is present in all viral mRNAs in HTLV-1. However, based on indirect observations, it is believed that nuclear export and expression of the doubly spliced tax/rex RNA are Rex independent. In this study, we demonstrate that Rex does stimulate Tax expression, through nuclear-cytoplasmic export of the tax/rex RNA, even though a Rex-independent basal export mechanism exists. This effect was dependent upon the RxRE element and the RNA-binding activity of Rex. In addition, Rex-mediated export of tax/rex RNA was CRM1 dependent and inhibited by leptomycin B treatment. RNA immunoprecipitation (RNA-IP) experiments confirmed Rex binding to the tax/rex RNA in both transfected cells with HTLV-1 molecular clones and HTLV-1-infected T cells. Since both Rex and p30 interact with the tax/rex RNA and with one another, this may offer a temporal and dynamic regulation of HTLV-1 replication. Our results shed light on HTLV-1 replication and reveal a more complex regulatory network than previously anticipated. PMID:22318152