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Sample records for nuclear export signals

  1. Nuclear Export Signal Masking Regulates HIV-1 Rev Trafficking and Viral RNA Nuclear Export

    PubMed Central

    Behrens, Ryan T.; Aligeti, Mounavya; Pocock, Ginger M.; Higgins, Christina A.

    2016-01-01

    ABSTRACT HIV-1's Rev protein forms a homo-oligomeric adaptor complex linking viral RNAs to the cellular CRM1/Ran-GTP nuclear export machinery through the activity of Rev's prototypical leucine-rich nuclear export signal (NES). In this study, we used a functional fluorescently tagged Rev fusion protein as a platform to study the effects of modulating Rev NES identity, number, position, or strength on Rev subcellular trafficking, viral RNA nuclear export, and infectious virion production. We found that Rev activity was remarkably tolerant of diverse NES sequences, including supraphysiological NES (SNES) peptides that otherwise arrest CRM1 transport complexes at nuclear pores. Rev's ability to tolerate a SNES was both position and multimerization dependent, an observation consistent with a model wherein Rev self-association acts to transiently mask the NES peptide(s), thereby biasing Rev's trafficking into the nucleus. Combined imaging and functional assays also indicated that NES masking underpins Rev's well-known tendency to accumulate at the nucleolus, as well as Rev's capacity to activate optimal levels of late viral gene expression. We propose that Rev multimerization and NES masking regulates Rev's trafficking to and retention within the nucleus even prior to RNA binding. IMPORTANCE HIV-1 infects more than 34 million people worldwide causing >1 million deaths per year. Infectious virion production is activated by the essential viral Rev protein that mediates nuclear export of intron-bearing late-stage viral mRNAs. Rev's shuttling into and out of the nucleus is regulated by the antagonistic activities of both a peptide-encoded N-terminal nuclear localization signal and C-terminal nuclear export signal (NES). How Rev and related viral proteins balance strong import and export activities in order to achieve optimal levels of viral gene expression is incompletely understood. We provide evidence that multimerization provides a mechanism by which Rev transiently masks

  2. Nuclear Export Signal Masking Regulates HIV-1 Rev Trafficking and Viral RNA Nuclear Export.

    PubMed

    Behrens, Ryan T; Aligeti, Mounavya; Pocock, Ginger M; Higgins, Christina A; Sherer, Nathan M

    2017-02-01

    HIV-1's Rev protein forms a homo-oligomeric adaptor complex linking viral RNAs to the cellular CRM1/Ran-GTP nuclear export machinery through the activity of Rev's prototypical leucine-rich nuclear export signal (NES). In this study, we used a functional fluorescently tagged Rev fusion protein as a platform to study the effects of modulating Rev NES identity, number, position, or strength on Rev subcellular trafficking, viral RNA nuclear export, and infectious virion production. We found that Rev activity was remarkably tolerant of diverse NES sequences, including supraphysiological NES (SNES) peptides that otherwise arrest CRM1 transport complexes at nuclear pores. Rev's ability to tolerate a SNES was both position and multimerization dependent, an observation consistent with a model wherein Rev self-association acts to transiently mask the NES peptide(s), thereby biasing Rev's trafficking into the nucleus. Combined imaging and functional assays also indicated that NES masking underpins Rev's well-known tendency to accumulate at the nucleolus, as well as Rev's capacity to activate optimal levels of late viral gene expression. We propose that Rev multimerization and NES masking regulates Rev's trafficking to and retention within the nucleus even prior to RNA binding.

  3. Chikungunya virus capsid protein contains nuclear import and export signals

    PubMed Central

    2013-01-01

    Background Chikungunya virus (CHIKV) is an alphavirus of the Togaviridae family. After autoproteolytic cleavage, the CHIKV capsid protein (CP) is involved in RNA binding and assembly of the viral particle. The monomeric CP is approximately 30 kDa in size and is small enough for passive transport through nuclear pores. Some alphaviruses are found to harbor nuclear localization signals (NLS) and transport of these proteins between cellular compartments was shown to be energy dependent. The active nuclear import of cytoplasmic proteins is mediated by karyopherins and their export by exportins. As nuclear and cytoplasmic trafficking may play a role in the life cycle of CHIKV, we have sought to identify nuclear localization and nuclear export signals in CHIKV CP in a virus-free system. Methods EGFP-fusion proteins of CHIKV CP and mutants thereof were created and used to monitor their intracellular localization. Binding of cellular proteins was confirmed in pull-down assays with purified CP using co-immuoprecipitation. Nuclear localization was demonstrated in a virus-free system using fluorescence microscopy. Results Here we show that CHIKV CP is a nuclear-cytoplasmic shuttling protein with an active NLS that binds to karyopherin α (Karα) for its nuclear translocation. We also found that the Karα4 C-terminal NLS binding site is sufficient for this interaction. We further demonstrate that CHIKV CP interacts directly with the export receptor CRM1 to transport this viral protein out of the nucleus via a nuclear export signal (NES). The CHIKV CP NES was mapped between amino acids 143 and 155 of CP. Deduced from in silico analyses we found that the NES has a mode of binding similar to the snurportin-1 CRM1 complex. Conclusions We were able to show that in a virus-free system that the CHIKV capsid protein contains both, a NLS and a NES, and that it is actively transported between the cytoplasma and the nucleus. We conclude that CHIKV CP has the ability to shuttle via

  4. Chikungunya virus capsid protein contains nuclear import and export signals.

    PubMed

    Thomas, Saijo; Rai, Jagdish; John, Lijo; Schaefer, Stephan; Pützer, Brigitte M; Herchenröder, Ottmar

    2013-08-28

    Chikungunya virus (CHIKV) is an alphavirus of the Togaviridae family. After autoproteolytic cleavage, the CHIKV capsid protein (CP) is involved in RNA binding and assembly of the viral particle. The monomeric CP is approximately 30 kDa in size and is small enough for passive transport through nuclear pores. Some alphaviruses are found to harbor nuclear localization signals (NLS) and transport of these proteins between cellular compartments was shown to be energy dependent. The active nuclear import of cytoplasmic proteins is mediated by karyopherins and their export by exportins. As nuclear and cytoplasmic trafficking may play a role in the life cycle of CHIKV, we have sought to identify nuclear localization and nuclear export signals in CHIKV CP in a virus-free system. EGFP-fusion proteins of CHIKV CP and mutants thereof were created and used to monitor their intracellular localization. Binding of cellular proteins was confirmed in pull-down assays with purified CP using co-immuoprecipitation. Nuclear localization was demonstrated in a virus-free system using fluorescence microscopy. Here we show that CHIKV CP is a nuclear-cytoplasmic shuttling protein with an active NLS that binds to karyopherin α (Karα) for its nuclear translocation. We also found that the Karα4 C-terminal NLS binding site is sufficient for this interaction. We further demonstrate that CHIKV CP interacts directly with the export receptor CRM1 to transport this viral protein out of the nucleus via a nuclear export signal (NES). The CHIKV CP NES was mapped between amino acids 143 and 155 of CP. Deduced from in silico analyses we found that the NES has a mode of binding similar to the snurportin-1 CRM1 complex. We were able to show that in a virus-free system that the CHIKV capsid protein contains both, a NLS and a NES, and that it is actively transported between the cytoplasma and the nucleus. We conclude that CHIKV CP has the ability to shuttle via interaction with karyopherins for its

  5. Nuclear export receptor CRM1 recognizes diverse conformations in nuclear export signals.

    PubMed

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

    2017-03-10

    Nuclear export receptor CRM1 binds highly variable nuclear export signals (NESs) in hundreds of different cargoes. Previously we have shown that CRM1 binds NESs in both polypeptide orientations (Fung et al., 2015). Here, we show crystal structures of CRM1 bound to eight additional NESs which reveal diverse conformations that range from loop-like to all-helix, which occupy different extents of the invariant NES-binding groove. Analysis of all NES structures show 5-6 distinct backbone conformations where the only conserved secondary structural element is one turn of helix that binds the central portion of the CRM1 groove. All NESs also participate in main chain hydrogen bonding with human CRM1 Lys568 side chain, which acts as a specificity filter that prevents binding of non-NES peptides. The large conformational range of NES backbones explains the lack of a fixed pattern for its 3-5 hydrophobic anchor residues, which in turn explains the large array of peptide sequences that can function as NESs.

  6. Nuclear export receptor CRM1 recognizes diverse conformations in nuclear export signals

    PubMed Central

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

    2017-01-01

    Nuclear export receptor CRM1 binds highly variable nuclear export signals (NESs) in hundreds of different cargoes. Previously we have shown that CRM1 binds NESs in both polypeptide orientations (Fung et al., 2015). Here, we show crystal structures of CRM1 bound to eight additional NESs which reveal diverse conformations that range from loop-like to all-helix, which occupy different extents of the invariant NES-binding groove. Analysis of all NES structures show 5-6 distinct backbone conformations where the only conserved secondary structural element is one turn of helix that binds the central portion of the CRM1 groove. All NESs also participate in main chain hydrogen bonding with human CRM1 Lys568 side chain, which acts as a specificity filter that prevents binding of non-NES peptides. The large conformational range of NES backbones explains the lack of a fixed pattern for its 3-5 hydrophobic anchor residues, which in turn explains the large array of peptide sequences that can function as NESs. DOI: http://dx.doi.org/10.7554/eLife.23961.001 PMID:28282025

  7. Inhibition of mRNA export in vertebrate cells by nuclear export signal conjugates.

    PubMed

    Pasquinelli, A E; Powers, M A; Lund, E; Forbes, D; Dahlberg, J E

    1997-12-23

    Leucine-rich nuclear export signals (NESs) are recognized by the NES receptor exportin 1 and are central to the export of multiple shuttling proteins and RNAs. The export of messenger RNA in vertebrates was, however, thought to occur by a different pathway, because inhibition by injection of a synthetic Rev NES conjugate could not be demonstrated. Here we find that peptide conjugates composed of the NES of either protein kinase A inhibitor protein (PKI) or the HIV-1 Rev protein, when coupled to human serum albumin, are potent inhibitors of mRNA and small nuclear RNA export. These results provide direct evidence that mRNA export in vertebrates depends on interactions between an NES and its cognate NES receptors. PKI NES conjugates are significantly more efficient at inhibiting RNA export than are REV NES conjugates, indicating that different NESs may have different abilities to promote protein and RNA export. Surprisingly, an expected control conjugate containing the mutant Rev NES sequence M10 strongly inhibited the export of intronless dihydrofolate reductase mRNA. Nuclear injection of NES peptide conjugates led to mislocalization to the nucleus of 10-20% of the cytoplasmic Ran GTPase-binding protein (RanBP1) indicating that RanBP1 shuttles between the nucleus and the cytoplasm via an NES pathway. These results demonstrate that in vertebrates the export of mRNA, like that of small nuclear RNA, 5S rRNA, and transport factors such as RanBP1, employs NES-mediated molecular machinery.

  8. Prediction of nuclear export signals using weighted regular expressions (Wregex).

    PubMed

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

    2014-05-01

    Leucine-rich nuclear export signals (NESs) are short amino acid motifs that mediate binding of cargo proteins to the nuclear export receptor CRM1, and thus contribute to regulate the localization and function of many cellular proteins. Computational prediction of NES motifs is of great interest, but remains a significant challenge. We have developed a novel approach for amino acid motif searching that can be used for NES prediction. This approach, termed Wregex (weighted regular expression), combines regular expressions with a position-specific scoring matrix (PSSM), and has been implemented in a web-based, freely available, software tool. By making use of a PSSM, Wregex provides a score to prioritize candidates for experimental testing. Key features of Wregex include its flexibility, which makes it useful for searching other types of protein motifs, and its fast execution time, which makes it suitable for large-scale analysis. In comparative tests with previously available prediction tools, Wregex is shown to offer a good rate of true-positive motifs, while keeping a smaller number of potential candidates.

  9. 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.

  10. Nuclear export signal-interacting protein forms complexes with lamin A/C-Nups to mediate the CRM1-independent nuclear export of large hepatitis delta antigen.

    PubMed

    Huang, Cheng; Jiang, Jia-Yin; Chang, Shin C; Tsay, Yeou-Guang; Chen, Mei-Ru; Chang, Ming-Fu

    2013-02-01

    Nuclear export is an important process that not only regulates the functions of cellular factors but also facilitates the assembly of viral nucleoprotein complexes. Chromosome region maintenance 1 (CRM1) that mediates the transport of proteins bearing the classical leucine-rich nuclear export signal (NES) is the best-characterized nuclear export receptor. Recently, several CRM1-independent nuclear export pathways were also identified. The nuclear export of the large form of hepatitis delta antigen (HDAg-L), a nucleocapsid protein of hepatitis delta virus (HDV), which contains a CRM1-independent proline-rich NES, is mediated by the host NES-interacting protein (NESI). The mechanism of the NESI protein in mediating nuclear export is still unknown. In this study, NESI was characterized as a highly glycosylated membrane protein. It interacted and colocalized well in the nuclear envelope with lamin A/C and nucleoporins. Importantly, HDAg-L could be coimmunoprecipitated with lamin A/C and nucleoporins. In addition, binding of the cargo HDAg-L to the C terminus of NESI was detected for the wild-type protein but not for the nuclear export-defective HDAg-L carrying a P205A mutation [HDAg-L(P205A)]. Knockdown of lamin A/C effectively reduced the nuclear export of HDAg-L and the assembly of HDV. These data indicate that by forming complexes with lamin A/C and nucleoporins, NESI facilitates the CRM1-independent nuclear export of HDAg-L.

  11. The signal sequence coding region promotes nuclear export of mRNA.

    PubMed

    Palazzo, Alexander F; Springer, Michael; Shibata, Yoko; Lee, Chung-Sheng; Dias, Anusha P; Rapoport, Tom A

    2007-12-01

    In eukaryotic cells, most mRNAs are exported from the nucleus by the transcription export (TREX) complex, which is loaded onto mRNAs after their splicing and capping. We have studied in mammalian cells the nuclear export of mRNAs that code for secretory proteins, which are targeted to the endoplasmic reticulum membrane by hydrophobic signal sequences. The mRNAs were injected into the nucleus or synthesized from injected or transfected DNA, and their export was followed by fluorescent in situ hybridization. We made the surprising observation that the signal sequence coding region (SSCR) can serve as a nuclear export signal of an mRNA that lacks an intron or functional cap. Even the export of an intron-containing natural mRNA was enhanced by its SSCR. Like conventional export, the SSCR-dependent pathway required the factor TAP, but depletion of the TREX components had only moderate effects. The SSCR export signal appears to be characterized in vertebrates by a low content of adenines, as demonstrated by genome-wide sequence analysis and by the inhibitory effect of silent adenine mutations in SSCRs. The discovery of an SSCR-mediated pathway explains the previously noted amino acid bias in signal sequences and suggests a link between nuclear export and membrane targeting of mRNAs.

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

    PubMed Central

    Onder, Zeynep; Chang, Vivian; Moroianu, Junona

    2014-01-01

    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), 76IRTFQELLF84, 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. PMID:25463601

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

    PubMed

    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), 76IRTFQELLF84, 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.

  14. 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.

  15. 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.

  16. A second CRM1-dependent nuclear export signal in the influenza A virus NS2 protein contributes to the nuclear export of viral ribonucleoproteins.

    PubMed

    Huang, Shengping; Chen, Jingjing; Chen, Quanjiao; Wang, Huadong; Yao, Yanfeng; Chen, Jianjun; Chen, Ze

    2013-01-01

    Influenza A virus NS2 protein, also called nuclear export protein (NEP), is crucial for the nuclear export of viral ribonucleoproteins. However, the molecular mechanisms of NEP mediation in this process remain incompletely understood. A leucine-rich nuclear export signal (NES2) in NEP, located at the predicted N2 helix of the N-terminal domain, was identified in the present study. NES2 was demonstrated to be a transferable NES, with its nuclear export activity depending on the nuclear export receptor chromosome region maintenance 1 (CRM1)-mediated pathway. The interaction between NEP and CRM1 is coordinately regulated by both the previously reported NES (NES1) and now the new NES2. Deletion of the NES1 enhances the interaction between NEP and CRM1, and deletion of the NES1 and NES2 motifs completely abolishes this interaction. Moreover, NES2 interacts with CRM1 in the mammalian two-hybrid system. Mutant viruses containing NES2 alterations generated by reversed genetics exhibit reduced viral growth and delay in the nuclear export of viral ribonucleoproteins (vRNPs). The NES2 motif is highly conserved in the influenza A and B viruses. The results demonstrate that leucine-rich NES2 is involved in the nuclear export of vRNPs and contributes to the understanding of nucleocytoplasmic transport of influenza virus vRNPs.

  17. 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.

  18. NESmapper: accurate prediction of leucine-rich nuclear export signals using activity-based profiles.

    PubMed

    Kosugi, Shunichi; Yanagawa, Hiroshi; Terauchi, Ryohei; Tabata, Satoshi

    2014-09-01

    The nuclear export of proteins is regulated largely through the exportin/CRM1 pathway, which involves the specific recognition of leucine-rich nuclear export signals (NESs) in the cargo proteins, and modulates nuclear-cytoplasmic protein shuttling by antagonizing the nuclear import activity mediated by importins and the nuclear import signal (NLS). Although the prediction of NESs can help to define proteins that undergo regulated nuclear export, current methods of predicting NESs, including computational tools and consensus-sequence-based searches, have limited accuracy, especially in terms of their specificity. We found that each residue within an NES largely contributes independently and additively to the entire nuclear export activity. We created activity-based profiles of all classes of NESs with a comprehensive mutational analysis in mammalian cells. The profiles highlight a number of specific activity-affecting residues not only at the conserved hydrophobic positions but also in the linker and flanking regions. We then developed a computational tool, NESmapper, to predict NESs by using profiles that had been further optimized by training and combining the amino acid properties of the NES-flanking regions. This tool successfully reduced the considerable number of false positives, and the overall prediction accuracy was higher than that of other methods, including NESsential and Wregex. This profile-based prediction strategy is a reliable way to identify functional protein motifs. NESmapper is available at http://sourceforge.net/projects/nesmapper.

  19. 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

    2016-06-02

    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.

  20. Nuclear export signal of PRRSV NSP1α is necessary for type I IFN inhibition.

    PubMed

    Chen, Zhi; Liu, Shaoning; Sun, Wenbo; Chen, Lei; Yoo, Dongwan; Li, Feng; Ren, Sufang; Guo, Lihui; Cong, Xiaoyan; Li, Jun; Zhou, Shun; Wu, Jiaqiang; Du, Yijun; Wang, Jinbao

    2016-12-01

    The nonstructural protein 1α (NSP1α) of porcine reproductive and respiratory syndrome virus (PRRSV) is a nucleo-cytoplasmic protein that suppresses the production of type I interferon (IFN). In this study, we investigated the relationship between the subcellular distribution of NSP1α and its inhibition of type I IFN. NSP1α was found to contain the classical nuclear export signal (NES) and NSP1α nuclear export was CRM-1-mediated. NSP1α was shuttling between the nucleus and cytoplasm. We also showed that the nuclear export of NSP1α was necessary for its ability for type I IFN inhibition. NSP1α was also found to interact with CBP, which implies a possible mechanism of CBP degradation by NSP1α. Taken together, our results describe a novel mechanism of PRRSV NSP1α for type I IFN inhibition and suppression of the host innate antiviral response. Copyright © 2016. Published by Elsevier Inc.

  1. 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.

  2. Multiple Novel Signals Mediate Thyroid Hormone Receptor Nuclear Import and Export*

    PubMed Central

    Mavinakere, Manohara S.; Powers, Jeremy M.; Subramanian, Kelly S.; Roggero, Vincent R.; Allison, Lizabeth A.

    2012-01-01

    Thyroid hormone receptor (TR) is a member of the nuclear receptor superfamily that shuttles between the cytosol and nucleus. The fine balance between nuclear import and export of TR has emerged as a critical control point for modulating thyroid hormone-responsive gene expression; however, sequence motifs of TR that mediate shuttling are not fully defined. Here, we characterized multiple signals that direct TR shuttling. Along with the known nuclear localization signal in the hinge domain, we identified a novel nuclear localization signal in the A/B domain of thyroid hormone receptor α1 that is absent in thyroid hormone receptor β1 and inactive in the oncoprotein v-ErbA. Our prior studies showed that thyroid hormone receptor α1 exits the nucleus through two pathways, one dependent on the export factor CRM1 and the other CRM1-independent. Here, we identified three novel CRM1-independent nuclear export signal (NES) motifs in the ligand-binding domain as follows: a highly conserved NES in helix 12 (NES-H12) and two additional NES sequences spanning helix 3 and helix 6, respectively. Mutations predicted to disrupt the α-helical structure resulted in a significant decrease in NES-H12 activity. The high degree of conservation of helix 12 suggests that this region may function as a key NES in other nuclear receptors. Furthermore, our mutagenesis studies on NES-H12 suggest that altered shuttling of thyroid hormone receptor β1 may be a contributing factor in resistance to thyroid hormone syndrome. Taken together, our findings provide a detailed mechanistic understanding of the multiple signals that work together to regulate TR shuttling and transcriptional activity, and they provide important insights into nuclear receptor function in general. PMID:22815488

  3. 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

  4. Identification of a functional, CRM-1-dependent nuclear export signal in hepatitis C virus core protein.

    PubMed

    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.

  5. The tight junction protein Z O-2 has several functional nuclear export signals

    SciTech Connect

    Gonzalez-Mariscal, Lorenza . E-mail: lorenza@fisio.cinvestav.mx; Ponce, Arturo; Alarcon, Lourdes; Jaramillo, Blanca Estela

    2006-10-15

    The tight junction (TJ) protein ZO-2 changes its subcellular distribution according to the state of confluency of the culture. Thus in confluent monolayers, it localizes at the TJ region whereas in sparse cultures it concentrates at the nucleus. The canine sequence of ZO-2 displays four putative nuclear export signals (NES), two at the second PDZ domain (NES-0 and NES-1) and the rest at the GK region (NES-2 and NES-3). The functionality of NES-0 and NES-3 was unknown, hence here we have explored it with a nuclear export assay, injecting into the nucleus of MDCK cells peptides corresponding to the ZO-2 NES sequences chemically coupled to ovalbumin. We show that both NES-0 and NES-3 are functional and sensitive to leptomycin B. We also demonstrate that NES-1, previously characterized as a non functional NES, is rendered capable of nuclear export upon the acquisition of a negative charge at its Ser369 residue. Experiments performed injecting at the nucleus WT and mutated ZO-2-GST fusion proteins revealed the need of both NES-0 and NES-1, and NES-2 and NES-3 for attaining an efficient nuclear exit of the respective amino and middle segments of ZO-2. Moreover, the transfection of MDCK cells with full-length ZO-2 revealed that the mutation of any of the NES present in the molecule was sufficient to induce nuclear accumulation of the protein.

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

    DOE PAGES

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

    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

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

    PubMed

    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). 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.

  8. 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.

  9. Acid Ceramidase Promotes Nuclear Export of PTEN through Sphingosine 1-Phosphate Mediated Akt Signaling

    PubMed Central

    Beckham, Thomas H.; Cheng, Joseph C.; Lu, Ping; Marrison, S. Tucker; Norris, James S.; Liu, Xiang

    2013-01-01

    The tumor suppressor PTEN is now understood to regulate cellular processes at the cytoplasmic membrane, where it classically regulates PI3K signaling, as well as in the nucleus where multiple roles in controlling cell cycle and genome stability have been elucidated. Mechanisms that dictate nuclear import and, less extensively, nuclear export of PTEN have been described, however the relevance of these processes in disease states, particularly cancer, remain largely unknown. We investigated the impact of acid ceramidase on the nuclear-cytoplasmic trafficking of PTEN. Immunohistochemical analysis of a human prostate tissue microarray revealed that nuclear PTEN was lost in patients whose tumors had elevated acid ceramidase. We found that acid ceramidase promotes a reduction in nuclear PTEN that is dependent upon sphingosine 1-phosphate-mediated activation of Akt. We were further able to show that sphingosine 1-phosphate promotes formation of a complex between Crm1 and PTEN, and that leptomycin B prevents acid ceramidase and sphingosine 1-phosphate mediated loss of nuclear PTEN, suggesting an active exportin-mediated event. To investigate whether the tumor promoting aspects of acid ceramidase in prostate cancer depend upon its ability to export PTEN from the nucleus, we used enforced nuclear expression of PTEN to study docetaxel-induced apoptosis and cell killing, proliferation, and xenoengraftment. Interestingly, while acid ceramidase was able to protect cells expressing wild type PTEN from docetaxel, promote proliferation and xenoengraftment, acid ceramidase had no impact in cells expressing PTEN-NLS. These findings suggest that acid ceramidase, through sphingosine 1-phosphate, promotes nuclear export of PTEN as a means of promoting tumor formation, cell proliferation, and resistance to therapy. PMID:24098536

  10. 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.

  11. A calreticulin-dependent nuclear export signal is involved in the regulation of liver receptor homologue-1 protein folding.

    PubMed

    Yang, Feng-Ming; Feng, Shan-Jung; Lai, Tsai-Chun; Hu, Meng-Chun

    2015-10-15

    As an orphan member of the nuclear receptor family, liver receptor homologue-1 (LRH-1) controls a tremendous range of transcriptional programmes that are essential for metabolism and hormone synthesis. Our previous studies have shown that nuclear localization of the LRH-1 protein is mediated by two nuclear localization signals (NLSs) that are karyopherin/importin-dependent. It is unclear whether LRH-1 can be actively exported from the nucleus to the cytoplasm. In the present study, we describe a nuclear export domain containing two leucine-rich motifs [named nuclear export signal (NES)1 and NES2] within the ligand-binding domain (LBD). Mutation of leucine residues in NES1 or NES2 abolished nuclear export, indicating that both NES1 and NES2 motifs are essential for full nuclear export activity. This NES-mediated nuclear export was insensitive to the chromosomal region maintenance 1 (CRM1) inhibitor leptomycin B (LMB) or to CRM1 knockdown. However, knockdown of calreticulin (CRT) prevented NES-mediated nuclear export. Furthermore, our data show that CRT interacts with LRH-1 and is involved in the nuclear export of LRH-1. With full-length LRH-1, mutation of NES1 led to perinuclear accumulation of the mutant protein. Immunofluorescence analysis showed that these perinuclear aggregates were co-localized with the centrosome marker, microtubule-associated protein 1 light chain 3 (LC3), ubiquitin and heat shock protein 70 (Hsp70), indicating that the mutant was misfolded and sequestered into aggresome-like structures via the autophagic clearance pathway. Our study demonstrates for the first time that LRH-1 has a CRT-dependent NES which is not only required for cytoplasmic trafficking, but also essential for correct protein folding to avoid misfolding-induced aggregation. © 2015 Authors; published by Portland Press Limited.

  12. Dual function of the nuclear export signal of the Borna disease virus nucleoprotein in nuclear export activity and binding to viral phosphoprotein.

    PubMed

    Yanai, Mako; Sakai, Madoka; Makino, Akiko; Tomonaga, Keizo

    2017-07-11

    Borna disease virus (BoDV), which has a negative-sense, single-stranded RNA genome, causes persistent infection in the cell nucleus. The nuclear export signal (NES) of the viral nucleoprotein (N) consisting of leucine at positions 128 and 131 and isoleucine at positions 133 and 136 overlaps with one of two predicted binding sites for the viral phosphoprotein (P). A previous study demonstrated that higher expression of BoDV-P inhibits nuclear export of N; however, the function of N NES in the interaction with P remains unclear. We examined the subcellular localization, viral polymerase activity, and P-binding ability of BoDV-N NES mutants. We also characterized a recombinant BoDV (rBoDV) harboring an NES mutation of N. BoDV-N with four alanine-substitutions in the leucine and isoleucine residues of the NES impaired its cytoplasmic localization and abolished polymerase activity and P-binding ability. Although an alanine-substitution at position 131 markedly enhanced viral polymerase activity as determined by a minigenome assay, rBoDV harboring this mutation showed expression of viral RNAs and proteins relative to that of wild-type rBoDV. Our results demonstrate that BoDV-N NES has a dual function in BoDV replication, i.e., nuclear export of N and an interaction with P, affecting viral polymerase activity in the nucleus.

  13. Subcellular trafficking signals of constitutive androstane receptor: evidence for a nuclear export signal in the DNA-binding domain.

    PubMed

    Xia, Jun; Kemper, Byron

    2007-09-01

    Translocation of constitutive androstane receptor (CAR) from the cytoplasm to the nucleus is induced by phenobarbital-like drugs. Nuclear localization signals (NLSs) and a sequence [xenochemical response signal (XRS)] required for xenobiotic-induced nuclear translocation have been defined in rat and human CAR, but a nuclear export signal (NES) has not been identified. To identify cellular localization signals of CAR, the localization of fragments and mutants of mouse CAR expressed in mouse hepatocytes in vivo was examined. Consistent with other studies, an NLS in the hinge region, a diffuse NLS in the ligand-binding domain, and a cytoplasmic retention sequence were identified, and mutation of the XRS blocked nuclear accumulation both in phenobarbital-treated mice in vivo and in untreated HepG2 cells. Fusing the simian virus 40 NLS to the mutant proteins reversed the localization defect resulting from mutation of the hinge NLS but not that from mutation of the XRS, indicating that the XRS is not simply a novel phenobarbital-responsive NLS. In the DNA-binding domain, a sequence in CAR is conserved with an NES identified in other nuclear receptors. Mutation of two conserved phenylalanines in this sequence resulted in increased nuclear localization of both full-length CAR and a CAR fragment containing the DNA-binding domain. The DNA-binding domain sequence, therefore, may contain an NES, which is consistent with nucleocytoplasmic shuttling of CAR. The results demonstrate that regulation of the cellular localization of CAR is complex, with multiple sequences mediating nuclear import and export and retention in the cytoplasm.

  14. 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%.

  15. A novel nuclear export signal and a REF interaction domain both promote mRNA export by the Epstein-Barr virus EB2 protein.

    PubMed

    Hiriart, Edwige; Farjot, Geraldine; Gruffat, Henri; Nguyen, Minh Vu Chuong; Sergeant, Alain; Manet, Evelyne

    2003-01-03

    A striking characteristic of mRNA export factors is that they shuttle continuously between the cytoplasm and the nucleus. This shuttling is mediated by specific factors interacting with peptide motifs called nuclear export signals (NES) and nuclear localization signals. We have identified a novel CRM-1-independent transferable NES and two nuclear localization signals in the Epstein-Barr virus mRNA export factor EB2 (also called BMLF1, Mta, or SM) localized at the N terminus of the protein between amino acids 61 and 146. We have also found that a previously described double NES (amino acids 213-236) does not mediate the nuclear shuttling of EB2, but is an interaction domain with the cellular export factor REF in vitro. This newly characterized REF interaction domain is essential for EB2-mediated mRNA export. Accordingly, in vivo, EB2 is found in complexes containing REF as well as the cellular factor TAP. However, these interactions are RNase-sensitive, suggesting that the RNA is an essential component of these complexes.

  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. In silico analyses of dystrophin Dp40 cellular distribution, nuclear export signals and structure modeling

    PubMed Central

    Martínez-Herrera, Alejandro; Aragón, Jorge; Bermúdez-Cruz, Rosa Ma.; Bazán, Ma. Luisa; Soid-Raggi, Gabriela; Ceja, Víctor; Santos Coy-Arechavaleta, Andrea; Alemán, Víctor; Depardón, Francisco; Montañez, Cecilia

    2015-01-01

    Dystrophin Dp40 is the shortest protein encoded by the DMD (Duchenne muscular dystrophy) gene. This protein is unique since it lacks the C-terminal end of dystrophins. In this data article, we describe the subcellular localization, nuclear export signals and the three-dimensional structure modeling of putative Dp40 proteins using bioinformatics tools. The Dp40 wild type protein was predicted as a cytoplasmic protein while the Dp40n4 was predicted to be nuclear. Changes L93P and L170P are involved in the nuclear localization of Dp40n4 protein. A close analysis of Dp40 protein scored that amino acids 93LEQEHNNLV101 and 168LLLHDSIQI176 could function as NES sequences and the scores are lost in Dp40n4. In addition, the changes L93/170P modify the tertiary structure of putative Dp40 mutants. The analysis showed that changes of residues 93 and 170 from leucine to proline allow the nuclear localization of Dp40 proteins. The data described here are related to the research article entitled “EF-hand domains are involved in the differential cellular distribution of dystrophin Dp40” (J. Aragón et al. Neurosci. Lett. 600 (2015) 115–120) [1]. PMID:26217814

  18. A novel transferable nuclear export signal mediates CRM1-independent nucleocytoplasmic shuttling of the human cytomegalovirus transactivator protein pUL69

    PubMed Central

    Lischka, Peter; Rosorius, Olaf; Trommer, Erik; Stamminger, Thomas

    2001-01-01

    The best studied nuclear export processes are mediated by classical leucine-rich nuclear export signals that specify recognition by the CRM1 export receptor. However, details concerning alternative nuclear export signals and pathways are beginning to emerge. Within the family of Herpesviridae, a set of homologous regulatory proteins that are exemplified by the ICP27 of herpes simplex virus were described recently as nucleocytoplasmic shuttling proteins. Here we report that pUL69 of the β-herpesvirus human cytomegalovirus is a nuclear protein that is able to shuttle between the nucleus and the cytoplasm independently of virus-encoded cofactors. In contrast to proteins containing a leucine-rich export signal, the shuttling activity of pUL69 was not affected by leptomycin B, indicating that pUL69 trafficking is not mediated by the export receptor CRM1. Importantly, we identified and characterized a novel type of transferable, leptomycin B-insensitive export signal that is distinct from other export signals described previously and is required for pUL69-mediated activation of gene expression. These data suggest that pUL69 is exported via a novel nuclear export pathway, based on a so far unique nuclear export signal of 28 amino acids. PMID:11743003

  19. A nuclear export signal within the structural Gag protein is required for prototype foamy virus replication

    PubMed Central

    2011-01-01

    Background The Gag polyproteins play distinct roles during the replication cycle of retroviruses, hijacking many cellular machineries to fulfill them. In the case of the prototype foamy virus (PFV), Gag structural proteins undergo transient nuclear trafficking after their synthesis, returning back to the cytoplasm for capsid assembly and virus egress. The functional role of this nuclear stage as well as the molecular mechanism(s) responsible for Gag nuclear export are not understood. Results We have identified a leptomycin B (LMB)-sensitive nuclear export sequence (NES) within the N-terminus of PFV Gag that is absolutely required for the completion of late stages of virus replication. Point mutations of conserved residues within this motif lead to nuclear redistribution of Gag, preventing subsequent virus egress. We have shown that a NES-defective PFV Gag acts as a dominant negative mutant by sequestrating its wild-type counterpart in the nucleus. Trans-complementation experiments with the heterologous NES of HIV-1 Rev allow the cytoplasmic redistribution of FV Gag, but fail to restore infectivity. Conclusions PFV Gag-Gag interactions are finely tuned in the cytoplasm to regulate their functions, capsid assembly, and virus release. In the nucleus, we have shown Gag-Gag interactions which could be involved in the nuclear export of Gag and viral RNA. We propose that nuclear export of unspliced and partially spliced PFV RNAs relies on two complementary mechanisms, which take place successively during the replication cycle. PMID:21255441

  20. Identification of a Nuclear Export Signal Sequence for Bovine Papillomavirus E1 Protein

    PubMed Central

    Rosas-Acosta, Germán; Wilson, Van G.

    2008-01-01

    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 the 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 pulldown assays. 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 wildtype E1, yet was defective for viral origin replication, implying that nucleocytoplasmic shuttling may be required to maintain E1 in a replication competent state. PMID:18201744

  1. 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.

  2. Epstein-Barr virus protein EB2 contains an N-terminal transferable nuclear export signal that promotes nucleocytoplasmic export by directly binding TAP/NXF1.

    PubMed

    Juillard, Franceline; Hiriart, Edwige; Sergeant, Nicolas; Vingtdeux-Didier, Valérie; Drobecq, Hervé; Sergeant, Alain; Manet, Evelyne; Gruffat, Henri

    2009-12-01

    The Epstein-Barr virus early protein EB2 (also called BMLF1, Mta, or SM), which allows the nuclear export of a subset of early and late viral mRNAs derived from intronless genes, is essential for the production of infectious virions. An important feature of mRNA export factors is their capacity to shuttle continuously between the nucleus and the cytoplasm. In a previous study, we identified a novel CRM1-independent transferable nuclear export signal (NES) at the N terminus of EB2, between amino acids 61 and 146. Here we show that this NES contains several small arginine-rich domains that cooperate to allow efficient interaction with TAP/NXF1. Recruitment of TAP/NXF1 correlates with this NES-mediated efficient nuclear export when it is fused to a heterologous protein. Moreover, the NES can export mRNAs bearing MS2 RNA-binding sites from the nucleus when tethered to the RNA via the MS2 phage coat protein RNA-binding domain.

  3. Epstein-Barr Virus Protein EB2 Contains an N-Terminal Transferable Nuclear Export Signal That Promotes Nucleocytoplasmic Export by Directly Binding TAP/NXF1▿

    PubMed Central

    Juillard, Franceline; Hiriart, Edwige; Sergeant, Nicolas; Vingtdeux-Didier, Valérie; Drobecq, Hervé; Sergeant, Alain; Manet, Evelyne; Gruffat, Henri

    2009-01-01

    The Epstein-Barr virus early protein EB2 (also called BMLF1, Mta, or SM), which allows the nuclear export of a subset of early and late viral mRNAs derived from intronless genes, is essential for the production of infectious virions. An important feature of mRNA export factors is their capacity to shuttle continuously between the nucleus and the cytoplasm. In a previous study, we identified a novel CRM1-independent transferable nuclear export signal (NES) at the N terminus of EB2, between amino acids 61 and 146. Here we show that this NES contains several small arginine-rich domains that cooperate to allow efficient interaction with TAP/NXF1. Recruitment of TAP/NXF1 correlates with this NES-mediated efficient nuclear export when it is fused to a heterologous protein. Moreover, the NES can export mRNAs bearing MS2 RNA-binding sites from the nucleus when tethered to the RNA via the MS2 phage coat protein RNA-binding domain. PMID:19793817

  4. Prediction of leucine-rich nuclear export signal containing proteins with NESsential

    PubMed Central

    Fu, Szu-Chin; Imai, Kenichiro; Horton, Paul

    2011-01-01

    The classical nuclear export signal (NES), also known as the leucine-rich NES, is a protein localization signal often involved in important processes such as signal transduction and cell cycle regulation. Although 15 years has passed since its discovery, limited structural information and high sequence diversity have hampered understanding of the NES. Several consensus sequences have been proposed to describe it, but they suffer from poor predictive power. On the other hand, the NetNES server provides the only computational method currently available. Although these two methods have been widely used to attempt to find the correct NES position within potential NES-containing proteins, their performance has not yet been evaluated on the basic task of identifying NES-containing proteins. We propose a new predictor, NESsential, which uses sequence derived meta-features, such as predicted disorder and solvent accessibility, in addition to primary sequence. We demonstrate that it can identify promising NES-containing candidate proteins (albeit at low coverage), but other methods cannot. We also quantitatively demonstrate that predicted disorder is a useful feature for prediction and investigate the different features of (predicted) ordered versus disordered NES’s. Finally, we list 70 recently discovered NES-containing proteins, doubling the number available to the community. PMID:21705415

  5. A nuclear export signal in the matrix protein of Influenza A virus is required for efficient virus replication.

    PubMed

    Cao, Shuai; Liu, Xiaoling; Yu, Maorong; Li, Jing; Jia, Xiaojuan; Bi, Yuhai; Sun, Lei; Gao, George F; Liu, Wenjun

    2012-05-01

    The influenza A virus matrix 1 protein (M1) shuttles between the cytoplasm and the nucleus during the viral life cycle and plays an important role in the replication, assembly, and budding of viruses. Here, a leucine-rich nuclear export signal (NES) was identified specifically for the nuclear export of the M1 protein. The predicted NES, designated the Flu-A-M1 NES, is highly conserved among all sequences from the influenza A virus subtype, but no similar NES motifs are found in the M1 sequences of influenza B or C viruses. The biological function of the Flu-A-M1 NES was demonstrated by its ability to translocate an enhanced green fluorescent protein (EGFP)-NES fusion protein from the nucleus to the cytoplasm in transfected cells, compared to the even nuclear and cytoplasmic distribution of EGFP. The translocation of EGFP-NES from the nucleus to the cytoplasm was not inhibited by leptomycin B. NES mutations in M1 caused a nuclear retention of the protein and an increased nuclear accumulation of NEP during transfection. Indeed, as shown by rescued recombinant viruses, the mutation of the NES impaired the nuclear export of M1 and significantly reduced the virus titer compared to titers of wild-type viruses. The NES-defective M1 protein was retained in the nucleus during infection, accompanied by a lowered efficiency of the nuclear export of viral RNPs (vRNPs). In conclusion, M1 nuclear export was specifically dependent on the Flu-A-M1 NES and critical for influenza A virus replication.

  6. Characterization of a nuclear export signal within the human T cell leukemia virus type I transactivator protein Tax.

    PubMed

    Alefantis, Timothy; Barmak, Kate; Harhaj, Edward W; Grant, Christian; Wigdahl, Brian

    2003-06-13

    Human T cell leukemia virus type I (HTLV-I) is the etiologic agent of adult T cell leukemia and HTLV-I-associated myelopathy/tropical spastic paraparesis. The HTLV-I transactivator protein Tax plays an integral role in the etiology of adult T cell leukemia, as expression of Tax in T lymphocytes has been shown to result in immortalization. In addition, Tax is known to interface with numerous transcription factor families, including activating transcription factor/cAMP response element-binding protein and nuclear factor-kappaB, requiring Tax to localize to both the nucleus and cytoplasm. In this report, the nucleocytoplasmic localization of Tax was examined in Jurkat, HeLa, and U-87 MG cells. The results reported herein indicate that Tax contains a leucine-rich nuclear export signal (NES) that, when fused to green fluorescent protein (GFP), can direct nuclear export via the CRM-1 pathway, as determined by leptomycin B inhibition of nuclear export. However, cytoplasmic localization of full-length Tax was not altered by treatment with leptomycin B, suggesting that native Tax utilizes another nuclear export pathway. Additional support for the presence of a functional NES has also been shown because the NES mutant Tax(L200A)-GFP localized to the nuclear membrane in the majority of U-87 MG cells. Evidence has also been provided suggesting that the Tax NES likely exists as a conditionally masked signal because the truncation mutant TaxDelta214-GFP localized constitutively to the cytoplasm. These results suggest that Tax localization may be directed by specific changes in Tax conformation or by specific interactions with cellular proteins leading to changes in the availability of the Tax NES and nuclear localization signal.

  7. A nuclear export signal within the high mobility group domain regulates the nucleocytoplasmic translocation of SOX9 during sexual determination.

    PubMed

    Gasca, Stephan; Canizares, Joaquin; De Santa Barbara, Pascal; Mejean, Catherine; Poulat, Francis; Berta, Philippe; Boizet-Bonhoure, Brigitte

    2002-08-20

    In mammals, male sex determination starts when the Y chromosome Sry gene is expressed within the undetermined male gonad. One of the earliest effect of Sry expression is to induce up-regulation of Sox9 gene expression in the developing gonad. SOX9, like SRY, contains a high mobility group domain and is sufficient to induce testis differentiation in transgenic XX mice. Before sexual differentiation, SOX9 protein is initially found in the cytoplasm of undifferentiated gonads from both sexes. At the time of testis differentiation and anti-Müllerian hormone expression, it becomes localized to the nuclear compartment in males whereas it is down-regulated in females. In this report, we used NIH 3T3 cells as a model to examine the regulation of SOX9 nucleo-cytoplasmic shuttling. SOX9-transfected cells expressed nuclear and cytoplasmic SOX9 whereas transfected cells treated with the nuclear export inhibitor leptomycin B, displayed an exclusive nuclear localization of SOX9. By using SOX9 deletion constructs in green fluorescent protein fusion proteins, we identified a functional nuclear export signal sequence between amino acids 134 and 147 of SOX9 high mobility group box. More strikingly, we show that inhibiting nuclear export with leptomycin B in mouse XX gonads cultured in vitro induced a sex reversal phenotype characterized by nuclear SOX9 and anti-Müllerian hormone expression. These results indicate that SOX9 nuclear export signal is essential for SOX9 sex-specific subcellular localization and could be part of a regulatory switch repressing (in females) or triggering (in males) male-specific sexual differentiation.

  8. A nuclear export signal within the high mobility group domain regulates the nucleocytoplasmic translocation of SOX9 during sexual determination

    PubMed Central

    Gasca, Stéphan; Cañizares, Joaquin; de Santa Barbara, Pascal; Méjean, Catherine; Poulat, Francis; Berta, Philippe; Boizet-Bonhoure, Brigitte

    2002-01-01

    In mammals, male sex determination starts when the Y chromosome Sry gene is expressed within the undetermined male gonad. One of the earliest effect of Sry expression is to induce up-regulation of Sox9 gene expression in the developing gonad. SOX9, like SRY, contains a high mobility group domain and is sufficient to induce testis differentiation in transgenic XX mice. Before sexual differentiation, SOX9 protein is initially found in the cytoplasm of undifferentiated gonads from both sexes. At the time of testis differentiation and anti-Müllerian hormone expression, it becomes localized to the nuclear compartment in males whereas it is down-regulated in females. In this report, we used NIH 3T3 cells as a model to examine the regulation of SOX9 nucleo-cytoplasmic shuttling. SOX9-transfected cells expressed nuclear and cytoplasmic SOX9 whereas transfected cells treated with the nuclear export inhibitor leptomycin B, displayed an exclusive nuclear localization of SOX9. By using SOX9 deletion constructs in green fluorescent protein fusion proteins, we identified a functional nuclear export signal sequence between amino acids 134 and 147 of SOX9 high mobility group box. More strikingly, we show that inhibiting nuclear export with leptomycin B in mouse XX gonads cultured in vitro induced a sex reversal phenotype characterized by nuclear SOX9 and anti-Müllerian hormone expression. These results indicate that SOX9 nuclear export signal is essential for SOX9 sex-specific subcellular localization and could be part of a regulatory switch repressing (in females) or triggering (in males) male-specific sexual differentiation. PMID:12169669

  9. Epac activation induces histone deacetylase nuclear export via a Ras-dependent signalling pathway.

    PubMed

    Métrich, Mélanie; Laurent, Anne-Coline; Breckler, Magali; Duquesnes, Nicolas; Hmitou, Isabelle; Courillau, Delphine; Blondeau, Jean-Paul; Crozatier, Bertrand; Lezoualc'h, Frank; Morel, Eric

    2010-10-01

    Epac (Exchange protein directly activated by cAMP) is a sensor for cAMP and represents a novel mechanism for governing cAMP signalling. Epac is a guanine nucleotide exchange factor (GEF) for the Ras family of small GTPases, Rap. Previous studies demonstrated that, in response to a prolonged beta-adrenergic stimulation Epac induced cardiac myocyte hypertrophy. The aim of our study was to further characterize Epac downstream effectors involved in cardiac myocyte growth. Here, we found that Epac led to the activation of the small G protein H-Ras in primary neonatal cardiac myocytes. A Rap GTPase activating protein (RapGAP) partially inhibited Epac-induced H-Ras activation. Interestingly, we found that H-Ras activation involved the GEF domain of Epac. However, Epac did not directly induce exchange activity on this small GTPase protein. Instead, the effect of Epac on H-Ras activation was dependent on a signalling cascade involving phospholipase C (PLC)/inositol 1,3,5 triphosphate receptor (IP3R) and an increase intracellular calcium. In addition, we found that Epac activation induced histone deacetylase type 4 (HDAC4) translocation. Whereas HDAC5 alone was unresponsive to Epac, it became responsive to Epac in the presence of HDAC4 in COS cells. Consistent with its effect on HDAC cytoplasmic shuttle, Epac activation also increased the prohypertrophic transcription factor MEF2 in a CaMKII dependent manner in primary cardiac myocytes. Thus, our data show that Epac activates a prohypertrophic signalling pathway which involves PLC, H-Ras, CaMKII and HDAC nuclear export. Copyright 2010 Elsevier Inc. All rights reserved.

  10. Canonical Wnt signalling regulates nuclear export of Setdb1 during skeletal muscle terminal differentiation.

    PubMed

    Beyer, Sophie; Pontis, Julien; Schirwis, Elija; Battisti, Valentine; Rudolf, Anja; Le Grand, Fabien; Ait-Si-Ali, Slimane

    2016-01-01

    The histone 3 lysine 9 methyltransferase Setdb1 is essential for both stem cell pluripotency and terminal differentiation of different cell types. To shed light on the roles of Setdb1 in these mutually exclusive processes, we used mouse skeletal myoblasts as a model of terminal differentiation. Ex vivo studies on isolated single myofibres showed that Setdb1 is required for adult muscle stem cells expansion following activation. In vitro studies in skeletal myoblasts confirmed that Setdb1 suppresses terminal differentiation. Genomic binding analyses showed a release of Setdb1 from selected target genes upon myoblast terminal differentiation, concomitant to a nuclear export of Setdb1 to the cytoplasm. Both genomic release and cytoplasmic Setdb1 relocalisation during differentiation were dependent on canonical Wnt signalling. Transcriptomic assays in myoblasts unravelled a significant overlap between Setdb1 and Wnt3a regulated genetic programmes. Together, our findings revealed Wnt-dependent subcellular relocalisation of Setdb1 as a novel mechanism regulating Setdb1 functions and myogenesis.

  11. Canonical Wnt signalling regulates nuclear export of Setdb1 during skeletal muscle terminal differentiation

    PubMed Central

    Beyer, Sophie; Pontis, Julien; Schirwis, Elija; Battisti, Valentine; Rudolf, Anja; Le Grand, Fabien; Ait-Si-Ali, Slimane

    2016-01-01

    The histone 3 lysine 9 methyltransferase Setdb1 is essential for both stem cell pluripotency and terminal differentiation of different cell types. To shed light on the roles of Setdb1 in these mutually exclusive processes, we used mouse skeletal myoblasts as a model of terminal differentiation. Ex vivo studies on isolated single myofibres showed that Setdb1 is required for adult muscle stem cells expansion following activation. In vitro studies in skeletal myoblasts confirmed that Setdb1 suppresses terminal differentiation. Genomic binding analyses showed a release of Setdb1 from selected target genes upon myoblast terminal differentiation, concomitant to a nuclear export of Setdb1 to the cytoplasm. Both genomic release and cytoplasmic Setdb1 relocalisation during differentiation were dependent on canonical Wnt signalling. Transcriptomic assays in myoblasts unravelled a significant overlap between Setdb1 and Wnt3a regulated genetic programmes. Together, our findings revealed Wnt-dependent subcellular relocalisation of Setdb1 as a novel mechanism regulating Setdb1 functions and myogenesis. PMID:27790377

  12. A nuclear export signal in the N-terminal regulatory domain of IκBα controls cytoplasmic localization of inactive NF-κB/IκBα complexes

    PubMed Central

    Huang, Tony T.; Kudo, Nobuaki; Yoshida, Minoru; Miyamoto, Shigeki

    2000-01-01

    Appropriate subcellular localization is crucial for regulation of NF-κB function. Herein, we show that latent NF-κB complexes can enter and exit the nucleus in preinduction states. The nuclear export inhibitor leptomycin B (LMB) sequestered NF-κB/IκBα complexes in the nucleus. Using deletion and site-directed mutagenesis, we identified a previously uncharacterized nuclear export sequence in residues 45–54 of IκBα that was required for cytoplasmic localization of inactive complexes. This nuclear export sequence also caused nuclear exclusion of heterologous proteins in a LMB-sensitive manner. Importantly, a LMB-insensitive CRM1 mutant (Crm1-K1) abolished LMB-induced nuclear accumulation of the inactive complexes. Moreover, a cell-permeable p50 NF-κB nuclear localization signal peptide also blocked these LMB effects. These results suggest that NF-κB/IκBα complexes shuttle between the cytoplasm and nucleus by a nuclear localization signal-dependent nuclear import and a CRM1-dependent nuclear export. The LMB-induced nuclear complexes could not bind DNA and were inaccessible to signaling events, because LMB inhibited NF-κB activation without affecting the subcellular localization of upstream kinases IKKβ and NIK. Our findings indicate that the dominant nuclear export over nuclear import contributes to the largely cytoplasmic localization of the inactive complexes to achieve efficient NF-κB activation by extracellular signals. PMID:10655476

  13. Identification of nuclear import and export signals within Fli-1: roles of the nuclear import signals in Fli-1-dependent activation of megakaryocyte-specific promoters.

    PubMed

    Hu, Wei; Philips, Alana S; Kwok, Juliana C; Eisbacher, Michael; Chong, Beng H

    2005-04-01

    The Ets factor Friend leukemia integration 1 (Fli-1) is an important regulator of megakaryocytic (Mk) differentiation. Here, we demonstrate two novel nuclear localization signals (NLSs) within Fli-1: one (NLS1) is located at the N terminus, and another (NLS2) is within the Ets domain. Nuclear accumulation of Fli-1 reflected the combined functional effects of the two discrete NLSs. Each NLS can independently direct nuclear transport of a carrier protein, with mutations within the NLSs affecting nuclear accumulation. NLS1 has a bipartite motif, whereas the NLS2 region contains a nonclassical NLS. Both NLSs bind importin alpha (IMPalpha) and IMPbeta, with NLS1 and NLS2 being predominantly recognized by IMPalpha and IMPbeta, respectively. Fli-1 also contains one nuclear export signal. Leptomycin B abolished its cytoplasmic accumulation, showing CRM1 dependency. We demonstrate that Ets domain binding to specific target DNA effectively blocks IMP binding, indicating that the targeted DNA binding plays a role in localizing Fli-1 to its destination and releasing IMPs for recycling back to the cytoplasm. Finally, by analyzing full-length Fli-1 carrying NLS1, NLS2, and combined NLS1-NLS2 mutations, we conclude that two functional NLSs exist in Fli-1 and that each NLS is sufficient to target Fli-1 to the nucleus for activation of Mk-specific genes.

  14. The yeast splicing factor Prp40p contains functional leucine-rich nuclear export signals that are essential for splicing.

    PubMed Central

    Murphy, Mark W; Olson, Brian L; Siliciano, Paul G

    2004-01-01

    To investigate the function of the essential U1 snRNP protein Prp40p, we performed a synthetic lethal screen in Saccharomyces cerevisiae. Using an allele of PRP40 that deletes 47 internal residues and causes only a slight growth defect, we identified aphenotypic mutations in three distinct complementation groups that conferred synthetic lethality. The synthetic phenotypes caused by these mutations were suppressed by wild-type copies of CRM1 (XPO1), YNL187w, and SME1, respectively. The strains whose synthetic phenotypes were suppressed by CRM1 contained no mutations in the CRM1 coding sequence or promoter. This indicates that overexpression of CRM1 confers dosage suppression of the synthetic lethality. Interestingly, PRP40 and YNL187w encode proteins with putative leucine-rich nuclear export signal (NES) sequences that fit the consensus sequence recognized by Crm1p. One of Prp40p's two NESs lies within the internal deletion. We demonstrate here that the NES sequences of Prp40p are functional for nuclear export in a leptomycin B-sensitive manner. Furthermore, mutation of these NES sequences confers temperature-sensitive growth and a pre-mRNA splicing defect. Although we do not expect that yeast snRNPs undergo compartmentalized biogenesis like their metazoan counterparts, our results suggest that Prp40p and Ynl187wp contain redundant NESs that aid in an important, Crm1p-mediated nuclear export event. PMID:15020406

  15. [Nuclear export signal of androgen receptor regulated of androgen receptor stability in prostate cancer].

    PubMed

    Gong, Y Q; Zhang, C J; He, S M; Li, X S; Zhou, L Q; Guo, Y L

    2017-08-18

    To investigate the mechanisms of nuclear export signal of androgen receptor (NES(AR)) in the regulation of androgen receptor (AR) protein expression and stability in prostate cancer. The green fluorescent protein fusion protein expression vectors pEGFP-AR(1-918aa), pEGFP-NES(AR) (743-817aa), pEGFP-NAR (1-665aa) and pEGFP-NAR-NES(AR), and lysine mutants of NES(AR) pEGFP-NES(AR) K776R, pEGFP-NES(AR) K807R and pEGFP-NES(AR) K776R/K807R, were transiently transfected into prostate cancer cell line PC3. Fluorescence microscopy, Western blot and immunoprecipitation were used to detect NES(AR) regulation of androgen receptor stability. Under the fluorescence microscope, NES(AR)-containing fusion proteins were cytoplasmic localization, and their fluorescence intensities were much weaker than those without NES(AR). The expression levels of NES(AR)-containing fusion proteins were significantly lower than those without NES(AR). The half-lives of GFP-NES(AR) and GFP-NAR-NES(AR) were less than 6 h, while the expression of GFP and GFP-NAR was relatively stable and the half-life was more than 24 h in the presence of cycloheximide. The expression levels of GFP-NES(AR) were significantly increased by proteasome inhibitor MG132 treatment in a dose-dependent manner; in contrast, MG132 did not show any significant effect on the protein levels of GFP. When new protein synthesis was blocked, MG132 could also prevent the degradation of GFP-NES(AR) in the transfected cells in the presence of cycloheximide, while it had no significant effect on GFP protein stability in the parallel experiment. GFP immunoprecipitation showed that the ubiquitination level of GFP-NES(AR) fusion protein was significantly higher than that of the GFP control. The mutations of lysine sites K776 and K807 in NES(AR) significantly reduced the level of ubiquitination, and showed increased protein stability, indicating that they were the key amino acid residues of NES(AR) ubiquitination. NES(AR) was unstable and

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. A novel CRM1-dependent nuclear export signal in adenoviral E1A protein regulated by phosphorylation.

    PubMed

    Jiang, Hong; Olson, Melissa V; Medrano, Diana R; Lee, Ok-Hee; Xu, Jing; Piao, Yuji; Alonso, Marta M; Gomez-Manzano, Candelaria; Hung, Mien-Chie; Yung, W K Alfred; Fueyo, Juan

    2006-12-01

    Adenoviral E1A is a versatile protein that can reprogram host cells for efficient viral replication. The nuclear import of E1A is mediated by a nuclear localization signal; however, whether E1A can be actively exported from the nucleus is unknown. We first reported a CRM1-dependent nuclear export signal (NES) in E1A that is conserved in the group C adenoviruses. We showed that CRM1 and E1A coimmunoprecipitated and that blockage of CRM1 function by leptomycin B or small interfering RNA resulted in the nuclear localization of E1A. Through mutational analyses, we identified an active canonical NES element within the E1A protein spanning amino acids 70-80. We further demonstrated that phosphorylation of adjacent serine (S)89 resulted in the cytoplasmic accumulation of E1A. Interestingly, coincident with the accumulation of cells in the S/G2/M phase and histone H1 phosphorylation, E1A was relocated to the cytoplasm at the late stage of the viral cycle, which was blocked by the CDC2/CDK2 inhibitor roscovitine. Importantly, titration of the progenies of the viruses in infected cells showed that the replication efficiency of the NES mutant adenovirus was up to 500-fold lower than that of the wild-type adenovirus. Collectively, our data demonstrate the existence of a NES in E1A that is modulated by the phosphorylation of the S89 residue and the NES plays a role for an efficient viral replication in the host cells.

  1. Nucleo-cytoplasmic shuttling of the endonuclease ankyrin repeats and LEM domain-containing protein 1 (Ankle1) is mediated by canonical nuclear export- and nuclear import signals.

    PubMed

    Zlopasa, Livija; Brachner, Andreas; Foisner, Roland

    2016-06-01

    Ankyrin repeats and LEM domain containing protein 1 (Ankle1) belongs to the LEM protein family, whose members share a chromatin-interacting LEM motif. Unlike most other LEM proteins, Ankle1 is not an integral protein of the inner nuclear membrane but shuttles between the nucleus and the cytoplasm. It contains a GIY-YIG-type nuclease domain, but its function is unknown. The mammalian genome encodes only one other GIY-YIG domain protein, termed Slx1. Slx1 has been described as a resolvase that processes Holliday junctions during homologous recombination-mediated DNA double strand break repair. Resolvase activity is regulated in a spatial and temporal manner during the cell cycle. We hypothesized that Ankle1 may have a similar function and its nucleo-cytoplasmic shuttling may contribute to the regulation of Ankle1 activity. Hence, we aimed at identifying the domains mediating Ankle1 shuttling and investigating whether cellular localization is affected during DNA damage response. Sequence analysis predicts the presence of two canonical nuclear import and export signals in Ankle1. Immunofluorescence microscopy of cells expressing wild-type and various mutated Ankle1-fusion proteins revealed a C-terminally located classical monopartite nuclear localization signal and a centrally located CRM1-dependent nuclear export signal that mediate nucleo-cytoplasmic shuttling of Ankle1. These sequences are also functional in heterologous proteins. The predominant localization of Ankle1 in the cytoplasm, however, does not change upon induction of several DNA damage response pathways throughout the cell cycle. We identified the domains mediating nuclear import and export of Ankle1. Ankle1's cellular localization was not affected following DNA damage.

  2. A leucine-rich nuclear export signal in the p53 tetramerization domain: regulation of subcellular localization and p53 activity by NES masking.

    PubMed Central

    Stommel, J M; Marchenko, N D; Jimenez, G S; Moll, U M; Hope, T J; Wahl, G M

    1999-01-01

    Appropriate subcellular localization is crucial for regulating p53 function. We show that p53 export is mediated by a highly conserved leucine-rich nuclear export signal (NES) located in its tetramerization domain. Mutation of NES residues prevented p53 export and hampered tetramer formation. Although the p53-binding protein MDM2 has an NES and has been proposed to mediate p53 export, we show that the intrinsic p53 NES is both necessary and sufficient for export. This report also demonstrates that the cytoplasmic localization of p53 in neuroblastoma cells is due to its hyperactive nuclear export: p53 in these cells can be trapped in the nucleus by the export-inhibiting drug leptomycin B or by binding a p53-tetramerization domain peptide that masks the NES. We propose a model in which regulated p53 tetramerization occludes its NES, thereby ensuring nuclear retention of the DNA-binding form. We suggest that attenuation of p53 function involves the conversion of tetramers into monomers or dimers, in which the NES is exposed to the proteins which mediate their export to the cytoplasm. PMID:10075936

  3. Comparative analysis of seven viral nuclear export signals (NESs) reveals the crucial role of nuclear export mediated by the third NES consensus sequence of nucleoprotein (NP) in influenza A virus replication.

    PubMed

    Chutiwitoonchai, Nopporn; Kakisaka, Michinori; Yamada, Kazunori; Aida, Yoko

    2014-01-01

    The assembly of influenza virus progeny virions requires machinery that exports viral genomic ribonucleoproteins from the cell nucleus. Currently, seven nuclear export signal (NES) consensus sequences have been identified in different viral proteins, including NS1, NS2, M1, and NP. The present study examined the roles of viral NES consensus sequences and their significance in terms of viral replication and nuclear export. Mutation of the NP-NES3 consensus sequence resulted in a failure to rescue viruses using a reverse genetics approach, whereas mutation of the NS2-NES1 and NS2-NES2 sequences led to a strong reduction in viral replication kinetics compared with the wild-type sequence. While the viral replication kinetics for other NES mutant viruses were also lower than those of the wild-type, the difference was not so marked. Immunofluorescence analysis after transient expression of NP-NES3, NS2-NES1, or NS2-NES2 proteins in host cells showed that they accumulated in the cell nucleus. These results suggest that the NP-NES3 consensus sequence is mostly required for viral replication. Therefore, each of the hydrophobic (Φ) residues within this NES consensus sequence (Φ1, Φ2, Φ3, or Φ4) was mutated, and its viral replication and nuclear export function were analyzed. No viruses harboring NP-NES3 Φ2 or Φ3 mutants could be rescued. Consistent with this, the NP-NES3 Φ2 and Φ3 mutants showed reduced binding affinity with CRM1 in a pull-down assay, and both accumulated in the cell nucleus. Indeed, a nuclear export assay revealed that these mutant proteins showed lower nuclear export activity than the wild-type protein. Moreover, the Φ2 and Φ3 residues (along with other Φ residues) within the NP-NES3 consensus were highly conserved among different influenza A viruses, including human, avian, and swine. Taken together, these results suggest that the Φ2 and Φ3 residues within the NP-NES3 protein are important for its nuclear export function during viral

  4. An N-terminal Nuclear Export Signal Regulates Trafficking and Aggregation of Huntingtin (Htt) Protein Exon 1*

    PubMed Central

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

    2013-01-01

    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

  5. 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.

  6. 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.

  7. 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

  8. Tetrachlorobenzoquinone induces Nrf2 activation via rapid Bach1 nuclear export/ubiquitination and JNK-P62 signaling.

    PubMed

    Su, Chuanyang; Shi, Qiong; Song, Xiufang; Fu, Juanli; Liu, Zixuan; Wang, Yawen; Wang, Yuxin; Xia, Xiaomin; Song, Erqun; Song, Yang

    2016-07-01

    Our previous studies demonstrated that tetrachlorobenzoquinone (TCBQ), an active metabolite of pentachlorophenol, has effects on the generation of reactive oxygen species (ROS) and oxidative stress in vitro and in vivo. Nuclear factor erythroid-derived 2-like 2 (Nrf2) is a cellular sensor of electrophilic or oxidative stress that regulates the expression of antioxidant enzymes and defensive proteins. We have illustrated that TCBQ activates Nrf2 signaling by promoting the formation of the Kelch-like ECH-associated protein 1 (Keap1) cross-linking dimer and the formation of an ubiquitination switch from Nrf2 to Keap1. The activation of Nrf2 by TCBQ may serve as an adaptive response to a TCBQ-induced oxidative insult. BTB and CNC homolog 1 (Bach1) compete with Nrf2, leading to the negative regulation of the antioxidant response element (ARE). In this report, we propose that TCBQ induces the dynamic inactivation of Bach1. We observed a rapid nuclear efflux of Bach1 and an accumulation of Nrf2 in nuclei upon TCBQ treatment that precedes the binding of Nrf2 with ARE. We found that the nuclear export of Bach1 is dependent on its chromosomal region maintenance 1 (Crm1) interaction and tyrosine phosphorylation. Although TCBQ induces the ubiquitination of Bach1, TCBQ also increases the mRNA and protein levels of Bach1, returning Bach1 to normal levels. Moreover, we found that TCBQ-induced activation of Nrf2 involves c-Jun N-terminal kinase (JNK)-P62 signaling.

  9. Nuclear Export of Messenger RNA

    PubMed Central

    Katahira, Jun

    2015-01-01

    Transport of messenger RNA (mRNA) from the nucleus to the cytoplasm is an essential step of eukaryotic gene expression. In the cell nucleus, a precursor mRNA undergoes a series of processing steps, including capping at the 5' ends, splicing and cleavage/polyadenylation at the 3' ends. During this process, the mRNA associates with a wide variety of proteins, forming a messenger ribonucleoprotein (mRNP) particle. Association with factors involved in nuclear export also occurs during transcription and processing, and thus nuclear export is fully integrated into mRNA maturation. The coupling between mRNA maturation and nuclear export is an important mechanism for providing only fully functional and competent mRNA to the cytoplasmic translational machinery, thereby ensuring accuracy and swiftness of gene expression. This review describes the molecular mechanism of nuclear mRNA export mediated by the principal transport factors, including Tap-p15 and the TREX complex. PMID:25836925

  10. Two nuclear export signals of Cdc6 are differentially associated with CDK-mediated phosphorylation residues for cytoplasmic translocation.

    PubMed

    Hwang, In Sun; Woo, Sang Uk; Park, Ji-Woong; Lee, Seung Ki; Yim, Hyungshin

    2014-02-01

    Cdc6 is cleaved at residues 442 and 290 by caspase-3 during apoptosis producing p49-tCdc6 and p32-tCdc6, respectively. While p32-tCdc6 is unable to translocate into the cytoplasm, p49-tCdc6 retains cytoplasmic translocation activity, but it has a lower efficiency than wild-type Cdc6. We hypothesized that a novel nuclear export signal (NES) sequence exists between amino acids 290 and 442. Cdc6 contains a novel NES in the region of amino acids 300-315 (NES2) that shares sequence similarity with NES1 at residues 462-476. In mutant versions of Cdc6, we replaced leucine with alanine in NES1 and NES2 and co-expressed the mutant constructs with cyclin A. We observed that the cytoplasmic translocation of these mutants was reduced in comparison to wild-type Cdc6. Moreover, the cytoplasmic translocation of a mutant in which all four leucine residues were mutated to alanine was significantly inhibited in comparison to the translocation of wild-type Cdc6. The Crm1 binding activities of Cdc6 NES mutants were consistent with the efficiency of its cytoplasmic translocation. Further studies have revealed that L468 and L470 of NES1 are required for cytoplasmic translocation of Cdc6 phosphorylated at S74, while L311 and L313 of NES2 accelerate the cytoplasmic translocation of Cdc6 phosphorylated at S54. These results suggest that the two NESs of Cdc6 work cooperatively and distinctly for the cytoplasmic translocation of Cdc6 phosphorylated at S74 and S54 by cyclin A/Cdk2.

  11. 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

    2016-05-12

    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.

  12. 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

  13. Nuclear Export Signal of Androgen Receptor (NESAR) Regulation of Androgen Receptor Level in Human Prostate Cell Lines via Ubiquitination and Proteasome-Dependent Degradation

    PubMed Central

    Gong, Yanqing; Wang, Dan; Dar, Javid A.; Singh, Prabhpreet; Graham, Lara; Liu, Weijun; Ai, Junkui; Xin, Zhongcheng

    2012-01-01

    Androgen receptor (AR) plays a key role in prostate development and carcinogenesis. Increased expression and/or stability of AR is associated with sensitization of prostate cancer cells to low levels of androgens, leading to castration resistance. Hence, understanding the mechanisms regulating AR protein stability is clinically relevant and may lead to new approaches to prevent and/or treat prostate cancer. Using fluorescence microscopy, Western blot, and pulse chase assay, we showed that nuclear export signal (NES)AR, a nuclear export signal in the ligand binding domain (LBD) of AR, can significantly enhance the degradation of fusion protein constructs in PC3 prostate cancer cells. The half-life of GFP-NESAR was less than 3 h, which was 10 times shorter than that of green fluorescent protein (GFP) control. Further analysis showed that NESAR can signal for polyubiquitination and that degradation of NESAR-containing fusion proteins can be blocked by proteasome inhibitor MG132. Ubiquitination of GFP-AR or GFP-LBD was suppressed in the presence of dihydrotestosterone, which is known to suppress NESAR while inducing nuclear localization signal 2 in AR or LBD, suggesting that the export activity of NESAR is required for NESAR-mediated polyubiquitination. Treatment with MG132 also induced aggresome formation of NESAR-containing fusion proteins in perinuclear regions of the transfected PC3 cells, indicating a role for NESAR in inducing unfolded protein responses. The above observations suggest that NESAR plays a key role in AR ubiquitination and proteasome-dependent degradation in prostate cancer cells. PMID:23041672

  14. 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

  15. Identification of the nuclear export signal and STAT-binding domains of the Nipah virus V protein reveals mechanisms underlying interferon evasion.

    PubMed

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

    2004-05-01

    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.

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

    PubMed

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

    2016-03-14

    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.

  17. Identification of CASZ1 nuclear export signal (NES) reveals potential mechanisms for loss of CASZ1 tumor suppressor activity in neuroblastoma

    PubMed Central

    Liu, Zhihui; Lam, Norris; Wang, Evelyn; Virden, Ryan A.; Pawel, Bruce; Attiyeh, Edward F.; Maris, John M.; Thiele, Carol J.

    2016-01-01

    As a transcription factor, localization to the nucleus and the recruitment of co-factors to regulate gene transcription is essential. Nuclear localization and nucleosome remodeling and histone deacetylase (NuRD) complex binding are required for the zinc finger transcription factor CASZ1 to function as neuroblastoma (NB) tumor suppressor. However, the critical amino acids (AAs) that are required for CASZ1 interaction with NuRD complex and the regulation of CASZ1 subcellular localization have not been characterized. Through alanine scanning, immunofluorescence cell staining and co-immunoprecipitation we define a critical region at the CASZ1 N-terminus (AA23-40) that mediates the CASZ1b nuclear localization and NuRD interaction. Furthermore, we identify a nuclear export signal (NES) at the N-terminus (AA176-192) that contributes to CASZ1 nuclear-cytoplasmic shuttling in a chromosomal maintenance 1 (CRM1)-dependent manner. An analysis of CASZ1 protein expression in a primary neuroblastoma tissue microarray shows that high nuclear CASZ1 staining is detected in tumors from NB patients with good prognoses. In contrast, cytoplasmic-restricted CASZ1 staining or low nuclear CASZ1 staining is found in tumors from patients with poor prognoses. These findings provide insight into mechanisms by which CASZ1 regulates transcription, and suggests that regulation of CASZ1 subcellular localization may impact its function in normal development and pathologic conditions such as neuroblastoma tumorigenesis. PMID:27270431

  18. Cooperativity among Rev-Associated Nuclear Export Signals Regulates HIV-1 Gene Expression and Is a Determinant of Virus Species Tropism

    PubMed Central

    Aligeti, Mounavya; Behrens, Ryan T.; Pocock, Ginger M.; Schindelin, Johannes; Dietz, Christian; Eliceiri, Kevin W.; Swanson, Chad M.; Malim, Michael H.; Ahlquist, Paul

    2014-01-01

    ABSTRACT Murine cells exhibit a profound block to HIV-1 virion production that was recently mapped to a species-specific structural attribute of the murine version of the chromosomal region maintenance 1 (mCRM1) nuclear export receptor and rescued by the expression of human CRM1 (hCRM1). In human cells, the HIV-1 Rev protein recruits hCRM1 to intron-containing viral mRNAs encoding the Rev response element (RRE), thereby facilitating viral late gene expression. Here we exploited murine 3T3 fibroblasts as a gain-of-function system to study hCRM1's species-specific role in regulating Rev's effector functions. We show that Rev is rapidly exported from the nucleus by mCRM1 despite only weak contributions to HIV-1's posttranscriptional stages. Indeed, Rev preferentially accumulates in the cytoplasm of murine 3T3 cells with or without hCRM1 expression, in contrast to human HeLa cells, where Rev exhibits striking en masse transitions between the nuclear and cytoplasmic compartments. Efforts to bias Rev's trafficking either into or out of the nucleus revealed that Rev encoding a second CRM1 binding domain (Rev-2xNES) or Rev-dependent viral gag-pol mRNAs bearing tandem RREs (GP-2xRRE), rescue virus particle production in murine cells even in the absence of hCRM1. Combined, these results suggest a model wherein Rev-associated nuclear export signals cooperate to regulate the number or quality of CRM1's interactions with viral Rev/RRE ribonucleoprotein complexes in the nucleus. This mechanism regulates CRM1-dependent viral gene expression and is a determinant of HIV-1's capacity to produce virions in nonhuman cell types. IMPORTANCE Cells derived from mice and other nonhuman species exhibit profound blocks to HIV-1 replication. Here we elucidate a block to HIV-1 gene expression attributable to the murine version of the CRM1 (mCRM1) nuclear export receptor. In human cells, hCRM1 regulates the nuclear export of viral intron-containing mRNAs through the activity of the viral Rev

  19. 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

  20. 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.

  1. p53 SUMOylation promotes its nuclear export by facilitating its release from the nuclear export receptor CRM1.

    PubMed

    Santiago, Aleixo; Li, Dawei; Zhao, Lisa Y; Godsey, Adam; Liao, Daiqing

    2013-09-01

    Chromosomal region maintenance 1 (CRM1) mediates p53 nuclear export. Although p53 SUMOylation promotes its nuclear export, the underlying mechanism is unclear. Here we show that tethering of a small, ubiquitin-like modifier (SUMO) moiety to p53 markedly increases its cytoplasmic localization. SUMO attachment to p53 does not affect its oligomerization, suggesting that subunit dissociation required for exposing p53's nuclear export signal (NES) is unnecessary for p53 nuclear export. Surprisingly, SUMO-mediated p53 nuclear export depends on the SUMO-interacting motif (SIM)-binding pocket of SUMO-1. The CRM1 C-terminal domain lacking the NES-binding groove interacts with tetrameric p53, and the proper folding of the p53 core domain, rather than the presence of the N- or C-terminal tails, appears to be important for p53-CRM1 interaction. The CRM1 Huntington, EF3, a subunit of PP2A, and TOR1 9 (HEAT9) loop, which regulates GTP-binding nuclear protein Ran binding and cargo release, contains a prototypical SIM. Remarkably, disruption of this SIM in conjunction with a mutated SIM-binding groove of SUMO-1 markedly enhances the binding of CRM1 to p53-SUMO-1 and their accumulation in the nuclear pore complexes (NPCs), as well as their persistent association in the cytoplasm. We propose that SUMOylation of a CRM1 cargo such as p53 at the NPCs unlocks the HEAT9 loop of CRM1 to facilitate the disassembly of the transporting complex and cargo release to the cytoplasm.

  2. Eukaryotic Ribosome Assembly and Nuclear Export.

    PubMed

    Nerurkar, Purnima; Altvater, Martin; Gerhardy, Stefan; Schütz, Sabina; Fischer, Ute; Weirich, Christine; Panse, Vikram Govind

    2015-01-01

    Accurate translation of the genetic code into functional polypeptides is key to cellular growth and proliferation. This essential process is carried out by the ribosome, a ribonucleoprotein complex of remarkable size and intricacy. Although the structure of the mature ribosome has provided insight into the mechanism of translation, our knowledge regarding the assembly, quality control, and intracellular targeting of this molecular machine is still emerging. Assembly of the eukaryotic ribosome begins in the nucleolus and requires more than 350 conserved assembly factors, which transiently associate with the preribosome at specific maturation stages. After accomplishing their tasks, early-acting assembly factors are released, preparing preribosomes for nuclear export. Export competent preribosomal subunits are transported through nuclear pore complexes into the cytoplasm, where they undergo final maturation steps, which are closely connected to quality control, before engaging in translation. In this chapter, we focus on the final events that commit correctly assembled ribosomal subunits for translation.

  3. Exportin-5 mediates nuclear export of SRP RNA in vertebrates.

    PubMed

    Takeiwa, Toshihiko; Taniguchi, Ichiro; Ohno, Mutsuhito

    2015-04-01

    The signal recognition particle is a ribonucleoprotein complex that is essential for the translocation of nascent proteins into the endoplasmic reticulum. It has been shown that the RNA component (SRP RNA) is exported from the nucleus by CRM1 in the budding yeast. However, how SRP RNA is exported in higher species has been elusive. Here, we show that SRP RNA does not use the CRM1 pathway in Xenopus oocytes. Instead, SRP RNA uses the same export pathway as pre-miRNA and tRNA as showed by cross-competition experiments. Consistently, the recombinant Exportin-5 protein specifically stimulated export of SRP RNA as well as of pre-miRNA and tRNA, whereas an antibody raised against Exportin-5 specifically inhibited export of the same RNA species. Moreover, biotinylated SRP RNA can pull down Exportin-5 but not CRM1 from HeLa cell nuclear extracts in a RanGTP-dependent manner. These results, taken together, strongly suggest that the principal export receptor for SRP RNA in vertebrates is Exportin-5 unlike in the budding yeast.

  4. Regulation of p53 tetramerization and nuclear export by ARC

    PubMed Central

    Foo, Roger S.-Y.; Nam, Young-Jae; Ostreicher, Marc Jason; Metzl, Mark D.; Whelan, Russell S.; Peng, Chang-Fu; Ashton, Anthony W.; Fu, Weimin; Mani, Kartik; Chin, Suet-Feung; Provenzano, Elena; Ellis, Ian; Figg, Nichola; Pinder, Sarah; Bennett, Martin R.; Caldas, Carlos; Kitsis, Richard N.

    2007-01-01

    Inactivation of the transcription factor p53 is central to carcinogenesis. Yet only approximately one-half of cancers have p53 loss-of-function mutations. Here, we demonstrate a mechanism for p53 inactivation by apoptosis repressor with caspase recruitment domain (ARC), a protein induced in multiple cancer cells. The direct binding in the nucleus of ARC to the p53 tetramerization domain inhibits p53 tetramerization. This exposes a nuclear export signal in p53, triggering Crm1-dependent relocation of p53 to the cytoplasm. Knockdown of endogenous ARC in breast cancer cells results in spontaneous tetramerization of endogenous p53, accumulation of p53 in the nucleus, and activation of endogenous p53 target genes. In primary human breast cancers with nuclear ARC, p53 is almost always WT. Conversely, nearly all breast cancers with mutant p53 lack nuclear ARC. We conclude that nuclear ARC is induced in cancer cells and negatively regulates p53. PMID:18087040

  5. Searching for nuclear export elements in hepatitis D virus RNA.

    PubMed

    Freitas, Natália; Cunha, Celso

    2013-08-12

    To search for the presence of cis elements in hepatitis D virus (HDV) genomic and antigenomic RNA capable of promoting nuclear export. We made use of a well characterized chloramphenicol acetyl-transferase reporter system based on plasmid pDM138. Twenty cDNA fragments corresponding to different HDV genomic and antigenomic RNA sequences were inserted in plasmid pDM138, and used in transfection experiments in Huh7 cells. The relative amounts of HDV RNA in nuclear and cytoplasmic fractions were then determined by real-time polymerase chain reaction and Northern blotting. The secondary structure of the RNA sequences that displayed nuclear export ability was further predicted using a web interface. Finally, the sensitivity to leptomycin B was assessed in order to investigate possible cellular pathways involved in HDV RNA nuclear export. Analysis of genomic RNA sequences did not allow identifying an unequivocal nuclear export element. However, two regions were found to promote the export of reporter mRNAs with efficiency higher than the negative controls albeit lower than the positive control. These regions correspond to nucleotides 266-489 and 584-920, respectively. In addition, when analyzing antigenomic RNA sequences a nuclear export element was found in positions 214-417. Export mediated by the nuclear export element of HDV antigenomic RNA is sensitive to leptomycin B suggesting a possible role of CRM1 in this transport pathway. A cis-acting nuclear export element is present in nucleotides 214-417 of HDV antigenomic RNA.

  6. 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.

  7. The influenza virus NEP (NS2 protein) mediates the nuclear export of viral ribonucleoproteins.

    PubMed Central

    O'Neill, R E; Talon, J; Palese, P

    1998-01-01

    Nuclear import and export of viral nucleic acids is crucial for the replication cycle of many viruses, and elucidation of the mechanism of these steps may provide a paradigm for understanding general biological processes. Influenza virus replicates its RNA genome in the nucleus of infected cells. The influenza virus NS2 protein, which had no previously assigned function, was shown to mediate the nuclear export of virion RNAs by acting as an adaptor between viral ribonucleoprotein complexes and the nuclear export machinery of the cell. A functional domain on the NS2 with characteristics of a nuclear export signal was mapped: it interacts with cellular nucleoporins, can functionally replace the effector domain of the human immunodeficiency virus type 1 (HIV-1) Rev protein and mediates rapid nuclear export when cross-linked to a reporter protein. Microinjection of anti-NS2 antibodies into infected cells inhibited nuclear export of viral ribonucleoproteins, suggesting that the Rev-like NS2 mediates this process. Therefore, we have renamed this Rev-like factor the influenza virus nuclear export protein or NEP. We propose a model by which NEP acts as a protein adaptor molecule bridging viral ribonucleoproteins and the nuclear pore complex. PMID:9427762

  8. Inhibition of CRM1-mediated nuclear export of influenza A nucleoprotein and nuclear export protein as a novel target for antiviral drug development.

    PubMed

    Chutiwitoonchai, Nopporn; Mano, Takafumi; Kakisaka, Michinori; Sato, Hirotaka; Kondoh, Yasumitsu; Osada, Hiroyuki; Kotani, Osamu; Yokoyama, Masaru; Sato, Hironori; Aida, Yoko

    2017-04-08

    An anti-influenza compound, DP2392-E10 based on inhibition of the nuclear export function of the viral nucleoprotein-nuclear export signal 3 (NP-NES3) domain was successfully identified by our previous high-throughput screening system. Here, we demonstrated that DP2392-E10 exerts its antiviral effect by inhibiting replication of a broad range of influenza A subtypes. In regard to the molecular mechanism, we revealed that DP2392-E10 inhibits nuclear export of both viral NP and nuclear export protein (NEP). More specifically, in vitro pull-down assays revealed that DP2392-E10 directly binds cellular CRM1, which mediates nuclear export of NP and NEP. In silico docking suggested that DP2392-E10 binds at a region close to the HEAT9 and HEAT10 domains of CRM1. Together, these results indicate that the CRM1-mediated nuclear export function of influenza virus represents a new potential target for antiviral drug development, and also provide a core structure for a novel class of inhibitors that target this function.

  9. The mechanism of nuclear export of Smad3 involves exportin 4 and Ran.

    PubMed

    Kurisaki, Akira; Kurisaki, Keiko; Kowanetz, Marcin; Sugino, Hiromu; Yoneda, Yoshihiro; Heldin, Carl-Henrik; Moustakas, Aristidis

    2006-02-01

    Transforming growth factor beta (TGF-beta) receptors phosphorylate Smad3 and induce its nuclear import so it can regulate gene transcription. Smad3 can return to the cytoplasm to propagate further cycles of signal transduction or to be degraded. We demonstrate that Smad3 is exported by a constitutive mechanism that is insensitive to leptomycin B. The Mad homology 2 (MH2) domain is responsible for Smad3 export, which requires the GTPase Ran. Inactive, GDP-locked RanT24N or nuclear microinjection of Ran GTPase activating protein 1 blocked Smad3 export. Inactivation of the Ran guanine nucleotide exchange factor RCC1 inhibited Smad3 export and led to nuclear accumulation of phosphorylated Smad3. A screen for importin/exportin family members that associate with Smad3 identified exportin 4, which binds a conserved peptide sequence in the MH2 domain of Smad3 in a Ran-dependent manner. Exportin 4 is sufficient for carrying the in vitro nuclear export of Smad3 in cooperation with Ran. Knockdown of endogenous exportin 4 completely abrogates the export of endogenous Smad3. A short peptide representing the minimal interaction domain in Smad3 effectively competes with Smad3 association to exportin 4 and blocks nuclear export of Smad3 in vivo. We thus delineate a novel nuclear export pathway for Smad3.

  10. Nuclear entry and CRM1-dependent nuclear export of the Rous sarcoma virus Gag polyprotein

    PubMed Central

    Scheifele, Lisa Z.; Garbitt, Rachel A.; Rhoads, Jonathan D.; Parent, Leslie J.

    2002-01-01

    The retroviral Gag polyprotein directs budding from the plasma membrane of infected cells. Until now, it was believed that Gag proteins of type C retroviruses, including the prototypic oncoretrovirus Rous sarcoma virus, were synthesized on cytosolic ribosomes and targeted directly to the plasma membrane. Here we reveal a previously unknown step in the subcellular trafficking of the Gag protein, that of transient nuclear localization. We have identified a targeting signal within the N-terminal matrix domain that facilitates active nuclear import of the Gag polyprotein. We also found that Gag is transported out of the nucleus through the CRM1 nuclear export pathway, based on observations that treatment of virus-expressing cells with leptomycin B resulted in the redistribution of Gag proteins from the cytoplasm to the nucleus. Internal deletion of the C-terminal portion of the Gag p10 region resulted in the nuclear sequestration of Gag and markedly diminished budding, suggesting that the nuclear export signal might reside within p10. Finally, we observed that a previously described matrix mutant, Myr1E, was insensitive to the effects of leptomycin B, apparently bypassing the nuclear compartment during virus assembly. Myr1E has a defect in genomic RNA packaging, implying that nuclear localization of Gag might be involved in viral RNA interactions. Taken together, these findings provide evidence that nuclear entry and egress of the Gag polyprotein are intrinsic components of the Rous sarcoma virus assembly pathway. PMID:11891341

  11. Enzymatically driven transport: a kinetic theory for nuclear export.

    PubMed

    Kim, Sanghyun; Elbaum, M

    2013-11-05

    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. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  12. Adducin family proteins possess different nuclear export potentials.

    PubMed

    Liu, Chia-Mei; Hsu, Wen-Hsin; Lin, Wan-Yi; Chen, Hong-Chen

    2017-05-10

    The adducin (ADD) family proteins, namely ADD1, ADD2, and ADD3, are actin-binding proteins that play important roles in the stabilization of membrane cytoskeleton and cell-cell junctions. All the ADD proteins contain a highly conserved bipartite nuclear localization signal (NLS) at the carboxyl termini, but only ADD1 can localize to the nucleus. The reason for this discrepancy is not clear. To avoid the potential effect of cell-cell junctions on the distribution of ADD proteins, HA epitope-tagged ADD proteins and mutants were transiently expressed in NIH3T3 fibroblasts and their distribution in the cytoplasm and nucleus was examined by immunofluorescence staining. Several nuclear proteins were identified to interact with ADD1 by mass spectrometry, which were further verified by co-immunoprecipitation. In this study, we found that ADD1 was detectable both in the cytoplasm and nucleus, whereas ADD2 and ADD3 were detected only in the cytoplasm. However, ADD2 and ADD3 were partially (~40%) sequestered in the nucleus by leptomycin B, a CRM1/exportin1 inhibitor. Upon the removal of leptomycin B, ADD2 and ADD3 re-distributed to the cytoplasm. These results indicate that ADD2 and ADD3 possess functional NLS and are quickly transported to the cytoplasm upon entering the nucleus. Indeed, we found that ADD2 and ADD3 possess much higher potential to counteract the activity of the NLS derived from Simian virus 40 large T-antigen than ADD1. All the ADD proteins appear to contain multiple nuclear export signals mainly in their head and neck domains. However, except for the leucine-rich motif ((377)FEALMRMLDWLGYRT(391)) in the neck domain of ADD1, no other classic nuclear export signal was identified in the ADD proteins. In addition, the nuclear retention of ADD1 facilitates its interaction with RNA polymerase II and zinc-finger protein 331. Our results suggest that ADD2 and ADD3 possess functional NLS and shuttle between the cytoplasm and nucleus. The discrepancy in the

  13. A Balance Between Two Nuclear Localization Sequences and a Nuclear Export Sequence Governs Extradenticle Subcellular Localization

    PubMed Central

    Stevens, Katherine E.; Mann, Richard S.

    2007-01-01

    During animal development, transcription factor activities are modulated by several means, including subcellular localization. The Hox cofactor Extradenticle (Exd) has a dynamic subcellular localization, such that Exd is cytoplasmic by default, but is nuclear when complexed with another homeodomain protein, Homothorax (Hth). These observations raise the question of whether dimerization with Hth simply induces Exd's nuclear localization or, alternatively, if Hth is also necessary for Exd activity. To address this question, we analyzed the nuclear transport signals in Exd, including a divergent nuclear export signal (NES) and two nuclear localization signals (NLSs). We show that, although these signals are weak compared to canonical signals, they balance each other in Exd. We also provide evidence that Exd contains an NLS mask that contributes to its cytoplasmic localization. With these signals characterized, we generated forms of Exd that are nuclear localized in the absence of Hth. Surprisingly, although these Exd forms are functional, they do not phenocopy Hth overexpression. These findings suggest that Hth is required for Exd activity, not simply for inducing its nuclear localization. PMID:17277370

  14. RNA Nuclear Export: From Neurological Disorders to Cancer.

    PubMed

    Hautbergue, Guillaume M

    2017-01-01

    The presence of a nuclear envelope, also known as nuclear membrane, defines the structural framework of all eukaryotic cells by separating the nucleus, which contains the genetic material, from the cytoplasm where the synthesis of proteins takes place. Translation of proteins in Eukaryotes is thus dependent on the active transport of DNA-encoded RNA molecules through pores embedded within the nuclear membrane. Several mechanisms are involved in this process generally referred to as RNA nuclear export or nucleocytoplasmic transport of RNA. The regulated expression of genes requires the nuclear export of protein-coding messenger RNA molecules (mRNAs) as well as non-coding RNAs (ncRNAs) together with proteins and pre-assembled ribosomal subunits. The nuclear export of mRNAs is intrinsically linked to the co-transcriptional processing of nascent transcripts synthesized by the RNA polymerase II. This functional coupling is essential for the survival of cells allowing for timely nuclear export of fully processed transcripts, which could otherwise cause the translation of abnormal proteins such as the polymeric repeat proteins produced in some neurodegenerative diseases. Alterations of the mRNA nuclear export pathways can also lead to genome instability and to various forms of cancer. This chapter will describe the molecular mechanisms driving the nuclear export of RNAs with a particular emphasis on mRNAs. It will also review their known alterations in neurological disorders and cancer, and the recent opportunities they offer for the potential development of novel therapeutic strategies.

  15. The interactions with Ro60 and La differentially affect nuclear export of hY1 RNA.

    PubMed

    Simons, F H; Rutjes, S A; van Venrooij, W J; Pruijn, G J

    1996-03-01

    Ro RNPs are evolutionarily conserved ribonucleoprotein particles that consist of a small RNA, known as Y RNA, associated with several proteins, such as La, Ro60, and Ro52. The Y RNAs (Y1-Y5), which are transcribed by RNA polymerase III, have been shown to reside almost exclusively in the cytoplasm as Ro RNPs. To obtain more insight into the nuclear export pathway of Y RNAs, hY1 RNA export was studied in Xenopus laevis oocytes. Injection of various hY1 RNA mutants showed that an intact Ro60 binding site is a prerequisite for nuclear export, whereas the presence of an intact La binding site resulted in strong nuclear retention of hY1 RNA. Competition studies with various classes of RNAs indicated that, in addition to Ro60, another titratable factor was necessary for nuclear export of hY1 RNA. This factor appears also to be involved in nuclear export of tRNA. Because export of hY1 RNA could not be blocked by a synthetic peptide containing the recently identified nuclear export signal of the HIV-1 Rev protein, nuclear export of hY1 RNA does not seem to be dependent on a Rev-like nuclear export signal.

  16. The interactions with Ro60 and La differentially affect nuclear export of hY1 RNA.

    PubMed Central

    Simons, F H; Rutjes, S A; van Venrooij, W J; Pruijn, G J

    1996-01-01

    Ro RNPs are evolutionarily conserved ribonucleoprotein particles that consist of a small RNA, known as Y RNA, associated with several proteins, such as La, Ro60, and Ro52. The Y RNAs (Y1-Y5), which are transcribed by RNA polymerase III, have been shown to reside almost exclusively in the cytoplasm as Ro RNPs. To obtain more insight into the nuclear export pathway of Y RNAs, hY1 RNA export was studied in Xenopus laevis oocytes. Injection of various hY1 RNA mutants showed that an intact Ro60 binding site is a prerequisite for nuclear export, whereas the presence of an intact La binding site resulted in strong nuclear retention of hY1 RNA. Competition studies with various classes of RNAs indicated that, in addition to Ro60, another titratable factor was necessary for nuclear export of hY1 RNA. This factor appears also to be involved in nuclear export of tRNA. Because export of hY1 RNA could not be blocked by a synthetic peptide containing the recently identified nuclear export signal of the HIV-1 Rev protein, nuclear export of hY1 RNA does not seem to be dependent on a Rev-like nuclear export signal. PMID:8608450

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

    PubMed

    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.

  18. 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

  19. Nuclear export of RNA: Different sizes, shapes and functions.

    PubMed

    Williams, Tobias; Ngo, Linh H; Wickramasinghe, Vihandha O

    2017-09-01

    Export of protein-coding and non-coding RNA molecules from the nucleus to the cytoplasm is critical for gene expression. This necessitates the continuous transport of RNA species of different size, shape and function through nuclear pore complexes via export receptors and adaptor proteins. Here, we provide an overview of the major RNA export pathways in humans, highlighting the similarities and differences between each. Its importance is underscored by the growing appreciation that deregulation of RNA export pathways is associated with human diseases like cancer. Copyright © 2017. Published by Elsevier Ltd.

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

    PubMed Central

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

    2016-01-01

    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

  1. Central Role of the Oxygen-dependent Degradation Domain of Drosophila HIFα/Sima in Oxygen-dependent Nuclear Export

    PubMed Central

    Irisarri, Maximiliano; Lavista-Llanos, Sofía; Romero, Nuria M.; Centanin, Lázaro; Dekanty, Andrés

    2009-01-01

    The Drosophila HIFα homologue, Sima, is localized mainly in the cytoplasm in normoxia and accumulates in the nucleus upon hypoxic exposure. We have characterized the mechanism governing Sima oxygen-dependent subcellular localization and found that Sima shuttles continuously between the nucleus and the cytoplasm. We have previously shown that nuclear import depends on an atypical bipartite nuclear localization signal mapping next to the C-terminus of the protein. We show here that nuclear export is mediated in part by a CRM1-dependent nuclear export signal localized in the oxygen-dependent degradation domain (ODDD). CRM1-dependent nuclear export requires both oxygen-dependent hydroxylation of a specific prolyl residue (Pro850) in the ODDD, and the activity of the von Hippel Lindau tumor suppressor factor. At high oxygen tension rapid nuclear export of Sima occurs, whereas in hypoxia, Sima nuclear export is largely inhibited. HIFα/Sima nucleo-cytoplasmic localization is the result of a dynamic equilibrium between nuclear import and nuclear export, and nuclear export is modulated by oxygen tension. PMID:19587118

  2. Central role of the oxygen-dependent degradation domain of Drosophila HIFalpha/Sima in oxygen-dependent nuclear export.

    PubMed

    Irisarri, Maximiliano; Lavista-Llanos, Sofía; Romero, Nuria M; Centanin, Lázaro; Dekanty, Andrés; Wappner, Pablo

    2009-09-01

    The Drosophila HIFalpha homologue, Sima, is localized mainly in the cytoplasm in normoxia and accumulates in the nucleus upon hypoxic exposure. We have characterized the mechanism governing Sima oxygen-dependent subcellular localization and found that Sima shuttles continuously between the nucleus and the cytoplasm. We have previously shown that nuclear import depends on an atypical bipartite nuclear localization signal mapping next to the C-terminus of the protein. We show here that nuclear export is mediated in part by a CRM1-dependent nuclear export signal localized in the oxygen-dependent degradation domain (ODDD). CRM1-dependent nuclear export requires both oxygen-dependent hydroxylation of a specific prolyl residue (Pro850) in the ODDD, and the activity of the von Hippel Lindau tumor suppressor factor. At high oxygen tension rapid nuclear export of Sima occurs, whereas in hypoxia, Sima nuclear export is largely inhibited. HIFalpha/Sima nucleo-cytoplasmic localization is the result of a dynamic equilibrium between nuclear import and nuclear export, and nuclear export is modulated by oxygen tension.

  3. Ran-dependent nuclear export mediators: a structural perspective.

    PubMed

    Güttler, Thomas; Görlich, Dirk

    2011-08-31

    Nuclear export is an essential eukaryotic activity. It proceeds through nuclear pore complexes (NPCs) and is mediated by soluble receptors that shuttle between nucleus and cytoplasm. RanGTPase-dependent export mediators (exportins) constitute the largest class of these carriers and are functionally highly versatile. All of these exportins load their substrates in response to RanGTP binding in the nucleus and traverse NPCs as ternary RanGTP-exportin-cargo complexes to the cytoplasm, where GTP hydrolysis leads to export complex disassembly. The different exportins vary greatly in their substrate range. Recent structural studies of both protein- and RNA-specific exporters have illuminated how exportins bind their cargoes, how Ran triggers cargo loading and how export complexes are disassembled in the cytoplasm. Here, we review the current state of knowledge and highlight emerging principles as well as prevailing questions.

  4. Ran-dependent nuclear export mediators: a structural perspective

    PubMed Central

    Güttler, Thomas; Görlich, Dirk

    2011-01-01

    Nuclear export is an essential eukaryotic activity. It proceeds through nuclear pore complexes (NPCs) and is mediated by soluble receptors that shuttle between nucleus and cytoplasm. RanGTPase-dependent export mediators (exportins) constitute the largest class of these carriers and are functionally highly versatile. All of these exportins load their substrates in response to RanGTP binding in the nucleus and traverse NPCs as ternary RanGTP–exportin–cargo complexes to the cytoplasm, where GTP hydrolysis leads to export complex disassembly. The different exportins vary greatly in their substrate range. Recent structural studies of both protein- and RNA-specific exporters have illuminated how exportins bind their cargoes, how Ran triggers cargo loading and how export complexes are disassembled in the cytoplasm. Here, we review the current state of knowledge and highlight emerging principles as well as prevailing questions. PMID:21878989

  5. Regulation of the Drosophila hypoxia-inducible factor alpha Sima by CRM1-dependent nuclear export.

    PubMed

    Romero, Nuria M; Irisarri, Maximiliano; Roth, Peggy; Cauerhff, Ana; Samakovlis, Christos; Wappner, Pablo

    2008-05-01

    Hypoxia-inducible factor alpha (HIF-alpha) proteins are regulated by oxygen levels through several different mechanisms that include protein stability, transcriptional coactivator recruitment, and subcellular localization. It was previously reported that these transcription factors are mainly nuclear in hypoxia and cytoplasmic in normoxia, but so far the molecular basis of this regulation is unclear. We show here that the Drosophila melanogaster HIF-alpha protein Sima shuttles continuously between the nucleus and the cytoplasm. We identified the relevant nuclear localization signal and two functional nuclear export signals (NESs). These NESs are in the Sima basic helix-loop-helix (bHLH) domain and promote CRM1-dependent nuclear export. Site-directed mutagenesis of either NES provoked Sima nuclear retention and increased transcriptional activity, suggesting that nuclear export contributes to Sima regulation. The identified NESs are conserved and probably functional in the bHLH domains of several bHLH-PAS proteins. We propose that rapid nuclear export of Sima regulates the duration of cellular responses to hypoxia.

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

    PubMed

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

    2014-12-08

    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-Ran(GTP) 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.

  7. Therapeutic Targeting of Nuclear Export Inhibition in Lung Cancer.

    PubMed

    Gupta, Arjun; Saltarski, Jessica M; White, Michael A; Scaglioni, Pier P; Gerber, David E

    2017-09-01

    Intracellular compartmentalization and trafficking of molecules plays a critical role in complex and essential cellular processes. In lung cancer and other malignancies, aberrant nucleocytoplasmic transport of tumor suppressor proteins and cell cycle regulators results in tumorigenesis and inactivation of apoptosis. Pharmacologic agents targeting this process, termed selective inhibitors of nuclear export (SINE), have demonstrated antitumor efficacy in preclinical models and human clinical trials. Exportin-1 (XPO1), which serves as the sole exporter of several tumor suppressor proteins and cell cycle regulators, including retinoblastoma, adenomatous polyposis coli, p53, p73, p21, p27, forkhead box O, signal transducer and activator of transcription 3, inhibitor of κB, topoisomerase II, and protease activated receptor 4-is the principal focus of development of SINE. The most extensively studied of the SINE to date, the exportin-1 inhibitor selinexor (KPT-330 [Karyopharm Therapeutics, Inc., Newton Centre, MA]), has demonstrated single-agent anticancer activity and synergistic effects in combination regimens against multiple cancer types, with principal toxicities of low-grade cytopenias and gastrointestinal effects. SINE may have particular relevance in KRAS-driven tumors, for which this treatment strategy demonstrates significant synthetic lethality. A multicenter phase 1/2 clinical trial of selinexor in previously treated advanced KRAS-mutant NSCLC is under way. Copyright © 2017 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

  8. Protein kinase A is part of a mechanism that regulates nuclear reimport of the nuclear tRNA export receptors Los1p and Msn5p.

    PubMed

    Pierce, Jacqueline B; van der Merwe, George; Mangroo, Dev

    2014-02-01

    The two main signal transduction mechanisms that allow eukaryotes to sense and respond to changes in glucose availability in the environment are the cyclic AMP (cAMP)/protein kinase A (PKA) and AMP-activated protein kinase (AMPK)/Snf1 kinase-dependent pathways. Previous studies have shown that the nuclear tRNA export process is inhibited in Saccharomyces cerevisiae deprived of glucose. However, the signal transduction pathway involved and the mechanism by which glucose availability regulates nuclear-cytoplasmic tRNA trafficking are not understood. Here, we show that inhibition of nuclear tRNA export is caused by a block in nuclear reimport of the tRNA export receptors during glucose deprivation. Cytoplasmic accumulation of the tRNA export receptors during glucose deprivation is not caused by activation of Snf1p. Evidence obtained suggests that PKA is part of the mechanism that regulates nuclear reimport of the tRNA export receptors in response to glucose availability. This mechanism does not appear to involve phosphorylation of the nuclear tRNA export receptors by PKA. The block in nuclear reimport of the tRNA export receptors appears to be caused by activation of an unidentified mechanism when PKA is turned off during glucose deprivation. Taken together, the data suggest that PKA facilitates return of the tRNA export receptors to the nucleus by inhibiting an unidentified activity that facilitates cytoplasmic accumulation of the tRNA export receptors when glucose in the environment is limiting. A PKA-independent mechanism was also found to regulate nuclear tRNA export in response to glucose availability. This mechanism, however, does not regulate nuclear reimport of the tRNA export receptors.

  9. Protein Kinase A Is Part of a Mechanism That Regulates Nuclear Reimport of the Nuclear tRNA Export Receptors Los1p and Msn5p

    PubMed Central

    Pierce, Jacqueline B.; van der Merwe, George

    2014-01-01

    The two main signal transduction mechanisms that allow eukaryotes to sense and respond to changes in glucose availability in the environment are the cyclic AMP (cAMP)/protein kinase A (PKA) and AMP-activated protein kinase (AMPK)/Snf1 kinase-dependent pathways. Previous studies have shown that the nuclear tRNA export process is inhibited in Saccharomyces cerevisiae deprived of glucose. However, the signal transduction pathway involved and the mechanism by which glucose availability regulates nuclear-cytoplasmic tRNA trafficking are not understood. Here, we show that inhibition of nuclear tRNA export is caused by a block in nuclear reimport of the tRNA export receptors during glucose deprivation. Cytoplasmic accumulation of the tRNA export receptors during glucose deprivation is not caused by activation of Snf1p. Evidence obtained suggests that PKA is part of the mechanism that regulates nuclear reimport of the tRNA export receptors in response to glucose availability. This mechanism does not appear to involve phosphorylation of the nuclear tRNA export receptors by PKA. The block in nuclear reimport of the tRNA export receptors appears to be caused by activation of an unidentified mechanism when PKA is turned off during glucose deprivation. Taken together, the data suggest that PKA facilitates return of the tRNA export receptors to the nucleus by inhibiting an unidentified activity that facilitates cytoplasmic accumulation of the tRNA export receptors when glucose in the environment is limiting. A PKA-independent mechanism was also found to regulate nuclear tRNA export in response to glucose availability. This mechanism, however, does not regulate nuclear reimport of the tRNA export receptors. PMID:24297441

  10. High-content screening of natural products reveals novel nuclear export inhibitors.

    PubMed

    Cautain, Bastien; de Pedro, Nuria; Murillo Garzón, Virginia; Muñoz de Escalona, María; González Menéndez, Victor; Tormo, José R; Martin, Jesús; El Aouad, Noureddine; Reyes, Fernando; Asensio, Francisco; Genilloud, Olga; Vicente, Francisca; Link, Wolfgang

    2014-01-01

    Natural products are considered an extremely valuable source for the discovery of new drugs against diverse pathologies. As yet, we have only explored a fraction of the diversity of bioactive compounds, and opportunities for discovering new natural products leading to new drugs are huge. In the present study, U2nesRELOC, a previously established cell-based imaging assay, was employed to screen a collection of extracts of microbial origin for nuclear export inhibition activity. The fluorescent signal of untreated U2nesRELOC cells localizes predominantly to the cytoplasm. Upon treatment with the nuclear export inhibitor leptomycin B, the fluorescent-tagged reporter proteins appear as speckles in the nucleus. A proprietary collection of extracts from fungi, actinomycetes, and unicellular bacteria that covers an uncommonly broad chemical space was used to interrogate this nuclear export assay system. A two-step image-based analysis allowed us to identify 12 extracts with biological activities that are not associated with previously known active metabolites. The fractionation and structural elucidation of active compounds revealed several chemical structures with nuclear export inhibition activity. Here we show that substrates of the nuclear export receptor CRM1, such as Rev, FOXO3a and NF-κB, accumulate in the nucleus in the presence of the fungal metabolite MDN-0105 with an IC50 value of 3.4 µM. Many important processes in tumor formation and progression, as well as in many viral infections, critically depend on the nucleocytoplasmic trafficking of proteins and RNA molecules. Therefore, the disruption of nuclear export is emerging as a novel therapeutic approach with enormous clinical potential. Our work highlights the potential of applying high-throughput phenotypic imaging on natural product extracts to identify novel nuclear export inhibitors.

  11. Nuclear Imprisonment: Viral Strategies to Arrest Host mRNA Nuclear Export

    PubMed Central

    Kuss, Sharon K.; Mata, Miguel A.; Zhang, Liang; Fontoura, Beatriz M. A.

    2013-01-01

    Viruses possess many strategies to impair host cellular responses to infection. Nuclear export of host messenger RNAs (mRNA) that encode antiviral factors is critical for antiviral protein production and control of viral infections. Several viruses have evolved sophisticated strategies to inhibit nuclear export of host mRNAs, including targeting mRNA export factors and nucleoporins to compromise their roles in nucleo-cytoplasmic trafficking of cellular mRNA. Here, we present a review of research focused on suppression of host mRNA nuclear export by viruses, including influenza A virus and vesicular stomatitis virus, and the impact of this viral suppression on host antiviral responses. PMID:23872491

  12. Stress-induced nuclear export of 5-lipoxygenase

    SciTech Connect

    Hanaka, Hiromi; Shimizu, Takao; Izumi, Takashi . E-mail: takizumi@med.gunma-u.ac.jp

    2005-12-09

    A key enzyme for leukotriene biosynthesis is 5-lipoxygenase (5-LO), which we found is exported from the nucleus when p38 MAPK is activated. CHO-K1 cells stably express green fluorescent protein-5-lipoxygenase fusion protein (GFP-5LO), which is located predominantly in the nucleus, and is exported by anisomycin, hydrogen peroxide, and sorbitol, with activation of p38 MAPK. SB203580, an inhibitor of p38 MAPK, and Leptomycin B, an inhibitor of the nuclear export, blocked the anisomycin-induced export of GFP-5LO. When HEK293 cells were transformed with plasmids for wild-type GFP-5LO, GFP-5LO-S271A or GFP-5LO-S271E mutants, most wild-type GFP-5LO and GFP-5LO-S271A localized in the nucleus, but GFP-5LO-S271E localized in the cytosol. Thus, phosphorylation at Ser-271 of 5-LO is important for its export. Endogenous 5-LO in RBL cells stimulated with anisomycin was also exported from the nucleus. These results suggest that the nuclear export of 5-LO depends on the stress-induced activation of the p38 MAPK pathway.

  13. 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

    ... / Wednesday, May 9, 2012 / Rules and Regulations#0;#0; ] NUCLEAR REGULATORY COMMISSION 10 CFR Part 110 RIN 3150-AJ04 Export and Import of Nuclear Equipment and Material; Export of International Atomic Energy Agency Safeguards Samples AGENCY: Nuclear Regulatory Commission. ACTION: Final rule. SUMMARY: The...

  14. Featured Article: Nuclear export of opioid growth factor receptor is CRM1 dependent.

    PubMed

    Kren, Nancy P; Zagon, Ian S; McLaughlin, Patricia J

    2016-02-01

    Opioid growth factor receptor (OGFr) facilitates growth inhibition in the presence of its specific ligand opioid growth factor (OGF), chemically termed [Met(5)]-enkephalin. The function of the OGF-OGFr axis requires the receptor to translocate to the nucleus. However, the mechanism of nuclear export of OGFr is unknown. In this study, endogenous OGFr, as well as exogenously expressed OGFr-EGFP, demonstrated significant nuclear accumulation in response to leptomycin B (LMB), an inhibitor of CRM1-dependent nuclear export, suggesting that OGFr is exported in a CRM1-dependent manner. One consensus sequence for a nuclear export signal (NES) was identified. Mutation of the associated leucines, L217 L220 L223 and L225, to alanine resulted in decreased nuclear accumulation. NES-EGFP responded to LMB, indicating that this sequence is capable of functioning as an export signal in isolation. To determine why the sequence functions differently in isolation than as a full length protein, the localization of subNES was evaluated in the presence and absence of MG132, a potent inhibitor of proteosomal degradation. MG132 had no effect of subNES localization. The role of tandem repeats located at the C-terminus of OGFr was examined for their role in nuclear trafficking. Six of seven tandem repeats were removed to form deltaTR. DeltaTR localized exclusively to the nucleus indicating that the tandem repeats may contribute to the localization of the receptor. Similar to the loss of cellular proliferation activity (i.e. inhibition) recorded with subNES, deltaTR also demonstrated a significant loss of inhibitory activity indicating that the repeats may be integral to receptor function. These experiments reveal that OGFr contains one functional NES, L217 L220 L223 and L225 and can be exported from the nucleus in a CRM1-dependent manner. © 2015 by the Society for Experimental Biology and Medicine.

  15. Multiple vesiculoviral matrix proteins inhibit both nuclear export and import

    PubMed Central

    Petersen, Jeannine M.; Her, Lu-Shiun; Dahlberg, James E.

    2001-01-01

    The matrix (M) protein of vesicular stomatitis virus inhibits both nuclear import and export. Here, we demonstrate that this inhibitory property is conserved between the M proteins from two other vesiculoviruses, chandipura virus and spring viremia carp virus. All three M proteins completely block nuclear transport of spliced mRNA, small nuclear RNAs, and small nuclear ribonucleoproteins and slow the nuclear transport of many other cargoes. In all cases where transport was merely slowed by the M proteins, the chandipura virus M protein had the strongest inhibitory activity. When expressed in transfected HeLa cells, active M proteins displayed prominent association with the nuclear rim. Moreover, mutation of a conserved methionine abolished both the inhibitory activity and efficient targeting of the M proteins to the nuclear rim. We propose that all of the vesiculoviral M proteins associate with the same nuclear target, which is likely to be a component of the nuclear pore complex. PMID:11447272

  16. Rev-mediated nuclear export of RNA is dominant over nuclear retention and is coupled to the Ran-GTPase cycle.

    PubMed

    Fischer, U; Pollard, V W; Lührmann, R; Teufel, M; Michael, M W; Dreyfuss, G; Malim, M H

    1999-11-01

    The human immunodeficiency virus type-1 Rev protein induces the nuclear export of intron-containing viral mRNAs that harbor its binding site, the Rev response element (RRE). A leucine-rich region of Rev, the activation domain, is essential for function and has been shown to be a nuclear export signal (NES). Although Rev exports viral RNAs that resemble cellular mRNAs, competition studies performed using microinjected Xenopus laevis oocytes have previously indicated that Rev utilizes a non-mRNA export pathway. Here, we show that Rev is able to induce the export of both spliceable and non-spliceable RRE-containing pre-mRNAs and that this activity is not dependent on the location of the RRE within the RNA. Importantly, even RNA molecules of different classes, such as U3 snoRNA and U6 snRNA, which are retained in the nucleus by non-pre-mRNA mechanisms, are exported to the cytoplasm in response to Rev. Consistent with the notion that Rev-mediated export of RRE-containing RNA is mechanistically distinct from the export of processed cellular mRNA, a chimeric Rev protein in which its NES is replaced by the NES of hnRNP A1 does not induce the export of a Rev-responsive mRNA. Finally, we demonstrate that Rev/RRE-activated RNA export is, like other nuclear export pathways, linked to the Ran-GTPase cycle.

  17. Tpr is localized within the nuclear basket of the pore complex and has a role in nuclear protein export.

    PubMed

    Frosst, Phyllis; Guan, Tinglu; Subauste, Cecilia; Hahn, Klaus; Gerace, Larry

    2002-02-18

    Tpr is a coiled-coil protein found near the nucleoplasmic side of the pore complex. Since neither the precise localization of Tpr nor its functions are well defined, we generated antibodies to three regions of Tpr to clarify these issues. Using light and EM immunolocalization, we determined that mammalian Tpr is concentrated within the nuclear basket of the pore complex in a distribution similar to Nup153 and Nup98. Antibody localization together with imaging of GFP-Tpr in living cells revealed that Tpr is in discrete foci inside the nucleus similar to several other nucleoporins but is not present in intranuclear filamentous networks (Zimowska et al., 1997) or in long filaments extending from the pore complex (Cordes et al., 1997) as proposed. Injection of anti-Tpr antibodies into mitotic cells resulted in depletion of Tpr from the nuclear envelope without loss of other pore complex basket proteins. Whereas nuclear import mediated by a basic amino acid signal was unaffected, nuclear export mediated by a leucine-rich signal was retarded significantly. Nuclear injection of anti-Tpr antibodies in interphase cells similarly yielded inhibition of protein export but not import. These results indicate that Tpr is a nucleoporin of the nuclear basket with a role in nuclear protein export.

  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 licensing...

  19. 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 licensing...

  20. 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 licensing...

  1. 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 licensing...

  2. 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...

  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 110.21 - 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....

  5. 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....

  6. 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....

  7. 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....

  8. Analysis of CRM1-Dependent Nuclear Export in Permeabilized Cells.

    PubMed

    Kehlenbach, Ralph H; Port, Sarah A

    2016-01-01

    Nuclear protein import and export assays in permeabilized cells have been instrumental for the identification of transport factors and for the molecular characterization of nucleocytoplasmic transport pathways. Our original assay to quantitatively analyze CRM1-dependent export was based on stably transfected cells expressing GFP-NFAT. We now present a simplified version of the assay using transiently transfected cells expressing GFP-NFAT or GFP-snurportin1 as a fluorescent export cargo and mCherry-emerin as a marker protein for transfected cells. CRM1- and Ran-dependent export is recapitulated in digitonin-permeabilized cells and quantified by flow cytometry. The assay should be applicable to other combinations of cargo and marker proteins.

  9. Nuclear export of the glucocorticoid receptor is accelerated by cell fusion-dependent release of calreticulin.

    PubMed

    Walther, Rhian F; Lamprecht, Claudia; Ridsdale, Andrew; Groulx, Isabelle; Lee, Stephen; Lefebvre, Yvonne A; Haché, Robert J G

    2003-09-26

    Nucleocytoplasmic exchange of nuclear hormone receptors is hypothesized to allow for rapid and direct interactions with cytoplasmic signaling factors. In addition to recycling between a naïve, chaperone-associated cytoplasmic complex and a liganded chaperone-free nuclear form, the glucocorticoid receptor (GR) has been observed to shuttle between nucleus and cytoplasm. Nuclear export of GR and other nuclear receptors has been proposed to depend on direct interactions with calreticulin, which is predominantly localized to the lumen of the endoplasmic reticulum. We show that rapid calreticulin-mediated nuclear export of GR is a specific response to transient disruption of the endoplasmic reticulum that occurs during polyethylene glycol-mediated cell fusion. Using live and digitonin-permeabilized cells we demonstrate that, in the absence of cell fusion, GR nuclear export occurs slowly over a period of many hours independent of direct interaction with calreticulin. Our findings temper expectations that nuclear receptors respond rapidly and directly to cytoplasmic signals in the absence of additional regulatory control. These results highlight the importance of verifying findings of nucleocytoplasmic trafficking using techniques in addition to heterokaryon cell fusion.

  10. Evaluating Russian Dual-Use Nuclear Exports

    DTIC Science & Technology

    2007-06-01

    Facilities: Bushehr-1.”. 80 Victor Mizin, “The Russia - Iran Nuclear Connection and U.S. Policy Options,” Middle East Review of Interenational Affairs...english/about/mission.html Accessed April 15, 2004. Mizin, Victor, “The Russia - Iran Nuclear Connection and U.S. Policy Options,” Middle East

  11. Nuclear export of the influenza virus ribonucleoprotein complex: Interaction of Hsc70 with viral proteins M1 and NS2.

    PubMed

    Watanabe, Ken; Shimizu, Teppei; Noda, Saiko; Tsukahara, Fujiko; Maru, Yoshiro; Kobayashi, Nobuyuki

    2014-01-01

    The influenza virus replicates in the host cell nucleus, and the progeny viral ribonucleoprotein complex (vRNP) is exported to the cytoplasm prior to maturation. NS2 has a nuclear export signal that mediates the nuclear export of vRNP by the vRNP-M1-NS2 complex. We previously reported that the heat shock cognate 70 (Hsc70) protein binds to M1 protein and mediates vRNP export. However, the interactions among M1, NS2, and Hsc70 are poorly understood. In the present study, we demonstrate that Hsc70 interacts with M1 more strongly than with NS2 and competes with NS2 for M1 binding, suggesting an important role of Hsc70 in the nuclear export of vRNP.

  12. Identification of multiple nuclear export sequences in Fanconi anemia group A protein that contribute to CRM1-dependent nuclear export.

    PubMed

    Ferrer, Miriam; Rodríguez, Jose A; Spierings, Ellen A; de Winter, Johan P; Giaccone, Giuseppe; Kruyt, Frank A E

    2005-05-15

    The Fanconi anemia (FA) pathway plays an important role in maintaining genomic stability, and defects in this pathway cause cancer susceptibility. The FA proteins have been found to function primarily in a nuclear complex, although a cytoplasmic localization and function for several FA proteins has also been reported. In this study, we investigated the possibility that FANCA, FANCC and FANCG are subjected to active export out of the nucleus. After treatment with leptomycin B, a specific inhibitor of CRM1-mediated nuclear export, the accumulation of epitope-tagged FANCA in the nucleus increased, whereas FANCC was affected to a lesser extent and FANCG showed no response. CRM1-mediated export of FANCA was further confirmed using CRM1 cotransfection, which led to a dramatic relocalization of FANCA to the cytoplasm. Five functional leucine-rich nuclear export sequences (NESs) distributed throughout the FANCA sequence were identified and characterized using an in vivo export assay. Simultaneous inactivation of three of these NESs resulted in a discrete but reproducible increase of FANCA nuclear accumulation. However, these NES mutations did not affect the ability of FANCA to complement the mitomycin C or cisplatin sensitivity of FA-A lymphoblasts. Surprisingly, mutations in the other two NESs resulted in an almost complete relocation of the protein to cytoplasm, suggesting that these motifs overlap with domains that are crucial for nuclear import. Taken together, these findings indicate that FANCA can be actively exported out of the nucleus by CRM1, revealing a new mechanism to regulate the function of the FA protein complex.

  13. Cofactor requirements for nuclear export of Rev response element (RRE)- and constitutive transport element (CTE)-containing retroviral RNAs. An unexpected role for actin.

    PubMed

    Hofmann, W; Reichart, B; Ewald, A; Müller, E; Schmitt, I; Stauber, R H; Lottspeich, F; Jockusch, B M; Scheer, U; Hauber, J; Dabauvalle, M C

    2001-03-05

    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).

  14. 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.

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

    PubMed

    Kang, WonKyung; Kurihara, Masaaki; Matsumoto, Shogo

    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.

  16. RanBP3 Regulates Melanoma Cell Proliferation via Selective Control of Nuclear Export.

    PubMed

    Pathria, Gaurav; Garg, Bhavuk; Wagner, Christine; Garg, Kanika; Gschaider, Melanie; Jalili, Ahmad; Wagner, Stephan N

    2016-01-01

    Chromosome region maintenance 1-mediated nucleocytoplasmic transport has been shown as a potential anticancer target in various malignancies. However, the role of the most characterized chromosome region maintenance 1 cofactor ran binding protein 3 (RanBP3) in cancer cell biology has never been investigated. Utilizing a loss-of-function experimental setting in a vast collection of genetically varied melanoma cell lines, we observed the requirement of RanBP3 in melanoma cell proliferation and survival. Mechanistically, we suggest the reinstatement of transforming growth factor-β (TGF-β)-Smad2/3-p21(Cip1) tumor-suppressor axis as part of the RanBP3 silencing-associated antiproliferative program. Employing extensive nuclear export sequence analyses and immunofluorescence-based protein localization studies, we further present evidence suggesting the requirement of RanBP3 function for the nuclear exit of the weak nuclear export sequence-harboring extracellular signal-regulated kinase protein, although it is dispensable for general CRM1-mediated nuclear export of strong nuclear export sequence-harboring cargoes. Rendering mechanistic support to RanBP3 silencing-mediated apoptosis, consequent to extracellular signal-regulated kinase nuclear entrapment, we observed increased levels of cytoplasmically restricted nonphosphorylated/active proapoptotic Bcl-2-antagonist of cell death (BAD) protein. Last, we present evidence suggesting the frequently activated mitogen-activated protein kinase signaling in melanoma as a potential founding basis for a deregulated post-translational control of RanBP3 activity. Collectively, the presented data suggest RanBP3 as a potential target for therapeutic intervention in human melanoma.

  17. Nuclear export of the yeast hexokinase 2 protein requires the Xpo1 (Crm1)-dependent pathway.

    PubMed

    Peláez, Rafael; Herrero, Pilar; Moreno, Fernando

    2009-07-31

    Hexokinase 2 (Hxk2) from Saccharomyces cerevisiae was one of the first metabolic enzymes described as a multifunctional protein. Hxk2 has a double subcellular localization; it functions as a glycolytic enzyme in the cytoplasm and as a regulator of gene transcription of several Mig1-regulated genes in the nucleus. However, the mechanism by which Hxk2 enters and leaves the nucleus is still unknown. In low glucose conditions, Hxk2 is phosphorylated at serine 14, but how this phosphorylation may affect glucose signaling is also unknown at the moment. Here we report that the Hxk2 protein is an export substrate of the carrier protein Xpo1 (Crm1). We also show that the Hxk2 nuclear export and the binding of Hxk2 and Xpo1 involve two leucine-rich nuclear export signals (NES) located between leucine 23 and isoleucine 33 (NES1) and between leucine 310 and leucine 318 (NES2). We also show that the Hxk2 phosphorylation at serine 14 promotes Hxk2 export by facilitating the association of Hxk2 with Xpo1. Our study uncovers a new cargo for the Xpo1 yeast exportin and identifies Hxk2 phosphorylation at serine 14 as a regulatory mechanism that controls its nuclear exit in function of the glucose levels.

  18. Nuclear export inhibition through covalent conjugation and hydrolysis of Leptomycin B by CRM1

    PubMed Central

    Sun, Qingxiang; Carrasco, Yazmin P.; Hu, Youcai; Guo, Xiaofeng; Mirzaei, Hamid; MacMillan, John; Chook, Yuh Min

    2013-01-01

    The polyketide natural product Leptomycin B inhibits nuclear export mediated by the karyopherin protein chromosomal region maintenance 1 (CRM1). Here, we present 1.8- to 2.0-Å-resolution crystal structures of CRM1 bound to Leptomycin B and related inhibitors Anguinomycin A and Ratjadone A. Structural and complementary chemical analyses reveal an unexpected mechanism of inhibition involving covalent conjugation and CRM1-mediated hydrolysis of the natural products’ lactone rings. Furthermore, mutagenesis reveals the mechanism of hydrolysis by CRM1. The nuclear export signal (NES)-binding groove of CRM1 is able to drive a chemical reaction in addition to binding protein cargos for transport through the nuclear pore complex. PMID:23297231

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

    PubMed

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

    2013-03-01

    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.

  20. CHD3 facilitates vRNP nuclear export by interacting with NES1 of influenza A virus NS2.

    PubMed

    Hu, Yong; Liu, Xiaokun; Zhang, Anding; Zhou, Hongbo; Liu, Ziduo; Chen, Huanchun; Jin, Meilin

    2015-03-01

    NS2 from influenza A virus mediates Crm1-dependent vRNP nuclear export through interaction with Crm1. However, even though the nuclear export signal 1 (NES1) of NS2 does not play a requisite role in NS2-Crm1 interaction, there is no doubt that NES1 is crucial for vRNP nuclear export. While the mechanism of the NES1 is still unclear, it is speculated that certain host partners might mediate the NES1 function through their interaction with NES1. In the present study, chromodomain-helicase-DNA-binding protein 3 (CHD3) was identified as a novel host nuclear protein for locating NS2 and Crm1 on dense chromatin for NS2 and Crm1-dependent vRNP nuclear export. CHD3 was confirmed to interact with NES1 in NS2, and a disruption to this interaction by mutation in NES1 significantly delayed viral vRNPs export and viral propagation. Further, the knockdown of CHD3 would affect the propagation of the wild-type virus but not the mutant with the weakened NS2-CHD3 interaction. Therefore, this study demonstrates that NES1 is required for maximal binding of NS2 to CHD3, and that the NS2-CHD3 interaction on the dense chromatin contributed to the NS2-mediated vRNP nuclear export.

  1. RanGTP-Binding Protein NXT1 Facilitates Nuclear Export of Different Classes of RNA In Vitro

    PubMed Central

    Ossareh-Nazari, Batool; Maison, Christèle; Black, Ben E.; Lévesque, Lyne; Paschal, Bryce M.; Dargemont, Catherine

    2000-01-01

    To better characterize the mechanisms responsible for RNA export from the nucleus, we developed an in vitro assay based on the use of permeabilized HeLa cells. This new assay supports nuclear export of U1 snRNA, tRNA, and mRNA in an energy- and Xenopus extract-dependent manner. U1 snRNA export requires a 5′ monomethylated cap structure, the nuclear export signal receptor CRM1, and the small GTPase Ran. In contrast, mRNA export does not require the participation of CRM1. We show here that NXT1, an NTF2-related protein that binds directly to RanGTP, strongly stimulates export of U1 snRNA, tRNA, and mRNA. The ability of NXT1 to promote export is dependent on its capacity to bind RanGTP. These results support the emerging view that NXT1 is a general export factor, functioning on both CRM1-dependent and CRM1-independent pathways of RNA export. PMID:10848583

  2. Mechanism for G2 phase-specific nuclear export of the kinetochore protein CENP-F.

    PubMed

    Loftus, Kyle M; Cui, Heying; Coutavas, Elias; King, David S; Ceravolo, Amanda; Pereiras, Dylan; Solmaz, Sozanne R

    2017-08-03

    Centromere protein F (CENP-F) is a component of the kinetochore and a regulator of cell cycle progression. CENP-F recruits the dynein transport machinery and orchestrates several cell cycle-specific transport events, including transport of the nucleus, mitochondria and chromosomes. A key regulatory step for several of these functions is likely the G2 phase-specific export of CENP-F from the nucleus to the cytosol, where the cytoplasmic dynein transport machinery resides; however, the molecular mechanism of this process is elusive. Here, we have identified 3 phosphorylation sites within the bipartite classical nuclear localization signal (cNLS) of CENP-F. These sites are specific for cyclin-dependent kinase 1 (Cdk1), which is active in G2 phase. Phosphomimetic mutations of these residues strongly diminish the interaction of the CENP-F cNLS with its nuclear transport receptor karyopherin α. These mutations also diminish nuclear localization of the CENP-F cNLS in cells. Notably, the cNLS is phosphorylated in the -1 position, which is important to orient the adjacent major motif for binding into its pocket on karyopherin α. We propose that localization of CENP-F is regulated by a cNLS, and a nuclear export pathway, resulting in nuclear localization during most of interphase. In G2 phase, the cNLS is weakened by phosphorylation through Cdk1, likely resulting in nuclear export of CENP-F via the still active nuclear export pathway. Once CENP-F resides in the cytosol, it can engage in pathways that are important for cell cycle progression, kinetochore assembly and the faithful segregation of chromosomes into daughter cells.

  3. 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 EQUIPMENT AND MATERIAL Review of License Applications § 110.46 Conduct resulting in termination of nuclear...

  4. 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 EQUIPMENT AND MATERIAL Review of License Applications § 110.46 Conduct resulting in termination of nuclear...

  5. 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 nuclear...

  6. 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 EQUIPMENT AND MATERIAL Review of License Applications § 110.46 Conduct resulting in termination of nuclear...

  7. 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... NUCLEAR EQUIPMENT AND MATERIAL Licenses § 110.26 General license for the export of nuclear reactor...) of this section any nuclear reactor component of U.S. origin described in paragraphs (5) through...

  8. 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... NUCLEAR EQUIPMENT AND MATERIAL Licenses § 110.26 General license for the export of nuclear reactor...) of this section any nuclear reactor component of U.S. origin described in paragraphs (5) through...

  9. 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... NUCLEAR EQUIPMENT AND MATERIAL Licenses § 110.26 General license for the export of nuclear reactor...) of this section any nuclear reactor component of U.S. origin described in paragraphs (5) through...

  10. 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... NUCLEAR EQUIPMENT AND MATERIAL Licenses § 110.26 General license for the export of nuclear reactor...) of this section any nuclear reactor component of U.S. origin described in paragraphs (5) through...

  11. 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... NUCLEAR EQUIPMENT AND MATERIAL Licenses § 110.26 General license for the export of nuclear reactor... nuclear power or research reactor in the United States: Austria Belgium Bulgaria Canada Czech...

  12. Novel nuclear localization signal regulated by ambient tonicity in vertebrates.

    PubMed

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

    2008-08-15

    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.

  13. 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

  14. 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.

  15. Regulation of the Drosophila Hypoxia-Inducible Factor α Sima by CRM1-Dependent Nuclear Export

    PubMed Central

    Romero, Nuria M.; Irisarri, Maximiliano; Roth, Peggy; Cauerhff, Ana; Samakovlis, Christos; Wappner, Pablo

    2008-01-01

    Hypoxia-inducible factor α (HIF-α) proteins are regulated by oxygen levels through several different mechanisms that include protein stability, transcriptional coactivator recruitment, and subcellular localization. It was previously reported that these transcription factors are mainly nuclear in hypoxia and cytoplasmic in normoxia, but so far the molecular basis of this regulation is unclear. We show here that the Drosophila melanogaster HIF-α protein Sima shuttles continuously between the nucleus and the cytoplasm. We identified the relevant nuclear localization signal and two functional nuclear export signals (NESs). These NESs are in the Sima basic helix-loop-helix (bHLH) domain and promote CRM1-dependent nuclear export. Site-directed mutagenesis of either NES provoked Sima nuclear retention and increased transcriptional activity, suggesting that nuclear export contributes to Sima regulation. The identified NESs are conserved and probably functional in the bHLH domains of several bHLH-PAS proteins. We propose that rapid nuclear export of Sima regulates the duration of cellular responses to hypoxia. PMID:18332128

  16. ATM induces MacroD2 nuclear export upon DNA damage

    PubMed Central

    Golia, Barbara; Moeller, Giuliana Katharina; Jankevicius, Gytis; Schmidt, Andreas; Hegele, Anna; Preißer, Julia; Tran, Mai Ly; Imhof, Axel; Timinszky, Gyula

    2017-01-01

    ADP-ribosylation is a dynamic post-translation modification that regulates the early phase of various DNA repair pathways by recruiting repair factors to chromatin. ADP-ribosylation levels are defined by the activities of specific transferases and hydrolases. However, except for the transferase PARP1/ARDT1 little is known about regulation of these enzymes. We found that MacroD2, a mono-ADP-ribosylhydrolase, is exported from the nucleus upon DNA damage, and that this nuclear export is induced by ATM activity. We show that the export is dependent on the phosphorylation of two SQ/TQ motifs, suggesting a novel direct interaction between ATM and ADP-ribosylation. Lastly, we show that MacroD2 nuclear export temporally restricts its recruitment to DNA lesions, which may decrease the net ADP-ribosylhydrolase activity at the site of DNA damage. Together, our results identify a novel feedback regulation between two crucial DNA damage-induced signaling pathways: ADP-ribosylation and ATM activation. PMID:28069995

  17. 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...

  18. 5-Flurouracil disrupts nuclear export and nuclear pore permeability in a calcium dependent manner.

    PubMed

    Higby, Kelly J; Bischak, Melissa M; Campbell, Christina A; Anderson, Rebecca G; Broskin, Sarah A; Foltz, Lauren E; Koper, Jarrett A; Nickle, Audrey C; Resendes, Karen K

    2017-03-01

    Regulation of nuclear transport is an essential component of apoptosis. As chemotherapy induced cell death progresses, nuclear transport and the nuclear pore complex (NPC) are slowly disrupted and dismantled. 5-Fluorouracil (5-FU) and the camptothecin derivatives irinotecan and topotecan, are linked to altered nuclear transport of specific proteins; however, their general effects on the NPC and transport during apoptosis have not been characterized. We demonstrate that 5-FU, but not topotecan, increases NPC permeability, and disrupts Ran-mediated nuclear transport before the disruption of the NPC. This increased permeability is dependent on increased cellular calcium, as the Ca(2+) chelator BAPTA-AM, abolishes the effect. Furthermore, increased calcium alone was sufficient to disrupt the Ran gradient. Combination treatments of 5-FU with topotecan or irinotecan, similarly disrupted nuclear transport before disassembly of the NPC. In both single and combination treatments nuclear transport was disrupted before caspase 9 activation, indicating that 5-FU induces an early caspase-independent increase in NPC permeability and alteration of nuclear transport. Because Crm1-mediated nuclear export of tumor suppressors is linked to drug resistance we also examined the effect of 5-FU on the nuclear export of a specific target, topoisomerase. 5-FU treatment led to accumulation of topoisomerase in the nucleus and recovered the loss nuclear topoisomerase induced by irinotecan or topotecan, a known cause of drug resistance. Furthermore, 5-FU retains its ability to cause nuclear accumulation of p53 in the presence of irinotecan or topotecan. Our results reveal a new mechanism of action for these therapeutics during apoptosis, opening the door to other potential combination chemotherapies that employ 5-FU as a calcium mediated inhibitor of Crm1-induced nuclear export of tumor suppressors.

  19. Brassinosteroid nuclear signaling recruits HSP90 activity.

    PubMed

    Samakovli, Despina; Margaritopoulou, Theoni; Prassinos, Constantinos; Milioni, Dimitra; Hatzopoulos, Polydefkis

    2014-08-01

    Heat shock protein 90 (HSP90) controls a number of developmental circuits, and serves a sophisticated and highly regulatory function in signaling pathways. Brassinosteroids (BRs) control many aspects of plant development. Genetic, physiological, cytological, gene expression, live cell imaging, and pharmacological approaches provide conclusive evidence for HSP90 involvement in Arabidopsis thalianaBR signaling. Nuclear-localized HSP90s translocate to cytoplasm when their activity is blocked by the HSP90 inhibitor geldanamycin (GDA). GDA treatment promoted the export of BIN2, a regulator of BR signaling, from the nucleus into the cytoplasm, indicating that active HSP90 is required to sustain BIN2 in the nucleus. HSP90 nuclear localization was inhibited by brassinolide (BL). HSP90s interact with BIN2 in the nucleus of untreated cells and in the cytoplasm of BL-treated cells, showing that the site-specific action of HSP90 on BIN2 is controlled by BRs. GDA and BL treatments change the expression of a common set of previously identified BR-responsive genes. This highlights the effect of active HSP90s on the regulation of BR-responsive genes. Our observations reveal that HSP90s have a central role in sustaining BIN2 nuclear function. We propose that BR signaling is mediated by HSP90 activity and via trafficking of BIN2-HSP90 complexes into the cytoplasm. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

  20. 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

  1. Efficient nuclear export of p65-IkappaBalpha complexes requires 14-3-3 proteins.

    PubMed

    Aguilera, Cristina; Fernández-Majada, Vanessa; Inglés-Esteve, Julia; Rodilla, Verónica; Bigas, Anna; Espinosa, Lluís

    2006-09-01

    IkappaB are responsible for maintaining p65 in the cytoplasm under non-stimulating conditions and promoting the active export of p65 from the nucleus following NFkappaB activation to terminate the signal. We now show that 14-3-3 proteins regulate the NFkappaB signaling pathway by physically interacting with p65 and IkappaBalpha proteins. We identify two functional 14-3-3 binding domains in the p65 protein involving residues 38-44 and 278-283, and map the interaction region of IkappaBalpha in residues 60-65. Mutation of these 14-3-3 binding domains in p65 or IkappaBalpha results in a predominantly nuclear distribution of both proteins. TNFalpha treatment promotes recruitment of 14-3-3 and IkappaBalpha to NFkappaB-dependent promoters and enhances the binding of 14-3-3 to p65. Disrupting 14-3-3 activity by transfection with a dominant-negative 14-3-3 leads to the accumulation of nuclear p65-IkappaBalpha complexes and the constitutive association of p65 with the chromatin. In this situation, NFkappaB-dependent genes become unresponsive to TNFalpha stimulation. Together our results indicate that 14-3-3 proteins facilitate the nuclear export of IkappaBalpha-p65 complexes and are required for the appropriate regulation of NFkappaB signaling.

  2. 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.

  3. IRAK2 directs stimulus-dependent nuclear export of inflammatory mRNAs.

    PubMed

    Zhou, Hao; Bulek, Katarzyna; Li, Xiao; Herjan, Tomasz; Yu, Minjia; Qian, Wen; Wang, Han; Zhou, Gao; Chen, Xing; Yang, Hui; Hong, Lingzi; Zhao, Junjie; Qin, Luke; Fukuda, Koichi; Flotho, Annette; Gao, Ji; Dongre, Ashok; Carman, Julie A; Kang, Zizhen; Su, Bing; Kern, Timothy S; Smith, Jonathan D; Hamilton, Thomas A; Melchior, Frauke; Fox, Paul L; Li, Xiaoxia

    2017-10-09

    Expression of inflammatory genes is determined in part by post-transcriptional regulation of mRNA metabolism but how stimulus- and transcript-dependent nuclear export influence is poorly understood. Here we report a novel pathway in which LPS/TLR4 engagement promotes nuclear localization of IRAK2 to facilitate nuclear export of a specific subset of inflammation related mRNAs for translation in murine macrophages. IRAK2 kinase activity is required for LPS-induced RanBP2-mediated IRAK2 sumoylation and subsequent nuclear translocation. Array analysis showed that an SRSF1 binding motif is enriched in mRNAs dependent on IRAK2 for nuclear export. Nuclear IRAK2 phosphorylates SRSF1 to reduce its binding to target mRNAs, which promotes the RNA binding of the nuclear export adaptor ALYREF and nuclear export receptor Nxf1 loading for the export of the mRNAs. In summary, LPS activates a nuclear function of IRAK2 that facilitates the assembly of nuclear export machinery to export selected inflammatory mRNAs to the cytoplasm for translation.

  4. Assessing mRNA nuclear export in mammalian cells by microinjection.

    PubMed

    Lee, Eliza S; Palazzo, Alexander F

    2017-08-15

    The nuclear export of mRNAs is an important yet little understood part of eukaryotic gene expression. One of the easiest methods for monitoring mRNA export in mammalian tissue culture cells is through the microinjection of DNA plasmids into the nucleus and monitoring the distribution of the transcribed product over time. Here we describe how to setup a microscope equipped with a micromanipulator used in cell microinjections, and we explain how to perform a nuclear mRNA export assay and obtain the nuclear export rate for any given mRNA. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. 37 CFR 5.20 - Export of technical data relating to sensitive nuclear technology.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 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 UNITED....20 Export of technical data relating to sensitive nuclear technology. Under regulations (10 CFR 810.7...

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

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 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 UNITED....20 Export of technical data relating to sensitive nuclear technology. Under regulations (10 CFR 810.7...

  7. 37 CFR 5.20 - Export of technical data relating to sensitive nuclear technology.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 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 UNITED....20 Export of technical data relating to sensitive nuclear technology. Under regulations (10 CFR 810.7...

  8. 37 CFR 5.20 - Export of technical data relating to sensitive nuclear technology.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 37 Patents, Trademarks, and Copyrights 1 2012-07-01 2012-07-01 false Export of technical data relating to sensitive nuclear technology. 5.20 Section 5.20 Patents, Trademarks, and Copyrights UNITED....20 Export of technical data relating to sensitive nuclear technology. Under regulations (10 CFR 810.7...

  9. 37 CFR 5.20 - Export of technical data relating to sensitive nuclear technology.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 37 Patents, Trademarks, and Copyrights 1 2010-07-01 2010-07-01 false Export of technical data relating to sensitive nuclear technology. 5.20 Section 5.20 Patents, Trademarks, and Copyrights UNITED....20 Export of technical data relating to sensitive nuclear technology. Under regulations (10 CFR...

  10. Autophagy requires poly(adp-ribosyl)ation-dependent AMPK nuclear export.

    PubMed

    Rodríguez-Vargas, José M; Rodríguez, María I; Majuelos-Melguizo, Jara; García-Diaz, Ángel; González-Flores, Ariannys; López-Rivas, Abelardo; Virág, László; Illuzzi, Giuditta; Schreiber, Valerie; Dantzer, Françoise; Oliver, F Javier

    2016-12-01

    AMPK is a central energy sensor linking extracellular milieu fluctuations with the autophagic machinery. In the current study we uncover that Poly(ADP-ribosyl)ation (PARylation), a post-translational modification (PTM) of proteins, accounts for the spatial and temporal regulation of autophagy by modulating AMPK subcellular localisation and activation. More particularly, we show that the minority AMPK pool needs to be exported to the cytosol in a PARylation-dependent manner for optimal induction of autophagy, including ULK1 phosphorylation and mTORC1 inactivation. PARP-1 forms a molecular complex with AMPK in the nucleus in non-starved cells. In response to nutrient deprivation, PARP-1 catalysed PARylation, induced the dissociation of the PARP-1/AMPK complex and the export of free PARylated nuclear AMPK to the cytoplasm to activate autophagy. PARP inhibition, its silencing or the expression of PARylation-deficient AMPK mutants prevented not only the AMPK nuclear-cytosolic export but also affected the activation of the cytosolic AMPK pool and autophagosome formation. These results demonstrate that PARylation of AMPK is a key early signal to efficiently convey extracellular nutrient perturbations with downstream events needed for the cell to optimize autophagic commitment before autophagosome formation.

  11. Autophagy requires poly(adp-ribosyl)ation-dependent AMPK nuclear export

    PubMed Central

    Rodríguez-Vargas, José M; Rodríguez, María I; Majuelos-Melguizo, Jara; García-Diaz, Ángel; González-Flores, Ariannys; López-Rivas, Abelardo; Virág, László; Illuzzi, Giuditta; Schreiber, Valerie; Dantzer, Françoise; Oliver, F Javier

    2016-01-01

    AMPK is a central energy sensor linking extracellular milieu fluctuations with the autophagic machinery. In the current study we uncover that Poly(ADP-ribosyl)ation (PARylation), a post-translational modification (PTM) of proteins, accounts for the spatial and temporal regulation of autophagy by modulating AMPK subcellular localisation and activation. More particularly, we show that the minority AMPK pool needs to be exported to the cytosol in a PARylation-dependent manner for optimal induction of autophagy, including ULK1 phosphorylation and mTORC1 inactivation. PARP-1 forms a molecular complex with AMPK in the nucleus in non-starved cells. In response to nutrient deprivation, PARP-1 catalysed PARylation, induced the dissociation of the PARP-1/AMPK complex and the export of free PARylated nuclear AMPK to the cytoplasm to activate autophagy. PARP inhibition, its silencing or the expression of PARylation-deficient AMPK mutants prevented not only the AMPK nuclear-cytosolic export but also affected the activation of the cytosolic AMPK pool and autophagosome formation. These results demonstrate that PARylation of AMPK is a key early signal to efficiently convey extracellular nutrient perturbations with downstream events needed for the cell to optimize autophagic commitment before autophagosome formation. PMID:27689873

  12. Cellular Nuclear Export Factors TAP and Aly Are Required for HDAg-L-mediated Assembly of Hepatitis Delta Virus.

    PubMed

    Huang, Hsiu-Chen; Lee, Chung-Pei; Liu, Hui-Kang; Chang, Ming-Fu; Lai, Yu-Heng; Lee, Yu-Ching; Huang, Cheng

    2016-12-09

    Hepatitis delta virus (HDV) is a satellite virus of hepatitis B virus (HBV). HDV genome encodes two forms of hepatitis delta antigen (HDAg), small HDAg (HDAg-S), which is required for viral replication, and large HDAg (HDAg-L), which is essential for viral assembly. HDAg-L is identical to HDAg-S except that it bears a 19-amino acid extension at the C terminus. Both HDAgs contain a nuclear localization signal (NLS), but only HDAg-L contains a CRM1-independent nuclear export signal at its C terminus. The nuclear export activity of HDAg-L is important for HDV particle formation. However, the mechanisms of HDAg-L-mediated nuclear export of HDV ribonucleoprotein are not clear. In this study, the host cellular RNA export complex TAP-Aly was found to form a complex with HDAg-L, but not with an export-defective HDAg-L mutant, in which Pro(205) was replaced by Ala. HDAg-L was found to colocalize with TAP and Aly in the nucleus. The C-terminal domain of HDAg-L was shown to directly interact with the N terminus of TAP, whereas an HDAg-L mutant lacking the NLS failed to interact with full-length TAP. In addition, small hairpin RNA-mediated down-regulation of TAP or Aly reduced nuclear export of HDAg-L and assembly of HDV virions. Furthermore, a peptide, TAT-HDAg-L(198-210), containing the 10-amino acid TAT peptide and HDAg-L(198-210), inhibited the interaction between HDAg-L and TAP and blocked HDV virion assembly and secretion. These data demonstrate that formation and release of HDV particles are mediated by TAP and Aly.

  13. 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.

  14. The nuclear export protein of H5N1 influenza A viruses recruits Matrix 1 (M1) protein to the viral ribonucleoprotein to mediate nuclear export.

    PubMed

    Brunotte, Linda; Flies, Joe; Bolte, Hardin; Reuther, Peter; Vreede, Frank; Schwemmle, Martin

    2014-07-18

    In influenza A virus-infected cells, replication and transcription of the viral genome occurs in the nucleus. To be packaged into viral particles at the plasma membrane, encapsidated viral genomes must be exported from the nucleus. Intriguingly, the nuclear export protein (NEP) is involved in both processes. Although NEP stimulates viral RNA synthesis by binding to the viral polymerase, its function during nuclear export implicates interaction with viral ribonucleoprotein (vRNP)-associated M1. The observation that both interactions are mediated by the C-terminal moiety of NEP raised the question whether these two features of NEP are linked functionally. Here we provide evidence that the interaction between M1 and the vRNP depends on the NEP C terminus and its polymerase activity-enhancing property for the nuclear export of vRNPs. This suggests that these features of NEP are linked functionally. Furthermore, our data suggest that the N-terminal domain of NEP interferes with the stability of the vRNP-M1-NEP nuclear export complex, probably mediated by its highly flexible intramolecular interaction with the NEP C terminus. On the basis of our data, we propose a new model for the assembly of the nuclear export complex of Influenza A vRNPs.

  15. Deciphering mechanisms of drug sensitivity and resistance to Selective Inhibitor of Nuclear Export (SINE) compounds.

    PubMed

    Crochiere, Marsha; Kashyap, Trinayan; Kalid, Ori; Shechter, Sharon; Klebanov, Boris; Senapedis, William; Saint-Martin, Jean-Richard; Landesman, Yosef

    2015-11-17

    Exportin 1 (XPO1) is a well-characterized nuclear export protein whose expression is up-regulated in many types of cancers and functions to transport key tumor suppressor proteins (TSPs) from the nucleus. Karyopharm Therapeutics has developed a series of small-molecule Selective Inhibitor of Nuclear Export (SINE) compounds, which have been shown to block XPO1 function both in vitro and in vivo. The drug candidate, selinexor (KPT-330), is currently in Phase-II/IIb clinical trials for treatment of both hematologic and solid tumors. The present study sought to decipher the mechanisms that render cells either sensitive or resistant to treatment with SINE compounds, represented by KPT-185, an early analogue of KPT-330. Using the human fibrosarcoma HT1080 cell line, resistance to SINE was acquired over a period of 10 months of constant incubation with increasing concentration of KPT-185. Cell viability was assayed by MTT. Immunofluorescence was used to compare nuclear export of TSPs. Fluorescence activated cell sorting (FACS), quantitative polymerase chain reaction (qPCR), and immunoblots were used to measure effects on cell cycle, gene expression, and cell death. RNA from naïve and drug treated parental and resistant cells was analyzed by Affymetrix microarrays. Treatment of HT1080 cells with gradually increasing concentrations of SINE resulted in >100 fold decrease in sensitivity to SINE cytotoxicity. Resistant cells displayed prolonged cell cycle, reduced nuclear accumulation of TSPs, and similar changes in protein expression compared to parental cells, however the magnitude of the protein expression changes were more significant in parental cells. Microarray analyses comparing parental to resistant cells indicate that a number of key signaling pathways were altered in resistant cells including expression changes in genes involved in adhesion, apoptosis, and inflammation. While the patterns of changes in transcription following drug treatment are similar in

  16. 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

  17. Murine leukemia virus uses TREX components for efficient nuclear export of unspliced viral transcripts.

    PubMed

    Sakuma, Toshie; Tonne, Jason M; Ikeda, Yasuhiro

    2014-03-10

    Previously we reported that nuclear export of both unspliced and spliced murine leukemia virus (MLV) transcripts depends on the nuclear export factor (NXF1) pathway. Although the mRNA export complex TREX, which contains Aly/REF, UAP56, and the THO complex, is involved in the NXF1-mediated nuclear export of cellular mRNAs, its contribution to the export of MLV mRNA transcripts remains poorly understood. Here, we studied the involvement of TREX components in the export of MLV transcripts. Depletion of UAP56, but not Aly/REF, reduced the level of both unspliced and spliced viral transcripts in the cytoplasm. Interestingly, depletion of THO components, including THOC5 and THOC7, affected only unspliced viral transcripts in the cytoplasm. Moreover, the RNA immunoprecipitation assay showed that only the unspliced viral transcript interacted with THOC5. These results imply that MLV requires UAP56, THOC5 and THOC7, in addition to NXF1, for nuclear export of viral transcripts. Given that naturally intronless mRNAs, but not bulk mRNAs, require THOC5 for nuclear export, it is plausible that THOC5 plays a key role in the export of unspliced MLV transcripts.

  18. Nuclear export of Far1p in response to pheromones requires the export receptor Msn5p/Ste21p

    PubMed Central

    Blondel, Marc; Alepuz, Paula M.; Huang, Linda S.; Shaham, Shai; Ammerer, Gustav; Peter, Matthias

    1999-01-01

    Far1p is a bifunctional protein that is required to arrest the cell cycle and to establish cell polarity during yeast mating. Far1p is localized predominantly in the nucleus but accumulates in the cytoplasm in cells exposed to pheromones. Here we show that Far1p functions in both subcellular compartments: nuclear Far1p is required to arrest the cell cycle, whereas cytoplasmic Far1p is involved in the establishment of cell polarity. The subcellular localization of Far1p is regulated by two mechanisms: (1) Far1p contains a functional bipartite nuclear localization signal (NLS), and (2) Far1p is exported from the nucleus by Msn5p/Ste21p, a member of the exportin family. Cells deleted for Msn5p/Ste21p failed to export Far1p in response to pheromones, whereas overexpression of Msn5p/Ste21p was sufficient to accumulate Far1p in the cytoplasm in the absence of pheromones. Msn5p/Ste21p was localized in the nucleus and interacted with Far1p in a manner dependent on GTP-bound Gsp1p. Two-hybrid analysis identified a small fragment within Far1p that is necessary and sufficient for binding to Msn5p/Ste21p, and is also required to export Far1p in vivo. Finally, similar to Δmsn5/ste21 strains, cells expressing a mutant Far1p, which can no longer be exported, exhibit a mating defect, but are able to arrest their cell cycle in response to pheromones. Taken together, our results suggest that nuclear export of Far1p by Msn5p/Ste21p coordinates the two separable functions of Far1p during mating. PMID:10485850

  19. 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.

  20. eEF1A mediates the nuclear export of SNAG-containing proteins via the Exportin5-aminoacyl-tRNA complex.

    PubMed

    Mingot, José Manuel; Vega, Sonia; Cano, Amparo; Portillo, Francisco; Nieto, M Angela

    2013-11-14

    Exportin5 mediates the nuclear export of double-stranded RNAs, including pre-microRNAs, adenoviral RNAs, and tRNAs. When tRNAs are aminoacylated, the Exportin5-aminoacyl (aa)-tRNA complex recruits and coexports the translation elongation factor eEF1A. Here, we show that eEF1A binds to Snail transcription factors when bound to their main target, the E-cadherin promoter, facilitating their export to the cytoplasm in association with the aa-tRNA-Exportin5 complex. Snail binds to eEF1A through the SNAG domain, a protein nuclear export signal present in several transcription factor families, and this binding is regulated by phosphorylation. Thus, we describe a nuclear role for eEF1A and provide a mechanism for protein nuclear export that attenuates the activity of SNAG-containing transcription factors.

  1. Murine leukemia virus uses NXF1 for nuclear export of spliced and unspliced viral transcripts.

    PubMed

    Sakuma, Toshie; Davila, Jaime I; Malcolm, Jessica A; Kocher, Jean-Pierre A; Tonne, Jason M; Ikeda, Yasuhiro

    2014-04-01

    Intron-containing mRNAs are subject to restricted nuclear export in higher eukaryotes. Retroviral replication requires the nucleocytoplasmic transport of both spliced and unspliced RNA transcripts, and RNA export mechanisms of gammaretroviruses are poorly characterized. Here, we report the involvement of the nuclear export receptor NXF1/TAP in the nuclear export of gammaretroviral RNA transcripts. We identified a conserved cis-acting element in the pol gene of gammaretroviruses, including murine leukemia virus (MLV) and xenotropic murine leukemia virus (XMRV), named the CAE (cytoplasmic accumulation element). The CAE enhanced the cytoplasmic accumulation of viral RNA transcripts and the expression of viral proteins without significantly affecting the stability, splicing, or translation efficiency of the transcripts. Insertion of the CAE sequence also facilitated Rev-independent HIV Gag expression. We found that the CAE sequence interacted with NXF1, whereas disruption of NXF1 ablated CAE function. Thus, the CAE sequence mediates the cytoplasmic accumulation of gammaretroviral transcripts in an NXF1-dependent manner. Disruption of NXF1 expression impaired cytoplasmic accumulations of both spliced and unspliced RNA transcripts of XMRV and MLV, resulting in their nuclear retention or degradation. Thus, our results demonstrate that gammaretroviruses use NXF1 for the cytoplasmic accumulation of both spliced and nonspliced viral RNA transcripts. Murine leukemia virus (MLV) has been studied as one of the classic models of retrovirology. Although unspliced host messenger RNAs are rarely exported from the nucleus, MLV actively exports unspliced viral RNAs to the cytoplasm. Despite extensive studies, how MLV achieves this difficult task has remained a mystery. Here, we have studied the RNA export mechanism of MLV and found that (i) the genome contains a sequence which supports the efficient nuclear export of viral RNAs, (ii) the cellular factor NXF1 is involved in the

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

    PubMed

    Li, Ping; Noegel, Angelika A

    2015-11-16

    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 l I: nker of N: ucleoskeleton and C: ytoskeleton (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.

  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. A Herpesvirus Protein Selectively Inhibits Cellular mRNA Nuclear Export.

    PubMed

    Gong, Danyang; Kim, Yong Hoon; Xiao, Yuchen; Du, Yushen; Xie, Yafang; Lee, Kevin K; Feng, Jun; Farhat, Nisar; Zhao, Dawei; Shu, Sara; Dai, Xinghong; Chanda, Sumit K; Rana, Tariq M; Krogan, Nevan J; Sun, Ren; Wu, Ting-Ting

    2016-11-09

    Nuclear mRNA export is highly regulated to ensure accurate cellular gene expression. Viral inhibition of cellular mRNA export can enhance viral access to the cellular translation machinery and prevent anti-viral protein production but is generally thought to be nonselective. We report that ORF10 of Kaposi's sarcoma-associated herpesvirus (KSHV), a nuclear DNA virus, inhibits mRNA export in a transcript-selective manner to control cellular gene expression. Nuclear export inhibition by ORF10 requires an interaction with an RNA export factor, Rae1. Genome-wide analysis reveals a subset of cellular mRNAs whose nuclear export is blocked by ORF10 with the 3' UTRs of ORF10-targeted transcripts conferring sensitivity to export inhibition. The ORF10-Rae1 interaction is important for the virus to express viral genes and produce infectious virions. These results suggest that a nuclear DNA virus can selectively interfere with RNA export to restrict host gene expression for optimal replication.

  5. Aiding and abetting cancer: mRNA export and the nuclear pore.

    PubMed

    Culjkovic-Kraljacic, Biljana; Borden, Katherine L B

    2013-07-01

    mRNA export is a critical step in gene expression. Export of transcripts can be modulated in response to cellular signaling or stress. Consistently, mRNA export is dysregulated in primary human specimens derived from many different forms of cancer. Aberrant expression of export factors can alter the export of specific transcripts encoding proteins involved in proliferation, survival, and oncogenesis. These specific factors, which are not used for bulk mRNA export, are obvious therapeutic targets. Indeed, given the emerging role of mRNA export in cancer, it is not surprising that efforts to target different aspects of this pathway have reached the clinical trial stage. Thus, like transcription and translation, mRNA export may also play a critical role in cancer genesis and maintenance.

  6. Aiding and Abetting Cancer: mRNA export and the nuclear pore

    PubMed Central

    Culjkovic-Kraljacic, Biljana; Borden, Katherine L.B

    2013-01-01

    mRNA export is a critical step in gene expression. Export of transcripts can be modulated in response to cellular signaling or stress. Consistently, mRNA export is dysregulated in primary human specimens derived from many different forms of cancer. Aberrant expression of export factors can alter export of specific transcripts encoding proteins involved in proliferation, survival and oncogenesis. These specific factors, which are not used for bulk mRNA export, are obvious therapeutic targets. Indeed, given the emerging role of mRNA export in cancer, it is not surprising that efforts to target different aspects of this pathway have reached the clinical trial stage. Thus, like transcription and translation, mRNA export may also play a critical role in cancer genesis and maintenance. PMID:23582887

  7. A subset of FG-nucleoporins is necessary for efficient Msn5-mediated nuclear protein export.

    PubMed

    Finn, Erin M; DeRoo, Elise P; Clement, George W; Rao, Sheila; Kruse, Sarah E; Kokanovich, Kate M; Belanger, Kenneth D

    2013-05-01

    The transport of proteins between the cytoplasm and nucleus requires interactions between soluble transport receptors (karyopherins) and phenylalanine-glycine (FG) repeat domains on nuclear pore complex proteins (nucleoporins). However, the role of specific FG repeat-containing nucleoporins in nuclear protein export has not been carefully investigated. We have developed a novel kinetic assay to investigate the relative export kinetics mediated by the karyopherin Msn5/Kap142 in yeast containing specific FG-Nup mutations. Using the Msn5 substrate Crz1 as a marker for Msn5-mediated protein export, we observe that deletions of NUP100 or NUP2 result in decreased rates of Crz1 export, while nup60Δ and nup42Δ mutants do not vary significantly from wild type. The decreased Msn5 export rate in nup100Δ was confirmed using Mig1-GFP as a transport substrate. A nup100ΔGLFG mutant shows defects in nuclear export kinetics similar to a nup100Δ deletion. Removal of FG-repeats from Nsp1 also decreases export kinetics, while a loss of Nup1 FXFGs does not. To confirm that our export data reflected functional differences in protein localization, we performed Crz1 transcription activation assays using a CDRE::LacZ reporter gene that is upregulated upon increased transcription activation by Crz1 in vivo. We observe that expression from this reporter increases in nup100ΔGLFG and nsp1ΔFGΔFXFG strains that exhibit decreased Crz1 export kinetics but resembles wild-type levels in nup1ΔFXFG strains that do not exhibit export defects. These data provide evidence that the export of Msn5 is likely mediated by a specific subset of FG-Nups and that the GLFG repeat domain of Nup100 is important for Msn5-mediated nuclear protein export.

  8. A subset of FG-nucleoporins is necessary for efficient Msn5-mediated nuclear protein export

    PubMed Central

    Finn, Erin M.; DeRoo, Elise P.; Clement, George W.; Rao, Sheila; Kruse, Sarah E.; Kokanovich, Kate M.; Belanger, Kenneth D.

    2013-01-01

    The transport of proteins between the cytoplasm and nucleus requires interactions between soluble transport receptors (karyopherins) and phenylalanine-glycine (FG) repeat domains on nuclear pore complex proteins (nucleoporins). However, the role of specific FG repeat-containing nucleoporins in nuclear protein export has not been carefully investigated. We have developed a novel kinetic assay to investigate the relative export kinetics mediated by the karyopherin Msn5/Kap142 in yeast containing specific FG-Nup mutations. Using the Msn5 substrate Crz1 as a marker for Msn5-mediated protein export, we observe that deletions of NUP100 or NUP2 result in decreased rates of Crz1 export, while nup60Δ and nup42Δ mutants do not vary significantly from wild type. The decreased Msn5 export rate in nup100Δ was confirmed using Mig1-GFP as a transport substrate. A nup100ΔGLFG mutant shows defects in nuclear export kinetics similar to a nup100Δ deletion. Removal of FG-repeats from Nsp1 also decreases export kinetics, while a loss of Nup1 FXFGs does not. To confirm that our export data reflected functional differences in protein localization, we performed Crz1 transcription activation assays using a CDRE::LacZ reporter gene that is upregulated upon increased transcription activation by Crz1 in vivo. We observe that expression from this reporter increases in nup100ΔGLFG and nsp1ΔFGΔFXFG strains that exhibit decreased Crz1 export kinetics but resembles wild-type levels in nup1ΔFXFG strains that do not exhibit export defects. These data provide evidence that the export of Msn5 is likely mediated by a specific subset of FG-Nups and that the GLFG repeat domain of Nup100 is important for Msn5-mediated nuclear protein export. PMID:23295456

  9. The CRM1 nuclear export protein in normal development and disease.

    PubMed

    Nguyen, Kevin T; Holloway, Michael P; Altura, Rachel A

    2012-01-01

    CRM1 (Chromosomal Maintenance 1, also known as Exportin 1) is the major mammalian export protein that facilitates the transport of large macromolecules including RNA and protein across the nuclear membrane to the cytoplasm. The gene encoding CRM1 was originally identified in yeast as required to maintain higher order chromosome structure. In mammalian cells, CRM1 was found to bind several nuclear pore proteins hence its role in nuclear-cytosolic transport was discovered. In addition to nuclear-cytosolic transport, CRM1 also plays a role in centrosome duplication and spindle assembly, especially in response to DNA damage. The crystal structure of CRM1 suggests a complex protein that binds the Ran protein bound to GTP, allowing for a conformational change that facilitates binding to different cargo proteins through a nuclear export signal (NES). Included in the cadre of cargo are multiple tumor suppressor and oncoproteins as p53, BRCA1, Survivin, NPM, and APC, which function in the nucleus to regulate transcription or aid in chromosomal assembly and movement. An imbalance in the cytosolic level of these proteins has been observed in cancer cells, resulting in either inactivation (tumor suppressor) or an excess of anti-apoptotic activity (oncoprotein). Thus, the concept of inhibiting CRM1 has been explored as a potential therapeutic intervention. Indeed, inhibition of CRM1 by a variety of small molecules that interfere with cargo-NES binding results in cancer cell death. Whether all of these proteins together are responsible for this phenotype or whether specific proteins are required for this effect is unclear at this time.

  10. Nuclear mRNA export requires specific FG nucleoporins for translocation through the nuclear pore complex.

    PubMed

    Terry, Laura J; Wente, Susan R

    2007-09-24

    Trafficking of nucleic acids and large proteins through nuclear pore complexes (NPCs) requires interactions with NPC proteins that harbor FG (phenylalanine-glycine) repeat domains. Specialized transport receptors that recognize cargo and bind FG domains facilitate these interactions. Whether different transport receptors utilize preferential FG domains in intact NPCs is not fully resolved. In this study, we use a large-scale deletion strategy in Saccharomyces cerevisiae to generate a new set of more minimal pore (mmp) mutants that lack specific FG domains. A comparison of messenger RNA (mRNA) export versus protein import reveals unique subsets of mmp mutants with functional defects in specific transport receptors. Thus, multiple functionally independent NPC translocation routes exist for different transport receptors. Our global analysis of the FG domain requirements in mRNA export also finds a requirement for two NPC substructures-one on the nuclear NPC face and one in the NPC central core. These results pinpoint distinct steps in the mRNA export mechanism that regulate NPC translocation efficiency.

  11. 77 FR 51581 - Request for a License To Export Nuclear Grade Graphite

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-08-24

    ... 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) ``Public Notice of Receipt of an Application,'' please take notice that the Nuclear Regulatory Commission...

  12. 10 CFR 110.8 - List of nuclear facilities and equipment under NRC export licensing authority.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... under NRC export licensing authority. (a) Nuclear reactors and especially designed or prepared equipment and components for nuclear reactors. (See Appendix A to this part.) (b) Plants for the separation of... plants. (See Appendix N to this part.) (d) Plants for the reprocessing of irradiated nuclear reactor...

  13. 10 CFR 110.8 - List of nuclear facilities and equipment under NRC export licensing authority.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... under NRC export licensing authority. (a) Nuclear reactors and especially designed or prepared equipment and components for nuclear reactors. (See Appendix A to this part.) (b) Plants for the separation of... plants. (See Appendix N to this part.) (d) Plants for the reprocessing of irradiated nuclear reactor...

  14. 10 CFR 110.8 - List of nuclear facilities and equipment under NRC export licensing authority.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... under NRC export licensing authority. (a) Nuclear reactors and especially designed or prepared equipment and components for nuclear reactors. (See Appendix A to this part.) (b) Plants for the separation of... plants. (See Appendix N to this part.) (d) Plants for the reprocessing of irradiated nuclear reactor...

  15. 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.

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

    PubMed

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

    2015-09-01

    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. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. 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

  18. Involvement of Nuclear Export in Human Papillomavirus Type 18 E6-Mediated Ubiquitination and Degradation of p53

    PubMed Central

    Stewart, Deborah; Ghosh, Anirban; Matlashewski, Greg

    2005-01-01

    The E6 protein from high-risk human papillomaviruses (HPVs) targets the p53 tumor suppressor for degradation by the proteasome pathway. This ability contributes to the oncogenic potential of these viruses. However, several aspects concerning the mechanism of E6-mediated p53 degradation at the cellular level remain to be clarified. This study therefore examined the role of cell localization and ubiquitination in the E6-mediated degradation of p53. As demonstrated within, following coexpression both p53 and high-risk HPV type 18 (HPV-18) E6 (18E6) shuttle from the nucleus to the cytoplasm. Mutation of the C-terminal nuclear export signal (NES) of p53 or treatment with leptomycin B inhibited the 18E6-mediated nuclear export of p53. Impairment of nuclear export resulted in only a partial reduction in 18E6-mediated degradation, suggesting that both nuclear and cytoplasmic proteasomes can target p53 for degradation. This was also consistent with the observation that 18E6 mediated the accumulation of polyubiquitinated p53 in the nucleus. In comparison, a p53 isoform that localizes predominantly to the cytoplasm was not targeted for degradation by 18E6 in vivo but could be degraded in vitro, arguing that nuclear p53 is the target for E6-mediated degradation. This study supports a model in which (i) E6 mediates the accumulation of polyubiquitinated p53 in the nucleus, (ii) E6 is coexported with p53 from the nucleus to the cytoplasm via a CRM1 nuclear export mechanism involving the C-terminal NES of p53, and (iii) E6-mediated p53 degradation can be mediated by both nuclear and cytoplasmic proteasomes. PMID:15994771

  19. Selective Inhibitor of Nuclear Export (SINE) Compounds Alter New World Alphavirus Capsid Localization and Reduce Viral Replication in Mammalian Cells.

    PubMed

    Lundberg, Lindsay; Pinkham, Chelsea; de la Fuente, Cynthia; Brahms, Ashwini; Shafagati, Nazly; Wagstaff, Kylie M; Jans, David A; Tamir, Sharon; Kehn-Hall, Kylene

    2016-11-01

    The capsid structural protein of the New World alphavirus, Venezuelan equine encephalitis virus (VEEV), interacts with the host nuclear transport proteins importin α/β1 and CRM1. Novel selective inhibitor of nuclear export (SINE) compounds, KPT-185, KPT-335 (verdinexor), and KPT-350, target the host's primary nuclear export protein, CRM1, in a manner similar to the archetypical inhibitor Leptomycin B. One major limitation of Leptomycin B is its irreversible binding to CRM1; which SINE compounds alleviate because they are slowly reversible. Chemically inhibiting CRM1 with these compounds enhanced capsid localization to the nucleus compared to the inactive compound KPT-301, as indicated by immunofluorescent confocal microscopy. Differences in extracellular versus intracellular viral RNA, as well as decreased capsid in cell free supernatants, indicated the inhibitors affected viral assembly, which led to a decrease in viral titers. The decrease in viral replication was confirmed using a luciferase-tagged virus and through plaque assays. SINE compounds had no effect on VEEV TC83_Cm, which encodes a mutated form of capsid that is unable to enter the nucleus. Serially passaging VEEV in the presence of KPT-185 resulted in mutations within the nuclear localization and nuclear export signals of capsid. Finally, SINE compound treatment also reduced the viral titers of the related eastern and western equine encephalitis viruses, suggesting that CRM1 maintains a common interaction with capsid proteins across the New World alphavirus genus.

  20. Selective Inhibitor of Nuclear Export (SINE) Compounds Alter New World Alphavirus Capsid Localization and Reduce Viral Replication in Mammalian Cells

    PubMed Central

    Lundberg, Lindsay; Pinkham, Chelsea; de la Fuente, Cynthia; Brahms, Ashwini; Shafagati, Nazly; Wagstaff, Kylie M.; Jans, David A.; Tamir, Sharon; Kehn-Hall, Kylene

    2016-01-01

    The capsid structural protein of the New World alphavirus, Venezuelan equine encephalitis virus (VEEV), interacts with the host nuclear transport proteins importin α/β1 and CRM1. Novel selective inhibitor of nuclear export (SINE) compounds, KPT-185, KPT-335 (verdinexor), and KPT-350, target the host’s primary nuclear export protein, CRM1, in a manner similar to the archetypical inhibitor Leptomycin B. One major limitation of Leptomycin B is its irreversible binding to CRM1; which SINE compounds alleviate because they are slowly reversible. Chemically inhibiting CRM1 with these compounds enhanced capsid localization to the nucleus compared to the inactive compound KPT-301, as indicated by immunofluorescent confocal microscopy. Differences in extracellular versus intracellular viral RNA, as well as decreased capsid in cell free supernatants, indicated the inhibitors affected viral assembly, which led to a decrease in viral titers. The decrease in viral replication was confirmed using a luciferase-tagged virus and through plaque assays. SINE compounds had no effect on VEEV TC83_Cm, which encodes a mutated form of capsid that is unable to enter the nucleus. Serially passaging VEEV in the presence of KPT-185 resulted in mutations within the nuclear localization and nuclear export signals of capsid. Finally, SINE compound treatment also reduced the viral titers of the related eastern and western equine encephalitis viruses, suggesting that CRM1 maintains a common interaction with capsid proteins across the New World alphavirus genus. PMID:27902702

  1. Interaction of HTLV-1 Tax protein with the calreticulin: Implications for Tax nuclear export and secretion

    PubMed Central

    Alefantis, Timothy; Flaig, Katherine E.; Wigdahl, Brian; Jain, Pooja

    2007-01-01

    Summary Human T cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 transcriptional transactivator protein Tax plays an integral role in virus replication and disease progression. Traditionally, Tax is described as a nuclear protein where it performs its primary role as a transcriptional transactivator. However, recent studies have clearly shown that Tax can also be localized to the cytoplasm where it has been shown to interact with a number of host transcription factors most notably NF-κB, constitutive expression of which is directly related to the T cell transforming properties of Tax in ATL patients. The presence of a functional nuclear export signal (NES) within Tax and the secretion of full-length Tax have also been demonstrated previously. Additionally, release of Tax from HTLV-1-infected cells and the presence of cell-free Tax was demonstrated in the CSF of HAM/TSP patients suggesting that the progression of HAM/TSP might be mediated by the ability of Tax to work as an extracellular cytokine. Therefore, in both ATL and HAM/TSP Tax nuclear export and nucleocytoplasmic shuttling may play a critical role, the mechanism of which remains unknown. In this study, we have demonstrated that the calcium binding protein calreticulin interacts with Tax by coimmunoprecipitation. This interaction was found to localize to a region at or near the nuclear membrane. In addition, differential expression of calreticulin was demonstrated in various cell types that correlated with their ability to retain cytoplasmic Tax, particularly in astrocytes. Finally, a comparison of a number of HTLV-1-infected T cell lines to non-infected T cells revealed higher expression of calreticulin in infected cells implicating a direct role for this protein in HTLV-1 infection. PMID:17395420

  2. 10 CFR 73.73 - Requirement for advance notice and protection of export shipments of special nuclear material of...

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... shipments of special nuclear material of low strategic significance. 73.73 Section 73.73 Energy NUCLEAR... Requirement for advance notice and protection of export shipments of special nuclear material of low strategic significance. (a) A licensee authorized to export special nuclear material of low strategic significance shall...

  3. 10 CFR 73.73 - Requirement for advance notice and protection of export shipments of special nuclear material of...

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... shipments of special nuclear material of low strategic significance. 73.73 Section 73.73 Energy NUCLEAR... Requirement for advance notice and protection of export shipments of special nuclear material of low strategic significance. (a) A licensee authorized to export special nuclear material of low strategic significance shall...

  4. 10 CFR 73.73 - Requirement for advance notice and protection of export shipments of special nuclear material of...

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... shipments of special nuclear material of low strategic significance. 73.73 Section 73.73 Energy NUCLEAR... Requirement for advance notice and protection of export shipments of special nuclear material of low strategic significance. (a) A licensee authorized to export special nuclear material of low strategic significance shall...

  5. 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

    ... shipments of special nuclear material of low strategic significance. 73.73 Section 73.73 Energy NUCLEAR... Requirement for advance notice and protection of export shipments of special nuclear material of low strategic significance. (a) A licensee authorized to export special nuclear material of low strategic significance shall...

  6. 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

    ... shipments of special nuclear material of low strategic significance. 73.73 Section 73.73 Energy NUCLEAR... Requirement for advance notice and protection of export shipments of special nuclear material of low strategic significance. (a) A licensee authorized to export special nuclear material of low strategic significance shall...

  7. Gastrin Induces Nuclear Export and Proteasome Degradation of Menin in Enteric Glial Cells.

    PubMed

    Sundaresan, Sinju; Meininger, Cameron A; Kang, Anthony J; Photenhauer, Amanda L; Hayes, Michael M; Sahoo, Nirakar; Grembecka, Jolanta; Cierpicki, Tomasz; Ding, Lin; Giordano, Thomas J; Else, Tobias; Madrigal, David J; Low, Malcolm J; Campbell, Fiona; Baker, Ann-Marie; Xu, Haoxing; Wright, Nicholas A; Merchant, Juanita L

    2017-08-28

    The multiple endocrine neoplasia, type 1 (MEN1) locus encodes the nuclear protein and tumor suppressor menin. MEN1 mutations frequently cause neuroendocrine tumors (NETs) such as gastrinomas, characterized by their predominant duodenal location and local metastasis at time of diagnosis. Diffuse gastrin cell hyperplasia precedes the appearance of MEN1 gastrinomas, which develop within submucosal Brunner's glands. We investigated how menin regulates expression of the gastrin gene and induces generation of submucosal gastrin-expressing cell hyperplasia. Primary enteric glial cultures were generated from the VillinCre:Men(1FL/FL):Sst(-/-) mice or C57BL/6 mice (controls), with or without inhibition of gastric acid by omeprazole. Primary enteric glial cells from VillinCre:Men1FL/FL:Sst(+/+) mice were incubated with gastrin and separated into nuclear and cytoplasmic fractions. Cells were incubated with forskolin and H89 to activate or inhibit protein kinase A (a family of enzymes whose activity depends on cellular levels of cyclic AMP). Gastrin was measured in blood, tissue, and cell cultures using an ELISA. Immunoprecipitation with menin or ubiquitin was used to demonstrate post-translational modification of menin. Primary glial cells were incubated with leptomycin b and MG132 to block nuclear export and proteasome activity, respectively. We obtained human duodenal, lymph node, and pancreatic gastrinoma samples, collected from patients who underwent surgery from 1996 through 2007 in the United States or the United Kingdom. Enteric glial cells that stained positive for glial fibrillary acidic protein (GFAP+) expressed gastrin de novo through a mechanism that required PKA. Gastrin-induced nuclear export of menin via cholecystokinin B receptor (CCKBR)-mediated activation of PKA. Once exported from the nucleus, menin was ubiquitinated and degraded by the proteasome. GFAP and other markers of enteric glial cells, e.g., p75 and S100B, colocalized with gastrin in human duodenal

  8. p54nrb/NonO and PSF promote U snRNA nuclear export by accelerating its export complex assembly.

    PubMed

    Izumi, Hiroto; McCloskey, Asako; Shinmyozu, Kaori; Ohno, Mutsuhito

    2014-04-01

    The assembly of spliceosomal U snRNPs in metazoans requires nuclear export of U snRNA precursors. Four factors, nuclear cap-binding complex (CBC), phosphorylated adaptor for RNA export (PHAX), the export receptor CRM1 and RanGTP, gather at the m(7)G-cap-proximal region and form the U snRNA export complex. Here we show that the multifunctional RNA-binding proteins p54nrb/NonO and PSF are U snRNA export stimulatory factors. These proteins, likely as a heterodimer, accelerate the recruitment of PHAX, and subsequently CRM1 and Ran onto the RNA substrates in vitro, which mediates efficient U snRNA export in vivo. Our results reveal a new layer of regulation for U snRNA export and, hence, spliceosomal U snRNP biogenesis.

  9. 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.

  10. An active nuclear retention signal in the glucocorticoid receptor functions as a strong inducer of transcriptional activation.

    PubMed

    Carrigan, Amanda; Walther, Rhian F; Salem, Houssein Abdou; Wu, Dongmei; Atlas, Ella; Lefebvre, Yvonne A; Haché, Robert J G

    2007-04-13

    The glucocorticoid receptor (GR) cycles between a naive chaperone-complexed form in the cytoplasm and a transcriptionally active steroid-bound nuclear form. Nuclear import of GR occurs rapidly and is mediated through the importin alpha/beta karyopherin import pathway. By contrast, nuclear export of GR occurs only slowly under most conditions, despite a dependence on active signaling. In this study we have defined a nuclear retention signal (NRS) in the hinge region of GR that actively opposes the nuclear export of GR as well as the nuclear export mediated through an ectopic CRM1-dependent nuclear export signal (NES). The GR NRS overlaps closely with the basic NL1 nuclear localization signal (NLS) but can be distinguished from NL1 by targeted mutagenesis. Substitution of the classical NLS from SV40 T antigen for the GR NL1 results in a receptor in which nuclear export is accelerated. Remarkably, although the SV40-modified GR remains predominantly nuclear in the presence of steroid and is recruited to transcriptional regulatory regions indistinguishably from wild-type GR, the substitution dramatically weakens the ability of GR to activate transcription of a mouse mammary tumor virus reporter gene. These results suggest that active nuclear retention of GR plays an integral role in glucocorticoid signaling.

  11. Structural basis for the nuclear export activity of Importin13

    PubMed Central

    Grünwald, Marlene; Lazzaretti, Daniela; Bono, Fulvia

    2013-01-01

    Importin13 (Imp13) is a bidirectional karyopherin that can mediate both import and export of cargoes. Imp13 recognizes several import cargoes, which include the exon junction complex components Mago-Y14 and the E2 SUMO-conjugating enzyme Ubc9, and one known export cargo, the translation initiation factor 1A (eIF1A). To understand how Imp13 can perform double duty, we determined the 3.6-Å crystal structure of Imp13 in complex with RanGTP and with eIF1A. eIF1A binds at the inner surface of the Imp13 C-terminal arch adjacent and concomitantly to RanGTP illustrating how eIF1A can be exported by Imp13. Moreover, the 3.0-Å structure of Imp13 in its unbound state reveals the existence of an open conformation in the cytoplasm that explains export cargo release and completes the export branch of the Imp13 pathway. Finally, we demonstrate that Imp13 is able to bind and export eIF1A in vivo and that its function is essential. PMID:23435562

  12. Somatic hypermutation is limited by CRM1-dependent nuclear export of activation-induced deaminase.

    PubMed

    McBride, Kevin M; Barreto, Vasco; Ramiro, Almudena R; Stavropoulos, Pete; Nussenzweig, Michel C

    2004-05-03

    Somatic hypermutation (SHM) and class switch recombination (CSR) are initiated in activated B lymphocytes by activation-induced deaminase (AID). AID is thought to make lesions in DNA by deaminating cytidine residues in single-stranded DNA exposed by RNA polymerase during transcription. Although this must occur in the nucleus, AID is found primarily in the cytoplasm. Here we show that AID is actively excluded from the nucleus by an exportin CRM1-dependent pathway. The AID nuclear export signal (NES) is found at the carboxyl terminus of AID in a region that overlaps a sequence required for CSR but not SHM. We find that AID lacking a functional NES causes more hypermutation of a nonphysiologic target gene in transfected fibroblasts. However, the NES does not impact on the rate of mutation of immunoglobulin genes in B lymphocytes, suggesting that the AID NES does not limit AID activity in these cells.

  13. Exportin-5-mediated nuclear export of eukaryotic elongation factor 1A and tRNA.

    PubMed

    Calado, Angelo; Treichel, Nathalie; Müller, Eva-Christina; Otto, Albrecht; Kutay, Ulrike

    2002-11-15

    Transport of proteins and RNA into and out of the cell nucleus is mediated largely by a family of RanGTP-binding transport receptors. Export receptors (exportins) need to bind RanGTP for efficient loading of their export cargo. We have identified eukaryotic elongation factor 1A (eEF1A) and tRNA as RanGTP-dependent binding partners of exportin-5 (Exp5). Exp5 stimulates nuclear export of eEF1A when microinjected into the nucleus of Xenopus laevis oocytes. Surprisingly, the interaction between eEF1A and Exp5 is dependent on tRNA that can interact directly with Exp5 and, if aminoacylated, recruits eEF1A into the export complex. These data suggested to us that Exp5 might support tRNA export. Indeed, not only the canonical tRNA export receptor, exportin-t, but also Exp5 can drive nuclear export of tRNA. Taken together, we show that there exists an alternative tRNA export pathway which can be exploited to keep eEF1A out of the cell nucleus.

  14. Influenza polymerase encoding mRNAs utilize atypical mRNA nuclear export.

    PubMed

    Larsen, Sean; Bui, Steven; Perez, Veronica; Mohammad, Adeba; Medina-Ramirez, Hilario; Newcomb, Laura L

    2014-08-28

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

  15. Nuclear export of single native mRNA molecules observed by light sheet fluorescence microscopy.

    PubMed

    Siebrasse, Jan Peter; Kaminski, Tim; Kubitscheck, Ulrich

    2012-06-12

    Nuclear export of mRNA is a key transport process in eukaryotic cells. To investigate it, we labeled native mRNP particles in living Chironomus tentans salivary gland cells with fluorescent hrp36, the hnRNP A1 homolog, and the nuclear envelope by fluorescent NTF2. Using light sheet microscopy, we traced single native mRNA particles across the nuclear envelope. The particles were observed to often probe nuclear pore complexes (NPC) at their nuclear face, and in only 25% of the cases yielded actual export. The complete export process took between 65 ms up to several seconds. A rate-limiting step was observed, which could be assigned to the nuclear basket of the pore and might correspond to a repositioning and unfolding of mRNPs before the actual translocation. Analysis of single fluorescent Dbp5 molecules, the RNA helicase essential for mRNA export, revealed that Dbp5 most often approached the cytoplasmic face of the NPC, and exhibited a binding duration of approximately 55 ms. Our results have allowed a refinement of the current models for mRNA export.

  16. Architecture of CRM1/Exportin1 suggests how cooperativity is achieved during formation of a nuclear export complex.

    PubMed

    Petosa, Carlo; Schoehn, Guy; Askjaer, Peter; Bauer, Ulrike; Moulin, Martine; Steuerwald, Ulrich; Soler-López, Montserrat; Baudin, Florence; Mattaj, Iain W; Müller, Christoph W

    2004-12-03

    CRM1/Exportin1 mediates the nuclear export of proteins bearing a leucine-rich nuclear export signal (NES) by forming a cooperative ternary complex with the NES-bearing substrate and the small GTPase Ran. We present a structural model of human CRM1 based on a combination of X-ray crystallography, homology modeling, and electron microscopy. The architecture of CRM1 resembles that of the import receptor transportin1, with 19 HEAT repeats and a large loop implicated in Ran binding. Residues critical for NES recognition are identified adjacent to the cysteine residue targeted by leptomycin B (LMB), a specific CRM1 inhibitor. We present evidence that a conformational change of the Ran binding loop accounts for the cooperativity of Ran- and substrate binding and for the selective enhancement of CRM1-mediated export by the cofactor RanBP3. Our findings indicate that a single architectural and mechanistic framework can explain the divergent effects of RanGTP on substrate binding by many import and export receptors.

  17. Nucleoporins nup98 and nup214 participate in nuclear export of human immunodeficiency virus type 1 Rev.

    PubMed

    Zolotukhin, A S; Felber, B K

    1999-01-01

    Human immunodeficiency virus type 1 (HIV-1) Rev contains a leucine-rich nuclear export signal that is essential for its nucleocytoplasmic export mediated by hCRM1. We examined the role of selected nucleoporins, which are located in peripheral structures of the nuclear pore complex and are thought to be involved in export, in Rev function in human cells. First, we found that upon actinomycin D treatment, Nup98, but not Nup214 or Nup153, is able to translocate to the cytoplasm of HeLa cells, demonstrating that Nup98 may act as a soluble factor. We further showed that Rev can recruit Nup98 and Nup214, but not Nup153, to the nucleolus. We also found that the isolated FG-containing repeat domains of Nup98 and Nup214, but not those of Nup153, competitively inhibit the Rev/RRE-mediated expression of HIV. Taken together, the recruitment of Nup98 and Nup214 by Rev and the competitive inhibition exhibited by their NP domains demonstrate direct participation of Nup98 and Nup214 in the Rev-hCRM1-mediated export.

  18. Nxt1 Is Necessary for the Terminal Step of Crm1-Mediated Nuclear Export

    PubMed Central

    Black, Ben E.; Holaska, James M.; Lévesque, Lyne; Ossareh-Nazari, Batool; Gwizdek, Carol; Dargemont, Catherine; Paschal, Bryce M.

    2001-01-01

    Soluble factors are required to mediate nuclear export of protein and RNA through the nuclear pore complex (NPC). These soluble factors include receptors that bind directly to the transport substrate and regulators that determine the assembly state of receptor–substrate complexes. We recently reported the identification of NXT1, an NTF2-related export factor that stimulates nuclear protein export in permeabilized cells and undergoes nucleocytoplasmic shuttling in vivo (Black, B.E., L. Lévesque, J.M. Holaska, T.C. Wood, and B.M. Paschal. 1999. Mol. Cell. Biol. 19:8616–8624). Here, we describe the molecular characterization of NXT1 in the context of the Crm1-dependent export pathway. We find that NXT1 binds directly to Crm1, and that the interaction is sensitive to the presence of Ran-GTP. Moreover, mutations in NXT1 that reduce binding to Crm1 inhibit the activity of NXT1 in nuclear export assays. We show that recombinant Crm1 and Ran are sufficient to reconstitute nuclear translocation of a Rev reporter protein from the nucleolus to an antibody accessible site on the cytoplasmic side of the NPC. Further progress on the export pathway, including the terminal step of Crm1 and Rev reporter protein release, requires NXT1. We propose that NXT1 engages with the export complex in the nucleoplasm, and that it facilitates delivery of the export complex to a site on the cytoplasmic side of NPC where the receptor and substrate are released into the cytoplasm. PMID:11149927

  19. Identification of Novel Saccharomyces cerevisiae Proteins with Nuclear Export Activity: Cell Cycle-Regulated Transcription Factor Ace2p Shows Cell Cycle-Independent Nucleocytoplasmic Shuttling

    PubMed Central

    Jensen, Torben Heick; Neville, Megan; Rain, Jean Christophe; McCarthy, Terri; Legrain, Pierre; Rosbash, Michael

    2000-01-01

    Nuclear export of proteins containing leucine-rich nuclear export signals (NESs) is mediated by the NES receptor CRM1/Crm1p. We have carried out a yeast two-hybrid screen with Crm1p as a bait. The Crm1p-interacting clones were subscreened for nuclear export activity in a visual assay utilizing the Crm1p-inhibitor leptomycin B (LMB). This approach identified three Saccharomyces cerevisiae proteins not previously known to have nuclear export activity. These proteins are the 5′ RNA triphosphatase Ctl1p, the cell cycle-regulated transcription factor Ace2p, and a protein encoded by the previously uncharacterized open reading frame YDR499W. Mutagenesis analysis show that YDR499Wp contains an NES that conforms to the consensus sequence for leucine-rich NESs. Mutagenesis of Ctl1p and Ace2p were unable to identify specific NES residues. However, a 29-amino-acid region of Ace2p, rich in hydrophobic residues, contains nuclear export activity. Ace2p accumulates in the nucleus at the end of mitosis and activates early-G1-specific genes. We now provide evidence that Ace2p is nuclear not only in late M-early G1 but also during other stages of the cell cycle. This feature of Ace2p localization explains its ability to activate genes such as CUP1, which are not expressed in a cell cycle-dependent manner. PMID:11027275

  20. 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. Dated...

  1. Murine Leukemia Virus Uses NXF1 for Nuclear Export of Spliced and Unspliced Viral Transcripts

    PubMed Central

    Sakuma, Toshie; Davila, Jaime I.; Malcolm, Jessica A.; Kocher, Jean-Pierre A.; Tonne, Jason M.

    2014-01-01

    ABSTRACT Intron-containing mRNAs are subject to restricted nuclear export in higher eukaryotes. Retroviral replication requires the nucleocytoplasmic transport of both spliced and unspliced RNA transcripts, and RNA export mechanisms of gammaretroviruses are poorly characterized. Here, we report the involvement of the nuclear export receptor NXF1/TAP in the nuclear export of gammaretroviral RNA transcripts. We identified a conserved cis-acting element in the pol gene of gammaretroviruses, including murine leukemia virus (MLV) and xenotropic murine leukemia virus (XMRV), named the CAE (cytoplasmic accumulation element). The CAE enhanced the cytoplasmic accumulation of viral RNA transcripts and the expression of viral proteins without significantly affecting the stability, splicing, or translation efficiency of the transcripts. Insertion of the CAE sequence also facilitated Rev-independent HIV Gag expression. We found that the CAE sequence interacted with NXF1, whereas disruption of NXF1 ablated CAE function. Thus, the CAE sequence mediates the cytoplasmic accumulation of gammaretroviral transcripts in an NXF1-dependent manner. Disruption of NXF1 expression impaired cytoplasmic accumulations of both spliced and unspliced RNA transcripts of XMRV and MLV, resulting in their nuclear retention or degradation. Thus, our results demonstrate that gammaretroviruses use NXF1 for the cytoplasmic accumulation of both spliced and nonspliced viral RNA transcripts. IMPORTANCE Murine leukemia virus (MLV) has been studied as one of the classic models of retrovirology. Although unspliced host messenger RNAs are rarely exported from the nucleus, MLV actively exports unspliced viral RNAs to the cytoplasm. Despite extensive studies, how MLV achieves this difficult task has remained a mystery. Here, we have studied the RNA export mechanism of MLV and found that (i) the genome contains a sequence which supports the efficient nuclear export of viral RNAs, (ii) the cellular factor NXF1 is

  2. Role of Mex67-Mtr2 in the Nuclear Export of 40S Pre-Ribosomes

    PubMed Central

    Occhipinti, Laura; Kemmler, Stefan; Panse, Vikram G.

    2012-01-01

    Nuclear export of mRNAs and pre-ribosomal subunits (pre40S and pre60S) is fundamental to all eukaryotes. While genetic approaches in budding yeast have identified bona fide export factors for mRNAs and pre60S subunits, little is known regarding nuclear export of pre40S subunits. The yeast heterodimeric transport receptor Mex67-Mtr2 (TAP-p15 in humans) binds mRNAs and pre60S subunits in the nucleus and facilitates their passage through the nuclear pore complex (NPC) into the cytoplasm by interacting with Phe-Gly (FG)-rich nucleoporins that line its transport channel. By exploiting a combination of genetic, cell-biological, and biochemical approaches, we uncovered an unanticipated role of Mex67-Mtr2 in the nuclear export of 40S pre-ribosomes. We show that recruitment of Mex67-Mtr2 to pre40S subunits requires loops emanating from its NTF2-like domains and that the C-terminal FG-rich nucleoporin interacting UBA-like domain within Mex67 contributes to the transport of pre40S subunits to the cytoplasm. Remarkably, the same loops also recruit Mex67-Mtr2 to pre60S subunits and to the Nup84 complex, the respective interactions crucial for nuclear export of pre60S subunits and mRNAs. Thus Mex67-Mtr2 is a unique transport receptor that employs a common interaction surface to participate in the nuclear export of both pre-ribosomal subunits and mRNAs. Mex67-Mtr2 could engage a regulatory crosstalk among the three major export pathways for optimal cellular growth and proliferation. PMID:22956913

  3. Nuclear export of the small ribosomal subunit requires the Ran–GTPase cycle and certain nucleoporins

    PubMed Central

    Moy, Terence I.; Silver, Pamela A.

    1999-01-01

    After their assembly in the nucleolus, ribosomal subunits are exported from the nucleus to the cytoplasm. After export, the 20S rRNA in the small ribosomal subunit is cleaved to yield 18S rRNA and the small 5′ ITS1 fragment. The 5′ ITS1 RNA is normally degraded by the cytoplasmic Xrn1 exonuclease, but in strains lacking XRN1, the 5′ ITS1 fragment accumulates in the cytoplasm. Using the cytoplasmic localization of the 5′ ITS1 fragment as an indicator for the export of the small ribosomal subunit, we have identified genes that are required for ribosome export. Ribosome export is dependent on the Ran–GTPase as mutations in Ran or its regulators caused 5′ ITS1 to accumulate in the nucleoplasm. Mutations in the genes encoding the nucleoporin Nup82 and in the NES exporter Xpo1/Crm1 also caused the nucleoplasmic accumulation of 5′ ITS1. Mutants in a subset of nucleoporins and in the nuclear transport factors Srp1, Kap95, Pse1, Cse1, and Mtr10 accumulate the 5′ ITS1 in the nucleolus and affect ribosome assembly. In contrast, we did not detect nuclear accumulation of 5′ ITS1 in 28 yeast strains that have mutations in other genes affecting nuclear trafficking. PMID:10465789

  4. The stability of AID and its function in class-switching are critically sensitive to the identity of its nuclear-export sequence.

    PubMed

    Geisberger, Roland; Rada, Cristina; Neuberger, Michael S

    2009-04-21

    The carboxyterminal region of activation-induced deaminase (AID) is required for its function in Ig class switch recombination (CSR) and also contains a nuclear-export sequence (NES). Here, based on an extensive fine-structure mutation analysis of the AID NES, as well as from AID chimeras bearing heterologous NESs, we show that while a functional NES is indeed essential for CSR, it is not sufficient. The precise nature of the NES is critical both for AID stabilization and CSR function: minor changes in the NES can perturb stabilization and CSR without jeopardizing nuclear export. The results indicate that the AID NES fulfills a function beyond simply providing a signal for nuclear export and suggest the possibility that the quality of exportin-binding may be critical to the stabilization of AID and its activity in CSR.

  5. Nuclear RNA Export and Packaging Functions of HIV-1 Rev Revisited▿

    PubMed Central

    Blissenbach, Maik; Grewe, Bastian; Hoffmann, Bianca; Brandt, Sabine; Überla, Klaus

    2010-01-01

    Although the viral Rev protein is necessary for HIV replication, its main function in the viral replication cycle has been controversial. Reinvestigating the effect of Rev on the HIV-1 RNA distribution in various cell lines and primary cells revealed that Rev enhanced cytoplasmic levels of the unspliced HIV-1 RNA, mostly 3- to 12-fold, while encapsidation of the RNA and viral infectivity could be stimulated >1,000-fold. Although this clearly questions the general notion that the nuclear export of viral RNAs is the major function of Rev, mechanistically encapsidation seems to be linked to nuclear export, since the tethering of the nuclear export factor TAP to the HIV-1 RNA also enhanced encapsidation. Interference with the formation of an inhibitory ribonucleoprotein complex in the nucleus could lead to enhanced accessibility of the cytoplasmic HIV-1 RNA for translation and encapsidation. This might explain why Rev and tethered TAP exert the same pattern of pleiotropic effects. PMID:20427541

  6. Three-Dimensional Mapping of mRNA Export through the Nuclear Pore Complex.

    PubMed

    Schnell, Steven J; Ma, Jiong; Yang, Weidong

    2014-11-11

    The locations of transcription and translation of mRNA in eukaryotic cells are spatially separated by the nuclear envelope (NE). Plenty of nuclear pore complexes (NPCs) embedded in the NE function as the major gateway for the export of transcribed mRNAs from the nucleus to the cytoplasm. Whereas the NPC, perhaps one of the largest protein complexes, provides a relatively large channel for macromolecules to selectively pass through it in inherently three-dimensional (3D) movements, this channel is nonetheless below the diffraction limit of conventional light microscopy. A full understanding of the mRNA export mechanism urgently requires real-time mapping of the 3D dynamics of mRNA in the NPC of live cells with innovative imaging techniques breaking the diffraction limit of conventional light microscopy. Recently, super-resolution fluorescence microscopy and single-particle tracking (SPT) techniques have been applied to the study of nuclear export of mRNA in live cells. In this review, we emphasize the necessity of 3D mapping techniques in the study of mRNA export, briefly summarize the feasibility of current 3D imaging approaches, and highlight the new features of mRNA nuclear export elucidated with a newly developed 3D imaging approach combining SPT-based super-resolution imaging and 2D-to-3D deconvolution algorithms.

  7. Three-Dimensional Mapping of mRNA Export through the Nuclear Pore Complex

    PubMed Central

    Schnell, Steven J.; Ma, Jiong; Yang, Weidong

    2014-01-01

    The locations of transcription and translation of mRNA in eukaryotic cells are spatially separated by the nuclear envelope (NE). Plenty of nuclear pore complexes (NPCs) embedded in the NE function as the major gateway for the export of transcribed mRNAs from the nucleus to the cytoplasm. Whereas the NPC, perhaps one of the largest protein complexes, provides a relatively large channel for macromolecules to selectively pass through it in inherently three-dimensional (3D) movements, this channel is nonetheless below the diffraction limit of conventional light microscopy. A full understanding of the mRNA export mechanism urgently requires real-time mapping of the 3D dynamics of mRNA in the NPC of live cells with innovative imaging techniques breaking the diffraction limit of conventional light microscopy. Recently, super-resolution fluorescence microscopy and single-particle tracking (SPT) techniques have been applied to the study of nuclear export of mRNA in live cells. In this review, we emphasize the necessity of 3D mapping techniques in the study of mRNA export, briefly summarize the feasibility of current 3D imaging approaches, and highlight the new features of mRNA nuclear export elucidated with a newly developed 3D imaging approach combining SPT-based super-resolution imaging and 2D-to-3D deconvolution algorithms. PMID:25393401

  8. Characterization of the ptr5+ gene involved in nuclear mRNA export in fission yeast.

    PubMed

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

    2012-02-03

    To analyze the mechanisms of mRNA export from the nucleus to the cytoplasm, we have isolated eleven mutants, ptr [poly(A)(+) RNA transport] 1 to 11, which accumulate poly(A)(+) 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)(+) RNA at the nuclear periphery after shifting to the nonpermissive temperature. We cloned the ptr5(+) 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.

  9. Influenza Virus-Induced Caspase-Dependent Enlargement of Nuclear Pores Promotes Nuclear Export of Viral Ribonucleoprotein Complexes

    PubMed Central

    Mühlbauer, Dirk; Dzieciolowski, Julia; Hardt, Martin; Hocke, Andreas; Schierhorn, Kristina L.; Mostafa, Ahmed; Müller, Christin; Wisskirchen, Christian; Herold, Susanne; Wolff, Thorsten; Ziebuhr, John

    2015-01-01

    ABSTRACT Influenza A viruses (IAV) replicate their segmented RNA genome in the nucleus of infected cells and utilize caspase-dependent nucleocytoplasmic export mechanisms to transport newly formed ribonucleoprotein complexes (RNPs) to the site of infectious virion release at the plasma membrane. In this study, we obtained evidence that apoptotic caspase activation in IAV-infected cells is associated with the degradation of the nucleoporin Nup153, an integral subunit of the nuclear pore complex. Transmission electron microscopy studies revealed a distinct enlargement of nuclear pores in IAV-infected cells. Transient expression and subcellular accumulation studies of multimeric marker proteins in virus-infected cells provided additional evidence for increased nuclear pore diameters facilitating the translocation of large protein complexes across the nuclear membrane. Furthermore, caspase 3/7 inhibition data obtained in this study suggest that active, Crm1-dependent IAV RNP export mechanisms are increasingly complemented by passive, caspase-induced export mechanisms at later stages of infection. IMPORTANCE In contrast to the process seen with most other RNA viruses, influenza virus genome replication occurs in the nucleus (rather than the cytoplasm) of infected cells. Therefore, completion of the viral replication cycle critically depends on intracellular transport mechanisms that ensure the translocation of viral ribonucleoprotein (RNP) complexes across the nuclear membrane. Here, we demonstrate that virus-induced cellular caspase activities cause a widening of nuclear pores, thereby facilitating nucleocytoplasmic translocation processes and, possibly, promoting nuclear export of newly synthesized RNPs. These passive transport mechanisms are suggested to complement Crm1-dependent RNP export mechanisms known to occur at early stages of the replication cycle and may contribute to highly efficient production of infectious virus progeny at late stages of the viral

  10. Overexpression of Ran GTPase Components Regulating Nuclear Export, but not Mitotic Spindle Assembly, Marks Chromosome Instability and Poor Prognosis in Breast Cancer.

    PubMed

    Vaidyanathan, Srividya; Thangavelu, Pulari U; Duijf, Pascal H G

    2016-10-01

    Ran GTPase regulates nuclear import, nuclear export, and mitotic spindle assembly. The multifunctional involvement of seventeen Ran GTPase components in these processes has complicated research into how each contributes to cancer development. To assess whether individual and process-specific misexpression of Ran GTPase components contribute to chromosome instability (CIN) and worsen breast cancer patient prognosis. Using publicly available datasets, we studied the degree of misexpression of all Ran GTPase signaling components in breast cancer, assessed their involvement in CIN and used four clinical tests to evaluate whether their misregulation may constitute independent prognostic predictors. A significant majority of Ran GTPase signaling components is overexpressed in breast cancer. Strikingly, spindle assembly components are overexpressed and associated with CIN with only marginal significance and four independent tests indicate that this does not worsen patient outcome. Overexpression of nuclear import components is neither CIN-associated nor clinically significant. In sharp contrast, overexpression of nuclear export components constitutes a strong independent marker for both CIN and poor patient prognosis. We identify Exportin 2/CSE1L, Exportin 3/XPOT, Exportin 5/XPO5, and RANBP1 as novel potential targets. We find that overexpression of Ran GTPase components involved in nuclear export, but not nuclear import or mitotic spindle assembly, is a strong CIN-associated marker for poor breast cancer prognosis. This could mean that increased nuclear export (of, for instance, pRb, p53, p73, BRCA1, p21, p27, E2F4, IκB, survivin), rather than spindle defects, mainly drives CIN and tumorigenesis. Hence, selective inhibitors of nuclear export may be effective for treating the most aggressive and chromosomally unstable breast cancers.

  11. Screening of nuclear targeting proteins in Acinetobacter baumannii based on nuclear localization signals.

    PubMed

    Moon, Dong Chan; Gurung, Mamata; Lee, Jung Hwa; Lee, Yong Seok; Choi, Chi Won; Kim, Seung Il; Lee, Je Chul

    2012-05-01

    Nuclear targeting of bacterial proteins is an emerging pathogenic mechanism in bacteria. However, due to the absence of an appropriate screening system for nuclear targeting proteins, systematic approaches to nuclear targeting of bacterial proteins and subsequent host cell pathology are limited. In this study, we developed a screening system for nuclear targeting proteins in Acinetobacter baumannii using a combination of bioinformatic analysis based on nuclear localization signal (NLS) and the Gateway(®) recombinational cloning system. Among 3367 open reading frames of A. baumannii ATCC 17978, 34 functional or hypothetical proteins were predicted to carry the putative NLS sequences. Of the 29 clones generated by the Gateway(®) recombinational cloning system, 14 proteins tagged with green fluorescent protein (GFP) were targeted to nuclei of host cells. Among the 14 nuclear targeting proteins, S21, L20, and L32 ribosomal proteins and transposase carried putative nuclear export signal (NES) sequences, but only transposase harbored the functional NES. After translocation to nuclei of host cells, four A. baumannii proteins induced cytotoxicity. In conclusion, we have developed a screening system for nuclear targeting proteins in A. baumannii. This system may open the way to a new field of bacterial pathogenesis.

  12. 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

  13. Identification of a new export signal in Plasmodium yoelii: identification of a new exportome.

    PubMed

    Siau, Anthony; Huang, Ximei; Yam, Xue Yan; Bob, Ndeye Sakha; Sun, Hequan; Rajapakse, Jagath C; Renia, Laurent; Preiser, Peter R

    2014-05-01

    Development of the erythrocytic malaria parasite requires targeting of parasite proteins into multiple compartments located within and beyond the parasite confine. Beyond the PEXEL/VTS pathway and its characterized players, increasing amount of evidence has highlighted the existence of proteins exported using alternative export-signal(s)/pathway(s); hence, the exportomes currently predicted are incomplete. The nature of these exported proteins which could have a prominent role in most of the Plasmodium species remains elusive. Using P.  yoelii variant proteins, we identified a signal associated to lipophilic region that mediates export of P.  yoelii proteins. This non-PEXEL signal termed PLASMED is defined by semi-conserved residues and possibly a secondary structure. In vivo characterization of exported-proteins indicated that PLASMED is a bona fide export-signal that allowed us to identify an unseen P.  yoelii exportome. The repertoire of the newly predicted exported proteins opens up perspectives for unravelling the remodelling of the host-cell by the parasite, against which new therapies could be elaborated.

  14. 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.

  15. A conserved CCCH-type zinc finger protein regulates mRNA nuclear adenylation and export.

    PubMed

    Hurt, Jessica A; Obar, Robert A; Zhai, Bo; Farny, Natalie G; Gygi, Steven P; Silver, Pamela A

    2009-04-20

    Coupling of messenger RNA (mRNA) nuclear export with prior processing steps aids in the fidelity and efficiency of mRNA transport to the cytoplasm. In this study, we show that the processes of export and polyadenylation are coupled via the Drosophila melanogaster CCCH-type zinc finger protein CG6694/dZC3H3 through both physical and functional interactions. We show that depletion of dZC3H3 from S2R+ cells results in transcript hyperadenylation. Using targeted coimmunoprecipitation and liquid chromatography mass spectrometry (MS)/MS techniques, we characterize interactions of known components of the mRNA nuclear export and polyadenylation machineries with dZC3H3. Furthermore, we demonstrate the functional conservation of this factor, as depletion of its human homologue ZC3H3 by small interfering RNA results in an mRNA export defect in human cells as well. Nuclear polyadenylated (poly(A)) RNA in ZC3H3-depleted cells is sequestered in foci removed from SC35-containing speckles, indicating a shift from the normal subnuclear distribution of poly(A) RNA. Our data suggest a model wherein ZC3H3 interfaces between the polyadenylation machinery, newly poly(A) mRNAs, and factors for transcript export.

  16. 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.

  17. Selective nuclear export of specific classes of mRNA from mammalian nuclei is promoted by GANP.

    PubMed

    Wickramasinghe, Vihandha O; Andrews, Robert; Ellis, Peter; Langford, Cordelia; Gurdon, John B; Stewart, Murray; Venkitaraman, Ashok R; Laskey, Ronald A

    2014-04-01

    The nuclear phase of the gene expression pathway culminates in the export of mature messenger RNAs (mRNAs) to the cytoplasm through nuclear pore complexes. GANP (germinal- centre associated nuclear protein) promotes the transfer of mRNAs bound to the transport factor NXF1 to nuclear pore complexes. Here, we demonstrate that GANP, subunit of the TRanscription-EXport-2 (TREX-2) mRNA export complex, promotes selective nuclear export of a specific subset of mRNAs whose transport depends on NXF1. Genome-wide gene expression profiling showed that half of the transcripts whose nuclear export was impaired following NXF1 depletion also showed reduced export when GANP was depleted. GANP-dependent transcripts were highly expressed, yet short-lived, and were highly enriched in those encoding central components of the gene expression machinery such as RNA synthesis and processing factors. After injection into Xenopus oocyte nuclei, representative GANP-dependent transcripts showed faster nuclear export kinetics than representative transcripts that were not influenced by GANP depletion. We propose that GANP promotes the nuclear export of specific classes of mRNAs that may facilitate rapid changes in gene expression.

  18. Selective nuclear export of specific classes of mRNA from mammalian nuclei is promoted by GANP

    PubMed Central

    Wickramasinghe, Vihandha O.; Andrews, Robert; Ellis, Peter; Langford, Cordelia; Gurdon, John B.; Stewart, Murray; Venkitaraman, Ashok R.; Laskey, Ronald A.

    2014-01-01

    The nuclear phase of the gene expression pathway culminates in the export of mature messenger RNAs (mRNAs) to the cytoplasm through nuclear pore complexes. GANP (germinal- centre associated nuclear protein) promotes the transfer of mRNAs bound to the transport factor NXF1 to nuclear pore complexes. Here, we demonstrate that GANP, subunit of the TRanscription-EXport-2 (TREX-2) mRNA export complex, promotes selective nuclear export of a specific subset of mRNAs whose transport depends on NXF1. Genome-wide gene expression profiling showed that half of the transcripts whose nuclear export was impaired following NXF1 depletion also showed reduced export when GANP was depleted. GANP-dependent transcripts were highly expressed, yet short-lived, and were highly enriched in those encoding central components of the gene expression machinery such as RNA synthesis and processing factors. After injection into Xenopus oocyte nuclei, representative GANP-dependent transcripts showed faster nuclear export kinetics than representative transcripts that were not influenced by GANP depletion. We propose that GANP promotes the nuclear export of specific classes of mRNAs that may facilitate rapid changes in gene expression. PMID:24510098

  19. Inhibition of CRM1-mediated nuclear export of transcription factors by leukemogenic NUP98 fusion proteins.

    PubMed

    Takeda, Akiko; Sarma, Nayan J; Abdul-Nabi, Anmaar M; Yaseen, Nabeel R

    2010-05-21

    NUP98 is a nucleoporin that plays complex roles in the nucleocytoplasmic trafficking of macromolecules. Rearrangements of the NUP98 gene in human leukemia result in the expression of numerous fusion oncoproteins whose effect on nucleocytoplasmic trafficking is poorly understood. The present study was undertaken to determine the effects of leukemogenic NUP98 fusion proteins on CRM1-mediated nuclear export. NUP98-HOXA9, a prototypic NUP98 fusion, inhibited the nuclear export of two known CRM1 substrates: mutated cytoplasmic nucleophosmin and HIV-1 Rev. In vitro binding assays revealed that NUP98-HOXA9 binds CRM1 through the FG repeat motif in a Ran-GTP-dependent manner similar to but stronger than the interaction between CRM1 and its export substrates. Two NUP98 fusions, NUP98-HOXA9 and NUP98-DDX10, whose fusion partners are structurally and functionally unrelated, interacted with endogenous CRM1 in myeloid cells as shown by co-immunoprecipitation. These leukemogenic NUP98 fusion proteins interacted with CRM1, Ran, and the nucleoporin NUP214 in a manner fundamentally different from that of wild-type NUP98. NUP98-HOXA9 and NUP98-DDX10 formed characteristic aggregates within the nuclei of a myeloid cell line and primary human CD34+ cells and caused aberrant localization of CRM1 to these aggregates. These NUP98 fusions caused nuclear accumulation of two transcription factors, NFAT and NFkappaB, that are regulated by CRM1-mediated export. The nuclear entrapment of NFAT and NFkappaB correlated with enhanced transcription from promoters responsive to these transcription factors. Taken together, the results suggest a new mechanism by which NUP98 fusions dysregulate transcription and cause leukemia, namely, inhibition of CRM1-mediated nuclear export with aberrant nuclear retention of transcriptional regulators.

  20. 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.

  1. Transfection of influenza A virus nuclear export protein induces the expression of tumor necrosis factor alpha.

    PubMed

    Lara-Sampablo, Alejandra; Flores-Alonso, Juan Carlos; De Jesús-Ortega, Nereyda; Santos-López, Gerardo; Vallejo-Ruiz, Verónica; Rosas-Murrieta, Nora; Reyes-Carmona, Sandra; Herrera-Camacho, Irma; Reyes-Leyva, Julio

    2014-06-24

    Influenza A virus genomic segments eight codes for non-structural 1 (NS1) protein that is involved in evasion of innate antiviral response, and nuclear export protein (NEP) that participates in the export of viral ribonucleoprotein (RNP) complexes, transcription and replication. Tumor necrosis factor alpha (TNF-α) is highly expressed during influenza virus infections and is considered an anti-infective cytokine. NS1 and NEP proteins were overexpressed and their role on TNF-α expression was evaluated. Both TNF-α mRNA and protein increased in cells transfected with NEP but not with NS1. We further investigate if NS1 or NEP regulates the activity of TNF-α promoter. In the presence of NEP the activity of TNF-α promoter increased significantly compared with the control (83.5±2.9 vs. 30.9±2.8, respectively; p=0.001). This effect decreased 15-fold when the TNF-α promoter distal region was deleted, suggesting the involvement of mitogen-activated protein kinases (MAPK) and NF-kB response elements. This was corroborated by testing the effect produced on TNF-α promoter by the treatment with Raf/MEK/ERK (U0126), NF-kB (Bay-11-7082) and PI3K (Ly294-002) cell signaling inhibitors. Treatment with U0126 and Bay-117082 reduced the activity of TNF-α promoter mediated by NEP (41.5±3.2, 70% inhibition; and 80.6±7.4, 35% inhibition, respectively) compared to mock-treated control. The results suggest a new role for NEP protein that participates in the transcriptional regulation of human TNF-α expression.

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

    PubMed

    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.

  3. 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

  4. 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.

  5. Thyroid Hormone Receptor α1 Follows a Cooperative CRM1/Calreticulin-mediated Nuclear Export Pathway*

    PubMed Central

    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.

    2008-01-01

    The thyroid hormone receptor α1 (TRα) exhibits a dual role as an activator or repressor of its target genes in response to thyroid hormone (T3). Previously, we have shown that TRα, 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 TRα is its ability to exit the nucleus through the nuclear pore complex. TRα 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 TRα. We show that, in addition to shuttling in heterokaryons, TRα shuttles rapidly in an unfused monokaryon system as well. Furthermore, our data show that TRα directly interacts with calreticulin, and point to the intriguing possibility that TRα follows a cooperative export pathway in which both calreticulin and CRM1 play a role in facilitating efficient translocation of TRα from the nucleus to cytoplasm. PMID:18641393

  6. An agent-based model for mRNA export through the nuclear pore complex.

    PubMed

    Azimi, Mohammad; Bulat, Evgeny; Weis, Karsten; Mofrad, Mohammad R K

    2014-11-05

    mRNA export from the nucleus is an essential step in the expression of every protein- coding gene in eukaryotes, but many aspects of this process remain poorly understood. The density of export receptors that must bind an mRNA to ensure export, as well as how receptor distribution affects transport dynamics, is not known. It is also unclear whether the rate-limiting step for transport occurs at the nuclear basket, in the central channel, or on the cytoplasmic face of the nuclear pore complex. Using previously published biophysical and biochemical parameters of mRNA export, we implemented a three-dimensional, coarse-grained, agent-based model of mRNA export in the nanosecond regime to gain insight into these issues. On running the model, we observed that mRNA export is sensitive to the number and distribution of transport receptors coating the mRNA and that there is a rate-limiting step in the nuclear basket that is potentially associated with the mRNA reconfiguring itself to thread into the central channel. Of note, our results also suggest that using a single location-monitoring mRNA label may be insufficient to correctly capture the time regime of mRNA threading through the pore and subsequent transport. This has implications for future experimental design to study mRNA transport dynamics. © 2014 Azimi, Bulat, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  7. 10 CFR Appendix A to Part 110 - Illustrative List of Nuclear Reactor Equipment Under NRC Export Licensing Authority

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 10 Energy 2 2014-01-01 2014-01-01 false Illustrative List of Nuclear Reactor Equipment Under NRC... List of Nuclear Reactor Equipment Under NRC Export Licensing Authority Note: A nuclear reactor... core of a nuclear reactor and capable of withstanding the operating pressure of the primary coolant....

  8. 10 CFR Appendix A to Part 110 - Illustrative List of Nuclear Reactor Equipment Under NRC Export Licensing Authority

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 10 Energy 2 2013-01-01 2013-01-01 false Illustrative List of Nuclear Reactor Equipment Under NRC... List of Nuclear Reactor Equipment Under NRC Export Licensing Authority Note—A nuclear reactor basically... nuclear reactor and capable of withstanding the operating pressure of the primary coolant. (2) On-line...

  9. 10 CFR Appendix A to Part 110 - Illustrative List of Nuclear Reactor Equipment Under NRC Export Licensing Authority

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 10 Energy 2 2012-01-01 2012-01-01 false Illustrative List of Nuclear Reactor Equipment Under NRC... List of Nuclear Reactor Equipment Under NRC Export Licensing Authority Note—A nuclear reactor basically... nuclear reactor and capable of withstanding the operating pressure of the primary coolant. (2) On-line...

  10. 10 CFR Appendix A to Part 110 - Illustrative List of Nuclear Reactor Equipment Under NRC Export Licensing Authority

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 2 2010-01-01 2010-01-01 false Illustrative List of Nuclear Reactor Equipment Under NRC... List of Nuclear Reactor Equipment Under NRC Export Licensing Authority Note—A nuclear reactor basically... nuclear reactor and capable of withstanding the operating pressure of the primary coolant. (2) On-line...

  11. 10 CFR Appendix A to Part 110 - Illustrative List of Nuclear Reactor Equipment Under NRC Export Licensing Authority

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 10 Energy 2 2011-01-01 2011-01-01 false Illustrative List of Nuclear Reactor Equipment Under NRC... List of Nuclear Reactor Equipment Under NRC Export Licensing Authority Note—A nuclear reactor basically... nuclear reactor and capable of withstanding the operating pressure of the primary coolant. (2) On-line...

  12. The RanGEF Bj1 promotes Prospero nuclear export and neuroblast self-renewal

    PubMed Central

    Joy, Tasha; Hirono, Keiko

    2014-01-01

    Drosophila larval neuroblasts are a model system for studying stem cell self-renewal and differentiation. Here we report a novel role for the Drosophila gene Bj1 in promoting larval neuroblast self-renewal. Bj1 is the guanine-nucleotide exchange factor for Ran GTPase, which regulates nuclear import/export. Bj1 transcripts are highly enriched in larval brain neuroblasts (in both central brain and optic lobe), while Bj1 protein is detected in both neuroblasts and their neuronal progeny. Loss of Bj1 using both mutants or RNAi causes a progressive loss of larval neuroblasts, showing that Bj1 is required to maintain neuroblast numbers. Loss of Bj1 does not result in neuroblast apoptosis, but rather leads to abnormal nuclear accumulation of the differentiation factor Prospero, and premature neuroblast differentiation. We conclude that the Bj1 RanGEF promotes Prospero nuclear export and neuroblast self-renewal. PMID:25312250

  13. The RanGEF Bj1 promotes prospero nuclear export and neuroblast self-renewal.

    PubMed

    Joy, Tasha; Hirono, Keiko; Doe, Chris Q

    2015-05-01

    Drosophila larval neuroblasts are a model system for studying stem cell self-renewal and differentiation. Here, we report a novel role for the Drosophila gene Bj1 in promoting larval neuroblast self-renewal. Bj1 is the guanine-nucleotide exchange factor for Ran GTPase, which regulates nuclear import/export. Bj1 transcripts are highly enriched in larval brain neuroblasts (in both central brain and optic lobe), while Bj1 protein is detected in both neuroblasts and their neuronal progeny. Loss of Bj1 using both mutants or RNAi causes a progressive loss of larval neuroblasts, showing that Bj1 is required to maintain neuroblast numbers. Loss of Bj1 does not result in neuroblast apoptosis, but rather leads to abnormal nuclear accumulation of the differentiation factor Prospero, and premature neuroblast differentiation. We conclude that the Bj1 RanGEF promotes Prospero nuclear export and neuroblast self-renewal. © 2014 Wiley Periodicals, Inc.

  14. Inhibition of the nuclear export of p65 and IQCG in leukemogenesis by NUP98-IQCG.

    PubMed

    Pan, Mengmeng; Zhang, Qiyao; Liu, Ping; Huang, Jinyan; Wang, Yueying; Chen, Saijuan

    2016-12-01

    NUP98 fuses with approximately 34 different partner genes via translocation in hematological malignancies. Transgenic or retrovirus-mediated bone marrow transplanted mouse models reveal the leukemogenesis of some NUP98-related fusion genes. We previously reported the fusion protein NUP98-IQ motif containing G (IQCG) in a myeloid/T lymphoid bi-phenoleukemia patient with t(3;11) and confirmed its leukemogenic ability. Herein, we demonstrated the association of NUP98-IQCG with CRM1, and found that NUP98-IQCG expression inhibits the CRM1-mediated nuclear export of p65 and enhances the transcriptional activity of nuclear factor-κB. Moreover, IQCG could be entrapped in the nucleus by NUP98-IQCG, and the fusion protein interacts with calmodulin via the IQ motif in a calcium-independent manner. Therefore, the inhibition of nuclear exports of p65 and IQCG might contribute to the leukemogenesis of NUP98-IQCG.

  15. The actin binding cytoskeletal protein Moesin is involved in nuclear mRNA export.

    PubMed

    Kristó, Ildikó; Bajusz, Csaba; Borsos, Barbara N; Pankotai, Tibor; Dopie, Joseph; Jankovics, Ferenc; Vartiainen, Maria K; Erdélyi, Miklós; Vilmos, Péter

    2017-10-01

    Current models imply that the evolutionarily conserved, actin-binding Ezrin-Radixin-Moesin (ERM) proteins perform their activities at the plasma membrane by anchoring membrane proteins to the cortical actin network. Here we show that beside its cytoplasmic functions, the single ERM protein of Drosophila, Moesin, has a novel role in the nucleus. The activation of transcription by heat shock or hormonal treatment increases the amount of nuclear Moesin, indicating biological function for the protein in the nucleus. The distribution of Moesin in the nucleus suggests a function in transcription and the depletion of mRNA export factors Nup98 or its interacting partner, Rae1, leads to the nuclear accumulation of Moesin, suggesting that the nuclear function of the protein is linked to mRNA export. Moesin localizes to mRNP particles through the interaction with the mRNA export factor PCID2 and knock down of Moesin leads to the accumulation of mRNA in the nucleus. Based on our results we propose that, beyond its well-known, manifold functions in the cytoplasm, the ERM protein of Drosophila is a new, functional component of the nucleus where it participates in mRNA export. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Unprecedented NES non-antagonistic inhibitor for nuclear export of Rev from Sida cordifolia.

    PubMed

    Tamura, Satoru; Kaneko, Masafumi; Shiomi, Atsushi; Yang, Guang-Ming; Yamaura, Toshiaki; Murakami, Nobutoshi

    2010-03-15

    Bioassay-guided separation from the MeOH extract of the South American medicinal plant Sida cordifolia resulted in isolation of (10E,12Z)-9-hydroxyoctadeca-10,12-dienoic acid (1) as an unprecedented NES non-antagonistic inhibitor for nuclear export of Rev. This mechanism of action was established by competitive experiment by the biotinylated probe derived from leptomycin B, the known NES antagonistic inhibitor. Additionally, structure-activity relationship analysis by use of the synthesized analogs clarified cooperation of several functionalities in the Rev-export inhibitory activity of 1.

  17. Structural insights into how Yrb2p accelerates the assembly of the Xpo1p nuclear export complex.

    PubMed

    Koyama, Masako; Shirai, Natsuki; Matsuura, Yoshiyuki

    2014-11-06

    Proteins and ribonucleoproteins containing a nuclear export signal (NES) assemble with the exportin Xpo1p (yeast CRM1) and Gsp1p-GTP (yeast Ran-GTP) in the nucleus and exit through the nuclear pore complex. In the cytoplasm, Yrb1p (yeast RanBP1) displaces NES from Xpo1p. Efficient export of NES-cargoes requires Yrb2p (yeast RanBP3), a primarily nuclear protein containing nucleoporin-like phenylalanine-glycine (FG) repeats and a low-affinity Gsp1p-binding domain (RanBD). Here, we show that Yrb2p strikingly accelerates the association of Gsp1p-GTP and NES to Xpo1p. We have solved the crystal structure of the Xpo1p-Yrb2p-Gsp1p-GTP complex, a key assembly intermediate that can bind cargo rapidly. Although the NES-binding cleft of Xpo1p is closed in this intermediate, our data suggest that preloading of Gsp1p-GTP onto Xpo1p by Yrb2p, conformational flexibility of Xpo1p, and the low affinity of RanBD enable active displacement of Yrb2p RanBD by NES to occur effectively. The structure also reveals the major binding sites for FG repeats on Xpo1p.

  18. A biochemical framework for eIF4E-dependent mRNA export and nuclear recycling of the export machinery.

    PubMed

    Volpon, Laurent; Culjkovic-Kraljacic, Biljana; Sohn, Hye Seon; Blanchet-Cohen, Alexis; Osborne, Michael J; Borden, Katherine L B

    2017-06-01

    The eukaryotic translation initiation factor eIF4E acts in the nuclear export and translation of a subset of mRNAs. Both of these functions contribute to its oncogenic potential. While the biochemical mechanisms that underlie translation are relatively well understood, the molecular basis for eIF4E's role in mRNA export remains largely unexplored. To date, over 3000 transcripts, many encoding oncoproteins, were identified as potential nuclear eIF4E export targets. These target RNAs typically contain a ∼50-nucleotide eIF4E sensitivity element (4ESE) in the 3' UTR and a 7-methylguanosine cap on the 5' end. While eIF4E associates with the cap, an unknown factor recognizes the 4ESE element. We previously identified cofactors that functionally interacted with eIF4E in mammalian cell nuclei including the leucine-rich pentatricopeptide repeat protein LRPPRC and the export receptor CRM1/XPO1. LRPPRC simultaneously interacts with both eIF4E bound to the 5' mRNA cap and the 4ESE element in the 3' UTR. In this way, LRPPRC serves as a specificity factor to recruit 4ESE-containing RNAs within the nucleus. Further, we show that CRM1 directly binds LRPPRC likely acting as the export receptor for the LRPPRC-eIF4E-4ESE RNA complex. We also found that Importin 8, the nuclear importer for cap-free eIF4E, imports RNA-free LRPPRC, potentially providing both coordinated nuclear recycling of the export machinery and an important surveillance mechanism to prevent futile export cycles. Our studies provide the first biochemical framework for the eIF4E-dependent mRNA export pathway. © 2017 Volpon et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  19. Structure and Function of the Nuclear Pore Complex Cytoplasmic mRNA Export Platform.

    PubMed

    Fernandez-Martinez, Javier; Kim, Seung Joong; Shi, Yi; Upla, Paula; Pellarin, Riccardo; Gagnon, Michael; Chemmama, Ilan E; Wang, Junjie; Nudelman, Ilona; Zhang, Wenzhu; Williams, Rosemary; Rice, William J; Stokes, David L; Zenklusen, Daniel; Chait, Brian T; Sali, Andrej; Rout, Michael P

    2016-11-17

    The last steps in mRNA export and remodeling are performed by the Nup82 complex, a large conserved assembly at the cytoplasmic face of the nuclear pore complex (NPC). By integrating diverse structural data, we have determined the molecular architecture of the native Nup82 complex at subnanometer precision. The complex consists of two compositionally identical multiprotein subunits that adopt different configurations. The Nup82 complex fits into the NPC through the outer ring Nup84 complex. Our map shows that this entire 14-MDa Nup82-Nup84 complex assembly positions the cytoplasmic mRNA export factor docking sites and messenger ribonucleoprotein (mRNP) remodeling machinery right over the NPC's central channel rather than on distal cytoplasmic filaments, as previously supposed. We suggest that this configuration efficiently captures and remodels exporting mRNP particles immediately upon reaching the cytoplasmic side of the NPC.

  20. Localization of Nucleoporin Tpr to the Nuclear Pore Complex Is Essential for Tpr Mediated Regulation of the Export of Unspliced RNA

    PubMed Central

    Rajanala, Kalpana; Nandicoori, Vinay Kumar

    2012-01-01

    Nucleoporin Tpr is a component of the nuclear pore complex (NPC) that localizes exclusively to intranuclear filaments. Tpr functions as a scaffolding element in the nuclear phase of the NPC and plays a role in mitotic spindle checkpoint signalling. Export of intron-containing mRNA in Mason Pfizer Monkey Virus is regulated by direct interaction of cellular proteins with the cis-acting Constitutive Transport Element (CTE). In mammalian cells, the transport of Gag/Pol-CTE reporter construct is not very efficient, suggesting a regulatory mechanism to retain this unspliced RNA. Here we report that the knockdown of Tpr in mammalian cells leads to a drastic enhancement in the levels of Gag proteins (p24) in the cytoplasm, which is rescued by siRNA resistant Tpr. Tpr's role in the retention of unspliced RNA is independent of the functions of Sam68 and Tap/Nxf1 proteins, which are reported to promote CTE dependent export. Further, we investigated the possible role for nucleoporins that are known to function in nucleocytoplasmic transport in modulating unspliced RNA export. Results show that depletion of Nup153, a nucleoporin required for NPC anchoring of Tpr, plays a role in regulating the export, while depletion of other FG repeat-containing nucleoporins did not alter the unspliced RNA export. Results suggest that Tpr and Nup153 both regulate the export of unspliced RNA and they are most likely functioning through the same pathway. Importantly, we find that localization of Tpr to the NPC is necessary for Tpr mediated regulation of unspliced RNA export. Collectively, the data indicates that perinuclear localization of Tpr at the nucleopore complex is crucial for regulating intron containing mRNA export by directly or indirectly participating in the processing and degradation of aberrant mRNA transcripts. PMID:22253824

  1. A cellular reporter to evaluate CRM1 nuclear export activity: functional analysis of the cancer-related mutant E571K.

    PubMed

    García-Santisteban, Iraia; Arregi, Igor; Alonso-Mariño, Marián; Urbaneja, María A; Garcia-Vallejo, Juan J; Bañuelos, Sonia; Rodríguez, Jose A

    2016-12-01

    The exportin CRM1 binds nuclear export signals (NESs), and mediates active transport of NES-bearing proteins from the nucleus to the cytoplasm. Structural and biochemical analyses have uncovered the molecular mechanisms underlying CRM1/NES interaction. CRM1 binds NESs through a hydrophobic cleft, whose open or closed conformation facilitates NES binding and release. Several cofactors allosterically modulate the conformation of the NES-binding cleft through intramolecular interactions involving an acidic loop and a C-terminal helix in CRM1. This current model of CRM1-mediated nuclear export has not yet been evaluated in a cellular setting. Here, we describe SRV100, a cellular reporter to interrogate CRM1 nuclear export activity. Using this novel tool, we provide evidence further validating the model of NES binding and release by CRM1. Furthermore, using both SRV100-based cellular assays and in vitro biochemical analyses, we investigate the functional consequences of a recurrent cancer-related mutation, which targets a residue near CRM1 NES-binding cleft. Our data indicate that this mutation does not necessarily abrogate the nuclear export activity of CRM1, but may increase its affinity for NES sequences bearing a more negatively charged C-terminal end.

  2. Antiviral activity of KR-23502 targeting nuclear export of influenza B virus ribonucleoproteins.

    PubMed

    Jang, Yejin; Lee, Hye Won; Shin, Jin Soo; Go, Yun Young; Kim, Chonsaeng; Shin, Daeho; Malpani, Yashwardhan; Han, Soo Bong; Jung, Young-Sik; Kim, Meehyein

    2016-10-01

    The spiro compound 5,6-dimethyl-3H,3'H-spiro(benzofuran-2,1'-isobenzofuran)-3,3'-dione (KR-23502) has antiviral activity against influenza A and more potently B viruses. The aim of this study is to elucidate its mechanism of action. Subcellular localization and time-course expression of influenza B viral proteins, nucleoprotein (NP) and matrix protein 1 (M1), showed that KR-23502 reduced their amounts within 5 h post-infection. Early steps of virus life cycle, including virus entry, nuclear localization of NP and viral RNA-dependent RNA replication, were not affected by KR-23502. Instead it interrupted a later event corresponding to nuclear export of NP and M1 proteins. Delivery of viral ribonucleoprotein (vRNP)-M1 complex has been known to be mediated by the viral nuclear export protein (NEP) through interaction with cellular chromosomal maintenance 1 (CRM1) protein. In this study, we experimentally demonstrated that the compound targets the nuclear export of vRNP. Moreover, a single mutation (aspartate to glycine) at amino acid position 54 in M1 [M1(D54G)] was detected after 18 passages in the presence of KR-23502 with a 2-fold increase in 50% effective concentration indicating that this compound has a relatively high genetic barrier to resistance. Interestingly, it was observed that proteasome-mediated degradation of M1(D54G) was attenuated by KR-23502. In conclusion, we suggest that KR-23502 shows its anti-influenza activity by downregulating NEP/CRM1-mediated nuclear export of influenza vRNP and M1. KR-23502 provides a core chemical skeleton for further structure-based design of novel antivirals against influenza viruses.

  3. 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

  4. Nuclear localization signals for four distinct karyopherin-β nuclear import systems.

    PubMed

    Soniat, Michael; Chook, Yuh Min

    2015-06-15

    The Karyopherin-β family of proteins mediates nuclear transport of macromolecules. Nuclear versus cytoplasmic localization of proteins is often suggested by the presence of NLSs (nuclear localization signals) or NESs (nuclear export signals). Import-Karyopherin-βs or Importins bind to NLSs in their protein cargos to transport them through nuclear pore complexes into the nucleus. Until recently, only two classes of NLS had been biochemically and structurally characterized: the classical NLS, which is recognized by the Importin-α/β heterodimer and the PY-NLS (proline-tyrosine NLS), which is recognized by Karyopherin-β2 or Transportin-1. Structures of two other Karyopherin-βs, Kap121 and Transportin-SR2, in complex with their respective cargos were reported for the first time recently, revealing two new distinct classes of NLSs. The present paper briefly describes the classical NLS, reviews recent literature on the PY-NLS and provides in-depth reviews of the two newly discovered classes of NLSs that bind Kap121p and Transportin-SR respectively.

  5. RNA-guided assembly of Rev-RRE nuclear export complexes.

    PubMed

    Bai, Yun; Tambe, Akshay; Zhou, Kaihong; Doudna, Jennifer A

    2014-08-27

    HIV replication requires nuclear export of unspliced and singly spliced viral transcripts. Although a unique RNA structure has been proposed for the Rev-response element (RRE) responsible for viral mRNA export, how it recruits multiple HIV Rev proteins to form an export complex has been unclear. We show here that initial binding of Rev to the RRE triggers RNA tertiary structural changes, enabling further Rev binding and the rapid formation of a viral export complex. Analysis of the Rev-RRE assembly pathway using SHAPE-Seq and small-angle X-ray scattering (SAXS) reveals two major steps of Rev-RRE complex formation, beginning with rapid Rev binding to a pre-organized region presenting multiple Rev binding sites. This step induces long-range remodeling of the RNA to expose a cryptic Rev binding site, enabling rapid assembly of additional Rev proteins into the RNA export complex. This kinetic pathway may help maintain the balance between viral replication and maturation.DOI: http://dx.doi.org/10.7554/eLife.03656.001.

  6. INSIDE-OUT SIGNALING PATHWAYS FROM NUCLEAR ROS CONTROL PULMONARY INNATE IMMUNITY

    PubMed Central

    Choudhary, Sanjeev; Brasier, Allan R.

    2016-01-01

    The airway mucosa is responsible for mounting a robust innate immune response (IIR) upon encountering pathogen-associated molecular patterns. The IIR produces protective gene networks that stimulate neighboring epithelia and components of the immune system to trigger adaptive immunity. Little is currently known about how cellular reactive oxygen species (ROS) signaling is produced and cooperates in the IIR. We discuss recent discoveries on two nuclear ROS signaling pathways controlling innate immunity. Nuclear ROS oxidize guanine bases to produce mutagenic 8-oxoguanine, a lesion excised by 8-oxoguanine DNA glycosylase1/AP-lyase (OGG1). OGG1 forms a complex with the excised base, inducing its nuclear export. The cytoplasmic OGG1•8-oxoG complex functions as a guanine nucleotide exchange factor, triggering small GTPase signaling and activating phosphorylation of the NFκB/RelA transcription factor to induce immediate early gene expression. In parallel, nuclear ROS are detected by ataxia telangiectasia mutated (ATM), a PI3 kinase activated by ROS, triggering its nuclear export. ATM forms a scaffold with ribosomal S6 kinases, inducing RelA phosphorylation and resulting in transcription-coupled synthesis of type -I and –III interferons and CC and CXC chemokines. We propose that ATM and OGG1 are endogenous nuclear ROS sensors that transmit nuclear signals that coordinate with outside-in PRR signaling, regulating the IIR. PMID:26756522

  7. Selective inhibitors of nuclear export (SINE) in hematological malignancies.

    PubMed

    Das, Arundhati; Wei, Guoqing; Parikh, Kaushal; Liu, Delong

    2015-01-01

    Regulated nucleo-cytoplasmic transport plays a major role in maintaining cellular homeostasis. CRM1 (chromosome region maintenance 1 or exportin 1 or XPO 1) is responsible for the nucleo-cytoplasmic transport of more than 200 proteins, including most of the tumor suppressor proteins (TSP). CRM1 is overexpressed in pancreatic cancer, osteosarcoma, glioma, cervical and hematological malignancies. This inspired the development of novel agents that selectively inhibit nuclear exportins (SINEs). In this review we focus on the significance of CRM1 in carcinogenesis and review the new development of SINE inhibitiors in hematological malignancies. Selinexor (KPT-330) as the first-in-human SINE agent represents this novel class of anti-cancer agents.

  8. Analysis of nuclear transport signals in the human apurinic/apyrimidinic endonuclease (APE1/Ref1)

    PubMed Central

    Jackson, Elias B.; Theriot, Corey A.; Chattopadhyay, Ranajoy; Mitra, Sankar; Izumi, Tadahide

    2005-01-01

    The mammalian abasic-endonuclease1/redox-factor1 (APE1/Ref1) is an essential protein whose subcellular distribution depends on the cellular physiological status. However, its nuclear localization signals have not been studied in detail. We examined nuclear translocation of APE1, by monitoring enhanced green fluorescent protein (EGFP) fused to APE1. APE1's nuclear localization was significantly decreased by deleting 20 amino acid residues from its N-terminus. Fusion of APE1's N-terminal 20 residues directed nuclear localization of EGFP. An APE1 mutant lacking the seven N-terminal residues (ND7 APE1) showed nearly normal nuclear localization, which was drastically reduced when the deletion was combined with the E12A/D13A double mutation. On the other hand, nearly normal nuclear localization of the full-length E12A/D13A mutant suggests that the first 7 residues and residues 8–13 can independently promote nuclear import. Both far-western analyses and immuno-pull-down assays indicate interaction of APE1 with karyopherin alpha 1 and 2, which requires the 20 N-terminal residues and implicates nuclear importins in APE1's nuclear translocation. Nuclear accumulation of the ND7 APE1(E12A/D13A) mutant after treatment with the nuclear export inhibitor leptomycin B suggests the presence of a previously unidentified nuclear export signal, and the subcellular distribution of APE1 may be regulated by both nuclear import and export. PMID:15942031

  9. The Selenocysteine-Specific Elongation Factor Contains Unique Sequences That Are Required for Both Nuclear Export and Selenocysteine Incorporation

    PubMed Central

    Dubey, Aditi

    2016-01-01

    Selenocysteine (Sec) is a critical residue in at least 25 human proteins that are essential for antioxidant defense and redox signaling in cells. Sec is inserted into proteins cotranslationally by the recoding of an in-frame UGA termination codon to a Sec codon. In eukaryotes, this recoding event requires several specialized factors, including a dedicated, Sec-specific elongation factor called eEFSec, which binds Sec-tRNASec with high specificity and delivers it to the ribosome for selenoprotein production. Unlike most translation factors, including the canonical elongation factor eEF1A, eEFSec readily localizes to the nucleus of mammalian cells and shuttles between the cytoplasmic and nuclear compartments. The functional significance of eEFSec’s nuclear localization has remained unclear. In this study, we have examined the subcellular localization of eEFSec in the context of altered Sec incorporation to demonstrate that reduced selenoprotein production does not correlate with changes in the nuclear localization of eEFSec. In addition, we identify several novel sequences of the protein that are essential for localization as well as Sec insertion activity, and show that eEFSec utilizes CRM1-mediated nuclear export pathway. Our findings argue for two distinct pools of eEFSec in the cell, where the cytoplasmic pool participates in Sec incorporation and the nuclear pool may be involved in an as yet unknown function. PMID:27802322

  10. 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).

  11. A putative N-terminal nuclear export sequence is sufficient for Mps1 nuclear exclusion during interphase.

    PubMed

    Jia, Haiwei; Zhang, Xiaojuan; Wang, Wenjun; Bai, Yuanyuan; Ling, Youguo; Cao, Cheng; Ma, Runlin Z; Zhong, Hui; Wang, Xue; Xu, Quanbin

    2015-02-27

    Mps1, an essential component of the mitotic checkpoint, is also an important interphase regulator and has roles in DNA damage response, cytokinesis and centrosome duplication. Mps1 predominantly resides in the cytoplasm and relocates into the nucleus at the late G2 phase. So far, the mechanism underlying the Mps1 translocation between the cytoplasm and nucleus has been unclear. In this work, a dynamic export process of Mps1 from the nucleus to cytoplasm in interphase was revealed- a process blocked by the Crm1 inhibitor, Leptomycin B, suggesting that export of Mps1 is Crm1 dependent. Consistent with this speculation, a direct association between Mps1 and Crm1 was found. Furthermore, a putative nuclear export sequence (pNES) motif at the N-terminal of Mps1 was identified by analyzing the motif of Mps1. This motif shows a high sequence similarity to the classic NES, a fusion of this motif with EGFP results in dramatic exclusion of the fusion protein from the nucleus. Additionally, Mps1 mutant loss of pNES integrity was shown by replacing leucine with alanine which produced a diffused subcellular distribution, compared to the wild type protein which resides predominantly in cytoplasm. Taken these findings together, it was concluded that the pNES sequence is sufficient for the Mps1 export from nucleus during interphase.

  12. Nuclear Successor States of the Soviet Union, Nuclear Weapon and Sensitive Export Status Report

    DTIC Science & Technology

    2016-03-24

    on Territory X X X X Nuclear Power Reactors X X X Nuclear Research Reactors X X X X Nuclear Weapons Design X Uranium Enrichment X Spent Fuel... nuclear power reactors . American inspectors will be allowed into Sverdlovsk-44 to verify that the HEU actually comes from dismantled warheads (see Nuclear ...fuel rod fabrication (Electrostal Machine Building Plant), Research reactors : Kurchatov Institute (5 kg HEU and 4 kg LEU) and Institute of Nuclear

  13. The Nuclear Export Receptor Xpo1p Forms Distinct Complexes with NES Transport Substrates and the Yeast Ran Binding Protein 1 (Yrb1p)

    PubMed Central

    Maurer, Patrick; Redd, Michael; Solsbacher, Jens; Bischoff, F. Ralf; Greiner, Markus; Podtelejnikov, Alexandre V.; Mann, Matthias; Stade, Katrin; Weis, Karsten; Schlenstedt, Gabriel

    2001-01-01

    Xpo1p (Crm1p) is the nuclear export receptor for proteins containing a leucine-rich nuclear export signal (NES). Xpo1p, the NES-containing protein, and GTP-bound Ran form a complex in the nucleus that translocates across the nuclear pore. We have identified Yrb1p as the major Xpo1p-binding protein in Saccharomyces cerevisiae extracts in the presence of GTP-bound Gsp1p (yeast Ran). Yrb1p is cytoplasmic at steady-state but shuttles continuously between the cytoplasm and the nucleus. Nuclear import of Yrb1p is mediated by two separate nuclear targeting signals. Export from the nucleus requires Xpo1p, but Yrb1p does not contain a leucine-rich NES. Instead, the interaction of Yrb1p with Xpo1p is mediated by Gsp1p-GTP. This novel type of export complex requires the acidic C-terminus of Gsp1p, which is dispensable for the binding to importin β-like transport receptors. A similar complex with Xpo1p and Gsp1p-GTP can be formed by Yrb2p, a relative of Yrb1p predominantly located in the nucleus. Yrb1p also functions as a disassembly factor for NES/Xpo1p/Gsp1p-GTP complexes by displacing the NES protein from Xpo1p/Gsp1p. This Yrb1p/Xpo1p/Gsp1p complex is then completely dissociated after GTP hydrolysis catalyzed by the cytoplasmic GTPase activating protein Rna1p. PMID:11251069

  14. Transflammation: Innate Immune Signaling in Nuclear Reprogramming.

    PubMed

    Meng, Shu; Chanda, Palas; Thandavarayan, Rajarajan A; Cooke, John P

    2017-09-12

    Induction of pluripotency in somatic cells by retroviral overexpression of four transcription factors has revolutionized the field of stem cell biology and regenerative medicine. The efficient induction of pluripotency requires the activation of innate immune signaling in a process termed "transflammation" [1]. Specifically, the stimulation of pattern recognition receptors (PRRs) causes global alterations in the expression and activity of epigenetic modifiers to favor an open chromatin configuration. Activation of toll-like receptors (TLR) or RIG-1-like receptors (RLR) [2] trigger signaling cascades that result in NFκB or IRF-3 mediated changes in epigenetic plasticity that facilitate reprogramming. Another form of nuclear reprogramming is so-called direct reprogramming or transdifferentiation of one somatic cell to another lineage. We have shown that transdifferentiation of human fibroblasts to endothelial cells also involves transflammation [3]. Recently, we also identified reactive oxygen species (ROS) [4] and reactive nitrogen species (RNS) [5] as mediators of innate immune signaling in nuclear reprogramming. Innate immune signaling plays a key role in nuclear reprogramming by regulating DNA accessibility (Figure 1). Here, we review recent progress of innate immunity signaling in nuclear reprogramming and epigenetic plasticity. Copyright © 2017. Published by Elsevier B.V.

  15. 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.

  16. 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

  17. 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

  18. Selective inhibitors of nuclear export (SINE) as novel therapeutics for prostate cancer

    PubMed Central

    Kim, Hae-Soo; Hammers, Hans; Meeker, Alan; De Marzo, Angelo; Carducci, Michael; Kauffman, Michael; Shacham, Sharon; Kachhap, Sushant

    2014-01-01

    Mislocalization of proteins is a common feature of cancer cells. Since localization of proteins is tightly linked to its function, cancer cells can inactivate function of a tumor suppressor protein through mislocalization. The nuclear exportin CRM1/XPO 1 is upregulated in many cancers. Targeting XPO 1 can lead to nuclear retention of cargo proteins such as p53, Foxo, and BRCA1 leading to cell cycle arrest and apoptosis. We demonstrate that selective inhibitors of nuclear export (SINE) can functionally inactivate XPO 1 in prostate cancer cells. Unlike the potent, but toxic, XPO 1 inhibitor leptomycin B, SINE inhibitors (KPT-185, KPT-330, and KPT-251) cause a decrease in XPO 1 protein level through the proteasomal pathway. Treatment of prostate cancer cells with SINE inhibitors lead to XPO 1 inhibition, as evaluated by RevGFP export assay, leading to nuclear retention of p53 and Foxo proteins, consequently, triggering apoptosis. Our data reveal that treatment with SINE inhibitors at nanomolar concentrations results in decrease in proliferation and colonogenic capacity of prostate cancer cells by triggering apoptosis without causing any cell cycle arrest. We further demonstrate that SINE inhibitors can be combined with other chemotherapeutics like doxorubicin to achieve enhanced growth inhibition of prostate cancer cells. Since SINE inhibitors offer increased bioavailability, reduced toxicity to normal cells, and are orally available they can serve as effective therapeutics against prostate cancer. In conclusion, our data reveals that nucleocytoplasmic transport in prostate cancer can be effectively targeted by SINE inhibitors. PMID:25026284

  19. Selective inhibitors of nuclear export (SINE) as novel therapeutics for prostate cancer.

    PubMed

    Mendonca, Janet; Sharma, Anup; Kim, Hae-Soo; Hammers, Hans; Meeker, Alan; De Marzo, Angelo; Carducci, Michael; Kauffman, Michael; Shacham, Sharon; Kachhap, Sushant

    2014-08-15

    Mislocalization of proteins is a common feature of cancer cells. Since localization of proteins is tightly linked to its function, cancer cells can inactivate function of a tumor suppressor protein through mislocalization. The nuclear exportin CRM1/XPO 1 is upregulated in many cancers. Targeting XPO 1 can lead to nuclear retention of cargo proteins such as p53, Foxo, and BRCA1 leading to cell cycle arrest and apoptosis. We demonstrate that selective inhibitors of nuclear export (SINE) can functionally inactivate XPO 1 in prostate cancer cells. Unlike the potent, but toxic, XPO 1 inhibitor leptomycin B, SINE inhibitors (KPT-185, KPT-330, and KPT-251) cause a decrease in XPO 1 protein level through the proteasomal pathway. Treatment of prostate cancer cells with SINE inhibitors lead to XPO 1 inhibition, as evaluated by RevGFP export assay, leading to nuclear retention of p53 and Foxo proteins, consequently, triggering apoptosis. Our data reveal that treatment with SINE inhibitors at nanomolar concentrations results in decrease in proliferation and colonogenic capacity of prostate cancer cells by triggering apoptosis without causing any cell cycle arrest. We further demonstrate that SINE inhibitors can be combined with other chemotherapeutics like doxorubicin to achieve enhanced growth inhibition of prostate cancer cells. Since SINE inhibitors offer increased bioavailability, reduced toxicity to normal cells, and are orally available they can serve as effective therapeutics against prostate cancer. In conclusion, our data reveals that nucleocytoplasmic transport in prostate cancer can be effectively targeted by SINE inhibitors.

  20. Novel selective inhibitors of nuclear export CRM1 antagonists for therapy in mantle cell lymphoma.

    PubMed

    Zhang, Kejie; Wang, Michael; Tamayo, Archito T; Shacham, Sharon; Kauffman, Michael; Lee, John; Zhang, Liang; Ou, Zhishuo; Li, Changping; Sun, Luhong; Ford, Richard J; Pham, Lan V

    2013-01-01

    Overexpression of the cellular nuclear exportin 1, more commonly called chromosomal region maintenance 1 (CRM1), has been associated with malignant progression and mortality. Therefore, activation of nuclear export can play a significant etiologic role in some forms of human neoplasia and serve as a novel target for the treatment of these cancers. Mantle cell lymphoma (MCL) is an aggressive histotype of B-cell non-Hodgkin lymphoma that remains incurable. The objective of this study was to investigate the functional significance of CRM1 in MCL by evaluating the therapeutic efficacy of CRM1 inhibition in MCL in vitro and in vivo. Our results showed that CRM1 is highly expressed in MCL cells and is involved in regulating growth and survival mechanisms through the critical nuclear factor-κB survival pathway, which is independent of p53 status. Inhibition of CRM1 by two novel selective inhibitors of nuclear export (SINE), KPT-185 and KPT-276, in MCL cells resulted in significant growth inhibition and apoptosis induction. KPT-185 also induced CRM1 accumulation in the nucleus, resulting in CRM1 degradation by the proteasome. Oral administration of KPT-276 significantly suppressed tumor growth in an MCL-bearing severe combined immunodeficient mouse model, without severe toxicity. Our data suggest that SINE CRM1 antagonists are a potential novel therapy for patients with MCL, particular in relapsed/refractory disease.

  1. Trypanosoma brucei RNA binding proteins p34 and p37 mediate NOPP44/46 cellular localization via the exportin 1 nuclear export pathway.

    PubMed

    Hellman, Kristina; Prohaska, Kimberly; Williams, Noreen

    2007-12-01

    We have previously identified and characterized two novel nuclear RNA binding proteins, p34 and p37, which have been shown to interact with a family of nucleolar phosphoproteins, NOPP44/46, in Trypanosoma brucei. These proteins are nearly identical, the major difference being an 18-amino-acid insert in the N terminus of p37. In order to characterize the interaction between p34 and p37 and NOPP44/46, we have utilized an RNA interference (RNAi) cell line that specifically targets p34 and p37. Within these RNAi cells, we detected a disruption of a higher-molecular-weight complex containing NOPP44/46, as well as a dramatic increase in nuclear NOPP44/46 protein levels. We demonstrated that no change occurred in NOPP44/46 mRNA steady-state levels or stability, nor was there a change in cellular protein levels. These results led us to investigate whether p34 and p37 regulate NOPP44/46 cellular localization. Examination of the p34 and p37 amino acid sequences revealed a leucine-rich nuclear export signal, which interacts with the nuclear export factor exportin 1. Immune capture experiments demonstrated that p34, p37, and NOPP44/46 associate with exportin 1. When these experiments were performed with p34/p37 RNAi cells, NOPP44/46 no longer associated with exportin 1. Sequential immune capture experiments demonstrated that p34, p37, NOPP44/46, and exportin 1 exist in a common complex. Inhibiting exportin 1-mediated nuclear export led to an increase in nuclear NOPP44/46 proteins, indicating that they are exported from the nucleus via this pathway. Together, our results demonstrate that p34 and p37 regulate NOPP44/46 cellular localization by facilitating their association with exportin 1.

  2. Identification and characterization of the mouse nuclear export factor (Nxf) family members

    PubMed Central

    Tan, Wei; Zolotukhin, Andrei S.; Tretyakova, Irina; Bear, Jenifer; Lindtner, Susan; Smulevitch, Sergey V.; Felber, Barbara K.

    2005-01-01

    TAP/hNXF1 is a key factor that mediates general cellular mRNA export from the nucleus, and its orthologs are structurally and functionally conserved from yeast to humans. Metazoans encode additional proteins that share homology and domain organization with TAP/hNXF1, suggesting their participation in mRNA metabolism; however, the precise role(s) of these proteins is not well understood. Here, we found that the human mRNA export factor hNXF2 is specifically expressed in the brain, suggesting a brain-specific role in mRNA metabolism. To address the roles of additional NXF factors, we have identified and characterized the two Nxf genes, Nxf2 and Nxf7, which together with the TAP/hNXF1's ortholog Nxf1 comprise the murine Nxf family. Both mNXF2 and mNXF7 have a domain structure typical of the NXF family. We found that mNXF2 protein is expressed during mouse brain development. Similar to TAP/hNXF1, the mNXF2 protein is found in the nucleus, the nuclear envelope and cytoplasm, and is an active mRNA export receptor. In contrast, mNXF7 localizes exclusively to cytoplasmic granules and, despite its overall conserved sequence, lacks mRNA export activity. We concluded that mNXF2 is an active mRNA export receptor similar to the prototype TAP/hNXF1, whereas mNXF7 may have a more specialized role in the cytoplasm. PMID:16027110

  3. Prediction of nuclear proteins using nuclear translocation signals proposed by probabilistic latent semantic indexing

    PubMed Central

    2012-01-01

    Background Identification of subcellular localization in proteins is crucial to elucidate cellular processes and molecular functions in a cell. However, given a tremendous amount of sequence data generated in the post-genomic era, determining protein localization based on biological experiments can be expensive and time-consuming. Therefore, developing prediction systems to analyze uncharacterised proteins efficiently has played an important role in high-throughput protein analyses. In a eukaryotic cell, many essential biological processes take place in the nucleus. Nuclear proteins shuttle between nucleus and cytoplasm based on recognition of nuclear translocation signals, including nuclear localization signals (NLSs) and nuclear export signals (NESs). Currently, only a few approaches have been developed specifically to predict nuclear localization using sequence features, such as putative NLSs. However, it has been shown that prediction coverage based on the NLSs is very low. In addition, most existing approaches only attained prediction accuracy and Matthew's correlation coefficient (MCC) around 54%~70% and 0.250~0.380 on independent test set, respectively. Moreover, no predictor can generate sequence motifs to characterize features of potential NESs, in which biological properties are not well understood from existing experimental studies. Results In this study, first we propose PSLNuc (Protein Subcellular Localization prediction for Nucleus) for predicting nuclear localization in proteins. First, for feature representation, a protein is represented by gapped-dipeptides and the feature values are weighted by homology information from a smoothed position-specific scoring matrix. After that, we incorporate probabilistic latent semantic indexing (PLSI) for feature reduction. Finally, the reduced features are used as input for a support vector machine (SVM) classifier. In addition to PSLNuc, we further identify gapped-dipeptide signatures for putative NLSs and NESs

  4. Nutrient export from catchments on forested landscapes reveals complex nonstationary and stationary climate signals

    NASA Astrophysics Data System (ADS)

    Mengistu, Samson G.; Quick, Christopher G.; Creed, Irena F.

    2013-06-01

    Headwater catchment hydrology and biogeochemistry are influenced by climate, including linear trends (nonstationary signals) and climate oscillations (stationary signals). We used an analytical framework to detect nonstationary and stationary signals in yearly time series of nutrient export [dissolved organic carbon (DOC), dissolved organic nitrogen (DON), nitrate (NO3--N), and total dissolved phosphorus (TDP)] in forested headwater catchments with differential water loading and water storage potential at the Turkey Lakes Watershed in Ontario, Canada. We tested the hypotheses that (1) climate has nonstationary and stationary effects on nutrient export, the combination of which explains most of the variation in nutrient export; (2) more metabolically active nutrients (e.g., DON, NO3--N, and TDP) are more sensitive to these signals; and (3) catchments with relatively low water loading and water storage capacity are more sensitive to these signals. Both nonstationary and stationary signals were identified, and the combination of both explained the majority of the variation in nutrient export data. More variation was explained in more labile nutrients (DON, NO3--N, and TDP), which were also more sensitive to climate signals. The catchment with low-water storage potential and low water loading was most sensitive to nonstationary and stationary climatic oscillations, suggesting that these hydrologic features are characteristic of the most effective sentinels of climate change. The observed complex links between climate change, climatic oscillations, and water nutrient fluxes in headwater catchments suggest that climate may have considerable influence on the productivity and biodiversity of surface waters, in addition to other drivers such as atmospheric pollution.

  5. Inhibition of the Nuclear Export Receptor XPO1 as a Therapeutic Target for Platinum-Resistant Ovarian Cancer.

    PubMed

    Chen, Ying; Camacho, Sandra Catalina; Silvers, Thomas R; Razak, Albiruni R A; Gabrail, Nashat Y; Gerecitano, John F; Kalir, Eva; Pereira, Elena; Evans, Brad R; Ramus, Susan J; Huang, Fei; Priedigkeit, Nolan; Rodriguez, Estefania; Donovan, Michael; Khan, Faisal; Kalir, Tamara; Sebra, Robert; Uzilov, Andrew; Chen, Rong; Sinha, Rileen; Halpert, Richard; Billaud, Jean-Noel; Shacham, Sharon; McCauley, Dilara; Landesman, Yosef; Rashal, Tami; Kauffman, Michael; Mirza, Mansoor R; Mau-Sørensen, Morten; Dottino, Peter; Martignetti, John A

    2017-03-15

    Purpose: The high fatality-to-case ratio of ovarian cancer is directly related to platinum resistance. Exportin-1 (XPO1) is a nuclear exporter that mediates nuclear export of multiple tumor suppressors. We investigated possible clinicopathologic correlations of XPO1 expression levels and evaluated the efficacy of XPO1 inhibition as a therapeutic strategy in platinum-sensitive and -resistant ovarian cancer.Experimental Design: XPO1 expression levels were analyzed to define clinicopathologic correlates using both TCGA/GEO datasets and tissue microarrays (TMA). The effect of XPO1 inhibition, using the small-molecule inhibitors KPT-185 and KPT-330 (selinexor) alone or in combination with a platinum agent on cell viability, apoptosis, and the transcriptome was tested in immortalized and patient-derived ovarian cancer cell lines (PDCL) and platinum-resistant mice (PDX). Seven patients with late-stage, recurrent, and heavily pretreated ovarian cancer were treated with an oral XPO1 inhibitor.Results: XPO1 RNA overexpression and protein nuclear localization were correlated with decreased survival and platinum resistance in ovarian cancer. Targeted XPO1 inhibition decreased cell viability and synergistically restored platinum sensitivity in both immortalized ovarian cancer cells and PDCL. The XPO1 inhibitor-mediated apoptosis occurred through both p53-dependent and p53-independent signaling pathways. Selinexor treatment, alone and in combination with platinum, markedly decreased tumor growth and prolonged survival in platinum-resistant PDX and mice. In selinexor-treated patients, tumor growth was halted in 3 of 5 patients, including one with a partial response, and was safely tolerated by all.Conclusions: Taken together, these results provide evidence that XPO1 inhibition represents a new therapeutic strategy for overcoming platinum resistance in women with ovarian cancer. Clin Cancer Res; 23(6); 1552-63. ©2016 AACR. ©2016 American Association for Cancer Research.

  6. Src subfamily kinases regulate nuclear export and degradation of transcription factor Nrf2 to switch off Nrf2-mediated antioxidant activation of cytoprotective gene expression.

    PubMed

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

    2011-08-19

    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.

  7. 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

  8. Functional and structural characterization of the mammalian TREX-2 complex that links transcription with nuclear messenger RNA export

    PubMed Central

    Jani, Divyang; Lutz, Sheila; Hurt, Ed; Laskey, Ronald A.; Stewart, Murray; Wickramasinghe, Vihandha O.

    2012-01-01

    Export of messenger RNA (mRNA) from the nucleus to the cytoplasm is a critical step in the gene expression pathway of eukaryotic cells. Here, we report the functional and structural characterization of the mammalian TREX-2 complex and show how it links transcription/processing with nuclear mRNA export. Mammalian TREX-2 is based on a germinal-centre associated nuclear protein (GANP) scaffold to which ENY2, PCID2 and centrins bind and depletion of any of these components inhibits mRNA export. The crystal structure of the GANP:ENY2 complex shows that two ENY2 chains interact directly with GANP, but they have different orientations from those observed on yeast Sac3. GANP is required to recruit ENY2 to nuclear pore complexes (NPCs), but ENY2 is not necessary to recruit GANP, which requires both its CID and MCM3AP domains, together with nucleoporin Nup153. GANP and ENY2 associate with RNA polymerase II and inhibition of mRNA processing redistributes GANP from NPCs into nuclear foci indicating that mammalian TREX-2 is associated with transcription. Thus, we implicate TREX-2 as an integral component of the mammalian mRNA export machinery where it links transcription and nuclear export by facilitating the transfer of mature mRNPs from the nuclear interior to NPCs. PMID:22307388

  9. Selective inhibitors of nuclear export avert progression in preclinical models of inflammatory demyelination

    PubMed Central

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

    2015-01-01

    Axonal damage has been associated with aberrant protein trafficking. This study characterizes a novel class of compounds targeting nucleo-cytoplasmic shuttling, by binding to the catalytic groove of the nuclear export protein XPO1/CRM1 (chromosome region maintenance protein1). Oral administration of novel 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 observed also in other mouse models of axonal damage (i.e. kainic acid injection) and detected in cultured neurons after knockdown of Xpo1, the gene encoding for 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

  10. Formation of Tap/NXT1 Heterodimers Activates Tap-Dependent Nuclear mRNA Export by Enhancing Recruitment to Nuclear Pore Complexes

    PubMed Central

    Wiegand, Heather L.; Coburn, Glen A.; Zeng, Yan; Kang, Yibin; Bogerd, Hal P.; Cullen, Bryan R.

    2002-01-01

    The Tap protein has been shown to activate the nuclear export of mRNA species bearing retroviral constitutive transport elements and is also believed to play an essential role in the sequence nonspecific export of cellular mRNAs. However, it has remained unclear how Tap activity is regulated in vivo. Here, we report that the small NXT1/p15-1 protein functions as a critical cofactor for Tap-mediated mRNA export in both human and invertebrate cells. In the absence of NXT1 binding, the Tap protein is unable to effectively interact with components of the nuclear pore complex and both Tap nucleocytoplasmic shuttling and the nuclear export of mRNA molecules tethered to Tap are therefore severely attenuated. Formation of a Tap/NXT1 heterodimer enhances nucleoporin binding both in vitro and in vivo and induces the formation of a Tap/NXT1/nucleoporin ternary complex that is likely to be a key intermediate in the process of nuclear mRNA export. The critical importance of NXT1 for the nuclear export of poly(A)+ RNA is emphasized by the finding that specific inhibition of the expression of the Drosophila homolog of human NXT1, by using RNA interference, results in the nuclear accumulation of poly(A)+ RNA in cultured insect cells. These data suggest that NXT1 may act as a molecular switch that regulates the ability of Tap to mediate nuclear mRNA export by controlling the interaction of Tap with components of the nuclear pore. PMID:11739738

  11. 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

  12. The function of the inner nuclear envelope protein SUN1 in mRNA export is regulated by phosphorylation.

    PubMed

    Li, Ping; Stumpf, Maria; Müller, Rolf; Eichinger, Ludwig; Glöckner, Gernot; Noegel, Angelika A

    2017-08-22

    SUN1, a component of the LINC (Linker of Nucleoskeleton and Cytoskeleton) complex, functions in mammalian mRNA export through the NXF1-dependent pathway. It associates with mRNP complexes by direct interaction with NXF1. It also binds to the NPC through association with the nuclear pore component Nup153, which is involved in mRNA export. The SUN1-NXF1 association is at least partly regulated by a protein kinase C (PKC) which phosphorylates serine 113 (S113) in the N-terminal domain leading to reduced interaction. The phosphorylation appears to be important for the SUN1 function in nuclear mRNA export since GFP-SUN1 carrying a S113A mutation was less efficient in restoring mRNA export after SUN1 knockdown as compared to the wild type protein. By contrast, GFP-SUN1-S113D resembling the phosphorylated state allowed very efficient export of poly(A)+RNA. Furthermore, probing a possible role of the LINC complex component Nesprin-2 in this process we observed impaired mRNA export in Nesprin-2 knockdown cells. This effect might be independent of SUN1 as expression of a GFP tagged SUN-domain deficient SUN1, which no longer can interact with Nesprin-2, did not affect mRNA export.

  13. Postage for the messenger: Designating routes for Nuclear mRNA Export

    PubMed Central

    Natalizio, Barbara J.; Wente, Susan R.

    2013-01-01

    Transcription of messenger(m) RNA occurs in the nucleus, making the translocation of mRNA across the nuclear envelope (NE) boundary a critical determinant of proper gene expression and cell survival. A major mRNA export route occurs via the NXF1-dependent pathway through the nuclear pore complexes (NPCs) embedded in the NE. However, recent findings have discovered new evidence supporting the existence of multiple mechanisms for crossing the NE, including both NPC-mediated and NE budding-mediated pathways. An analysis of the trans-acting factors and cis components that define these pathways reveals shared elements as well as mechanistic differences. We review here the current understanding of the mechanisms that characterize each pathway and highlight the determinants that influence mRNA transport fate. PMID:23583578

  14. 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.

  15. Serine residue 115 of MAPK-activated protein kinase MK5 is crucial for its PKA-regulated nuclear export and biological function.

    PubMed

    Kostenko, Sergiy; Shiryaev, Alexey; Gerits, Nancy; Dumitriu, Gianina; Klenow, Helle; Johannessen, Mona; Moens, Ugo

    2011-03-01

    The mitogen-activated protein kinase-activated protein kinase-5 (MK5) resides predominantly in the nucleus of resting cells, but p38(MAPK), extracellular signal-regulated kinases-3 and -4 (ERK3 and ERK4), and protein kinase A (PKA) induce nucleocytoplasmic redistribution of MK5. The mechanism by which PKA causes nuclear export remains unsolved. In the study reported here we demonstrated that Ser-115 is an in vitro PKA phosphoacceptor site, and that PKA, but not p38(MAPK), ERK3 or ERK4, is unable to redistribute MK5 S115A to the cytoplasm. However, the phospho-mimicking MK5 S115D mutant resides in the cytoplasm in untreated cells. While p38(MAPK), ERK3 and ERK4 fail to trigger nuclear export of the kinase dead T182A and K51E MK5 mutants, S115D/T182A and K51E/S115D mutants were able to enter the cytoplasm of resting cells. Finally, we demonstrated that mutations in Ser-115 affect the biological properties of MK5. Taken together, our results suggest that Ser-115 plays an essential role in PKA-regulated nuclear export of MK5, and that it also may regulate the biological functions of MK5.

  16. 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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. 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.

  18. YTHDC1 Mediates Nuclear Export of N(6)-methyladenosine Methylated mRNAs.

    PubMed

    Roundtree, Ian A; Luo, Guan-Zheng; Zhang, Zijie; Wang, Xiao; Zhou, Tao; Cui, Yiquang; Sha, Jiahao; Huang, Xingxu; Guerrero, Laura; Xie, Phil; He, Emily; Shen, Bin; He, Chuan

    2017-10-06

    N(6)-methyladenosine (m(6)A) is the most abundant internal modification in eukaryotic messenger RNA (mRNA), and plays critical roles in RNA biology. The function of this modification is mediated by m(6)A-selective 'reader' proteins of the YTH family, which incorporate m(6)A-modified mRNAs into pathways of RNA metabolism. Here, we show that the m(6)A-binding protein YTHDC1 mediates export of methylated mRNA from the nucleus to the cytoplasm in HeLa cells. Knockdown of YTHDC1 results in an extended residence time for nuclear m(6)A-containing mRNA, with an accumulation of transcripts in the nucleus and accompanying depletion within the cytoplasm. YTHDC1 interacts with the splicing factor and nuclear export adaptor protein SRSF3, and facilitates RNA binding to both SRSF3 and NXF1. This role for YTHDC1 expands the potential utility of chemical modification of mRNA, and supports an emerging paradigm of m(6)A as a distinct biochemical entity for selective processing and metabolism of mammalian mRNAs.

  19. Cyclophilin A Peptidyl-Prolyl Isomerase Activity Promotes Zpr1 Nuclear Export

    PubMed Central

    Ansari, Husam; Greco, Giampaolo; Luban, Jeremy

    2002-01-01

    The peptidyl-prolyl isomerase (PPIase) cyclophilin A (Cpr1p) is conserved from eubacteria to mammals, yet its biological function has resisted elucidation. Unable to identify a phenotype that is suggestive of Cpr1p's function in a cpr1Δ Saccharomyces cerevisiae strain, we screened for CPR1-dependent strains. In all cases, dependence was conferred by mutations in ZPR1, a gene encoding an essential zinc finger protein. CPR1 dependence was suppressed by overexpression of EF1α (a translation factor that binds Zpr1p), Cpr6p (another cyclophilin), or Fpr1p (a structurally unrelated PPIase). Suppression by a panel of cyclophilin A mutants correlated with PPIase activity, confirming the relevance of this activity in CPR1-dependent strains. In CPR1+ cells, wild-type Zpr1p was distributed equally between the nucleus and cytoplasm. In contrast, proteins encoded by CPR1-dependent alleles of ZPR1 accumulated in the nucleus, as did wild-type Zpr1p in cpr1Δ cells. Transport kinetic studies indicated that nuclear export of Zpr1p was defective in cpr1Δ cells, and rescue of this defect correlated with PPIase activity. Our results demonstrate a functional interaction between Cpr1p, Zpr1p, and EF1α, a role for Cpr1p in Zpr1p nuclear export, and a biological function for Cpr1p PPIase activity. PMID:12242280

  20. Molecular components of the signal sequence that function in the initiation of protein export

    PubMed Central

    1982-01-01

    We are studying the mechanism by which the LamB protein is exported to the outer membrane of Escherichia coli. Using two selection procedures based on gene fusions, we have identified a number of mutations that cause alterations in the LamB signal sequence. Characterization of the mutant strains revealed that although many such mutations block LamB export to greater than 95%, others have essentially no effect. These results allow an analysis of the functions performed by the various molecular components of the signal sequence. Our results suggest that a critical subset of four amino acids is contained within the central hydrophobic core of the LamB signal sequence. If this core can assume an alpha-helical conformation, these four amino acids comprise a recognition site that interacts with a component of the cellular export machinery. Since mechanisms of protein localization appear to have been conserved during evolution, the principles established by these results should be applicable to similar studies in eukaryotic cells. PMID:6759512

  1. Ribosome-stalk biogenesis is coupled with recruitment of nuclear-export factor to the nascent 60S subunit.

    PubMed

    Sarkar, Anshuk; Pech, Markus; Thoms, Matthias; Beckmann, Roland; Hurt, Ed

    2016-12-01

    Nuclear export of preribosomal subunits is a key step during eukaryotic ribosome formation. To efficiently pass through the FG-repeat meshwork of the nuclear pore complex, the large pre-60S subunit requires several export factors. Here we describe the mechanism of recruitment of the Saccharomyces cerevisiae RNA-export receptor Mex67-Mtr2 to the pre-60S subunit at the proper time. Mex67-Mtr2 binds at the premature ribosomal-stalk region, which later during translation serves as a binding platform for translational GTPases on the mature ribosome. The assembly factor Mrt4, a structural homolog of cytoplasmic-stalk protein P0, masks this site, thus preventing untimely recruitment of Mex67-Mtr2 to nuclear pre-60S particles. Subsequently, Yvh1 triggers Mrt4 release in the nucleus, thereby creating a narrow time window for Mex67-Mtr2 association at this site and facilitating nuclear export of the large subunit. Thus, a spatiotemporal mark on the ribosomal stalk controls the recruitment of an RNA-export receptor to the nascent 60S subunit.

  2. Functional Characterization of Nuclear Trafficking Signals in Pseudorabies Virus pUL31

    PubMed Central

    Paßvogel, Lars; Klupp, Barbara G.; Granzow, Harald; Fuchs, Walter

    2014-01-01

    ABSTRACT The herpesviral nuclear egress complex (NEC), consisting of pUL31 and pUL34 homologs, mediates efficient translocation of newly synthesized capsids from the nucleus to the cytosol. The tail-anchored membrane protein pUL34 is autonomously targeted to the nuclear envelope, while pUL31 is recruited to the inner nuclear membrane (INM) by interaction with pUL34. A nuclear localization signal (NLS) in several pUL31 homologs suggests importin-mediated translocation of the protein. Here we demonstrate that deletion or mutation of the NLS in pseudorabies virus (PrV) pUL31 resulted in exclusively cytosolic localization, indicating active nuclear export. Deletion or mutation of a predicted nuclear export signal (NES) in mutant constructs lacking a functional NLS resulted in diffuse nuclear and cytosolic localization, indicating that both signals are functional. pUL31 molecules lacking the complete NLS or NES were not recruited to the INM by pUL34, while site-specifically mutated proteins formed the NEC and partially complemented the defect of the UL31 deletion mutant. Our data demonstrate that the N terminus of pUL31, encompassing the NLS, is required for efficient nuclear targeting but not for pUL34 interaction, while the C terminus, containing the NES but not necessarily the NES itself, is required for complex formation and efficient budding of viral capsids at the INM. Moreover, pUL31-ΔNLS displayed a dominant negative effect on wild-type PrV replication, probably by diverting pUL34 to cytoplasmic membranes. IMPORTANCE The molecular details of nuclear egress of herpesvirus capsids are still enigmatic. Although the key players, homologs of herpes simplex virus pUL34 and pUL31, which interact and form the heterodimeric nuclear egress complex, are well known, the molecular basis of this interaction and the successive budding, vesicle formation, and scission from the INM, as well as capsid release into the cytoplasm, remain largely obscure. Here we show that

  3. Flow-induced HDAC1 phosphorylation and nuclear export in angiogenic sprouting

    PubMed Central

    Bazou, Despina; Ng, Mei Rosa; Song, Jonathan W.; Chin, Shan Min; Maimon, Nir; Munn, Lance L.

    2016-01-01

    Angiogenesis requires the coordinated growth and migration of endothelial cells (ECs), with each EC residing in the vessel wall integrating local signals to determine whether to remain quiescent or undergo morphogenesis. These signals include vascular endothelial growth factor (VEGF) and flow-induced mechanical stimuli such as interstitial flow, which are both elevated in the tumor microenvironment. However, it is not clear how VEGF signaling and mechanobiological activation due to interstitial flow cooperate during angiogenesis. Here, we show that endothelial morphogenesis is histone deacetylase-1- (HDAC1) dependent and that interstitial flow increases the phosphorylation of HDAC1, its activity, and its export from the nucleus. Furthermore, we show that HDAC1 inhibition decreases endothelial morphogenesis and matrix metalloproteinase-14 (MMP14) expression. Our results suggest that HDAC1 modulates angiogenesis in response to flow, providing a new target for modulating vascularization in the clinic. PMID:27669993

  4. Influenza A Virus NS1 Protein Promotes Efficient Nuclear Export of Unspliced Viral M1 mRNA.

    PubMed

    Pereira, Carina F; Read, Eliot K C; Wise, Helen M; Amorim, Maria J; Digard, Paul

    2017-08-01

    Influenza A virus mRNAs are transcribed by the viral RNA-dependent RNA polymerase in the cell nucleus before being exported to the cytoplasm for translation. Segment 7 produces two major transcripts: an unspliced mRNA that encodes the M1 matrix protein and a spliced transcript that encodes the M2 ion channel. Export of both mRNAs is dependent on the cellular NXF1/TAP pathway, but it is unclear how they are recruited to the export machinery or how the intron-containing but unspliced M1 mRNA bypasses the normal quality-control checkpoints. Using fluorescent in situ hybridization to monitor segment 7 mRNA localization, we found that cytoplasmic accumulation of unspliced M1 mRNA was inefficient in the absence of NS1, both in the context of segment 7 RNPs reconstituted by plasmid transfection and in mutant virus-infected cells. This effect was independent of any major effect on steady-state levels of segment 7 mRNA or splicing but corresponded to a ∼5-fold reduction in the accumulation of M1. A similar defect in intronless hemagglutinin (HA) mRNA nuclear export was seen with an NS1 mutant virus. Efficient export of M1 mRNA required both an intact NS1 RNA-binding domain and effector domain. Furthermore, while wild-type NS1 interacted with cellular NXF1 and also increased the interaction of segment 7 mRNA with NXF1, mutant NS1 polypeptides unable to promote mRNA export did neither. Thus, we propose that NS1 facilitates late viral gene expression by acting as an adaptor between viral mRNAs and the cellular nuclear export machinery to promote their nuclear export.IMPORTANCE Influenza A virus is a major pathogen of a wide variety of mammalian and avian species that threatens public health and food security. A fuller understanding of the virus life cycle is important to aid control strategies. The virus has a small genome that encodes relatively few proteins that are often multifunctional. Here, we characterize a new function for the NS1 protein, showing that, as well as

  5. Nuclear Receptor Signaling: a home for nuclear receptor and coregulator signaling research.

    PubMed

    McKenna, Neil J; Evans, Ronald M; O'Malley, Bert W

    2014-01-01

    The field of nuclear receptor and coregulator signaling has grown into one of the most active and interdisciplinary in eukaryotic biology. Papers in this field are spread widely across a vast number of journals, which complicates the task of investigators in keeping current with the literature in the field. In 2003, we launched Nuclear Receptor Signaling as an Open Access reviews, perspectives and methods journal for the nuclear receptor signaling field. Building on its success and impact on the community, we have added primary research and dataset articles to this list of article categories, and we now announce the re-launch of the journal this month. Here we will summarize the rationale that informed the creation and expansion of the journal, and discuss the possibilities for its future development.

  6. The principal mRNA nuclear export factor NXF1:NXT1 forms a symmetric binding platform that facilitates export of retroviral CTE-RNA

    PubMed Central

    Aibara, Shintaro; Katahira, Jun; Valkov, Eugene; Stewart, Murray

    2015-01-01

    The NXF1:NXT1 complex (also known as TAP:p15) is a general mRNA nuclear export factor that is conserved from yeast to humans. NXF1 is a modular protein constructed from four domains (RRM, LRR, NTF2-like and UBA domains). It is currently unclear how NXF1:NXT1 binds transcripts and whether there is higher organization of the NXF1 domains. We report here the 3.4 Å resolution crystal structure of the first three domains of human NXF1 together with NXT1 that has two copies of the complex in the asymmetric unit arranged to form an intimate domain-swapped dimer. In this dimer, the linkers between the NXF1 LRR and NTF2-like domains interact with NXT1, generating a 2-fold symmetric platform in which the RNA-binding RRM, LRR and NTF2-like domains are arranged on one face. In addition to bulk transcripts, NXF1:NXT1 also facilitates the export of unspliced retroviral genomic RNA from simple type-D retroviruses such as SRV-1 that contain a constitutive transport element (CTE), a cis-acting 2-fold symmetric RNA stem–loop motif. Complementary structural, biochemical and cellular techniques indicated that the formation of a symmetric RNA binding platform generated by dimerization of NXF1:NXT1 facilitates the recognition of CTE-RNA and promotes its nuclear export. PMID:25628361

  7. The principal mRNA nuclear export factor NXF1:NXT1 forms a symmetric binding platform that facilitates export of retroviral CTE-RNA.

    PubMed

    Aibara, Shintaro; Katahira, Jun; Valkov, Eugene; Stewart, Murray

    2015-02-18

    The NXF1:NXT1 complex (also known as TAP:p15) is a general mRNA nuclear export factor that is conserved from yeast to humans. NXF1 is a modular protein constructed from four domains (RRM, LRR, NTF2-like and UBA domains). It is currently unclear how NXF1:NXT1 binds transcripts and whether there is higher organization of the NXF1 domains. We report here the 3.4 Å resolution crystal structure of the first three domains of human NXF1 together with NXT1 that has two copies of the complex in the asymmetric unit arranged to form an intimate domain-swapped dimer. In this dimer, the linkers between the NXF1 LRR and NTF2-like domains interact with NXT1, generating a 2-fold symmetric platform in which the RNA-binding RRM, LRR and NTF2-like domains are arranged on one face. In addition to bulk transcripts, NXF1:NXT1 also facilitates the export of unspliced retroviral genomic RNA from simple type-D retroviruses such as SRV-1 that contain a constitutive transport element (CTE), a cis-acting 2-fold symmetric RNA stem-loop motif. Complementary structural, biochemical and cellular techniques indicated that the formation of a symmetric RNA binding platform generated by dimerization of NXF1:NXT1 facilitates the recognition of CTE-RNA and promotes its nuclear export. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  8. Exosome cofactor hMTR4 competes with export adaptor ALYREF to ensure balanced nuclear RNA pools for degradation and export.

    PubMed

    Fan, Jing; Kuai, Bin; Wu, Guifen; Wu, Xudong; Chi, Binkai; Wang, Lantian; Wang, Ke; Shi, Zhubing; Zhang, Heng; Chen, She; He, Zhisong; Wang, Siyuan; Zhou, Zhaocai; Li, Guohui; Cheng, Hong

    2017-10-02

    The exosome is a key RNA machine that functions in the degradation of unwanted RNAs. Here, we found that significant fractions of precursors and mature forms of mRNAs and long noncoding RNAs are degraded by the nuclear exosome in normal human cells. Exosome-mediated degradation of these RNAs requires its cofactor hMTR4. Significantly, hMTR4 plays a key role in specifically recruiting the exosome to its targets. Furthermore, we provide several lines of evidence indicating that hMTR4 executes this role by directly competing with the mRNA export adaptor ALYREF for associating with ARS2, a component of the cap-binding complex (CBC), and this competition is critical for determining whether an RNA is degraded or exported to the cytoplasm. Together, our results indicate that the competition between hMTR4 and ALYREF determines exosome recruitment and functions in creating balanced nuclear RNA pools for degradation and export. © 2017 The Authors.

  9. 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.

  10. 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

  11. 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. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  12. Research Resources for Nuclear Receptor Signaling Pathways

    PubMed Central

    2016-01-01

    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

  13. Histone deacetylase 6 (HDAC6) deacetylates survivin for its nuclear export in breast cancer.

    PubMed

    Riolo, Matthew T; Cooper, Zachary A; Holloway, Michael P; Cheng, Yan; Bianchi, Cesario; Yakirevich, Evgeny; Ma, Li; Chin, Y Eugene; Altura, Rachel A

    2012-03-30

    Survivin is an oncogenic protein that is highly expressed in breast cancer and has a dual function that is dependent on its subcellular localization. In the cytosol, survivin blocks programmed cell death by inactivating caspase proteins; however, in the nucleus it facilitates cell division by regulating chromosomal movement and cytokinesis. In prior work, we showed that survivin is acetylated by CREB-binding protein (CBP), which restricts its localization to the nuclear compartment and thereby inhibits its anti-apoptotic function. Here, we identify histone deacetylase 6 (HDAC6) as responsible for abrogating CBP-mediated survivin acetylation in the estrogen receptor (ER)-positive breast cancer cell line, MCF-7. HDAC6 directly binds survivin, an interaction that is enhanced by CBP. In quiescent breast cancer cells in culture and in malignant tissue sections from ER+ breast tumors, HDAC6 localizes to a perinuclear region of the cell, undergoing transport to the nucleus following CBP activation where it then deacetylates survivin. Genetically modified mouse embryonic fibroblasts that lack mhdac6 localize survivin predominantly to the nuclear compartment, whereas wild-type mouse embryonic fibroblasts localize survivin to distinct cytoplasmic structures. Together, these data imply that HDAC6 deacetylates survivin to regulate its nuclear export, a feature that may provide a novel target for patients with ER+ breast cancer.

  14. 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.

  15. CRM1 Blockade by Selective Inhibitors of Nuclear Export (SINE) attenuates Kidney Cancer Growth

    PubMed Central

    Inoue, Hiromi; Kauffman, Michael; Shacham, Sharon; Landesman, Yosef; Yang, Joy; Evans, Christopher P.; Weiss, Robert H.

    2015-01-01

    Since renal cell carcinoma (RCC) often presents asymptomatically, patients are commonly diagnosed at the metastatic stage when treatment options are limited and survival is poor. Given that progression-free survival with current therapies for metastatic RCC is only one to two years and existing drugs are associated with a high rate of resistance, new pharmacological targets are desperately needed. We identified and evaluated the nuclear exporter protein, chromosome region maintenance protein 1 (CRM1), as a novel potential therapeutic for RCC. Purpose To evaluate novel, selective inhibitors of nuclear export as potential RCC therapeutics. Materials and Methods Efficacy of the CRM1 inhibitors, KPT-185 and -251, was tested in several RCC cell lines and in a RCC xenograft model. Apoptosis and cell cycle arrest were quantified, and localization of p53 family proteins was assessed using standard techniques. Results KPT-185 attenuated CRM1 and showed increased cytotoxicity in RCC cells in vitro, with evidence of increased apoptosis as well as cell cycle arrest. KPT-185 caused both p53 and p21 to remain primarily in the nucleus in all RCC cell lines, suggesting a mechanism of action of these compounds dependent upon tumor-suppressor protein localization. Furthermore, when administered orally in a high-grade RCC xenograft model, the bioavailable CRM1 inhibitor KPT-251 significantly inhibited tumor growth in vivo with the expected on-target effects and with no obvious toxicity. Conclusions The CRM1 inhibitor family of proteins are novel therapeutic targets RCC and deserve further intensive investigation in this and other urologic malignancies. PMID:23079374

  16. 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

    2016-08-08

    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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Identification of a New Lipoprotein Export Signal in Gram-Negative Bacteria.

    PubMed

    Lauber, Frédéric; Cornelis, Guy Richard; Renzi, Francesco

    2016-10-25

    Bacteria of the phylum Bacteroidetes, including commensal organisms and opportunistic pathogens, harbor abundant surface-exposed multiprotein membrane complexes (Sus-like systems) involved in carbohydrate acquisition. These complexes have been mostly linked to commensalism, and in some instances, they have also been shown to play a role in pathogenesis. Sus-like systems are mainly composed of lipoproteins anchored to the outer membrane and facing the external milieu. This lipoprotein localization is uncommon in most studied Gram-negative bacteria, while it is widespread in Bacteroidetes Little is known about how these complexes assemble and particularly about how lipoproteins reach the bacterial surface. Here, by bioinformatic analyses, we identify a lipoprotein export signal (LES) at the N termini of surface-exposed lipoproteins of the human pathogen Capnocytophaga canimorsus corresponding to K-(D/E)2 or Q-A-(D/E)2 We show that, when introduced in sialidase SiaC, an intracellular lipoprotein, this signal is sufficient to target the protein to the cell surface. Mutational analysis of the LES in this reporter system showed that the amino acid composition, position of the signal sequence, and global charge are critical for lipoprotein surface transport. These findings were further confirmed by the analysis of the LES of mucinase MucG, a naturally surface-exposed C. canimorsus lipoprotein. Furthermore, we identify a LES in Bacteroides fragilis and Flavobacterium johnsoniae surface lipoproteins that allow C. canimorsus surface protein exposure, thus suggesting that Bacteroidetes share a new bacterial lipoprotein export pathway that flips lipoproteins across the outer membrane. Bacteria of the phylum Bacteroidetes are important human commensals and pathogens. Understanding their biology is therefore a key question for human health. A main feature of these bacteria is the presence of abundant lipoproteins at their surface that play a role in nutrient acquisition. To

  18. Role of nuclear bodies in apoptosis signalling.

    PubMed

    Krieghoff-Henning, Eva; Hofmann, Thomas G

    2008-11-01

    Promyelocytic leukemia nuclear bodies (PML NBs) are dynamic macromolecular multiprotein complexes that recruit and release a plethora of proteins. A considerable number of PML NB components play vital roles in apoptosis, senescence regulation and tumour suppression. The molecular basis by which PML NBs control these cellular responses is still just beginning to be understood. In addition to PML itself, numerous further tumour suppressors including transcriptional regulator p53, acetyl transferase CBP (CREB binding protein) and protein kinase HIPK2 (homeodomain interacting protein kinase 2) are recruited to PML NBs in response to genotoxic stress or oncogenic transformation and drive the senescence and apoptosis response by regulating p53 activity. Moreover, in response to death-receptor activation, PML NBs may act as nuclear depots that release apoptotic factors, such as the FLASH (FLICE-associated huge) protein, to amplify the death signal. PML NBs are also associated with other nuclear domains including Cajal bodies and nucleoli and share apoptotic regulators with these domains, implying crosstalk between NBs in apoptosis regulation. In conclusion, PML NBs appear to regulate cell death decisions through different, pathway-specific molecular mechanisms.

  19. XPO1-dependent nuclear export is a druggable vulnerability in KRAS-mutant lung cancer

    PubMed Central

    Kim, Jimi; McMillan, Elizabeth; Kim, Hyun Seok; Venkateswaran, Niranjan; Makkar, Gurbani; Rodriguez-Canales, Jaime; Villalobos, Pamela; Neggers, Jasper Edgar; Mendiratta, Saurabh; Wei, Shuguang; Landesman, Yosef; Senapedis, William; Baloglu, Erkan; Chow, Chi-Wan B.; Frink, Robin E.; Gao, Boning; Roth, Michael; Minna, John D.; Daelemans, Dirk; Wistuba, Ignacio I.; Posner, Bruce A.; Scaglioni, PierPaolo; White, Michael A.

    2016-01-01

    The common participation of oncogenic KRAS proteins in many of the most lethal human cancers, together with the ease of detecting somatic KRAS mutant alleles in patient samples, has spurred persistent and intensive efforts to develop drugs that inhibit KRAS activity1. However, advances have been hindered by the pervasive inter- and intra-lineage diversity in the targetable mechanisms that underlie KRAS-driven cancers, limited pharmacological accessibility of many candidate synthetic-lethal interactions and the swift emergence of unanticipated resistance mechanisms to otherwise effective targeted therapies. Here we demonstrate the acute and specific cell-autonomous addiction of KRAS-mutant non-small-cell lung cancer cells to receptor-dependent nuclear export. A multi-genomic, data-driven approach, utilizing 106 human non-small-cell lung cancer cell lines, was used to interrogate 4,725 biological processes with 39,760 short interfering RNA pools for those selectively required for the survival of KRAS-mutant cells that harbour a broad spectrum of phenotypic variation. Nuclear transport machinery was the sole process-level discriminator of statistical significance. Chemical perturbation of the nuclear export receptor XPO1 (also known as CRM1), with a clinically available drug, revealed a robust synthetic-lethal interaction with native or engineered oncogenic KRAS both in vitro and in vivo. The primary mechanism underpinning XPO1 inhibitor sensitivity was intolerance to the accumulation of nuclear IκBα (also known as NFKBIA), with consequent inhibition of NFκB transcription factor activity. Intrinsic resistance associated with concurrent FSTL5 mutations was detected and determined to be a consequence of YAP1 activation via a previously unappreciated FSTL5–Hippo pathway regulatory axis. This occurs in approximately 17% of KRAS-mutant lung cancers, and can be overcome with the co-administration of a YAP1–TEAD inhibitor. These findings indicate that clinically

  20. XPO1-dependent nuclear export is a druggable vulnerability in KRAS-mutant lung cancer.

    PubMed

    Kim, Jimi; McMillan, Elizabeth; Kim, Hyun Seok; Venkateswaran, Niranjan; Makkar, Gurbani; Rodriguez-Canales, Jaime; Villalobos, Pamela; Neggers, Jasper Edgar; Mendiratta, Saurabh; Wei, Shuguang; Landesman, Yosef; Senapedis, William; Baloglu, Erkan; Chow, Chi-Wan B; Frink, Robin E; Gao, Boning; Roth, Michael; Minna, John D; Daelemans, Dirk; Wistuba, Ignacio I; Posner, Bruce A; Scaglioni, Pier Paolo; White, Michael A

    2016-10-06

    The common participation of oncogenic KRAS proteins in many of the most lethal human cancers, together with the ease of detecting somatic KRAS mutant alleles in patient samples, has spurred persistent and intensive efforts to develop drugs that inhibit KRAS activity. However, advances have been hindered by the pervasive inter- and intra-lineage diversity in the targetable mechanisms that underlie KRAS-driven cancers, limited pharmacological accessibility of many candidate synthetic-lethal interactions and the swift emergence of unanticipated resistance mechanisms to otherwise effective targeted therapies. Here we demonstrate the acute and specific cell-autonomous addiction of KRAS-mutant non-small-cell lung cancer cells to receptor-dependent nuclear export. A multi-genomic, data-driven approach, utilizing 106 human non-small-cell lung cancer cell lines, was used to interrogate 4,725 biological processes with 39,760 short interfering RNA pools for those selectively required for the survival of KRAS-mutant cells that harbour a broad spectrum of phenotypic variation. Nuclear transport machinery was the sole process-level discriminator of statistical significance. Chemical perturbation of the nuclear export receptor XPO1 (also known as CRM1), with a clinically available drug, revealed a robust synthetic-lethal interaction with native or engineered oncogenic KRAS both in vitro and in vivo. The primary mechanism underpinning XPO1 inhibitor sensitivity was intolerance to the accumulation of nuclear IκBα (also known as NFKBIA), with consequent inhibition of NFκB transcription factor activity. Intrinsic resistance associated with concurrent FSTL5 mutations was detected and determined to be a consequence of YAP1 activation via a previously unappreciated FSTL5-Hippo pathway regulatory axis. This occurs in approximately 17% of KRAS-mutant lung cancers, and can be overcome with the co-administration of a YAP1-TEAD inhibitor. These findings indicate that clinically available

  1. Biogenesis of the signal recognition particle (SRP) involves import of SRP proteins into the nucleolus, assembly with the SRP-RNA, and Xpo1p-mediated export.

    PubMed

    Grosshans, H; Deinert, K; Hurt, E; Simos, G

    2001-05-14

    The signal recognition particle (SRP) targets nascent secretory proteins to the ER, but how and where the SRP assembles is largely unknown. Here we analyze the biogenesis of yeast SRP, which consists of an RNA molecule (scR1) and six proteins, by localizing all its components. Although scR1 is cytoplasmic in wild-type cells, nuclear localization was observed in cells lacking any one of the four SRP "core proteins" Srp14p, Srp21p, Srp68p, or Srp72p. Consistently, a major nucleolar pool was detected for these proteins. Sec65p, on the other hand, was found in both the nucleoplasm and the nucleolus, whereas Srp54p was predominantly cytoplasmic. Import of the core proteins into the nucleolus requires the ribosomal protein import receptors Pse1p and Kap123p/Yrb4p, which might, thus, constitute a nucleolar import pathway. Nuclear export of scR1 is mediated by the nuclear export signal receptor Xpo1p, is distinct from mRNA transport, and requires, as evidenced by the nucleolar accumulation of scR1 in a dis3/rrp44 exosome component mutant, an intact scR1 3' end. A subset of nucleoporins, including Nsp1p and Nup159p (Rat7p), are also necessary for efficient translocation of scR1 from the nucleus to the cytoplasm. We propose that assembly of the SRP requires import of all SRP core proteins into the nucleolus, where they assemble into a pre-SRP with scR1. This particle can then be targeted to the nuclear pores and is subsequently exported to the cytoplasm in an Xpo1p-dependent way.

  2. Dissection of a nuclear localization signal.

    PubMed

    Hodel, M R; Corbett, A H; Hodel, A E

    2001-01-12

    The regulated process of protein import into the nucleus of a eukaryotic cell is mediated by specific nuclear localization signals (NLSs) that are recognized by protein import receptors. This study seeks to decipher the energetic details of NLS recognition by the receptor importin alpha through quantitative analysis of variant NLSs. The relative importance of each residue in two monopartite NLS sequences was determined using an alanine scanning approach. These measurements yield an energetic definition of a monopartite NLS sequence where a required lysine residue is followed by two other basic residues in the sequence K(K/R)X(K/R). In addition, the energetic contributions of the second basic cluster in a bipartite NLS ( approximately 3 kcal/mol) as well as the energy of inhibition of the importin alpha importin beta-binding domain ( approximately 3 kcal/mol) were also measured. These data allow the generation of an energetic scale of nuclear localization sequences based on a peptide's affinity for the importin alpha-importin beta complex. On this scale, a functional NLS has a binding constant of approximately 10 nm, whereas a nonfunctional NLS has a 100-fold weaker affinity of 1 microm. Further correlation between the current in vitro data and in vivo function will provide the foundation for a comprehensive quantitative model of protein import.

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

    PubMed Central

    Cigdem, Sadik; Okuwaki, Mitsuru; Nagata, Kyosuke

    2016-01-01

    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

  4. 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. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  5. The ubiquitin ligase MuRF1 regulates PPARα activity in the heart by enhancing nuclear export via monoubiquitination

    PubMed Central

    Rodríguez, Jessica E.; Liao, Jie-Ying; He, Jun; Schisler, Jonathan C.; Newgard, Christopher B.; Drujan, Doreen; Glass, David L.; Frederick, C.Brandon; Yoder, Bryan C.; Lalush, David S.; Patterson, Cam; Willis, Monte S.

    2015-01-01

    The transcriptional regulation of peroxisome proliferator-activated receptor (PPAR) α by post-translational modification, such as ubiquitin, has not been described. We report here for the first time an ubiquitin ligase (muscle ring finger-1/MuRF1) that inhibits fatty acid oxidation by inhibiting PPARα, but not PPARβ/δ or PPARγ in cardiomyocytes in vitro. Similarly, MuRF1 Tg+ hearts showed significant decreases in nuclear PPARα activity and acyl-carnitine intermediates, while MuRF1−/− hearts exhibited increased PPARα activity and acyl-carnitine intermediates. MuRF1 directly interacts with PPARα, mono-ubiquitinates it, and targets it for nuclear export to inhibit fatty acid oxidation in a proteasome independent manner. We then identified a previously undescribed nuclear export sequence in PPARα, along with three specific lysines (292, 310, 388) required for MuRF1s targeting of nuclear export. These studies identify the role of ubiquitination in regulating cardiac PPARα, including the ubiquitin ligase that may be responsible for this critical regulation of cardiac metabolism in heart failure. PMID:26116825

  6. TNF Stimulates Nuclear Export and Secretion of IL-15 by Acting on CRM1 and ARF6

    PubMed Central

    Ouyang, Suidong; Hsuchou, Hung; Kastin, Abba J.; Pan, Weihong

    2013-01-01

    Interleukin (IL)-15 is a ubiquitously expressed cytokine that in the basal state is mainly localized intracellularly, including the nucleus. Unexpectedly, tumor necrosis factor-α (TNF) time-dependently induced nuclear export of IL-15Rα and IL15. This process was inhibited by leptomycine B (LMB), a specific inhibitor of nuclear export receptor chromosomal region maintenance 1 (CRM1). In the presence of TNF, LMB co-treatment led to accumulation of both IL-15Rα and IL-15 in the nucleus of HeLa cells, suggesting that CRM1 facilitates nuclear export and that TNF enhances CRM1 activity. Once in the cytoplasm, IL-15 showed partial co-localization with late endosomes but very little with other organelles tested 4 h after TNF treatment. IL-15Rα showed co-localization with both early and late endosomes, and to a lesser extent with endoplasmic reticulum and Golgi. This indicates different kinetics and possibly different trafficking routes of IL-15 from its specific receptor. The TNF-induced secretion of IL-15 was attenuated by pretreatment of cells by brefeldin A that inhibits ER-to-Golgi transport, or by use of domain negative ADP-ribosylation factor 6 (ARF6) that interferes with exocytotic sorting. We conclude that TNF abolishes nuclear localization of IL-15 and IL-15Rα by acting on CRM1, and it facilitates exocytosis of IL-15 with the involvement of ARF6. PMID:23950892

  7. TNF stimulates nuclear export and secretion of IL-15 by acting on CRM1 and ARF6.

    PubMed

    Ouyang, Suidong; Hsuchou, Hung; Kastin, Abba J; Pan, Weihong

    2013-01-01

    Interleukin (IL)-15 is a ubiquitously expressed cytokine that in the basal state is mainly localized intracellularly, including the nucleus. Unexpectedly, tumor necrosis factor-α (TNF) time-dependently induced nuclear export of IL-15Rα and IL15. This process was inhibited by leptomycine B (LMB), a specific inhibitor of nuclear export receptor chromosomal region maintenance 1 (CRM1). In the presence of TNF, LMB co-treatment led to accumulation of both IL-15Rα and IL-15 in the nucleus of HeLa cells, suggesting that CRM1 facilitates nuclear export and that TNF enhances CRM1 activity. Once in the cytoplasm, IL-15 showed partial co-localization with late endosomes but very little with other organelles tested 4 h after TNF treatment. IL-15Rα showed co-localization with both early and late endosomes, and to a lesser extent with endoplasmic reticulum and Golgi. This indicates different kinetics and possibly different trafficking routes of IL-15 from its specific receptor. The TNF-induced secretion of IL-15 was attenuated by pretreatment of cells by brefeldin A that inhibits ER-to-Golgi transport, or by use of domain negative ADP-ribosylation factor 6 (ARF6) that interferes with exocytotic sorting. We conclude that TNF abolishes nuclear localization of IL-15 and IL-15Rα by acting on CRM1, and it facilitates exocytosis of IL-15 with the involvement of ARF6.

  8. Identification of a New Lipoprotein Export Signal in Gram-Negative Bacteria

    PubMed Central

    Lauber, Frédéric; Cornelis, Guy Richard

    2016-01-01

    ABSTRACT Bacteria of the phylum Bacteroidetes, including commensal organisms and opportunistic pathogens, harbor abundant surface-exposed multiprotein membrane complexes (Sus-like systems) involved in carbohydrate acquisition. These complexes have been mostly linked to commensalism, and in some instances, they have also been shown to play a role in pathogenesis. Sus-like systems are mainly composed of lipoproteins anchored to the outer membrane and facing the external milieu. This lipoprotein localization is uncommon in most studied Gram-negative bacteria, while it is widespread in Bacteroidetes. Little is known about how these complexes assemble and particularly about how lipoproteins reach the bacterial surface. Here, by bioinformatic analyses, we identify a lipoprotein export signal (LES) at the N termini of surface-exposed lipoproteins of the human pathogen Capnocytophaga canimorsus corresponding to K-(D/E)2 or Q-A-(D/E)2. We show that, when introduced in sialidase SiaC, an intracellular lipoprotein, this signal is sufficient to target the protein to the cell surface. Mutational analysis of the LES in this reporter system showed that the amino acid composition, position of the signal sequence, and global charge are critical for lipoprotein surface transport. These findings were further confirmed by the analysis of the LES of mucinase MucG, a naturally surface-exposed C. canimorsus lipoprotein. Furthermore, we identify a LES in Bacteroides fragilis and Flavobacterium johnsoniae surface lipoproteins that allow C. canimorsus surface protein exposure, thus suggesting that Bacteroidetes share a new bacterial lipoprotein export pathway that flips lipoproteins across the outer membrane. PMID:27795390

  9. 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

  10. Erythropoietin and carbamylated erythropoietin promote histone deacetylase 5 phosphorylation and nuclear export in rat hippocampal neurons

    SciTech Connect

    Jo, Hye-Ryeong; Kim, Yong-Seok; Son, Hyeon

    2016-01-29

    Erythropoietin (EPO) produces neurotrophic effects in animal model of neurodegeneration. However, clinical use of EPO is limited due to thrombotic risk. Carbamylated EPO (cEPO), devoid of thrombotic risk, has been proposed as a novel neuroprotective and neurotrophic agent although the molecular mechanisms of cEPO remain incomplete. Here, we show a previously unidentified role of histone deacetylase 5 (HDAC5) in the actions of EPO and cEPO. EPO and cEPO regulate the HDAC5 phosphorylation at two critical sites, Ser259 and Ser498 through a protein kinase D (PKD) dependent pathway. In addition, EPO and cEPO rapidly stimulates nuclear export of HDAC5 in rat hippocampal neurons which expressing HDAC5-GFP. Consequently, EPO and cEPO enhanced the myocyte enhancer factor-2 (MEF2) target gene expression. Taken together, our results reveal that EPO and cEPO mediate MEF2 target gene expression via the regulation of HDAC5 phosphorylation at Ser259/498, and suggest that HDAC5 could be a potential mechanism contributing to the therapeutic actions of EPO and cEPO.

  11. 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

  12. 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

  13. China's mixed signals on nuclear weapons

    SciTech Connect

    Fieldhouse, R. )

    1991-05-01

    Ultimately, it is nuclear whether the Chinese leadership has made up its collective mind on practical nuclear weapons. It is known from Chinese official sources, including articles in Communist Party and military publications and histories of the Chinese nuclear program, that an internal debate has proceeded for more than two decades, punctuated by occasional nuclear exercises or low-yield warhead tests. But China presumably has less reason now to pursue development of tactical nuclear weapons than in previous decades: relations with the Soviet Union have improved and military confrontation has eased; China's relations with India and Vietnam are also improving. The decision may already have been made, however, and the weapons built. The mystery surrounding Chinese tactical nuclear weapons is itself interesting, but it is also symbolic of the difficulty of understanding China's nuclear weapons program and policies. The West has accumulated a considerable body of knowledge about China's nuclear forces, especially historical material. But important aspects of China's nuclear behavior and its future as a nuclear power are hard to discern. A key question is China's future role in the spread of nuclear-capable weapons to other countries. China might add to international efforts to stem the proliferation of nuclear related technology, or it might become the world's missile merchant. It could make a constructive contribution to arms control efforts in general, or it could act as a spoiler.

  14. 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

  15. 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

  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.

  17. Heterozygous mutation of cysteine528 in XPO1 is sufficient for resistance to selective inhibitors of nuclear export.

    PubMed

    Neggers, Jasper Edgar; Vanstreels, Els; Baloglu, Erkan; Shacham, Sharon; Landesman, Yosef; Daelemans, Dirk

    2016-10-18

    Exportin-1 (CRM1/XPO1) is a crucial nuclear export protein that transports a wide variety of proteins from the nucleus to the cytoplasm. These cargo proteins include tumor suppressors and growth-regulatory factors and as such XPO1 is considered a potential anti-cancer target. From this perspective, inhibition of the XPO1-mediated nuclear export by selective inhibitor of nuclear export (SINE) compounds has shown broad-spectrum anti-cancer activity. Furthermore, the clinical candidate SINE, selinexor, is currently in multiple phase I/II/IIb trials for treatment of cancer. Resistance against selinexor has not yet been observed in the clinic, but in vitro selection of resistance did not reveal any mutations in the target protein, XPO1. However, introduction of a homozygous mutation at the drug's target site, the cysteine 528 residue inside the XPO1 cargo-binding pocket, by genetic engineering, confers resistance to selinexor. Here we investigated whether this resistance to selinexor is recessive or dominant. For this purpose we have engineered multiple leukemia cell lines containing heterozygous or homozygous C528S substitutions using CRISPR/Cas9-mediated genome editing. Our findings show that heterozygous mutation confers similar resistance against selinexor as homozygous substitution, demonstrating that SINE resistance can be obtained by a single and dominant mutation of the cysteine528 residue in XPO1.

  18. 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.

  19. [Polyadenylated RNA and mRNA export factors in extrachromosomal nuclear domains of vitellogenic oocytes of the insect Tenebrio molitor].

    PubMed

    Bogoliubov, D S; Kiselev, A M; Shabel'nikov, S V; Parfenov, V N

    2012-01-01

    The nucleus ofvitellogenic oocytes of the yellow mealworm, Tenebrio molitor, contains a karyosphere that consists of the condensed chromatin embedded in an extrachromosomal fibrogranular material. Numerous nuclear bodies located freely in the nucleoplasm are also observed. Amongst these bodies, counterparts of nuclear speckles (= interchromatin granule clusters, IGCs) can be identified by the presence of the marker protein SC35. Microinjections of fluorescently tagged methyloligoribonucleotide probes 2'-O-Me(U)22, complementary to poly(A) tails of RNAs, revealed poly(A)+ RNA in the vast majority of IGCs. We found that all T. molitor oocyte IGCs contain heterogeneous ribonucleoprotein (hnRNP) core protein Al that localizes to IGCs in an RNA-dependent manner. The extrachromosomal material of the karyosphere and a part of nucleoplasmic IGCs also contain the adapter protein Aly that is known to provide a link between pre-mRNA splicing and mRNA export. The essential mRNA export factor/receptor NXF1 was observed to colocalize with Aly. In nucleoplasmic IGCs, NXF1 was found to localize in an RNA-dependent manner whereas it is RNA-independently located in the extrachromosomal material of the karyosphere. We believe our data suggest on a role of the nucleoplasmic IGCs in mRNA biogenesis and retention in a road to nuclear export.

  20. Nup100 regulates Saccharomyces cerevisiae replicative life span by mediating the nuclear export of specific tRNAs.

    PubMed

    Lord, Christopher L; Ospovat, Ophir; Wente, Susan R

    2017-03-01

    Nuclear pore complexes (NPCs), which are composed of nucleoporins (Nups) and regulate transport between the nucleus and cytoplasm, significantly impact the replicative life span (RLS) of Saccharomyces cerevisiae We previously reported that deletion of the nonessential gene NUP100 increases RLS, although the molecular basis for this effect was unknown. In this study, we find that nuclear tRNA accumulation contributes to increased longevity in nup100Δ cells. Fluorescence in situ hybridization (FISH) experiments demonstrate that several specific tRNAs accumulate in the nuclei of nup100Δ mutants. Protein levels of the transcription factor Gcn4 are increased when NUP100 is deleted, and GCN4 is required for the elevated life spans of nup100Δ mutants, similar to other previously described tRNA export and ribosomal mutants. Northern blots indicate that tRNA splicing and aminoacylation are not significantly affected in nup100Δ cells, suggesting that Nup100 is largely required for nuclear export of mature, processed tRNAs. Distinct tRNAs accumulate in the nuclei of nup100Δ and msn5Δ mutants, while Los1-GFP nucleocytoplasmic shuttling is unaffected by Nup100. Thus, we conclude that Nup100 regulates tRNA export in a manner distinct from Los1 or Msn5. Together, these experiments reveal a novel Nup100 role in the tRNA life cycle that impacts the S. cerevisiae life span.

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

    PubMed

    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.

  2. Remnant signal peptides on non-exported enzymes: implications for the evolution of prokaryotic respiratory chains.

    PubMed

    Ize, Bérengère; Coulthurst, Sarah J; Hatzixanthis, Kostas; Caldelari, Isabelle; Buchanan, Grant; Barclay, Elaine C; Richardson, David J; Palmer, Tracy; Sargent, Frank

    2009-12-01

    The twin-arginine translocation (Tat) pathway is a prokaryotic protein targeting system dedicated to the transmembrane translocation of folded proteins. Substrate proteins are directed to the Tat translocase by signal peptides bearing a conserved SRRxFLK 'twin-arginine' motif. In Escherichia coli, most of the 27 periplasmically located Tat substrates are cofactor-containing respiratory enzymes, and many of these harbour a molybdenum cofactor at their active site. Molybdenum cofactor-containing proteins are not exclusively located in the periplasm, however, with the major respiratory nitrate reductase (NarG) and the biotin sulfoxide reductase (BisC), for example, being located at the cytoplasmic side of the membrane. Interestingly, both NarG and BisC contain 'N-tail' regions that bear some sequence similarity to twin-arginine signal peptides. In this work, we have examined the relationship between the non-exported N-tails and the Tat system. Using a sensitive genetic screen for Tat transport, variant N-tails were identified that displayed Tat transport activity. For the NarG 36-residue N-tail, six amino acid changes were needed to induce transport activity. However, these changes interfered with binding by the NarJ biosynthetic chaperone and impaired biosynthesis of the native enzyme. For the BisC 36-residue N-tail, only five amino acid substitutions were needed to restore Tat transport activity. These modifications also impaired in vivo BisC activity, but it was not possible to identify a biosynthetic chaperone for this enzyme. These data highlight an intimate genetic and evolutionary link between some non-exported redox enzymes and those transported across membranes by the Tat translocation system.

  3. The Gpn3 Q279* cancer-associated mutant inhibits Gpn1 nuclear export and is deficient in RNA polymerase II nuclear targeting.

    PubMed

    Barbosa-Camacho, Angel A; Méndez-Hernández, Lucía E; Lara Chacón, Bárbara; Peña-Gómez, Sonia G; Romero, Violeta; González-González, Rogelio; Guerra-Moreno, José A; Robledo-Rivera, Angélica Y; Sánchez-Olea, Roberto; Calera, Mónica R

    2017-09-23

    Gpn3 is required for RNA polymerase II (RNAPII) nuclear targeting. Here, we investigated the effect of a cancer-associated Q279* nonsense mutation in Gpn3 cellular function. Employing RNAi, we replaced endogenous Gpn3 by wt or Q279* RNAi-resistant Gpn3R in epithelial model cells. RNAPII nuclear accumulation and transcriptional activity are markedly decreased in cells expressing only Gpn3R Q279*. Wild-type Gpn3R localizes to the cytoplasm but a fraction of Gpn3R Q279* enters the cell nucleus and inhibits Gpn1-EYFP nuclear export. This property and the transcriptional deficit in Gpn3R Q279*-expressing cells requires a PDZ-binding motif generated by the Q279* mutation. We conclude that this PDZ-binding motif resulting from the Q279* mutation causes Gpn3 nuclear entry, inhibits Gpn1 nuclear export and Gpn3-mediated RNAPII nuclear targeting. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  4. Infectious salmon anaemia virus nuclear export protein is encoded by a spliced gene product of genomic segment 7.

    PubMed

    Ramly, Rimatulhana Binti; Olsen, Christel M; Braaen, Stine; Rimstad, Espen

    2013-10-01

    Infectious salmon anaemia virus (ISAV) is an orthomyxovirus causing anaemia and circulatory disease with high mortality in farmed Atlantic salmon (Salmo salar). Orthomyxoviruses are unusual as RNA viruses as they replicate in the nucleus and some viral transcripts undergo splicing. The nuclear replication necessitates a tightly controlled nuclear import and export of viral proteins. From ISAV genomic segment 7 two known mRNAs are transcribed; one collinear with the genomic segment, coding for the non-structural protein, and one spliced transcript, S7ORF2, coding for a protein with unknown function. Here we report initial functional analysis of the S7ORF2 protein. The results indicate that S7ORF2 protein gradually accumulates in the host cell during virus replication cycle, locates predominantly in the cytoplasm and is a part of purified virus particles. Trapping of S7ORF2 in the nucleus was obtained by treatment with leptomycin B, an inhibitor of CRM1-mediated nuclear export, indicating that S7ORF2 use CRM1 for the nuclear exit. Immunofluorescent staining of cells over-expressing both S7ORF2 and matrix protein (M) showed co-localization in the nucleus. However, S7ORF2 protein was found to interact with both the viral nucleoprotein (NP) and M proteins in ISAV infected cells as well as in purified viral particles. These results indicate that the S7ORF2 could be called the ISAV nuclear export protein, ISAV/NEP. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. 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

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

    PubMed

    Chutiwitoonchai, Nopporn; Aida, Yoko

    2016-07-28

    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.

  7. 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. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. 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

  9. 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

  10. CRM1 blockade by selective inhibitors of nuclear export attenuates kidney cancer growth.

    PubMed

    Inoue, Hiromi; Kauffman, Michael; Shacham, Sharon; Landesman, Yosef; Yang, Joy; Evans, Christopher P; Weiss, Robert H

    2013-06-01

    Renal cell carcinoma often presents asymptomatically and patients are commonly diagnosed at the metastatic stage, when treatment options are limited and survival is poor. Since progression-free survival using current therapy for metastatic renal cell carcinoma is only 1 to 2 years and existing drugs are associated with a high resistance rate, new pharmacological targets are needed. We identified and evaluated the nuclear exporter protein CRM1 as a novel potential therapy for renal cell carcinoma. We tested the efficacy of the CRM1 inhibitors KPT-185 and 251 in several renal cell carcinoma cell lines and in a renal cell carcinoma xenograft model. Apoptosis and cell cycle arrest were quantified and localization of p53 family proteins was assessed using standard techniques. KPT-185 attenuated CRM1 and showed increased cytotoxicity in renal cell carcinoma cells in vitro with evidence of increased apoptosis as well as cell cycle arrest. KPT-185 caused p53 and p21 to remain primarily in the nucleus in all renal cell carcinoma cell lines, suggesting that the mechanism of action of these compounds depends on tumor suppressor protein localization. Furthermore, when administered orally in a high grade renal cell carcinoma xenograft model, the bioavailable CRM1 inhibitor KPT-251 significantly inhibited tumor growth in vivo with the expected on target effects and no obvious toxicity. The CRM1 inhibitor protein family is a novel therapeutic target for renal cell carcinoma that deserves further intensive investigation for this and other urological malignancies. Copyright © 2013 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

  11. Nuclear DNA damage signalling to mitochondria in ageing

    PubMed Central

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

    2016-01-01

    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

  12. The Mex67p-mediated nuclear mRNA export pathway is conserved from yeast to human.

    PubMed Central

    Katahira, J; Strässer, K; Podtelejnikov, A; Mann, M; Jung, J U; Hurt, E

    1999-01-01

    Human TAP is an orthologue of the yeast mRNA export factor Mex67p. In mammalian cells, TAP has a preferential intranuclear localization, but can also be detected at the nuclear pores and shuttles between the nucleus and the cytoplasm. TAP directly associates with mRNA in vivo, as it can be UV-crosslinked to poly(A)+ RNA in HeLa cells. Both the FG-repeat domain of nucleoporin CAN/Nup214 and a novel human 15 kDa protein (p15) with homology to NTF2 (a nuclear transport factor which associates with RanGDP), directly bind to TAP. When green fluorescent protein (GFP)-tagged TAP and p15 are expressed in yeast, they localize to the nuclear pores. Strikingly, co-expression of human TAP and p15 restores growth of the otherwise lethal mex67::HIS3/mtr2::HIS3 double knockout strain. Thus, the human TAP-p15 complex can functionally replace the Mex67p-Mtr2p complex in yeast and thus performs a conserved role in nuclear mRNA export. PMID:10228171

  13. Cocaine induces nuclear export and degradation of neuronal retinoid X receptor-γ via a TNF-α/JNK- mediated mechanism.

    PubMed

    Kovalevich, Jane; Yen, William; Ozdemir, Ahmet; Langford, Dianne

    2015-03-01

    Cocaine abuse represents an immense societal health and economic burden for which no effective treatment currently exists. Among the numerous intracellular signaling cascades impacted by exposure to cocaine, increased and aberrant production of pro-inflammatory cytokines in the CNS has been observed. Additionally, we have previously reported a decrease in retinoid-X-receptor-gamma (RXR-γ) in brains of mice chronically exposed to cocaine. Through obligate heterodimerization with a number of nuclear receptors, RXRs serve as master regulatory transcription factors, which can potentiate or suppress expression of a wide spectrum of genes. Little is known about the regulation of RXR levels, but previous studies indicate cellular stressors such as cytokines negatively regulate levels of RXRs in vitro. To evaluate the mechanism underlying the cocaine-induced decreases in RXR-γ levels observed in vivo, we exposed neurons to cocaine in vitro and examined pathways which may contribute to disruption in RXR signaling, including activation of stress pathways by cytokine induction. In these studies, we provide the first evidence that cocaine exposure disrupts neuronal RXR-γ signaling in vitro by promoting its nuclear export and degradation. Furthermore, we demonstrate this effect may be mediated, at least in part, by cocaine-induced production of TNF-α and its downstream effector c-Jun-NH-terminal kinase (JNK). Findings from this study are therefore applicable to both cocaine abuse and to pathological conditions characterized by neuroinflammatory factors, such as neurodegenerative disease.

  14. Dual requirement for yeast hnRNP Nab2p in mRNA poly(A) tail length control and nuclear export

    PubMed Central

    Hector, Ronald E.; Nykamp, Keith R.; Dheur, Sonia; Anderson, James T.; Non, Priscilla J.; Urbinati, Carl R.; Wilson, Scott M.; Minvielle-Sebastia, Lionel; Swanson, Maurice S.

    2002-01-01

    Recent studies of mRNA export factors have provided additional evidence for a mechanistic link between mRNA 3′-end formation and nuclear export. Here, we identify Nab2p as a nuclear poly(A)-binding protein required for both poly(A) tail length control and nuclear export of mRNA. Loss of NAB2 expression leads to hyperadenylation and nuclear accumulation of poly(A)+ RNA but, in contrast to mRNA export mutants, these defects can be uncoupled in a nab2 mutant strain. Previous studies have implicated the cytoplasmic poly(A) tail-binding protein Pab1p in poly(A) tail length control during polyadenylation. Although cells are viable in the absence of NAB2 expression when PAB1 is overexpressed, Pab1p fails to resolve the nab2Δ hyperadenylation defect even when Pab1p is tagged with a nuclear localization sequence and targeted to the nucleus. These results indicate that Nab2p is essential for poly(A) tail length control in vivo, and we demonstrate that Nab2p activates polyadenylation, while inhibiting hyperadenylation, in the absence of Pab1p in vitro. We propose that Nab2p provides an important link between the termination of mRNA polyadenylation and nuclear export. PMID:11927564

  15. 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.

  16. Nuclear import/export of hRPF1/Nedd4 regulates the ubiquitin-dependent degradation of its nuclear substrates.

    PubMed

    Hamilton, M H; Tcherepanova, I; Huibregtse, J M; McDonnell, D P

    2001-07-13

    The ubiquitin-protein ligase (E3), hRPF1/Nedd4, is a component of the ubiquitin-proteasome pathway responsible for substrate recognition and specificity. Although previously characterized as a regulator of the stability of cytoplasmic proteins, hRPF1/Nedd4 has also been suggested to have a role in the nucleus. However, in light of the cytoplasmic localization of hRPF1/Nedd4, it is unclear whether bona fide nuclear substrates of hRPF1/Nedd4 exist, and if so, what mechanism may allow a cytoplasmic ubiquitin ligase to manifest nuclear activity. Our search for nuclear substrates led to the identification of the human proline-rich transcript, brain-expressed (hPRTB) protein, the ubiquitination and degradation of which is regulated by hRPF1/Nedd4. Interestingly, hPRTB colocalizes with the splicing factor SC35 in nuclear speckles. Finally, we demonstrate that hRPF1/Nedd4 is indeed capable of entering the nucleus; however, the presence of a functional Rev-like nuclear export sequence in hRPF1/Nedd4 ensures a predominant cytoplasmic localization. Cumulatively, these findings highlight a nuclear role for the ubiquitin ligase hRPF1/Nedd4 and underscore cytoplasmic/nuclear localization as an important regulatory component of hRPF1/Nedd4-substrate recognition.

  17. Sphingosine Kinase 1 Serves as a Pro-Viral Factor by Regulating Viral RNA Synthesis and Nuclear Export of Viral Ribonucleoprotein Complex upon Influenza Virus Infection

    PubMed Central

    Seo, Young-Jin; Pritzl, Curtis J.; Vijayan, Madhuvanthi; Bomb, Kavita; McClain, Mariah E.; Alexander, Stephen; Hahm, Bumsuk

    2013-01-01

    Influenza continues to pose a threat to humans by causing significant morbidity and mortality. Thus, it is imperative to investigate mechanisms by which influenza virus manipulates the function of host factors and cellular signal pathways. In this study, we demonstrate that influenza virus increases the expression and activation of sphingosine kinase (SK) 1, which in turn regulates diverse cellular signaling pathways. Inhibition of SK suppressed virus-induced NF-κB activation and markedly reduced the synthesis of viral RNAs and proteins. Further, SK blockade interfered with activation of Ran-binding protein 3 (RanBP3), a cofactor of chromosome region maintenance 1 (CRM1), to inhibit CRM1-mediated nuclear export of the influenza viral ribonucleoprotein complex. In support of this observation, SK inhibition altered the phosphorylation of ERK, p90RSK, and AKT, which is the upstream signal of RanBP3/CRM1 activation. Collectively, these results indicate that SK is a key pro-viral factor regulating multiple cellular signal pathways triggered by influenza virus infection. PMID:24137500

  18. GSK-3beta acts upstream of Fyn kinase in regulation of nuclear export and degradation of NF-E2 related factor 2.

    PubMed

    Jain, Abhinav K; Jaiswal, Anil K

    2007-06-01

    NF-E2-related factor 2 (Nrf2) regulates expression and coordinated induction of a battery of chemoprotective genes in response to oxidative and electrophilic stress. This leads to protection against oxidative stress and neoplastic diseases. Nuclear import and export of Nrf2 play a significant role in control of nuclear levels of Nrf2 and thus the expression of Nrf2 down-stream genes. Tyrosine kinase Fyn phosphorylates tyrosine 568 of Nrf2 that leads to the nuclear export of Nrf2. In this study, we investigated the upstream factor(s) in regulation of Fyn and Fyn-mediated nuclear export of Nrf2. The investigations shed light on a novel mechanism of Nrf2 regulation in response to oxidative stress. We demonstrate that GSK-3beta acts upstream of Fyn kinase in control of nuclear export of Nrf2. Chemical and short interfering RNA-mediated inhibition of GSK-3beta led to nuclear accumulation of Nrf2 and transcriptional activation of the Nrf2 downstream gene nqo1. Chemical and short interfering RNA inhibition of GSK-3beta and Fyn individually and in combination revealed that both kinases follow the same pathway to regulate nuclear export of Nrf2. We further demonstrate that hydrogen peroxide phosphorylates tyrosine 216 of GSK-3beta. This leads to activation of GSK-3beta. The activated GSK-3beta phosphorylates Fyn at threonine residue(s). Phosphorylated Fyn accumulates in the nucleus and phosphorylates Nrf2 at tyrosine 568. This leads to nuclear export, ubiquitination, and degradation of Nrf2.

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

    SciTech Connect

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

    2016-03-04

    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. - Highlights: • The plant-derived natural product TGBG has broad-spectrum antiviral activity against seasonal influenza A and B viruses. • TGBG has a novel anti-viral mechanism of action that from differs from the currently available anti-influenza drugs. • TGBG hinders nuclear export of the influenza virus ribonucleoprotein (v

  20. Non-FG mediated transport of the large pre-ribosomal subunit through the nuclear pore complex by the mRNA export factor Gle2

    PubMed Central

    Occhipinti, Laura; Chang, Yiming; Altvater, Martin; Menet, Anna M.; Kemmler, Stefan; Panse, Vikram G.

    2013-01-01

    Multiple export receptors passage bound pre-ribosomes through nuclear pore complexes (NPCs) by transiently interacting with the Phe-Gly (FG) meshwork of their transport channels. Here, we reveal how the non-FG interacting yeast mRNA export factor Gly-Leu-FG lethal 2 (Gle2) functions in the export of the large pre-ribosomal subunit (pre-60S). Structure-guided studies uncovered conserved platforms used by Gle2 to export pre-60S: an uncharacterized basic patch required to bind pre-60S, and a second surface that makes non-FG contacts with the nucleoporin Nup116. A basic patch mutant of Gle2 is able to function in mRNA export, but not pre-60S export. Thus, Gle2 provides a distinct interaction platform to transport pre-60S to the cytoplasm. Notably, Gle2’s interaction platforms become crucial for pre-60S export when FG-interacting receptors are either not recruited to pre-60S or are impaired. We propose that large complex cargos rely on non-FG as well as FG-interactions for their efficient translocation through the nuclear pore complex channel. PMID:23907389

  1. Non-FG mediated transport of the large pre-ribosomal subunit through the nuclear pore complex by the mRNA export factor Gle2.

    PubMed

    Occhipinti, Laura; Chang, Yiming; Altvater, Martin; Menet, Anna M; Kemmler, Stefan; Panse, Vikram G

    2013-09-01

    Multiple export receptors passage bound pre-ribosomes through nuclear pore complexes (NPCs) by transiently interacting with the Phe-Gly (FG) meshwork of their transport channels. Here, we reveal how the non-FG interacting yeast mRNA export factor Gly-Leu-FG lethal 2 (Gle2) functions in the export of the large pre-ribosomal subunit (pre-60S). Structure-guided studies uncovered conserved platforms used by Gle2 to export pre-60S: an uncharacterized basic patch required to bind pre-60S, and a second surface that makes non-FG contacts with the nucleoporin Nup116. A basic patch mutant of Gle2 is able to function in mRNA export, but not pre-60S export. Thus, Gle2 provides a distinct interaction platform to transport pre-60S to the cytoplasm. Notably, Gle2's interaction platforms become crucial for pre-60S export when FG-interacting receptors are either not recruited to pre-60S or are impaired. We propose that large complex cargos rely on non-FG as well as FG-interactions for their efficient translocation through the nuclear pore complex channel.

  2. Discriminating anthropogenic climate change from natural climate oscillation signals in dissolved organic matter export from headwater catchments.

    NASA Astrophysics Data System (ADS)

    Creed, I. F.; Girma, S.; Quick, C.

    2012-12-01

    Headwater catchments are potential sentinels for climate change because their hydrology and biogeochemistry respond to changing environmental conditions at scales appropriate to changes in both weather and climate. Scientific investigations focusing on changes in climate-driven environmental change would benefit by understanding changes in the export pattern of dissolved organic matter, "DOM signatures", from headwater catchments. However, climatic change has complex influences on headwater catchment dynamics, and to determine the effects of climate change, we must be able to discriminate between anthropogenic climate warming (non-stationary linear trends) and natural climate oscillation (stationary oscillating cycles) signals within catchment DOM yields from catchments. We present an analytical framework based on wavelet theory for analyzing non-stationary and stationary signals in yearly time series of DOM yields. This analytical framework was used to test the following hypotheses: That yearly DOM yields in headwater catchments contain both non-stationary and stationary climate signals; That non-stationary signals are greater than stationary ones; And that the effects of these signals are greater in catchments with lower rates of change in water loading and lower water storage capacity (i.e., are more directly linked to atmospheric conditions). We applied this framework on the yearly time series of DOM yield from catchments in the Turkey Lakes Watershed in Ontario, Canada and show that DOM signals appear "decoupled". In all catchments, dissolved organic carbon (DOC) showed no non-stationary (declining trends) despite showing declining yearly water yields, but catchments with relatively lower water loading (lower elevation on windward side of mountain) showed stronger and larger DOC export than those with higher water loading (high elevation on same side of mountain). In contrast, catchments showed both significant declining trends and oscillating cycles in yearly

  3. Crystal structure of the Xpo1p nuclear export complex bound to the SxFG/PxFG repeats of the nucleoporin Nup42p.

    PubMed

    Koyama, Masako; Hirano, Hidemi; Shirai, Natsuki; Matsuura, Yoshiyuki

    2017-08-08

    Xpo1p (yeast CRM1) is the major nuclear export receptor that carries a plethora of proteins and ribonucleoproteins from the nucleus to cytoplasm. The passage of the Xpo1p nuclear export complex through nuclear pore complexes (NPCs) is facilitated by interactions with nucleoporins (Nups) containing extensive repeats of phenylalanine-glycine (so-called FG repeats), although the precise role of each Nup in the nuclear export reaction remains incompletely understood. Here we report structural and biochemical characterization of the interactions between the Xpo1p nuclear export complex and the FG repeats of Nup42p, a nucleoporin localized at the cytoplasmic face of yeast NPCs and has characteristic SxFG/PxFG sequence repeat motif. The crystal structure of Xpo1p-PKI-Nup42p-Gsp1p-GTP complex identified three binding sites for the SxFG/PxFG repeats on HEAT repeats 14-20 of Xpo1p. Mutational analyses of Nup42p showed that the conserved serines and prolines in the SxFG/PxFG repeats contribute to Xpo1p-Nup42p binding. Our structural and biochemical data suggest that SxFG/PxFG-Nups such as Nup42p and Nup159p at the cytoplasmic face of NPCs provide high-affinity docking sites for the Xpo1p nuclear export complex in the terminal stage of NPC passage and that subsequent disassembly of the nuclear export complex facilitates recycling of free Xpo1p back to the nucleus. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

  4. 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.

  5. Influenza A Virus Polymerase Recruits the RNA Helicase DDX19 to Promote the Nuclear Export of Viral mRNAs

    PubMed Central

    Diot, Cédric; Fournier, Guillaume; Dos Santos, Mélanie; Magnus, Julie; Komarova, Anastasia; van der Werf, Sylvie; Munier, Sandie; Naffakh, Nadia

    2016-01-01

    Enhancing the knowledge of host factors that are required for efficient influenza A virus (IAV) replication is essential to address questions related to pathogenicity and to identify targets for antiviral drug development. Here we focused on the interplay between IAV and DExD-box RNA helicases (DDX), which play a key role in cellular RNA metabolism by remodeling RNA-RNA or RNA-protein complexes. We performed a targeted RNAi screen on 35 human DDX proteins to identify those involved in IAV life cycle. DDX19 was a major hit. In DDX19-depleted cells the accumulation of viral RNAs and proteins was delayed, and the production of infectious IAV particles was strongly reduced. We show that DDX19 associates with intronless, unspliced and spliced IAV mRNAs and promotes their nuclear export. In addition, we demonstrate an RNA-independent association between DDX19 and the viral polymerase, that is modulated by the ATPase activity of DDX19. Our results provide a model in which DDX19 is recruited to viral mRNAs in the nucleus of infected cells to enhance their nuclear export. Information gained from this virus-host interaction improves the understanding of both the IAV replication cycle and the cellular function of DDX19. PMID:27653209

  6. 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

  7. 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

  8. Anti-tumor activity of selective inhibitor of nuclear export (SINE) compounds, is enhanced in non-Hodgkin lymphoma through combination with mTOR inhibitor and dexamethasone.

    PubMed

    Muqbil, Irfana; Aboukameel, Amro; Elloul, Sivan; Carlson, Robert; Senapedis, William; Baloglu, Erkan; Kauffman, Michael; Shacham, Sharon; Bhutani, Divaya; Zonder, Jeffrey; Azmi, Asfar S; Mohammad, Ramzi M

    2016-12-28

    In previous studies we demonstrated that targeting the nuclear exporter protein exportin-1 (CRM1/XPO1) by a selective inhibitor of nuclear export (SINE) compound is a viable therapeutic strategy against Non-Hodgkin Lymphoma (NHL). Our studies along with pre-clinical work from others led to the evaluation of the lead SINE compound, selinexor, in a phase 1 trial in patients with CLL or NHL (NCT02303392). Continuing our previous work, we studied combinations of selinexor-dexamethasone (DEX) and selinexor-everolimus (EVER) in NHL. Combination of selinexor with DEX or EVER resulted in enhanced cytotoxicity in WSU-DLCL2 and WSU-FSCCL cells which was consistent with enhanced apoptosis. Molecular analysis showed enhancement in the activation of apoptotic signaling and down-regulation of XPO1. This enhancement is consistent with the mechanism of action of these drugs in that both selinexor and DEX antagonize NF-κB (p65) and mTOR (EVER target) is an XPO1 cargo protein. SINE compounds, KPT-251 and KPT-276, showed activities similar to CHOP (cyclophosphamide-hydroxydaunorubicin-oncovin-prednisone) regimen in subcutaneous and disseminated NHL xenograft models in vivo. In both animal models the anti-lymphoma activity of selinexor is enhanced through combination with DEX or EVER. The in vivo activity of selinexor and related SINE compounds relative to 'standard of care' treatment is consistent with the objective responses observed in Phase I NHL patients treated with selinexor. Our pre-clinical data provide a rational basis for testing these combinations in Phase II NHL trials.

  9. Nuclear receptor signaling and cardiac energetics.

    PubMed

    Huss, Janice M; Kelly, Daniel P

    2004-09-17

    The heart has a tremendous capacity for ATP generation, allowing it to function as an efficient pump throughout the life of the organism. The adult myocardium uses either fatty acid or glucose oxidation as its main energy source. Under normal conditions, the adult heart derives most of its energy through oxidation of fatty acids in mitochondria. However, the myocardium has a remarkable ability to switch between carbohydrate and fat fuel sources so that ATP production is maintained at a constant rate in diverse physiological and dietary conditions. This fuel selection flexibility is important for normal cardiac function. Although cardiac energy conversion capacity and metabolic flux is modulated at many levels, an important mechanism of regulation occurs at the level of gene expression. The expression of genes involved in multiple energy transduction pathways is dynamically regulated in response to developmental, physiological, and pathophysiological cues. This review is focused on gene transcription pathways involved in short- and long-term regulation of myocardial energy metabolism. Much of our knowledge about cardiac metabolic regulation comes from studies focused on mitochondrial fatty acid oxidation. The genes involved in this key energy metabolic pathway are transcriptionally regulated by members of the nuclear receptor superfamily, specifically the fatty acid-activated peroxisome proliferator-activated receptors (PPARs) and the nuclear receptor coactivator, PPARgamma coactivator-1alpha (PGC-1alpha). The dynamic regulation of the cardiac PPAR/PGC-1 complex in accordance with physiological and pathophysiological states will be described.

  10. A set of enhanced green fluorescent protein concatemers for quantitative determination of nuclear localization signal strength.

    PubMed

    Böhm, Jennifer; Thavaraja, Ramya; Giehler, Susanne; Nalaskowski, Marcus M

    2017-09-15

    Regulated transport of proteins between nucleus and cytoplasm is an important process in the eukaryotic cell. In most cases, active nucleo-cytoplasmic protein transport is mediated by nuclear localization signal (NLS) and/or nuclear export signal (NES) motifs. In this study, we developed a set of vectors expressing enhanced GFP (EGFP) concatemers ranging from 2 to 12 subunits (2xEGFP to 12xEGFP) for analysis of NLS strength. As shown by in gel GFP fluorescence analysis and αGFP Western blotting, EGFP concatemers are expressed as fluorescent full-length proteins in eukaryotic cells. As expected, nuclear localization of concatemeric EGFPs decreases with increasing molecular weight. By oligonucleotide ligation this set of EGFP concatemers can be easily fused to NLS motifs. After determination of intracellular localization of EGFP concatemers alone and fused to different NLS motifs we calculated the size of a hypothetic EGFP concatemer showing a defined distribution of EGFP fluorescence between nucleus and cytoplasm (n/c ratio = 2). Clear differences of the size of the hypothetic EGFP concatemer depending on the fused NLS motif were observed. Therefore, we propose to use the size of this hypothetic concatemer as quantitative indicator for comparing strength of different NLS motifs. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. The Oncogenic Fusion Proteins SET-Nup214 and Sequestosome-1 (SQSTM1)-Nup214 Form Dynamic Nuclear Bodies and Differentially Affect Nuclear Protein and Poly(A)+ RNA Export.

    PubMed

    Port, Sarah A; Mendes, Adélia; Valkova, Christina; Spillner, Christiane; Fahrenkrog, Birthe; Kaether, Christoph; Kehlenbach, Ralph H

    2016-10-28

    Genetic rearrangements are a hallmark of several forms of leukemia and can lead to oncogenic fusion proteins. One example of an affected chromosomal region is the gene coding for Nup214, a nucleoporin that localizes to the cytoplasmic side of the nuclear pore complex (NPC). We investigated two such fusion proteins, SET-Nup214 and SQSTM1 (sequestosome)-Nup214, both containing C-terminal portions of Nup214. SET-Nup214 nuclear bodies containing the nuclear export receptor CRM1 were observed in the leukemia cell lines LOUCY and MEGAL. Overexpression of SET-Nup214 in HeLa cells leads to the formation of similar nuclear bodies that recruit CRM1, export cargo proteins, and certain nucleoporins and concomitantly affect nuclear protein and poly(A)(+) RNA export. SQSTM1-Nup214, although mostly cytoplasmic, also forms nuclear bodies and inhibits nuclear protein but not poly(A)(+) RNA export. The interaction of the fusion proteins with CRM1 is RanGTP-dependent, as shown in co-immunoprecipitation experiments and binding assays. Further analysis revealed that the Nup214 parts mediate the inhibition of nuclear export, whereas the SET or SQSTM1 part determines the localization of the fusion protein and therefore the extent of the effect. SET-Nup214 nuclear bodies are highly mobile structures, which are in equilibrium with the nucleoplasm in interphase and disassemble during mitosis or upon treatment of cells with the CRM1-inhibitor leptomycin B. Strikingly, we found that nucleoporins can be released from nuclear bodies and reintegrated into existing NPC. Our results point to nuclear bodies as a means of preventing the formation of potentially insoluble and harmful protein aggregates that also may serve as storage compartments for nuclear transport factors. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Nuclear export of human hepatitis B virus core protein and pregenomic RNA depends on the cellular NXF1-p15 machinery.

    PubMed

    Yang, Ching-Chun; Huang, Er-Yi; Li, Hung-Cheng; Su, Pei-Yi; Shih, Chiaho

    2014-01-01

    Hepatitis B virus (HBV) core protein (HBc) can shuttle between nucleus and cytoplasm. Cytoplasm-predominant HBc is clinically associated with severe liver inflammation. Previously, we found that HBc arginine-rich domain (ARD) can associate with a host factor NXF1 (TAP) by coimmunoprecipitation. It is well known that NXF1-p15 heterodimer can serve as a major export receptor of nuclear mRNA as a ribonucleoprotein complex (RNP). In the NXF1-p15 pathway, TREX (transcription/export) complex plays an important role in coupling nuclear pre-mRNA processing with mRNA export in mammalian cells. Here, we tested the hypothesis whether HBc and HBV specific RNA can be exported via the TREX and NXF1-p15 mediated pathway. We demonstrated here that HBc can physically and specifically associate with TREX components, and the NXF1-p15 export receptor by coimmunoprecipitation. Accumulation of HBc protein in the nucleus can be induced by the interference with TREX and NXF1-p15 mediated RNA export machinery. HBV transcripts encodes a non-spliced 3.5 kb pregenomic RNA (pgRNA) which can serve as a template for reverse transcription. Cytoplasmic HBV pgRNA appeared to be reduced by siRNA treatment specific for the NXF1-p15 complex by quantitative RT-qPCR and Northern blot analyses. This result suggests that the pgRNA was also exported via the NXF1-p15 machinery. We entertain the hypothesis that HBc protein can be exported as an RNP cargo via the mRNA export pathway by hijacking the TREX and NXF1-p15 complex. In our current and previous studies, HBc is not required for pgRNA accumulation in the cytoplasm. Furthermore, HBc ARD can mediate nuclear export of a chimeric protein containing HBc ARD in a pgRNA-independent manner. Taken together, it suggests that while both pgRNA and HBc protein exports are dependent on NXF1-p15, they are using the same export machinery in a manner independent of each other.

  13. Elevated expression of the nuclear export protein, Crm1 (exportin 1), associates with human oesophageal squamous cell carcinoma.

    PubMed

    van der Watt, Pauline J; Zemanay, Widaad; Govender, Dhirendra; Hendricks, Denver T; Parker, M I; Leaner, Virna D

    2014-08-01

    The nuclear export receptor, Crm1 (exportin 1), is involved in the nuclear translocation of proteins and certain RNAs from the nucleus to the cytoplasm and is thus crucial for the correct localisation of cellular components. Crm1 has recently been reported to be highly expressed in certain types of cancers, yet its expression in oesophageal cancer has not been investigated to date. We investigated the expression of Crm1 in normal and tumour tissues derived from 56 patients with human oesophageal squamous cell carcinoma and its functional significance in oesophageal cancer cell line models. Immunohistochemistry revealed that Crm1 expression was significantly elevated in oesophageal tumour tissues compared to normal tissues and its localisation shifted from predominantly nuclear to nuclear and cytoplasmic. Real‑time RT‑PCR revealed that Crm1 expression was elevated at the mRNA level. To determine the functional significance of elevated Crm1 expression in oesophageal cancer, its expression was inhibited using siRNA, and a significant decrease in cell proliferation was observed associated with G1 cell cycle arrest and the induction of apoptosis. Similarly, leptomycin B (LMB) treatment resulted in the effective killing of oesophageal cancer cells at nanomolar concentrations. Normal oesophageal epithelial cells, however, were much less sensitive to Crm1 inhibition with siRNA and LMB. Together, this study reveals that Crm1 expression is increased in oesophageal cancer and is required for the proliferation and survival of oesophageal cancer cells.

  14. 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

  15. Integration of light and photoperiodic signaling in transcriptional nuclear foci

    PubMed Central

    Kaiserli, Eirini; Paldi, Katalin; O'Donnell, Liz; Batalov, Olga; Pedmale, Ullas V.; Nusinow, Dmitri A.; Kay, Steve A.; Chory, Joanne

    2015-01-01

    Summary Light regulates major plant developmental transitions by orchestrating a series of nuclear events. This study uncovers the molecular function of the natural variant, TZP (Tandem Zinc-finger-Plus3), as a novel signal integrator of light and photoperiodic pathways in transcriptional nuclear foci. We report that TZP acts as a positive regulator of photoperiodic flowering via physical interactions with the red-light receptor phytochrome B (phyB). We demonstrate that TZP localizes in dynamic nuclear domains regulated by light quality and photoperiod. This study shows that phyB is indispensible not only for localizing TZP to transcriptionally active nuclear photobodies, but also for recruiting TZP on the promoter of the floral inducer FLOWERING LOCUS T (FT). Our findings signify a unique transcriptional regulatory role to the highly enigmatic plant nuclear photobodies, where TZP directly activates FT gene expression and promotes flowering. PMID:26555051

  16. 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. Copyright © 2015 by the Genetics Society of America.

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

    PubMed Central

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

    2015-01-01

    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

  18. RNA-binding proteins of the NXF (nuclear export factor) family and their connection with the cytoskeleton.

    PubMed

    Mamon, L A; Ginanova, V R; Kliver, S F; Yakimova, A O; Atsapkina, A A; Golubkova, E V

    2017-04-01

    The mutual relationship between mRNA and the cytoskeleton can be seen from two points of view. On the one hand, the cytoskeleton is necessary for mRNA trafficking and anchoring to subcellular domains. On the other hand, cytoskeletal growth and rearrangement require the translation of mRNAs that are connected to the cytoskeleton. β-actin mRNA localization may influence dynamic changes in the actin cytoskeleton. In the cytoplasm, long-lived mRNAs exist in the form of RNP (ribonucleoprotein) complexes, where they interact with RNA-binding proteins, including NXF (Nuclear eXport Factor). Dm NXF1 is an evolutionarily conserved protein in Drosophila melanogaster that has orthologs in different animals. The universal function of nxf1 genes is the nuclear export of different mRNAs in various organisms. In this mini-review, we briefly discuss the evidence demonstrating that Dm NXF1 fulfils not only universal but also specialized cytoplasmic functions. This protein is detected not only in the nucleus but also in the cytoplasm. It is a component of neuronal granules. Dm NXF1 marks nuclear division spindles during early embryogenesis and the dense body on one side of the elongated spermatid nuclei. The characteristic features of sbr mutants (sbr(10) and sbr(5) ) are impairment of chromosome segregation and spindle formation anomalies during female meiosis. sbr(12) mutant sterile males with immobile spermatozoa exhibit disturbances in the axoneme, mitochondrial derivatives and cytokinesis. These data allow us to propose that the Dm NXF1 proteins transport certain mRNAs in neurites and interact with localized mRNAs that are necessary for dynamic changes of the cytoskeleton.

  19. Expanded polyglutamine domain possesses nuclear export activity which modulates subcellular localization and toxicity of polyQ disease protein via exportin-1.

    PubMed

    Chan, Wing Man; Tsoi, Ho; Wu, Chi Chung; Wong, Chi Hang; Cheng, Tat Cheung; Li, Hoi Yeung; Lau, Kwok Fai; Shaw, Pang Chui; Perrimon, Norbert; Chan, Ho Yin Edwin

    2011-05-01

    Polyglutamine (polyQ) diseases are a group of late-onset, progressive neurodegenerative disorders caused by CAG trinucleotide repeat expansion in the coding region of disease genes. The cell nucleus is an important site of pathology in polyQ diseases, and transcriptional dysregulation is one of the pathologic hallmarks observed. In this study, we showed that exportin-1 (Xpo1) regulates the nucleocytoplasmic distribution of expanded polyQ protein. We found that expanded polyQ protein, but not its unexpanded form, possesses nuclear export activity and interacts with Xpo1. Genetic manipulation of Xpo1 expression levels in transgenic Drosophila models of polyQ disease confirmed the specific nuclear export role of Xpo1 on expanded polyQ protein. Upon Xpo1 knockdown, the expanded polyQ protein was retained in the nucleus. The nuclear disease protein enhanced polyQ toxicity by binding to heat shock protein (hsp) gene promoter and abolished hsp gene induction. Further, we uncovered a developmental decline of Xpo1 protein levels in vivo that contributes to the accumulation of expanded polyQ protein in the nucleus of symptomatic polyQ transgenic mice. Taken together, we first showed that Xpo1 is a nuclear export receptor for expanded polyQ domain, and our findings establish a direct link between protein nuclear export and the progressive nature of polyQ neurodegeneration.

  20. The Mtr2-Mex67 NTF2-like domain complex. Structural insights into a dual role of Mtr2 for yeast nuclear export.

    PubMed

    Senay, Claire; Ferrari, Paul; Rocher, Corinne; Rieger, Klaus-Jörg; Winter, Jacques; Platel, Denis; Bourne, Yves

    2003-11-28

    The formation of the Mtr2-Mex67 heterodimer is essential for yeast mRNA export as it constitutes a key nuclear component for shuttling mRNA between the nuclear and cytoplasm compartments through the nuclear pore complex. We report the crystal structures of apo-Mtr2 from the human pathogen Candida albicans and of its complex with the Mex67 NTF2-like domain. Compared with other members of the NTF2 fold family, Mtr2 displays novel structural features involved in the nuclear export of the large ribosomal subunit and consistent with a dual functional role of Mtr2 during yeast nuclear export events. The structure of the Mtr2-Mex67 NTF2-like domain complex, which overall is similar to those of the human and Saccharomyces cerevisiae homologs, unveils three putative Phe-Gly repeat binding sites, of which one contributes to the heterodimer interface. These structures exemplify an unrecognized adaptability of the NTF2 building block in evolution, identify novel structural determinants associated with key biological functions at the molecular surface of the yeast Mtr2-Mex67 complex, and suggest that the yeast and human mRNA export machineries may differ.

  1. 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.

  2. 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. Copyright © 2015. Published by Elsevier Ltd.

  3. NXF-2, REF-1, and REF-2 affect the choice of nuclear export pathway for tra-2 mRNA in C. elegans.

    PubMed

    Kuersten, Scott; Segal, Scott P; Verheyden, Jamie; LaMartina, Sarah M; Goodwin, Elizabeth B

    2004-06-04

    In C. elegans, tra-2 mRNA nuclear export is controlled by a 3'UTR element, the TRE. In the absence of TRA-1, the TRE retains tra-2 mRNA in the nucleus. The binding of TRA-1 to the 3'UTR overcomes this retention resulting in export of a TRA-1/tra-2 mRNA complex. Here, we find that, unlike most mRNAs, tra-2 mRNA exits the nucleus via an alternative pathway to NXF-1 that requires CRM1 activity. Inhibition of export by NXF-1 depends upon the TRE, CeNXF-2, CeREF-1, and CeREF-2. Removal of the TRE or any one of these factors results in export of tra-2 by NXF-1. NXF-2 and REF-1 specifically bind the TRE, suggesting that they directly control tra-2 mRNA export. Furthermore, choice of proper export pathway affects tra-2 translational control. Therefore, tra-2 mRNA export is highly regulated and plays an important role in development by regulating the activity of tra-2 mRNA in the cytoplasm.

  4. Golgi Export of the Kir2.1 Channel is Driven by a Trafficking Signal Located within Tertiary Structure

    PubMed Central

    Ma, Donghui; Taneja, Tarvinder Kaur; Hagen, Brian M.; Kim, Bo-Young; Ortega, Bernardo; Lederer, W. Jonathan; Welling, Paul A.

    2011-01-01

    Mechanisms responsible for sorting newly synthesized proteins for traffic to the cell surface from the Golgi are poorly understood. Here we show that the potassium channel Kir2.1, mutations in which are associated with Andersen-Tawil Syndrome, is selected as cargo into Golgi export carriers in an unusual signal-dependent manner. Unlike conventional trafficking signals, which are typically comprised of short linear peptide sequences, Golgi exit of Kir2.1 is dictated by residues embedded within the confluence of two separate domains. This signal patch forms a recognition site for interaction with the AP1 adaptor complex, thereby marking Kir2.1 for incorporation into clathrin-coated vesicles at the trans-Golgi. The identification of a trafficking signal in the tertiary structure of Kir2.1 reveals a quality control step that couples protein conformation to Golgi export and provides molecular insight into how mutations in Kir2.1 arrest the channels at the Golgi. PMID:21703452

  5. 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

  6. 75 FR 44072 - Export and Import of Nuclear Equipment and Material; Updates and Clarifications

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-07-28

    ... Clarifications AGENCY: Nuclear Regulatory Commission. ACTION: Final rule. SUMMARY: The United States Nuclear... Sec. Sec. 110.32, 110.43, and 110.50. Imports of radioactive material into the United States under a... the United States would be considered ``radioactive waste'' under 10 CFR part 110. Exclusion two...

  7. Sumoylation regulates nuclear accumulation and signaling activity of the soluble intracellular domain of the erbb4 receptor tyrosine kinase.

    PubMed

    Knittle, Anna Maria; Helkkula, Maria; Johnson, Mark S; Sundvall, Maria; Elenius, Klaus

    2017-10-03

    Erb-B2 receptor tyrosine kinase 4 (ErbB4) is a kinase that can signal via a proteolytically released intracellular domain (ICD) in addition to classical receptor tyrosine kinase-activated signaling cascades. Previously, we have demonstrated that ErbB4 ICD is posttranslationally modified by the small ubiquitin-like modifier (SUMO) and functionally interacts with the PIAS3 SUMO E3 ligase. However, direct evidence of SUMO modification in ErbB4 signaling has remained elusive. Here, we report that the conserved lysine residue 714 in the ErbB4 ICD undergoes SUMO modification, which was reversed by sentrin-specific proteases (SENPs) 1, 2 and 5. Although ErbB4 kinase activity was not necessary for the SUMOylation, the SUMOylated ErbB4 ICD was tyrosine phosphorylated to a higher extent than unmodified ErbB4 ICD. Mutation of the SUMOylation site neither compromised ErbB4-induced phosphorylation of the canonical signaling pathway effectors Erk1/2, Akt, or STAT5 nor ErbB4 stability. In contrast, SUMOylation was required for nuclear accumulation of the ErbB4 ICD. We also found that Lys-714 was located within a leucine-rich stretch, which resembles a nuclear export signal, and could be inactivated by site-directed mutagenesis. Furthermore, SUMOylation modulated the interaction of ErbB4 with chromosomal region maintenance 1 (CRM1), the major nuclear export receptor for proteins. Finally, the SUMO acceptor lysine was functionally required for ErbB4 ICD-mediated inhibition of mammary epithelial cell differentiation in a three-dimensional cell culture model. Our findings indicate that a SUMOylation-mediated mechanism regulates nuclear localization and function of the ICD of ErbB4 receptor tyrosine kinase. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  8. HuR and GRSF1 modulate the nuclear export and mitochondrial localization of the lncRNA RMRP

    PubMed Central

    Noh, Ji Heon; Kim, Kyoung Mi; Abdelmohsen, Kotb; Yoon, Je-Hyun; Panda, Amaresh C.; Munk, Rachel; Kim, Jiyoung; Curtis, Jessica; Moad, Christopher A.; Wohler, Christina M.; Indig, Fred E.; de Paula, Wilson; Dudekula, Dawood B.; De, Supriyo; Piao, Yulan; Yang, Xiaoling; Martindale, Jennifer L.; de Cabo, Rafael; Gorospe, Myriam

    2016-01-01

    Some mitochondrial long noncoding RNAs (lncRNAs) are encoded by nuclear DNA, but the mechanisms that mediate their transport to mitochondria are poorly characterized. Using affinity RNA pull-down followed by mass spectrometry analysis, we found two RNA-binding proteins (RBPs), HuR (human antigen R) and GRSF1 (G-rich RNA sequence-binding factor 1), that associated with the nuclear DNA-encoded lncRNA RMRP and mobilized it to mitochondria. In cultured human cells, HuR bound RMRP in the nucleus and mediated its CRM1 (chromosome region maintenance 1)-dependent export to the cytosol. After RMRP was imported into mitochondria, GRSF1 bound RMRP and increased its abundance in the matrix. Loss of GRSF1 lowered the mitochondrial levels of RMRP, in turn suppressing oxygen consumption rates and modestly reducing mitochondrial DNA replication priming. Our findings indicate that RBPs HuR and GRSF1 govern the cytoplasmic and mitochondrial localization of the lncRNA RMRP, which is encoded by nuclear DNA but has key functions in mitochondria. PMID:27198227

  9. Impaired nuclear export of tumor-derived c-terminal truncated cyclin D1 mutant in ESCC cancer.

    PubMed

    Hao, Meili; Chen, Xiangmei; Zhang, Ting; Shen, Tao; Xie, Qing; Xing, Xiujuan; Gu, Hongxi; Lu, Fengmin

    2011-11-01

    Cyclin D1 is a significant regulator of the G1- to S-phase transition and is often aberrant in human tumors of various origins. Although cancer-derived cyclin D1 mutants are potent oncogenes in vitro and in vivo, the mechanisms by which they contribute to neoplasia remaind to be elucidated. We previously identified a cyclin D1 mutation (Δ266-295) in esophageal cancer with deleted codons from 266 to 295 of wild-type cyclin D1, the critical COOH-terminal regulatory sequences necessary for cyclin D1 nuclear export. In the present study, this cancer-derived cyclin D1-Δ266-295 was shown to be a constitutively nuclear cyclin D1 protein with a significantly increased oncogenic potential. Moreover, the cancer-derived cyclin D1-Δ266-295 mutant was found to retain its ability to bind to and activate CDK4, which in turn phosphorylates and inactivates the pRb protein and promotes cell cycle progression. In comparison to wild-type cyclin D1a, D1-Δ266-295 exhibited enforced nuclear accumulation. In addition, the transient transfection and ectopic expression of this nuclear localized D1-Δ266-295 up-regulated endogenous Notch1 expression, indicating that the mutant retained its ability as a transcriptional regulator. Furthermore, data from the flow cytometry assay showed that D1-Δ266-295 fractionally increased >4N cell accumulation, and further analysis suggested the retriggering of DNA replication relevant to its inhibition of Cdt1 proteolysis. Therefore, the inappropriate nuclear localization of this cyclin D1 mutant may interfere with DNA replication in cultured cells, thereby contributing to genomic instability.

  10. RNA-binding protein PSPC1 promotes the differentiation-dependent nuclear export of adipocyte RNAs.

    PubMed

    Wang, Jiexin; Rajbhandari, Prashant; Damianov, Andrey; Han, Areum; Sallam, Tamer; Waki, Hironori; Villanueva, Claudio J; Lee, Stephen D; Nielsen, Ronni; Mandrup, Susanne; Reue, Karen; Young, Stephen G; Whitelegge, Julian; Saez, Enrique; Black, Douglas L; Tontonoz, Peter

    2017-03-01

    A highly orchestrated gene expression program establishes the properties that define mature adipocytes, but the contribution of posttranscriptional factors to the adipocyte phenotype is poorly understood. Here we have shown that the RNA-binding protein PSPC1, a component of the paraspeckle complex, promotes adipogenesis in vitro and is important for mature adipocyte function in vivo. Cross-linking and immunoprecipitation followed by RNA sequencing revealed that PSPC1 binds to intronic and 3'-untranslated regions of a number of adipocyte RNAs, including the RNA encoding the transcriptional regulator EBF1. Purification of the paraspeckle complex from adipocytes further showed that PSPC1 associates with the RNA export factor DDX3X in a differentiation-dependent manner. Remarkably, PSPC1 relocates from the nucleus to the cytoplasm during differentiation, coinciding with enhanced export of adipogenic RNAs. Mice lacking PSPC1 in fat displayed reduced lipid storage and adipose tissue mass and were resistant to diet-induced obesity and insulin resistance due to a compensatory increase in energy expenditure. These findings highlight a role for PSPC1-dependent RNA maturation in the posttranscriptional control of adipose development and function.

  11. 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

  12. A Sub-Element in PRE enhances nuclear export of intronless mRNAs by recruiting the TREX complex via ZC3H18.

    PubMed

    Chi, Binkai; Wang, Ke; Du, Yanhua; Gui, Bin; Chang, Xingya; Wang, Lantian; Fan, Jing; Chen, She; Wu, Xudong; Li, Guohui; Cheng, Hong

    2014-06-01

    Viral RNA elements that facilitate mRNA export are useful tools for identifying cellular RNA export factors. Here we show that hepatitis B virus post-transcriptional element (PRE) is one such element, and using PRE several new cellular mRNA export factors were identified. We found that PRE drastically enhances the cytoplasmic accumulation of cDNA transcripts independent of any viral protein. Systematic deletion analysis revealed the existence of a 116 nt functional Sub-Element of PRE (SEP1). The RNP that forms on the SEP1 RNA was affinity purified, in which TREX components as well as several other proteins were identified. TREX components and the SEP1-associating protein ZC3H18 are required for SEP1-mediated mRNA export. Significantly, ZC3H18 directly binds to the SEP1 RNA, interacts with TREX and is required for stable association of TREX with the SEP1-containing mRNA. Requirements for SEP1-mediated mRNA export are similar to those for splicing-dependent mRNA export. Consistent with these similarities, several SEP1-interacting proteins, including ZC3H18, ARS2, Acinus and Brr2, are required for efficient nuclear export of polyA RNAs. Together, our data indicate that SEP1 enhances mRNA export by recruiting TREX via ZC3H18. The new mRNA export factors that we identified might be involved in cap- and splicing-dependent TREX recruitment to cellular mRNAs.

  13. A Sub-Element in PRE enhances nuclear export of intronless mRNAs by recruiting the TREX complex via ZC3H18

    PubMed Central

    Chi, Binkai; Wang, Ke; Du, Yanhua; Gui, Bin; Chang, Xingya; Wang, Lantian; Fan, Jing; Chen, She; Wu, Xudong; Li, Guohui; Cheng, Hong

    2014-01-01

    Viral RNA elements that facilitate mRNA export are useful tools for identifying cellular RNA export factors. Here we show that hepatitis B virus post-transcriptional element (PRE) is one such element, and using PRE several new cellular mRNA export factors were identified. We found that PRE drastically enhances the cytoplasmic accumulation of cDNA transcripts independent of any viral protein. Systematic deletion analysis revealed the existence of a 116 nt functional Sub-Element of PRE (SEP1). The RNP that forms on the SEP1 RNA was affinity purified, in which TREX components as well as several other proteins were identified. TREX components and the SEP1-associating protein ZC3H18 are required for SEP1-mediated mRNA export. Significantly, ZC3H18 directly binds to the SEP1 RNA, interacts with TREX and is required for stable association of TREX with the SEP1-containing mRNA. Requirements for SEP1-mediated mRNA export are similar to those for splicing-dependent mRNA export. Consistent with these similarities, several SEP1-interacting proteins, including ZC3H18, ARS2, Acinus and Brr2, are required for efficient nuclear export of polyA RNAs. Together, our data indicate that SEP1 enhances mRNA export by recruiting TREX via ZC3H18. The new mRNA export factors that we identified might be involved in cap- and splicing-dependent TREX recruitment to cellular mRNAs. PMID:24782531

  14. Distinctive Nuclear Localization Signals in the Oomycete Phytophthora sojae

    PubMed Central

    Fang, Yufeng; Jang, Hyo Sang; Watson, Gregory W.; Wellappili, Dulani P.; Tyler, Brett M.

    2017-01-01

    To date, nuclear localization signals (NLSs) that target proteins to nuclei in oomycetes have not been defined, but have been assumed to be the same as in higher eukaryotes. Here, we use the soybean pathogen Phytophthora sojae as a model to investigate these sequences in oomycetes. By establishing a reliable in vivo NLS assay based on confocal microscopy, we found that many canonical monopartite and bipartite classical NLSs (cNLSs) mediated nuclear import poorly in P. sojae. We found that efficient localization of P. sojae nuclear proteins by cNLSs requires additional basic amino acids at distal sites or collaboration with other NLSs. We found that several representatives of another well-characterized NLS, proline-tyrosine NLS (PY-NLS) also functioned poorly in P. sojae. To characterize PY-NLSs in P. sojae, we experimentally defined the residues required by functional PY-NLSs in three P. sojae nuclear-localized proteins. These results showed that functional P. sojae PY-NLSs include an additional cluster of basic residues for efficient nuclear import. Finally, analysis of several highly conserved P. sojae nuclear proteins including ribosomal proteins and core histones revealed that these proteins exhibit a similar but stronger set of sequence requirements for nuclear targeting compared with their orthologs in mammals or yeast. PMID:28210240

  15. Distinctive Nuclear Localization Signals in the Oomycete Phytophthora sojae.

    PubMed

    Fang, Yufeng; Jang, Hyo Sang; Watson, Gregory W; Wellappili, Dulani P; Tyler, Brett M

    2017-01-01

    To date, nuclear localization signals (NLSs) that target proteins to nuclei in oomycetes have not been defined, but have been assumed to be the same as in higher eukaryotes. Here, we use the soybean pathogen Phytophthora sojae as a model to investigate these sequences in oomycetes. By establishing a reliable in vivo NLS assay based on confocal microscopy, we found that many canonical monopartite and bipartite classical NLSs (cNLSs) mediated nuclear import poorly in P. sojae. We found that efficient localization of P. sojae nuclear proteins by cNLSs requires additional basic amino acids at distal sites or collaboration with other NLSs. We found that several representatives of another well-characterized NLS, proline-tyrosine NLS (PY-NLS) also functioned poorly in P. sojae. To characterize PY-NLSs in P. sojae, we experimentally defined the residues required by functional PY-NLSs in three P. sojae nuclear-localized proteins. These results showed that functional P. sojae PY-NLSs include an additional cluster of basic residues for efficient nuclear import. Finally, analysis of several highly conserved P. sojae nuclear proteins including ribosomal proteins and core histones revealed that these proteins exhibit a similar but stronger set of sequence requirements for nuclear targeting compared with their orthologs in mammals or yeast.

  16. Light signaling controls nuclear architecture reorganization during seedling establishment.

    PubMed

    Bourbousse, Clara; Mestiri, Imen; Zabulon, Gerald; Bourge, Mickaël; Formiggini, Fabio; Koini, Maria A; Brown, Spencer C; Fransz, Paul; Bowler, Chris; Barneche, Fredy

    2015-05-26

    The spatial organization of chromatin can be subject to extensive remodeling in plant somatic cells in response to developmental and environmental signals. However, the mechanisms controlling these dynamic changes and their functional impact on nuclear activity are poorly understood. Here, we determined that light perception triggers a switch between two different nuclear architectural schemes during Arabidopsis postembryonic development. Whereas progressive nucleus expansion and heterochromatin rearrangements in cotyledon cells are achieved similarly under light and dark conditions during germination, the later steps that lead to mature nuclear phenotypes are intimately associated with the photomorphogenic transition in an organ-specific manner. The light signaling integrators DE-ETIOLATED 1 and CONSTITUTIVE PHOTOMORPHOGENIC 1 maintain heterochromatin in a decondensed state in etiolated cotyledons. In contrast, under light conditions cryptochrome-mediated photoperception releases nuclear expansion and heterochromatin compaction within conspicuous chromocenters. For all tested loci, chromatin condensation during photomorphogenesis does not detectably rely on DNA methylation-based processes. Notwithstanding, the efficiency of transcriptional gene silencing may be impacted during the transition, as based on the reactivation of transposable element-driven reporter genes. Finally, we report that global engagement of RNA polymerase II in transcription is highly increased under light conditions, suggesting that cotyledon photomorphogenesis involves a transition from globally quiescent to more active transcriptional states. Given these findings, we propose that light-triggered changes in nuclear architecture underlie interplays between heterochromatin reorganization and transcriptional reprogramming associated with the establishment of photosynthesis.

  17. Light signaling controls nuclear architecture reorganization during seedling establishment

    PubMed Central

    Bourbousse, Clara; Mestiri, Imen; Zabulon, Gerald; Bourge, Mickaël; Formiggini, Fabio; Koini, Maria A.; Brown, Spencer C.; Fransz, Paul; Bowler, Chris; Barneche, Fredy

    2015-01-01

    The spatial organization of chromatin can be subject to extensive remodeling in plant somatic cells in response to developmental and environmental signals. However, the mechanisms controlling these dynamic changes and their functional impact on nuclear activity are poorly understood. Here, we determined that light perception triggers a switch between two different nuclear architectural schemes during Arabidopsis postembryonic development. Whereas progressive nucleus expansion and heterochromatin rearrangements in cotyledon cells are achieved similarly under light and dark conditions during germination, the later steps that lead to mature nuclear phenotypes are intimately associated with the photomorphogenic transition in an organ-specific manner. The light signaling integrators DE-ETIOLATED 1 and CONSTITUTIVE PHOTOMORPHOGENIC 1 maintain heterochromatin in a decondensed state in etiolated cotyledons. In contrast, under light conditions cryptochrome-mediated photoperception releases nuclear expansion and heterochromatin compaction within conspicuous chromocenters. For all tested loci, chromatin condensation during photomorphogenesis does not detectably rely on DNA methylation-based processes. Notwithstanding, the efficiency of transcriptional gene silencing may be impacted during the transition, as based on the reactivation of transposable element-driven reporter genes. Finally, we report that global engagement of RNA polymerase II in transcription is highly increased under light conditions, suggesting that cotyledon photomorphogenesis involves a transition from globally quiescent to more active transcriptional states. Given these findings, we propose that light-triggered changes in nuclear architecture underlie interplays between heterochromatin reorganization and transcriptional reprogramming associated with the establishment of photosynthesis. PMID:25964332

  18. Exportin 5 is a RanGTP-dependent dsRNA-binding protein that mediates nuclear export of pre-miRNAs.

    PubMed

    Bohnsack, Markus T; Czaplinski, Kevin; Gorlich, Dirk

    2004-02-01

    microRNAs (miRNAs) are widespread among eukaryotes, and studies in several systems have revealed that miRNAs can regulate expression of specific genes. Primary miRNA transcripts are initially processed to approximately 70-nucleotide (nt) stem-loop structures (pre-miRNAs), exported to the cytoplasm, further processed to yield approximately 22-nt dsRNAs, and finally incorporated into ribonucleoprotein particles, which are thought to be the active species. Here we study nuclear export of pre-miRNAs and show that the process is saturable and thus carrier-mediated. Export is sensitive to depletion of nuclear RanGTP and, according to this criterion, mediated by a RanGTP-dependent exportin. An unbiased affinity chromatography approach with immobilized pre-miRNAs identified exportin 5 as the pre-miRNA-specific export carrier. We have cloned exportin 5 from Xenopus and demonstrate that antibodies raised against the Xenopus receptor specifically block pre-miRNA export from nuclei of Xenopus oocytes. We further show that exportin 5 interacts with double-stranded RNA in a sequence-independent manner.

  19. 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.

  20. Optimal ROS Signaling Is Critical for Nuclear Reprogramming.

    PubMed

    Zhou, Gang; Meng, Shu; Li, Yanhui; Ghebre, Yohannes T; Cooke, John P

    2016-05-03

    Efficient nuclear reprogramming of somatic cells to pluripotency requires activation of innate immunity. Because innate immune activation triggers reactive oxygen species (ROS) signaling, we sought to determine whether there was a role of ROS signaling in nuclear reprogramming. We examined ROS production during the reprogramming of doxycycline (dox)-inducible mouse embryonic fibroblasts (MEFs) carrying the Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc [OSKM]) into induced pluripotent stem cells (iPSCs). ROS generation was substantially increased with the onset of reprogramming. Depletion of ROS via antioxidants or Nox inhibitors substantially decreased reprogramming efficiency. Similarly, both knockdown and knockout of p22(phox)-a critical subunit of the Nox (1-4) complex-decreased reprogramming efficiency. However, excessive ROS generation using genetic and pharmacological approaches also impaired reprogramming. Overall, our data indicate that ROS signaling is activated early with nuclear reprogramming, and optimal levels of ROS signaling are essential to induce pluripotency. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  1. Analysis of nuclear export using photoactivatable GFP fusion proteins and interspecies heterokaryons.

    PubMed

    Nakrieko, Kerry-Ann; Ivanova, Iordanka A; Dagnino, Lina

    2010-01-01

    In this chapter, we review protocols for the analysis of nucleocytoplasmic shuttling of transcription factors and nuclear proteins, using two different approaches. The first involves the use of photoactivatable forms of the protein of interest by fusion to photoactivatable green fluorescent protein to follow its movement out of the nucleus by live-cell confocal microscopy. This methodology allows for the kinetic characterization of protein movements as well as measurement of steady-state levels. In a second procedure to assess the ability of a nuclear protein to move into and out of the nucleus, we describe the use of interspecies heterokaryon assays, which provide a measurement of steady-state distribution. These technologies are directly applicable to the analysis of nucleocytoplasmic movements not only of transcription factors, but also other nuclear proteins.

  2. Dual Loss of ER Export and Endocytic Signals with Altered Melanosome Morphology in the silver Mutation of Pmel17

    PubMed Central

    Theos, Alexander C.; Berson, Joanne F.; Theos, Sarah C.; Herman, Kathryn E.; Harper, Dawn C.; Tenza, Danièle; Sviderskaya, Elena V.; Lamoreux, M. Lynn; Bennett, Dorothy C.; Raposo, Graça

    2006-01-01

    Pmel17 is a pigment cell-specific integral membrane protein that participates in the formation of the intralumenal fibrils upon which melanins are deposited in melanosomes. The Pmel17 cytoplasmic domain is truncated by the mouse silver mutation, which is associated with coat hypopigmentation in certain strain backgrounds. Here, we show that the truncation interferes with at least two steps in Pmel17 intracellular transport, resulting in defects in melanosome biogenesis. Human Pmel17 engineered with the truncation found in the mouse silver mutant (hPmel17si) is inefficiently exported from the endoplasmic reticulum (ER). Localization and metabolic pulse-chase analyses with site-directed mutants and chimeric proteins show that this effect is due to the loss of a conserved C-terminal valine that serves as an ER exit signal. hPmel17si that exits the ER accumulates abnormally at the plasma membrane due to the loss of a di-leucine–based endocytic signal. The combined effects of reduced ER export and endocytosis significantly deplete Pmel17 within endocytic compartments and delay proteolytic maturation required for premelanosome-like fibrillogenesis. The ER export delay and cell surface retention are also observed for endogenous Pmel17si in melanocytes from silver mice, within which Pmel17 accumulation in premelanosomes is dramatically reduced. Mature melanosomes in these cells are larger, rounder, more highly pigmented, and less striated than in control melanocytes. These data reveal a dual sorting defect in a natural mutant of Pmel17 and support a requirement of endocytic trafficking in Pmel17 fibril formation. PMID:16760433

  3. Dual loss of ER export and endocytic signals with altered melanosome morphology in the silver mutation of Pmel17.

    PubMed

    Theos, Alexander C; Berson, Joanne F; Theos, Sarah C; Herman, Kathryn E; Harper, Dawn C; Tenza, Danièle; Sviderskaya, Elena V; Lamoreux, M Lynn; Bennett, Dorothy C; Raposo, Graça; Marks, Michael S

    2006-08-01

    Pmel17 is a pigment cell-specific integral membrane protein that participates in the formation of the intralumenal fibrils upon which melanins are deposited in melanosomes. The Pmel17 cytoplasmic domain is truncated by the mouse silver mutation, which is associated with coat hypopigmentation in certain strain backgrounds. Here, we show that the truncation interferes with at least two steps in Pmel17 intracellular transport, resulting in defects in melanosome biogenesis. Human Pmel17 engineered with the truncation found in the mouse silver mutant (hPmel17si) is inefficiently exported from the endoplasmic reticulum (ER). Localization and metabolic pulse-chase analyses with site-directed mutants and chimeric proteins show that this effect is due to the loss of a conserved C-terminal valine that serves as an ER exit signal. hPmel17si that exits the ER accumulates abnormally at the plasma membrane due to the loss of a di-leucine-based endocytic signal. The combined effects of reduced ER export and endocytosis significantly deplete Pmel17 within endocytic compartments and delay proteolytic maturation required for premelanosome-like fibrillogenesis. The ER export delay and cell surface retention are also observed for endogenous Pmel17si in melanocytes from silver mice, within which Pmel17 accumulation in premelanosomes is dramatically reduced. Mature melanosomes in these cells are larger, rounder, more highly pigmented, and less striated than in control melanocytes. These data reveal a dual sorting defect in a natural mutant of Pmel17 and support a requirement of endocytic trafficking in Pmel17 fibril formation.

  4. Combining dehydration, construct optimization and improved data collection to solve the crystal structure of a CRM1-RanGTP-SPN1-Nup214 quaternary nuclear export complex.

    PubMed

    Monecke, Thomas; Dickmanns, Achim; Weiss, Manfred S; Port, Sarah A; Kehlenbach, Ralph H; Ficner, Ralf

    2015-12-01

    High conformational flexibility is an intrinsic and indispensable property of nuclear transport receptors, which makes crystallization and structure determination of macromolecular complexes containing exportins or importins particularly challenging. Here, the crystallization and structure determination of a quaternary nuclear export complex consisting of the exportin CRM1, the small GTPase Ran in its GTP-bound form, the export cargo SPN1 and an FG repeat-containing fragment of the nuclear pore complex component nucleoporin Nup214 fused to maltose-binding protein is reported. Optimization of constructs, seeding and the development of a sophisticated protocol including successive PEG-mediated crystal dehydration as well as additional post-mounting steps were essential to obtain well diffracting crystals.

  5. RNA helicase MOV10 functions as a co-factor of HIV-1 Rev to facilitate Rev/RRE-dependent nuclear export of viral mRNAs

    SciTech Connect

    Huang, Feng; Zhang, Junsong; Zhang, Yijun; Geng, Guannan; Liang, Juanran; Li, Yingniang; Chen, Jingliang; Liu, Chao; Zhang, Hui

    2015-12-15

    Human immunodeficiency virus type 1 (HIV-1) exploits multiple host factors during its replication. The REV/RRE-dependent nuclear export of unspliced/partially spliced viral transcripts needs the assistance of host proteins. Recent studies have shown that MOV10 overexpression inhibited HIV-1 replication at various steps. However, the endogenous MOV10 was required in certain step(s) of HIV-1 replication. In this report, we found that MOV10 potently enhances the nuclear export of viral mRNAs and subsequently increases the expression of Gag protein and other late products through affecting the Rev/RRE axis. The co-immunoprecipitation analysis indicated that MOV10 interacts with Rev in an RNA-independent manner. The DEAG-box of MOV10 was required for the enhancement of Rev/RRE-dependent nuclear export and the DEAG-box mutant showed a dominant-negative activity. Our data propose that HIV-1 utilizes the anti-viral factor MOV10 to function as a co-factor of Rev and demonstrate the complicated effects of MOV10 on HIV-1 life cycle. - Highlights: • MOV10 can function as a co-factor of HIV-1 Rev. • MOV10 facilitates Rev/RRE-dependent transport of viral mRNAs. • MOV10 interacts with Rev in an RNA-independent manner. • The DEAG-box of MOV10 is required for the enhancement of Rev/RRE-dependent export.

  6. Inhibition of Cellular Protein Secretion by Norwalk Virus Nonstructural Protein p22 Requires a Mimic of an Endoplasmic Reticulum Export Signal

    PubMed Central

    Sharp, Tyler M.; Guix, Susana; Katayama, Kazuhiko; Crawford, Sue E.; Estes, Mary K.

    2010-01-01

    Protein trafficking between the endoplasmic reticulum (ER) and Golgi apparatus is central to cellular homeostasis. ER export signals are utilized by a subset of proteins to rapidly exit the ER by direct uptake into COPII vesicles for transport to the Golgi. Norwalk virus nonstructural protein p22 contains a YXΦESDG motif that mimics a di-acidic ER export signal in both sequence and function. However, unlike normal ER export signals, the ER export signal mimic of p22 is necessary for apparent inhibition of normal COPII vesicle trafficking, which leads to Golgi disassembly and antagonism of Golgi-dependent cellular protein secretion. This is the first reported function for p22. Disassembly of the Golgi apparatus was also observed in cells replicating Norwalk virus, which may contribute to pathogenesis by interfering with cellular processes that are dependent on an intact secretory pathway. These results indicate that the ER export signal mimic is critical to the antagonistic function of p22, shown herein to be a novel antagonist of ER/Golgi trafficking. This unique and well-conserved human norovirus motif is therefore an appealing target for antiviral drug development. PMID:20976190

  7. 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

  8. 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.

  9. 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.

  10. The unique signal concept for detonation safety in nuclear weapons

    SciTech Connect

    Spray, S.D.; Cooper, J.A.

    1993-06-01

    The purpose of a unique signal (UQS) in a nuclear weapon system is to provide an unambiguous communication of intent to detonate from the UQS information input source device to a stronglink safety device in the weapon in a manner that is highly unlikely to be duplicated or simulated in normal environments and in a broad range of ill-defined abnormal environments. This report presents safety considerations for the design and implementation of UQSs in the context of the overall safety system.

  11. 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.

  12. A translocator-specific export signal establishes the translocator-effector secretion hierarchy that is important for type III secretion system function.

    PubMed

    Tomalka, Amanda G; Stopford, Charles M; Lee, Pei-Chung; Rietsch, Arne

    2012-12-01

    Type III secretion systems are used by many Gram-negative pathogens to directly deliver effector proteins into the cytoplasm of host cells. To accomplish this, bacteria secrete translocator proteins that form a pore in the host-cell membrane through which the effector proteins are then introduced into the host cell. Evidence from multiple systems indicates that the pore-forming translocator proteins are exported before effectors, but how this secretion hierarchy is established is unclear. Here we used the Pseudomonas aeruginosa translocator protein PopD as a model to identify its export signals. The N-terminal secretion signal and chaperone, PcrH, are required for export under all conditions. Two novel signals in PopD, one proximal to the chaperone binding site and one at the very C-terminus of the protein, are required for export of PopD before effector proteins. These novel export signals establish the translocator-effector secretion hierarchy, which in turn, is critical for the delivery of effectors into host cells. © 2012 Blackwell Publishing Ltd.

  13. A translocator-specific export signal establishes the translocator-effector secretion hierarchy that is important for type III secretion system function

    PubMed Central

    Tomalka, Amanda G.; Stopford, Charles M.; Lee, Pei-Chung; Rietsch, Arne

    2012-01-01

    Summary Type III secretion systems are used by many Gram-negative pathogens to directly deliver effector proteins into the cytoplasm of host cells. To accomplish this, bacteria secrete translocator proteins that form a pore in the host-cell membrane through which the effector proteins are then introduced into the host cell. Evidence from multiple systems indicates that the pore-forming translocator proteins are exported before effectors, but how this secretion hierarchy is established is unclear. Here we used the P. aeruginosa translocator protein PopD as a model to identify its export signals. The amino-terminal secretion signal and chaperone, PcrH, are required for export under all conditions. Two novel signals in PopD, one proximal to the chaperone-binding site and one at the very C-terminus of the protein, are required for export of PopD before effector proteins. These novel export signals establish the translocator-effector secretion hierarchy, which in turn, is critical for the delivery of effectors into host cells. PMID:23121689

  14. Nuclear pore localization and nucleocytoplasmic transport of eIF-5A: evidence for direct interaction with the export receptor CRM1.

    PubMed

    Rosorius, O; Reichart, B; Krätzer, F; Heger, P; Dabauvalle, M C; Hauber, J

    1999-07-01

    Eukaryotic initiation factor 5A (eIF-5A) is the only cellular protein known to contain the unusual amino acid hypusine. The exact in vivo function of eIF-5A, however, is to date unknown. The finding that eIF-5A is an essential cofactor of the human immunodeficiency virus type 1 (HIV-1) Rev RNA transport factor suggested that eIF-5A is part of a specific nuclear export pathway. In this study we used indirect immunofluorescence and immunogold electron microscopy to demonstrate that eIF-5A accumulates at nuclear pore-associated intranuclear filaments in mammalian cells and Xenopus oocytes. We are able to show that eIF-5A interacts with the general nuclear export receptor, CRM1. Furthermore, microinjection studies in somatic cells revealed that eIF-5A is transported from the nucleus to the cytoplasm, and that this nuclear export is blocked by leptomycin B. Our data demonstrate that eIF-5A is a nucleocytoplasmic shuttle protein.

  15. Explaining state autonomy and state capacity: A comparison of US and West German policies on arms and nuclear exports and East-West trade

    SciTech Connect

    Hofhansel, C.

    1988-01-01

    Two central theoretical concerns of the recent statist literature, state autonomy and state capacity are analyzed in the context of policy making in two states (West Germany and the United States) across three different issue areas (nuclear exports, conventional arms exports, and East-West trade). The empirical core of the thesis consists of six case studies involving a West German reactor export to Argentina, an American reactor export to the Philippines, U.S. and West German arms exports to Saudi Arabia, the West German participation in the Siberian gas pipeline deal of 1982, and the attempt by the Reagan administration to block this deal. The larger theoretical goal was to make a contribution towards an overall evaluation of the liberal, statist, and neo-Marxist approaches to state autonomy and state capacity. The evidence presented suggests that the strong/weak state distinction has limited utility for understanding foreign economic policy making and is in fact flawed. Institutional factors, which are at the core of the statist approach, did not have the predicted effects.

  16. Characterization of nuclear localization signals and cytoplasmic retention region in the nuclear receptor CAR.

    PubMed

    Kanno, Yuichiro; Suzuki, Motoyoshi; Nakahama, Takayuki; Inouye, Yoshio

    2005-09-10

    The constitutive androstane receptor (CAR) is a ligand/activator-dependent transactivation factor that resides in the cytoplasm and forms part of an as yet unidentified protein complex. Upon stimulation, CAR translocates into the nucleus where it modulates the transactivation of target genes. However, CAR exogenously expressed in rat liver RL-34 cells is located in the nucleus even in the absence of activators. By transiently transfecting RL-34 cells with various mutated rat CAR segments, we identified two nuclear localization signals: a basic amino acid-rich sequence (RRARQARRR) between amino acids 100 and 108; and an assembly of noncontiguous residues widely spread over amino acid residues 111 to 320 within the ligand binding domain. A C-terminal leucine-rich segment corresponding to a previously reported murine xenochemical response signal was not found to exhibit nuclear import activity in cultured cells. Using rat primary hepatocytes transfected with various CAR segments, we identified the region required for the cytoplasmic retention of CAR. Based on these results, the intracellular localization of CAR would be determined by the combined effects of nuclear localization signals, the xenochemical response signal, and the cytoplasmic retention region.

  17. Classic Nuclear Localization Signals and a Novel Nuclear Localization Motif Are Required for Nuclear Transport of Porcine Parvovirus Capsid Proteins

    PubMed Central

    Boisvert, Maude; Bouchard-Lévesque, Véronique; Fernandes, Sandra

    2014-01-01

    ABSTRACT Nuclear targeting of capsid proteins (VPs) is important for genome delivery and precedes assembly in the replication cycle of porcine parvovirus (PPV). Clusters of basic amino acids, corresponding to potential nuclear localization signals (NLS), were found only in the unique region of VP1 (VP1up, for VP1 unique part). Of the five identified basic regions (BR), three were important for nuclear localization of VP1up: BR1 was a classic Pat7 NLS, and the combination of BR4 and BR5 was a classic bipartite NLS. These NLS were essential for viral replication. VP2, the major capsid protein, lacked these NLS and contained no region with more than two basic amino acids in proximity. However, three regions of basic clusters were identified in the folded protein, assembled into a trimeric structure. Mutagenesis experiments showed that only one of these three regions was involved in VP2 transport to the nucleus. This structural NLS, termed the nuclear localization motif (NLM), is located inside the assembled capsid and thus can be used to transport trimers to the nucleus in late steps of infection but not for virions in initial infection steps. The two NLS of VP1up are located in the N-terminal part of the protein, externalized from the capsid during endosomal transit, exposing them for nuclear targeting during early steps of infection. Globally, the determinants of nuclear transport of structural proteins of PPV were different from those of closely related parvoviruses. IMPORTANCE Most DNA viruses use the nucleus for their replication cycle. Thus, structural proteins need to be targeted to this cellular compartment at two distinct steps of the infection: in early steps to deliver viral genomes to the nucleus and in late steps to assemble new viruses. Nuclear targeting of proteins depends on the recognition of a stretch of basic amino acids by cellular transport proteins. This study reports the identification of two classic nuclear localization signals in the minor

  18. Expanding the definition of the classical bipartite nuclear localization signal.

    PubMed

    Lange, Allison; McLane, Laura M; Mills, Ryan E; Devine, Scott E; Corbett, Anita H

    2010-03-01

    Nuclear localization signals (NLSs) are amino acid sequences that target cargo proteins into the nucleus. Rigorous characterization of NLS motifs is essential to understanding and predicting pathways for nuclear import. The best-characterized NLS is the classical NLS (cNLS), which is recognized by the cNLS receptor, importin-alpha. cNLSs are conventionally defined as having one (monopartite) or two clusters of basic amino acids separated by a 9-12 aa linker (bipartite). Motivated by the finding that Ty1 integrase, which contains an unconventional putative bipartite cNLS with a 29 aa linker, exploits the classical nuclear import machinery, we assessed the functional boundaries for linker length within a bipartite cNLS. We confirmed that the integrase cNLS is a bona fide bipartite cNLS, then carried out a systematic analysis of linker length in an obligate bipartite cNLS cargo, which revealed that some linkers longer than conventionally defined can function in nuclear import. Linker function is dependent on the sequence and likely the inherent flexibility of the linker. Subsequently, we interrogated the Saccharomyces cerevisiae proteome to identify cellular proteins containing putative long bipartite cNLSs. We experimentally confirmed that Rrp4 contains a bipartite cNLS with a 25 aa linker. Our studies show that the traditional definition of bipartite cNLSs is too restrictive and linker length can vary depending on amino acid composition.

  19. Adaptive mutation in nuclear export protein allows stable transgene expression in a chimaeric influenza A virus vector.

    PubMed

    Kuznetsova, Irina; Shurygina, Anna-Polina; Wolf, Brigitte; Wolschek, Markus; Enzmann, Florian; Sansyzbay, Abylay; Khairullin, Berik; Sandybayev, Nurlan; Stukova, Marina; Kiselev, Oleg; Egorov, Andrej; Bergmann, Michael

    2014-02-01

    The development of influenza virus vectors with long insertions of foreign sequences remains difficult due to the small size and instable nature of the virus. Here, we used the influenza virus inherent property of self-optimization to generate a vector stably expressing long transgenes from the NS1 protein ORF. This was achieved by continuous selection of bright fluorescent plaques of a GFP-expressing vector during multiple passages in mouse B16f1 cells. The newly generated vector acquired stability in IFN-competent cell lines and in vivo in murine lungs. Although improved vector fitness was associated with the appearance of four coding mutations in the polymerase (PB2), haemagglutinin and non-structural (NS) segments, the stability of the transgene expression was dependent primarily on the single mutation Q20R in the nuclear export protein (NEP). Importantly, a longer insert, such as a cassette of 1299 nt encoding two Mycobacterium tuberculosis Esat6 and Ag85A proteins, could substitute for the GFP transgene. Thus, the inherent property of the influenza virus to adapt can also be used to adjust a vector backbone to give stable expression of long transgenes.

  20. The dependence of Ig class-switching on the nuclear export sequence of AID likely reflects interaction with factors additional to Crm1 exportin.

    PubMed

    Ellyard, Julia I; Benk, Amelie S; Taylor, Benjamin; Rada, Cristina; Neuberger, Michael S

    2011-02-01

    Activation-induced deaminase (AID) is a B lymphocyte-specific DNA deaminase that triggers Ig class-switch recombination (CSR) and somatic hypermutation. It shuttles between cytoplasm and nucleus, containing a nuclear export sequence (NES) at its carboxyterminus. Intriguingly, the precise nature of this NES is critical to AID's function in CSR, though not in somatic hypermutation. Many alterations to the NES, while preserving its nuclear export function, destroy CSR ability. We have previously speculated that AID's ability to potentiate CSR may critically depend on the affinity of interaction between its NES and Crm1 exportin. Here, however, by comparing multiple AID NES mutants, we find that - beyond a requirement for threshold Crm1 binding - there is little correlation between CSR and Crm1 binding affinity. The results suggest that CSR, as well as the stabilisation of AID, depend on an interaction between the AID C-terminal decapeptide and factor(s) additional to Crm1.

  1. The dependence of Ig class-switching on the nuclear export sequence of AID likely reflects interaction with factors additional to Crm1 exportin

    PubMed Central

    Ellyard, Julia I; Benk, Amelie S; Taylor, Benjamin; Rada, Cristina; Neuberger, Michael S

    2011-01-01

    Activation-induced deaminase (AID) is a B lymphocyte-specific DNA deaminase that triggers Ig class-switch recombination (CSR) and somatic hypermutation. It shuttles between cytoplasm and nucleus, containing a nuclear export sequence (NES) at its carboxyterminus. Intriguingly, the precise nature of this NES is critical to AID's function in CSR, though not in somatic hypermutation. Many alterations to the NES, while preserving its nuclear export function, destroy CSR ability. We have previously speculated that AID's ability to potentiate CSR may critically depend on the affinity of interaction between its NES and Crm1 exportin. Here, however, by comparing multiple AID NES mutants, we find that – beyond a requirement for threshold Crm1 binding – there is little correlation between CSR and Crm1 binding affinity. The results suggest that CSR, as well as the stabilisation of AID, depend on an interaction between the AID C-terminal decapeptide and factor(s) additional to Crm1. PMID:21268017

  2. Multiple nuclear localization signals function in the nuclear import of the transcription factor Nrf2.

    PubMed

    Theodore, Melanie; Kawai, Yumiko; Yang, Jianqi; Kleshchenko, Yuliya; Reddy, Sekhar P; Villalta, Fernando; Arinze, Ifeanyi J

    2008-04-04

    Nuclear factor erythroid 2-related factor 2 (Nrf2) mediates the transcriptional response of cells to oxidative stress and is translocated into the nucleus following, or concomitant with, its activation by electrophiles or reactive oxygen species. The mechanism of its translocation into the nucleus is not entirely elucidated. Here we have identified two novel nuclear localization signal (NLS) motifs in murine Nrf2, one located near the N-terminal region (amino acid residues 42-53) and the other (residues 587-593) located near the C-terminal region. Imaging of green fluorescent protein (GFP)-tagged Nrf2 revealed that mutation(s) in any of these sequences resulted in decreased nuclear fluorescence intensity compared with the wild-type Nrf2 when Nrf2 activation was induced with the electrophile tert-butylhydroquinone. The mutations also impaired Nrf2-induced transactivation of antioxidant response element-driven reporter gene expression to the same extent as the Nrf2 construct bearing mutation in a previously identified bipartite NLS that maps at residues 494-511. When linked to GFP or to GFP-PEPCK-C each of the novel NLS motifs was sufficient to drive nuclear translocation of the fusion proteins. Co-immunoprecipitation assays demonstrated that importins alpha5 and beta1 associate with Nrf2, an interaction that was blocked by the nuclear import inhibitor SN50. SN50 also blocked tert-butylhydroquinone-induced nuclear fluorescence of GFP-Nrf2 in cells transfected with wild-type GFP-Nrf2. Overall these results reveal that multiple NLS motifs in Nrf2 function in its nuclear translocation in response to pro-oxidant stimuli and that the importin alpha-beta heterodimer nuclear import receptor system plays a critical role in the import process.

  3. THOC5, a member of the mRNA export complex: a novel link between mRNA export machinery and signal transduction pathways in cell proliferation and differentiation.

    PubMed

    Tran, Doan D H; Koch, Alexandra; Tamura, Teruko

    2014-01-10

    Cell growth, differentiation, and commitment to a restricted lineage are guided by a timely expressed set of growth factor/cytokine receptors and their down-stream transcription factor genes. Transcriptional control mechanisms of gene expression during differentiation have been mainly studied by focusing on the cis- and trans-elements in promoters however, the role of mRNA export machinery during differentiation has not been adequately examined. THO (Suppressors of the transcriptional defects of hpr1 delta by overexpression) complex 5 (THOC5) is a member of THO complex which is a subcomplex of the transcription/export complex (TREX). THOC5 is evolutionarily conserved in higher eukaryotes, however the exact roles of THOC5 in transcription and mRNA export are still unclear. In this review, we focus on recently uncovered aspects of the role of THOC5 in signal transduction induced by extracellular stimuli. THOC5 is phosphorylated by several protein kinases at multiple residues upon extracellular stimuli. These include stimulation with growth factors/cytokines/chemokines, or DNA damage reagents. Furthermore, THOC5 is a substrate for several oncogenic tyrosine kinases, suggesting that THOC5 may be involved in cancer development. Recent THOC5 knockout mouse data reveal that THOC5 is an essential element in the maintenance of stem cells and growth factor/cytokine-mediated differentiation/proliferation. Furthermore, depletion of THOC5 influences less than 1% of total mRNA export in the steady state, however it influences more than 90% of growth factor/cytokine induced genes. THOC5, thereby contributes to the 3' processing and/or export of immediate-early genes induced by extracellular stimuli. These studies bring new insight into the link between the mRNA export complex and immediate-early gene response. The data from these studies also suggest that THOC5 may be a useful tool for studying stem cell biology, for modifying the differentiation processes and for cancer therapy.

  4. Use of Synthetic Signal Sequences to Explore the Protein Export Machinery

    PubMed Central

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

    2010-01-01

    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

  5. The role of nuclear localization signal in parvovirus life cycle.

    PubMed

    Liu, Peng; Chen, Shun; Wang, Mingshu; Cheng, Anchun

    2017-04-14

    Parvoviruses are small, non-enveloped viruses with an approximately 5.0 kb, single-stranded DNA genome. Usually, the parvovirus capsid gene contains one or more nuclear localization signals (NLSs), which are required for guiding the virus particle into the nucleus through the nuclear pore. However, several classical NLSs (cNLSs) and non-classical NLSs (ncNLSs) have been identified in non-structural genes, and the ncNLSs can also target non-structural proteins into the nucleus. In this review, we have summarized recent research findings on parvovirus NLSs. The capsid protein of the adeno-associated virus has four potential nuclear localization sequences, named basic region 1 (BR), BR2, BR3 and BR4. BR3 was identified as an NLS by fusing it with green fluorescent protein. Moreover, BR3 and BR4 are required for infectivity and virion assembly. In Protoparvovirus, the canine parvovirus has a common cNLS located in the VP1 unique region, similar to parvovirus minute virus of mice (MVM) and porcine parvovirus. Moreover, an ncNLS is found in the C-terminal region of MVM VP1/2. Parvovirus B19 also contains an ncNLS in the C-terminal region of VP1/2, which is essential for the nuclear transport of VP1/VP2. Approximately 1 or 2 cNLSs and 1 ncNLS have been reported in the non-structural protein of bocaviruses. Understanding the role of the NLS in the process of parvovirus infection and its mechanism of nuclear transport will contribute to the development of therapeutic vaccines and novel antiviral medicines.

  6. RNA helicase MOV10 functions as a co-factor of HIV-1 Rev to facilitate Rev/RRE-dependent nuclear export of viral mRNAs.

    PubMed

    Huang, Feng; Zhang, Junsong; Zhang, Yijun; Geng, Guannan; Liang, Juanran; Li, Yingniang; Chen, Jingliang; Liu, Chao; Zhang, Hui

    2015-12-01

    Human immunodeficiency virus type 1 (HIV-1) exploits multiple host factors during its replication. The REV/RRE-dependent nuclear export of unspliced/partially spliced viral transcripts needs the assistance of host proteins. Recent studies have shown that MOV10 overexpression inhibited HIV-1 replication at various steps. However, the endogenous MOV10 was required in certain step(s) of HIV-1 replication. In this report, we found that MOV10 potently enhances the nuclear export of viral mRNAs and subsequently increases the expression of Gag protein and other late products through affecting the Rev/RRE axis. The co-immunoprecipitation analysis indicated that MOV10 interacts with Rev in an RNA-independent manner. The DEAG-box of MOV10 was required for the enhancement of Rev/RRE-dependent nuclear export and the DEAG-box mutant showed a dominant-negative activity. Our data propose that HIV-1 utilizes the anti-viral factor MOV10 to function as a co-factor of Rev and demonstrate the complicated effects of MOV10 on HIV-1 life cycle. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Altered RNA processing and export lead to retention of mRNAs near transcription sites and nuclear pore complexes or within the nucleolus

    PubMed Central

    Paul, Biplab; Montpetit, Ben

    2016-01-01

    Many protein factors are required for mRNA biogenesis and nuclear export, which are central to the eukaryotic gene expression program. It is unclear, however, whether all factors have been identified. Here we report on a screen of >1000 essential gene mutants in Saccharomyces cerevisiae for defects in mRNA processing and export, identifying 26 mutants with defects in this process. Single-molecule FISH data showed that the majority of these mutants accumulated mRNA within specific regions of the nucleus, which included 1) mRNAs within the nucleolus when nucleocytoplasmic transport, rRNA biogenesis, or RNA processing and surveillance was disrupted, 2) the buildup of mRNAs near transcription sites in 3′-end processing and chromosome segregation mutants, and 3) transcripts being enriched near nuclear pore complexes when components of the mRNA export machinery were mutated. These data show that alterations to various nuclear processes lead to the retention of mRNAs at discrete locations within the nucleus. PMID:27385342

  8. Regulation of RNA-binding proteins affinity to export receptors enables the nuclear basket proteins to distinguish and retain aberrant mRNAs

    PubMed Central

    Soheilypour, M.; Mofrad, M. R. K.

    2016-01-01

    Export of messenger ribonucleic acids (mRNAs) into the cytoplasm is a fundamental step in gene regulation processes, which is meticulously quality controlled by highly efficient mechanisms in eukaryotic cells. Yet, it remains unclear how the aberrant mRNAs are recognized and retained inside the nucleus. Using a new modelling approach for complex systems, namely the agent-based modelling (ABM) approach, we develop a minimal model of the mRNA quality control (QC) mechanism. Our results demonstrate that regulation of the affinity of RNA-binding proteins (RBPs) to export receptors along with the weak interaction between the nuclear basket protein (Mlp1 or Tpr) and RBPs are the minimum requirements to distinguish and retain aberrant mRNAs. Our results show that the affinity between Tpr and RBPs is optimized to maximize the retention of aberrant mRNAs. In addition, we demonstrate how the length of mRNA affects the QC process. Since longer mRNAs spend more time in the nuclear basket to form a compact conformation and initiate their export, nuclear basket proteins could more easily capture and retain them inside the nucleus. PMID:27805000

  9. Nuclear movement regulated by non-Smad Nodal signaling via JNK is associated with Smad signaling during zebrafish endoderm specification.

    PubMed

    Hozumi, Shunya; Aoki, Shun; Kikuchi, Yutaka

    2017-09-25

    Although asymmetric nuclear positioning is observed during animal development, the regulation and significance of this nuclear positioning in cell differentiation remains poorly understood. Using zebrafish blastulae, we provide evidence that nuclear movement toward the yolk syncytial layer, which comprises extraembryonic tissue, occurs in the first endoderm specified cells during endoderm specification. Nodal signaling is essential for nuclear movement, whereas nuclear envelope proteins are involved in the movement through the microtubule formation. The positioning of the microtubule organizing center, which is proposed to be critical for nuclear movement, is regulated by Nodal signaling and nuclear envelope proteins. The non-Smad JNK signaling pathway, which is downstream of Nodal signaling, regulates nuclear movement independent of the Smad pathway, and this nuclear movement is associated with Smad signal transduction toward the nucleus. Our study provides insights into the function of nuclear movement in Smad signaling toward the nucleus, and could be applied to the control of Transforming Growth Factor-β signaling. © 2017. Published by The Company of Biologists Ltd.

  10. Specific inhibition of the nuclear exporter exportin-1 attenuates kidney cancer growth.

    PubMed

    Wettersten, Hiromi I; Landesman, Yosef; Friedlander, Sharon; Shacham, Sharon; Kauffman, Michael; Weiss, Robert H

    2014-01-01

    Despite the advent of FDA-approved therapeutics to a limited number of available targets (kinases and mTOR), PFS of kidney cancer (RCC) has been extended only one to two years due to the development of drug resistance. Here, we evaluate a novel therapeutic for RCC which targets the exportin-1 (XPO1) inhibitor. RCC cells were treated with the orally available XPO1 inhibitor, KPT-330, and cell viability and Annexin V (apoptosis) assays, and cell cycle analyses were performed to evaluate the efficacy of KPT-330 in two RCC cell lines. Immunoblotting and immunofluorescence analysis were performed to validate mechanisms of XPO1 inhibition. The efficacy and on-target effects of KPT-330 were further analyzed in vivo in RCC xenograft mice, and KPT-330-resistant cells were established to evaluate potential mechanisms of KPT-330 resistance. KPT-330 attenuated RCC viability through growth inhibition and apoptosis induction both in vitro and in vivo, a process in which increased nuclear localization of p21 by XPO1 inhibition played a major role. In addition, KPT-330 resistant cells remained sensitive to the currently approved for RCC multi-kinase inhibitors (sunitinib, sorafenib) and mTOR inhibitors (everolimus, temsirolimus), suggesting that these targeted therapeutics would remain useful as second line therapeutics following KPT-330 treatment. The orally-available XPO1 inhibitor, KPT-330, represents a novel target for RCC whose in vivo efficacy approaches that of sunitinib. In addition, cells resistant to KPT-330 retain their ability to respond to available RCC therapeutics suggesting a novel approach for treatment in KPT-330-naïve as well as -resistant RCC patients.

  11. Membrane-anchored prolyl hydroxylase with an export signal from the endoplasmic reticulum.

    PubMed

    Yuasa, Koji; Toyooka, Kiminori; Fukuda, Hiroo; Matsuoka, Ken

    2005-01-01

    We cloned a novel prolyl 4-hydroxylase (PH; EC 1.14.11.2) homolog cDNA from tobacco (Nicotiana tabacum) BY-2 cells based on expression sequence tag information. Like other PHs, this tobacco PH polypeptide has two conserved histidine residues, and it comprises 286 amino acids with a calculated molecular mass of 32 kDa. Interestingly, this protein and homologs in Arabidopsis and rice have predicted transmembrane sequences in their N-terminal regions. This PH homolog was expressed in BY-2 cells as a His-tagged protein, and the expressed protein showed PH activity. Incubation of membranes with high salt, urea, and protease with or without detergents indicated that this protein is an integral membrane protein with a type II configuration. Its membrane-anchored nature is specific for plants because no integral membrane PH has been found in animals. A membrane fractionation study and immunocytochemical studies indicate that this protein localizes in both the endoplasmic reticulum (ER) and Golgi apparatus. Analysis of this protein fused to green fluorescent protein indicated that basic amino acids in the cytoplasmic, N-terminal region of the PH play a role in its export from the ER.

  12. Cellular stress stimulates nuclear localization signal (NLS) independent nuclear transport of MRJ

    SciTech Connect

    Andrews, Joel F.; Sykora, Landon J.; Barik Letostak, Tiasha; Menezes, Mitchell E.; Mitra, Aparna; Barik, Sailen; Shevde, Lalita A.; Samant, Rajeev S.

    2012-06-10

    HSP40 family member MRJ (DNAJB6) has been in the spot light for its relevance to Huntington's, Parkinson's diseases, limb-girdle muscular dystrophy, placental development, neural stem cells, cell cycle and malignancies such as breast cancer and melanoma. This gene has two spliced variants coding for 2 distinct proteins with significant homology. However, MRJ(L) (large variant) is predominantly localized to the nucleus whereas MRJ(S) (small variant) is predominantly cytoplasmic. Interestingly MRJ(S) translocates to the nucleus in response to heat shock. The classical heat shock proteins respond to crises (stress) by increasing the number of molecules, usually by transcriptional up-regulation. Our studies imply that a quick increase in the molar concentration of MRJ in the nuclear compartment is a novel method by which MRJ responds to stress. We found that MRJ(S) shows NLS (nuclear localization signal) independent nuclear localization in response to heat shock and hypoxia. The specificity of this response is realized due to lack of such response by MRJ(S) when challenged by other stressors, such as some cytokines or UV light. Deletion analysis has allowed us to narrow down on a 20 amino acid stretch at the C-terminal region of MRJ(S) as a potential stress sensing region. Functional studies indicated that constitutive nuclear localization of MRJ(S) promoted attributes of malignancy such as proliferation and invasiveness overall indicating distinct phenotypic characteristics of nuclear MRJ(S).

  13. Cellular stress stimulates nuclear localization signal (NLS) independent nuclear transport of MRJ

    PubMed Central

    Andrews, Joel F.; Sykora, Landon J.; Barik-Letostak, Tiasha; Menezes, Mitchell E.; Mitra, Aparna; Barik, Sailen; Shevde, Lalita A.; Samant, Rajeev S.

    2012-01-01

    HSP40 family member MRJ (DNAJB6) has been in the spot light for its relevance to Huntington’s, Parkinson’s diseases, limb-girdle muscular dystrophy, placental development, neural stem cells, cell cycle and malignancies such as breast cancer and melanoma. This gene has two spliced variants coding for 2 distinct proteins with significant homology. However, MRJ(L) (large variant) is predominantly localized to the nucleus whereas MRJ(S) (small variant) is predominantly cytoplasmic. Interestingly MRJ(S) translocates to the nucleus in response to heat shock. The classical heat shock proteins respond to crises (stress) by increasing the number of molecules, usually by transcriptional up-regulation. Our studies imply that a quick increase in the molar concentration of MRJ in the nuclear compartment is a novel method by which MRJ responds to stress. We found that MRJ(S) shows NLS (nuclear localization signal) independent nuclear localization in response to heat shock and hypoxia. The specificity of this response is realized due to lack of such response by MRJ(S) when challenged by other stressors, such as some cytokines or UV light. Deletion analysis has allowed us to narrow down on a 20 amino acid stretch at the C-terminal region of MRJ(S) as a potential stress sensing region. Functional studies indicated that constitutive nuclear localization of MRJ(S) promoted attributes of malignancy such as proliferation and invasiveness overall indicating distinct phenotypic characteristics of nuclear MRJ(S). PMID:22504047

  14. Nuclear BK Channels Regulate Gene Expression via the Control of Nuclear Calcium Signaling

    PubMed Central

    Li, Boxing; Jie, Wei; Huang, Lianyan; Wei, Peng; Li, Shuji; Luo, Zhengyi; Friedman, Allyson K.; Meredith, Andrea L.; Han, Ming-Hu; Zhu, Xin-Hong; Gao, Tian-Ming

    2014-01-01

    Ion channels are essential for the regulation of neuronal functions. The significance of plasma membrane, mitochondrial, endoplasmic reticulum, and lysosomal ion channels in the regulation of Ca2+ is well established. In contrast, surprisingly less is known about the function of ion channels on the nuclear envelope (NE). Here we demonstrate the presence of functional large-conductance, calcium-activated potassium channels (BK channels) on the NE of rodent hippocampal neurons. Functionally blockade of nuclear BK channels (nBK channels) induces NE-derived Ca2+ release, nucleoplasmic Ca2+ elevation, and cAMP response element binding protein (CREB)-dependent transcription. More importantly, blockade of nBK channels regulates nuclear Ca2+-sensitive gene expression and promotes dendritic arborization in a nuclear Ca2+-dependent manner. These results suggest that nBK channel functions as a molecular linker between neuronal activity and nuclear Ca2+ to convey the signals from synapse to nucleus and is a new modulator for synaptic activity-dependent neuronal functions at the NE level. PMID:24952642

  15. Nuclear Export and Expression of Human T-Cell Leukemia Virus Type 1 tax/rex mRNA Are RxRE/Rex Dependent

    PubMed Central

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

    2012-01-01

    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

  16. Nuclear phytochrome A signaling promotes phototropism in Arabidopsis.

    PubMed

    Kami, Chitose; Hersch, Micha; Trevisan, Martine; Genoud, Thierry; Hiltbrunner, Andreas; Bergmann, Sven; Fankhauser, Christian

    2012-02-01

    Phototropin photoreceptors (phot1 and phot2 in Arabidopsis thaliana) enable responses to directional light cues (e.g., positive phototropism in the hypocotyl). In Arabidopsis, phot1 is essential for phototropism in response to low light, a response that is also modulated by phytochrome A (phyA), representing a classical example of photoreceptor coaction. The molecular mechanisms underlying promotion of phototropism by phyA remain unclear. Most phyA responses require nuclear accumulation of the photoreceptor, but interestingly, it has been proposed that cytosolic phyA promotes phototropism. By comparing the kinetics of phototropism in seedlings with different subcellular localizations of phyA, we show that nuclear phyA accelerates the phototropic response, whereas in the fhy1 fhl mutant, in which phyA remains in the cytosol, phototropic bending is slower than in the wild type. Consistent with this data, we find that transcription factors needed for full phyA responses are needed for normal phototropism. Moreover, we show that phyA is the primary photoreceptor promoting the expression of phototropism regulators in low light (e.g., PHYTOCHROME KINASE SUBSTRATE1 [PKS1] and ROOT PHOTO TROPISM2 [RPT2]). Although phyA remains cytosolic in fhy1 fhl, induction of PKS1 and RPT2 expression still occurs in fhy1 fhl, indicating that a low level of nuclear phyA signaling is still present in fhy1 fhl.

  17. 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. Copyright © 2016. Published by Elsevier Inc.

  18. A novel class of secA alleles that exert a signal-sequence-dependent effect on protein export in Escherichia coli.

    PubMed

    Khatib, Karim; Belin, Dominique

    2002-11-01

    The murine plasminogen activator inhibitor 2 (PAI2) signal sequence inefficiently promotes the export of E. coli alkaline phosphatase (AP). High-level expression of PAI2::AP chimeric proteins from the arabinose P(BAD) promoter is toxic and confers an Ara(S) phenotype. Most Ara(R) suppressors map to secA, as determined by sequencing 21 independent alleles. Mutations occur throughout the gene, including both nucleotide binding domains (NBDI and NBDII) and the putative signal sequence binding domain (SSBD). Using malE and phoA signal sequence mutants, we showed that the vast majority of these secA suppressors exhibit weak Sec phenotypes. Eight of these secA mutations were further characterized in detail. Phenotypically, these eight suppressors can be divided into three groups, each localized to one domain of SecA. Most mutations allow near-normal levels of wild-type preprotein export, but they enhance the secretion defect conferred by signal sequence mutations. Interestingly, one group exerts a selective effect on the export of PAI2::AP when compared to that of AP. In conclusion, this novel class of secA mutations, selected as suppressors of a toxic signal sequence, differs from the classical secA (prlD) mutations, selected as suppressors of defective signal sequences, although both types of mutations affect signal sequence recognition.

  19. The zinc finger cluster domain of RanBP2 is a specific docking site for the nuclear export factor, exportin-1.

    PubMed

    Singh, B B; Patel, H H; Roepman, R; Schick, D; Ferreira, P A

    1999-12-24

    The Ran-binding protein 2 (RanBP2) is a large scaffold cyclophilin-related protein expressed in photoreceptor cells. Red/green opsin, Ran-GTPase, and the 19 S regulatory complex of the proteasome associate with specific RanBP2 structural modules. Some of these play a role in chaperoning the functional expression of opsin. RanBP2 localization at cytoplasmic fibrils emanating from the nuclear pore complex and interaction with the Ran-GTPase support also its role in nucleocytoplasmic transport processes. The degenerate nucleoporin repeat motifs FXFG, GLFG, and XXFG have been proposed to mediate the movement of nucleocytoplasmic transport factors. In particular, RanBP2 has been implicated in nuclear import processes. Here, we show the zinc fingers of RanBP2 associate with high specificity to the nuclear export factor, exportin-1 (CRM1). The bovine RanBP2 transcript contained only five of the eight zinc fingers reported in the human counterpart and are sufficient for exportin-1 association with RanBP2. In contrast to Ran interaction with RanBP2-exportin-1 complex, exportin-1 binding to the zinc finger cluster domain of RanBP2 is insensitive to leptomycin B and nucleotide-bound state of Ran-GTPase. Our results indicate that the zinc finger-rich domain of RanBP2 constitutes a docking site for exportin-1 during nuclear export. Thus, RanBP2 emerges as a key component of the nuclear export pathway.

  20. Crystal structure of importin-α3 bound to the nuclear localization signal of Ran-binding protein 3.

    PubMed

    Koyama, Masako; Matsuura, Yoshiyuki

    2017-09-23

    Ran-binding protein 3 (RanBP3) is a primarily nuclear Ran-binding protein that functions as an accessory factor in the Ran GTPase system. RanBP3 associates with Ran-specific nucleotide exchange factor RCC1 and enhances its catalytic activity towards Ran. RanBP3 also promotes CRM1-mediated nuclear export as well as CRM1-independent nuclear export of β-catenin, Smad2, and Smad3. Nuclear import of RanBP3 is dependent on the nuclear import adaptor protein importin-α and, RanBP3 is imported more efficiently by importin-α3 than by other members of the importin-α family. Protein kinase signaling pathways control nucleocytoplasmic transport through phosphorylation of RanBP3 at Ser58, immediately C-terminal to the nuclear localization signal (NLS) in the N-terminal region of RanBP3. Here we report the crystal structure of human importin-α3 bound to an N-terminal fragment of human RanBP3 containing the NLS sequence that is necessary and sufficient for nuclear import. The structure reveals that RanBP3 binds to importin-α3 residues that are strictly conserved in all seven isoforms of human importin-α at the major NLS-binding site, indicating that the region of importin-α outside the NLS-binding site, possibly the autoinhibotory importin-β1-binding domain, may be the key determinant for the preferential binding of RanBP3 to importin-α3. Computational docking simulation indicates that phosphorylation of RanBP3 at Ser58 could potentially stabilize the association of RanBP3 with importin-α through interactions between the phosphate moiety of phospho-Ser58 of RanBP3 and a cluster of basic residues (Arg96 and Lys97 in importin-α3) on armadillo repeat 1 of importin-α. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Differential IFN-gamma stimulation of HLA-A gene expression through CRM-1-dependent nuclear RNA export.

    PubMed

    Browne, Sarah K; Roesser, James R; Zhu, Sheng Zu; Ginder, Gordon D

    2006-12-15

    IFNs regulate most MHC class I genes by stimulating transcription initiation. As shown previously, IFN-gamma controls HLA-A expression primarily at the posttranscriptional level. We have defined two 8-base sequences in a 39-nucleotide region in the 3'-transcribed region of the HLA-A gene that are required for the posttranscriptional response to IFN-gamma. Stimulation of HLA-A expression by IFN-gamma requires nuclear export of HLA-A mRNA by chromosome maintenance region 1 (CRM-1). Treatment of cells with leptomycin B, a specific inhibitor of CRM-1, completely inhibited IFN-gamma induction of HLA-A. Expression of a truncated, dominant-negative form of the nucleoporin NUP214/CAN, DeltaCAN, that specifically interacts with CRM-1, also prevented IFN-gamma stimulation of HLA-A, providing confirmation of the role of CRM-1. Increased expression of HLA-A induced by IFN-gamma also requires protein methylation, as shown by the fact that treatment of SK-N-MC cells or HeLa cells with the PRMT1 inhibitor 5'-methyl-5'-thioadenosine abolished the cellular response to IFN-gamma. In contrast with HLA-A, IFN-gamma-induced expression of the HLA class Ib gene, HLA-E, was not affected by either 5'-methyl-5'-thioadenosine or leptomycin B. These results provide proof of principle that it is possible to differentially modulate the IFN-gamma-induced expression of the HLA-E and HLA-A genes, whose products often mediate opposing effects on cellular immunity to tumor cells, pathogens, and autoantigens.

  2. Alternative exon definition events control the choice between nuclear retention and cytoplasmic export of U11/U12-65K mRNA

    PubMed Central

    Verbeeren, Jens; Verma, Bhupendra

    2017-01-01

    Cellular homeostasis of the minor spliceosome is regulated by a negative feed-back loop that targets U11-48K and U11/U12-65K mRNAs encoding essential components of the U12-type intron-specific U11/U12 di-snRNP. This involves interaction of the U11 snRNP with an evolutionarily conserved splicing enhancer giving rise to unproductive mRNA isoforms. In the case of U11/U12-65K, this mechanism controls the length of the 3′ untranslated region (3′UTR). We show that this process is dynamically regulated in developing neurons and some other cell types, and involves a binary switch between translation-competent mRNAs with a short 3′UTR to non-productive isoforms with a long 3′UTR that are retained in the nucleus or/and spliced to the downstream amylase locus. Importantly, the choice between these alternatives is determined by alternative terminal exon definition events regulated by conserved U12- and U2-type 5′ splice sites as well as sequence signals used for pre-mRNA cleavage and polyadenylation. We additionally show that U11 snRNP binding to the U11/U12-65K mRNA species with a long 3′UTR is required for their nuclear retention. Together, our studies uncover an intricate molecular circuitry regulating the abundance of a key spliceosomal protein and shed new light on the mechanisms limiting the export of non-productively spliced mRNAs from the nucleus to the cytoplasm. PMID:28549066

  3. Association of CD147 and Calcium Exporter PMCA4 Uncouples IL-2 Expression from Early TCR Signaling.

    PubMed

    Supper, Verena; Schiller, Herbert B; Paster, Wolfgang; Forster, Florian; Boulègue, Cyril; Mitulovic, Goran; Leksa, Vladimir; Ohradanova-Repic, Anna; Machacek, Christian; Schatzlmaier, Philipp; Zlabinger, Gerhard J; Stockinger, Hannes

    2016-02-01

    The Ig superfamily member CD147 is upregulated following T cell activation and was shown to serve as a negative regulator of T cell proliferation. Thus, Abs targeting CD147 are being tested as new treatment strategies for cancer and autoimmune diseases. How CD147 mediates immunosuppression and whether association with other coreceptor complexes is needed have remained unknown. In the current study, we show that silencing of CD147 in human T cells increases IL-2 production without affecting the TCR proximal signaling components. We mapped the immunosuppressive moieties of CD147 to its transmembrane domain and Ig-like domain II. Using affinity purification combined with mass spectrometry, we determined the domain specificity of CD147 interaction partners and identified the calcium exporter plasma membrane calcium ATPase isoform 4 (PMCA4) as the interaction partner of the immunosuppressive moieties of CD147. CD147 does not control the proper membrane localization of PMCA4, but PMCA4 is essential for the CD147-dependent inhibition of IL-2 expression via a calcium-independent mechanism. In summary, our data show that CD147 interacts via its immunomodulatory domains with PMCA4 to bypass TCR proximal signaling and inhibit IL-2 expression.

  4. The Chlamydomonas reinhardtii BBSome is an IFT cargo required for export of specific signaling proteins from flagella.

    PubMed

    Lechtreck, Karl-Ferdinand; Johnson, Eric C; Sakai, Tsuyoshi; Cochran, Deborah; Ballif, Bryan A; Rush, John; Pazour, Gregory J; Ikebe, Mitsuo; Witman, George B

    2009-12-28

    In humans, seven evolutionarily conserved genes that cause the cilia-related disorder Bardet-Biedl syndrome (BBS) encode proteins that form a complex termed the BBSome. The function of the BBSome in the cilium is not well understood. We purified a BBSome-like complex from Chlamydomonas reinhardtii flagella and found that it contains at least BBS1, -4, -5, -7, and -8 and undergoes intraflagellar transport (IFT) in association with a subset of IFT particles. C. reinhardtii insertional mutants defective in BBS1, -4, and -7 assemble motile, full-length flagella but lack the ability to phototax. In the bbs4 mutant, the assembly and transport of IFT particles are unaffected, but the flagella abnormally accumulate several signaling proteins that may disrupt phototaxis. We conclude that the BBSome is carried by IFT but is an adapter rather than an integral component of the IFT machinery. C. reinhardtii BBS4 may be required for the export of signaling proteins from the flagellum via IFT.

  5. Rho-kinase signaling controls nucleocytoplasmic shuttling of class IIa histone deacetylase (HDAC7) and transcriptional activation of orphan nuclear receptor NR4A1.

    PubMed

    Compagnucci, Claudia; Barresi, Sabina; Petrini, Stefania; Bertini, Enrico; Zanni, Ginevra

    2015-04-03

    Rho-kinase (ROCK) has been well documented to play a key role in RhoA-induced actin remodeling. ROCK activation results in myosin light chain (MLC) phosphorylation either by direct action on MLC kinase (MLCK) or by inhibition of MLC phosphatase (MLCP), modulating actin-myosin contraction. We found that inhibition of the ROCK pathway in induced pluripotent stem cells, leads to nuclear export of HDAC7 and transcriptional activation of the orphan nuclear receptor NR4A1 while in cells with constitutive ROCK hyperactivity due to loss of function of the RhoGTPase activating protein Oligophrenin-1 (OPHN1), the orphan nuclear receptor NR4A1 is downregulated. Our study identify a new target of ROCK signaling via myosin phosphatase subunit (MYPT1) and Histone Deacetylase (HDAC7) at the nuclear level and provide new insights in the cellular functions of ROCK. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. A conserved RNA structural element within the hepatitis B virus post-transcriptional regulatory element enhance nuclear export of intronless transcripts and repress the splicing mechanism.

    PubMed

    Visootsat, Akasit; Payungporn, Sunchai; T-Thienprasert, Nattanan P

    2015-12-01

    Hepatitis B virus (HBV) infection is a primary cause of hepatocellular carcinoma and liver cirrhosis worldwide. To develop novel antiviral drugs, a better understanding of HBV gene expression regulation is vital. One important aspect is to understand how HBV hijacks the cellular machinery to export unspliced RNA from the nucleus. The HBV post-transcriptional regulatory element (HBV PRE) has been proposed to be the HBV RNA nuclear export element. However, the function remains controversial, and the core element is unclear. This study, therefore, aimed to identify functional regulatory elements within the HBV PRE and investigate their functions. Using bioinformatics programs based on sequence conservation and conserved RNA secondary structures, three regulatory elements were predicted, namely PRE 1151-1410, PRE 1520-1620 and PRE 1650-1684. PRE 1151-1410 significantly increased intronless and unspliced luciferase activity in both HepG2 and COS-7 cells. Likewise, PRE 1151-1410 significantly elevated intronless and unspliced HBV surface transcripts in liver cancer cells. Moreover, motif analysis predicted that PRE 1151-1410 contains several regulatory motifs. This study reported the roles of PRE 1151-1410 in intronless transcript nuclear export and the splicing mechanism. Additionally, these results provide knowledge in the field of HBV RNA regulation. Moreover, PRE 1151-1410 may be used to enhance the expression of other mRNAs in intronless reporter plasmids.

  7. Regulation of the nuclear export of the transcription factor NFATc1 by protein kinases after slow fibre type electrical stimulation of adult mouse skeletal muscle fibres.

    PubMed

    Shen, Tiansheng; Cseresnyés, Zoltán; Liu, Yewei; Randall, William R; Schneider, Martin F

    2007-03-01

    The transcription factor nuclear factor of activated T cells (NFAT)c1 has been shown to be involved in turning on slow skeletal muscle fibre gene expression. Previous studies from our laboratory have characterized the stimulation pattern-dependent nuclear import and resting shuttling of NFATc1-green fluorescent protein (GFP) in flexor digitorum brevis (FDB) muscle fibres from adult mouse. In this study, we use viral expression of the transcription factor NFATc1-GFP fusion protein to investigate the mechanisms underlying the nuclear export of the NFATc1-GFP that accumulated in the nuclei of cultured dissociated adult mouse FDB muscle fibres during slow-twitch fibre type electrical stimulation. In these studies, we found that inhibition of either glycogen synthase kinase 3beta (GSK3beta) or casein kinase 1 or 2 (CK1/2) markedly slowed the decay of nuclear NFATc1-GFP after cessation of muscle fibre electrical stimulation, whereas inhibition of casein kinase 1delta, p38 mitogen-activated protein kinase, c-Jun N-terminal kinase and protein kinase A had little effect. Simultaneous inhibition of GSK3beta and CK1/2 completely blocked the nuclear export of NFATc1-GFP after muscle activity. We also developed a simplified model of NFATc1 phosphorylation/dephosphorylation and nuclear fluxes, and used this model to simulate the observed time courses of nuclear NFATc1-GFP with and without NFATc1 kinase inhibition. Our results suggest that GSK3beta and CK1/2 are the major protein kinases that contribute to the removal of NFATc1 that accumulates in muscle fibre nuclei during muscle activity, and that GSK3beta and CK1/2 are responsible for phosphorylating NFATc1 in muscle nuclei in a complementary or synergistic fashion.

  8. Nuclear calcium sensors reveal that repetition of trains of synaptic stimuli boosts nuclear calcium signaling in CA1 pyramidal neurons.

    PubMed

    Bengtson, C Peter; Freitag, H Eckehard; Weislogel, Jan-Marek; Bading, Hilmar

    2010-12-15

    Nuclear calcium is a key signal in the dialogue between synapse and nucleus that controls the genomic responses required for persistent adaptations, including memory and acquired neuroprotection. The amplitude and duration of nuclear calcium transients specify activity-induced transcriptional changes. However, the precise relationship between synaptic input and nuclear calcium output is unknown. Here, we used stereotaxic delivery to the rat brain of recombinant adeno-associated viruses encoding nuclear-targeted calcium sensors to assess nuclear calcium transients in CA1 pyramidal neurons after stimulation of the Schaffer collaterals. We show that in acute hippocampal slices, a burst of synaptic activity elicits a nuclear calcium signal with a regenerative component at above-threshold stimulation intensities. Using classical stimulation paradigms (i.e., high-frequency stimulation (HFS) and θ burst stimulation (TBS)) to induce early LTP (E-LTP) and transcription-dependent late LTP (L-LTP), we found that the magnitude of nuclear calcium signals and the number of action potentials activated by synaptic stimulation trains are greatly amplified by their repetition. Nuclear calcium signals and action potential generation were reduced by blockade of either NMDA receptors or L-type voltage-gated calcium channels, but not by procedures that lead to internal calcium store depletion or by blockade of metabotropic glutamate receptors. These findings identify a repetition-induced switch in nuclear calcium signaling that correlates with the transition from E-LTP to L-LTP, and may explain why the transcription-dependent phase of L-LTP is not induced by a single HFS or TBS but requires repeated trains of activity. Recombinant, nuclear-targeted indicators may prove useful for further analysis of nuclear calcium signaling in vivo.

  9. Nuclear Calcium Sensors Reveal that Repetition of Trains of Synaptic Stimuli Boosts Nuclear Calcium Signaling in CA1 Pyramidal Neurons

    PubMed Central

    Bengtson, C. Peter; Freitag, H. Eckehard; Weislogel, Jan-Marek; Bading, Hilmar

    2010-01-01

    Nuclear calcium is a key signal in the dialogue between synapse and nucleus that controls the genomic responses required for persistent adaptations, including memory and acquired neuroprotection. The amplitude and duration of nuclear calcium transients specify activity-induced transcriptional changes. However, the precise relationship between synaptic input and nuclear calcium output is unknown. Here, we used stereotaxic delivery to the rat brain of recombinant adeno-associated viruses encoding nuclear-targeted calcium sensors to assess nuclear calcium transients in CA1 pyramidal neurons after stimulation of the Schaffer collaterals. We show that in acute hippocampal slices, a burst of synaptic activity elicits a nuclear calcium signal with a regenerative component at above-threshold stimulation intensities. Using classical stimulation paradigms (i.e., high-frequency stimulation (HFS) and θ burst stimulation (TBS)) to induce early LTP (E-LTP) and transcription-dependent late LTP (L-LTP), we found that the magnitude of nuclear calcium signals and the number of action potentials activated by synaptic stimulation trains are greatly amplified by their repetition. Nuclear calcium signals and action potential generation were reduced by blockade of either NMDA receptors or L-type voltage-gated calcium channels, but not by procedures that lead to internal calcium store depletion or by blockade of metabotropic glutamate receptors. These findings identify a repetition-induced switch in nuclear calcium signaling that correlates with the transition from E-LTP to L-LTP, and may explain why the transcription-dependent phase of L-LTP is not induced by a single HFS or TBS but requires repeated trains of activity. Recombinant, nuclear-targeted indicators may prove useful for further analysis of nuclear calcium signaling in vivo. PMID:21156150

  10. Rho-kinase signaling controls nucleocytoplasmic shuttling of class IIa Histone Deacetylase (HDAC7) and transcriptional activation of orphan nuclear receptor NR4A1

    SciTech Connect

    Compagnucci, Claudia; Barresi, Sabina; Petrini, Stefania; Bertini, Enrico; Zanni, Ginevra

    2015-04-03

    Rho-kinase (ROCK) has been well documented to play a key role in RhoA-induced actin remodeling. ROCK activation results in myosin light chain (MLC) phosphorylation either by direct action on MLC kinase (MLCK) or by inhibition of MLC phosphatase (MLCP), modulating actin–myosin contraction. We found that inhibition of the ROCK pathway in induced pluripotent stem cells, leads to nuclear export of HDAC7 and transcriptional activation of the orphan nuclear receptor NR4A1 while in cells with constitutive ROCK hyperactivity due to loss of function of the RhoGTPase activating protein Oligophrenin-1 (OPHN1), the orphan nuclear receptor NR4A1 is downregulated. Our study identify a new target of ROCK signaling via myosin phosphatase subunit (MYPT1) and Histone Deacetylase (HDAC7) at the nuclear level and provide new insights in the cellular functions of ROCK. - Highlights: • ROCK regulates nucleocytoplasmic shuttling of HDAC7 via phosphorylation of MYPT1. • Nuclear export of HDAC7 and upregulation of NR4A1 occurs with low ROCK activity. • High levels of ROCK activity due to OPHN1 loss of function downregulate NR4A1.

  11. Importin