FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2085248380> ?p ?o ?g. }

• W2085248380 endingPage "31990" @default.
• W2085248380 startingPage "31981" @default.
• W2085248380 abstract "In eukaryotes, the nuclear export of mRNA is mediated by nuclear export factor 1 (NXF1) receptors. Metazoans encode additional NXF1-related proteins of unknown function, which share homology and domain organization with NXF1. Some mammalian NXF1-related genes are expressed preferentially in the brain and are thought to participate in neuronal mRNA metabolism. To address the roles of NXF1-related factors, we studied the two mouse NXF1 homologues, mNXF2 and mNXF7. In neuronal cells, mNXF2, but not mNXF7, exhibited mRNA export activity similar to that of Tip-associated protein/NXF1. Surprisingly, mNXF7 incorporated into mobile particles in the neurites that contained poly(A) and ribosomal RNA and colocalized with Staufen1-containing transport granules, indicating a role in neuronal mRNA trafficking. Yeast two-hybrid interaction, coimmunoprecipitation, and in vitro binding studies showed that NXF proteins bound to brain-specific microtubule-associated proteins (MAP) such as MAP1B and the WD repeat protein Unrip. Both in vitro and in vivo, MAP1B also bound to NXF export cofactor U2AF as well as to Staufen1 and Unrip. These findings revealed a network of interactions likely coupling the export and cytoplasmic trafficking of mRNA. We propose a model in which MAP1B tethers the NXF-associated mRNA to microtubules and facilitates their translocation along dendrites while Unrip provides a scaffold for the assembly of these transport intermediates. In eukaryotes, the nuclear export of mRNA is mediated by nuclear export factor 1 (NXF1) receptors. Metazoans encode additional NXF1-related proteins of unknown function, which share homology and domain organization with NXF1. Some mammalian NXF1-related genes are expressed preferentially in the brain and are thought to participate in neuronal mRNA metabolism. To address the roles of NXF1-related factors, we studied the two mouse NXF1 homologues, mNXF2 and mNXF7. In neuronal cells, mNXF2, but not mNXF7, exhibited mRNA export activity similar to that of Tip-associated protein/NXF1. Surprisingly, mNXF7 incorporated into mobile particles in the neurites that contained poly(A) and ribosomal RNA and colocalized with Staufen1-containing transport granules, indicating a role in neuronal mRNA trafficking. Yeast two-hybrid interaction, coimmunoprecipitation, and in vitro binding studies showed that NXF proteins bound to brain-specific microtubule-associated proteins (MAP) such as MAP1B and the WD repeat protein Unrip. Both in vitro and in vivo, MAP1B also bound to NXF export cofactor U2AF as well as to Staufen1 and Unrip. These findings revealed a network of interactions likely coupling the export and cytoplasmic trafficking of mRNA. We propose a model in which MAP1B tethers the NXF-associated mRNA to microtubules and facilitates their translocation along dendrites while Unrip provides a scaffold for the assembly of these transport intermediates. The proteins of the NXF 1The abbreviations used are: NXF, nuclear export factor; mNXF, mouse nuclear export factor; mRNP, messenger ribonucleoproteins; SR, serine/arginine-rich; CAT, chloramphenicol acetyltransferase; Stau1, Staufen1 protein; snRNP, small nuclear ribonucleoprotein; L chain, light chain; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; GFP, green fluorescent protein; PBS, phosphate-buffered saline; HA, hemagglutinin; GST, glutathione S-transferase; MAP, microtubule-associated protein; HIV, human immunodeficiency virus. family share homology and domain structure and are conserved from yeast to humans. The best studied family members are the metazoan TAP/NXF1 and their Saccharomyces cerevisiae orthologue Mex67p, which are essential for general mRNA export from the nucleus and act as direct export receptors by linking their mRNA cargo to the nuclear pore complex. TAP/NXF1 is added to the export-ready messenger ribonucleoproteins (mRNP) in a manner that is coupled to splicing via the interactions of its N-terminal region with cofactors such as exonic junction complex components (1Strasser K. Hurt E. EMBO J. 2000; 19: 410-420Crossref PubMed Google Scholar, 2Stutz F. Bachi A. Doerks T. Braun I.C. Seraphin B. Wilm M. Bork P. Izaurralde E. RNA (N. Y.). 2000; 6: 638-650Crossref PubMed Scopus (308) Google Scholar), including Y14/MAGOH (3Kataoka N. Diem M.D. Kim V.N. Yong J. Dreyfuss G. EMBO J. 2001; 20: 6424-6433Crossref PubMed Scopus (171) Google Scholar, 4Kataoka N. Yong J. Kim V.N. Velazquez F. Perkinson R.A. Wang F. Dreyfuss G. Mol. Cell. 2000; 6: 673-682Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar), as well as with shuttling SR (serine/arginine-rich) splicing factors such as U2AF (5Zolotukhin A.S. Tan W. Bear J. Smulevitch S. Felber B.K. J. Biol. Chem. 2002; 277: 3935-3942Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), 9G8, SRp20, and ASF/SF2 (6Lai M.C. Tarn W.Y. J. Biol. Chem. 2004; 279: 31745-31749Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 7Huang Y. Steitz J.A. Mol. Cell. 2001; 7: 899-905Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 8Huang Y. Yario T.A. Steitz J.A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9666-9670Crossref PubMed Scopus (195) Google Scholar) (for recent reviews see Refs. 9Izaurralde E. Nat. Struct. Mol. Biol. 2004; 11: 210-212Crossref PubMed Scopus (17) Google Scholar, 10Izaurralde E. Results Probl. Cell Differ. 2002; 35: 133-150Crossref PubMed Scopus (24) Google Scholar, 11Dreyfuss G. Kim V.N. Kataoka N. Nat. Rev. Mol. Cell. Biol. 2002; 3: 195-205Crossref PubMed Scopus (1122) Google Scholar, 12Vinciguerra P. Stutz F. Curr. Opin. Cell Biol. 2004; 16: 285-292Crossref PubMed Scopus (149) Google Scholar). Subsequently, TAP/NXF1 docks the mRNP to the nuclear pore complex via interactions of its C-terminal region with the phenylalanine/glycine-rich repeat domains of nucleoporins, resulting in their translocation to the cytoplasm. This step is thought to be facilitated by the binding factor of TAP/NXF1 (p15/NXT1) (13Levesque L. Guzik B. Guan T. Coyle J. Black B.E. Rekosh D. Hammarskjold M.L. Paschal B.M. J. Biol. Chem. 2001; 276: 44953-44962Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 14Herold A. Klymenko T. Izaurralde E. RNA (N. Y.). 2001; 7: 1768-1780PubMed Google Scholar, 15Guzik B.W. Levesque L. Prasad S. Bor Y.C. Black B.E. Paschal B.M. Rekosh D. Hammarskjold M.L. Mol. Cell. Biol. 2001; 21: 2545-2554Crossref PubMed Scopus (87) Google Scholar, 16Forler D. Rabut G. Ciccarelli F.D. Herold A. Kocher T. Niggeweg R. Bork P. Ellenberg J. Izaurralde E. Mol. Cell. Biol. 2004; 24: 1155-1167Crossref PubMed Scopus (79) Google Scholar). Besides NXF1 orthologues, metazoans encode additional NXF-like proteins. Based on the extensive structural similarity, both within and across species, they are thought, like TAP/NXF1, to be involved in mRNA metabolism. For some of them, such activity has been confirmed by mRNA export assays (human NXF2), whereas the others (such as human NXF3) were inactive (17Herold A. Suyama M. Rodrigues J.P. Braun I.C. Kutay U. Carmo-Fonseca M. Bork P. Izaurralde E. Mol. Cell. Biol. 2000; 20: 8996-9008Crossref PubMed Scopus (183) Google Scholar). Several proteins were also shown by loss-of-function experiments in Drosophila cultured cells to be nonessential for general mRNA export, suggesting roles that are more specialized than that of TAP/NXF1 (14Herold A. Klymenko T. Izaurralde E. RNA (N. Y.). 2001; 7: 1768-1780PubMed Google Scholar). Indeed, the nonessential Caenorhabditis elegans Ce-NXF2 (18Tan W. Zolotukhin A.S. Bear J. Patenaude D.J. Felber B.K. RNA (N. Y.). 2000; 6: 1762-1772Crossref PubMed Scopus (96) Google Scholar) participates in posttranscriptional regulation of tra-2 mRNA, which is required for female development (19Kuersten S. Segal S.P. Verheyden J. LaMartina S.M. Goodwin E.B. Mol. Cell. 2004; 14: 599-610Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), and a human NXF5 nullisomy was linked to mental retardation (20Jun L. Frints S. Duhamel H. Herold A. Abad-Rodrigues J. Dotti C. Izaurralde E. Marynen P. Froyen G. Curr. Biol. 2001; 11: 1381-1391Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). In mouse, there are two genes encoding such additional factors, mNXF2 and mNXF7 (20Jun L. Frints S. Duhamel H. Herold A. Abad-Rodrigues J. Dotti C. Izaurralde E. Marynen P. Froyen G. Curr. Biol. 2001; 11: 1381-1391Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 21Tan W. Zolotukhin A.S. Tretyakova I. Bear J. Lindtner S. Smulevitch S. Felber B.K. Nucleic Acids Res. 2005; 33: 3855-3865Crossref PubMed Scopus (30) Google Scholar). Like their human analogues, both mNXF2 and mNXF7 mRNAs are preferentially expressed in the brain (20Jun L. Frints S. Duhamel H. Herold A. Abad-Rodrigues J. Dotti C. Izaurralde E. Marynen P. Froyen G. Curr. Biol. 2001; 11: 1381-1391Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Here, we show that mNXF2 has properties similar to these of TAP/NXF1. In contrast, mNXF7 has properties of a cytoplasmic RNA transport factor. We further show that TAP/NXF1, mNXF2, and mNXF7 bind to the light chain (L chain) of brain-specific microtubule-associated protein MAP1B, which interacts with cytoplasmic microtubules and actin filaments and participates in the development and function of the nervous system (22Meixner A. Haverkamp S. Wassle H. Fuhrer S. Thalhammer J. Kropf N. Bittner R.E. Lassmann H. Wiche G. Propst F. J. Cell Biol. 2000; 151: 1169-1178Crossref PubMed Scopus (160) Google Scholar, 23Noiges R. Eichinger R. Kutschera W. Fischer I. Nemeth Z. Wiche G. Propst F. J. Neurosci. 2002; 22: 2106-2114Crossref PubMed Google Scholar, 24Togel M. Wiche G. Propst F. J. Cell Biol. 1998; 143: 695-707Crossref PubMed Scopus (135) Google Scholar, 25Gonzalez-Billault C. Jimenez-Mateos E.M. Caceres A. Diaz-Nido J. Wandosell F. Avila J. J. Neurobiol. 2004; 58: 48-59Crossref PubMed Scopus (88) Google Scholar, 26Opal P. Garcia J.J. Propst F. Matilla A. Orr H.T. Zoghbi H.Y. J. Biol. Chem. 2003; 278: 34691-34699Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Supporting a role after export, we found that mNXF7 colocalizes with Staufen1 (Stau1) protein, which is a marker of neuronal RNA transport granules (27Kiebler M.A. Hemraj I. Verkade P. Kohrmann M. Fortes P. Marion R.M. Ortin J. Dotti C.G. J. Neurosci. 1999; 19: 288-297Crossref PubMed Google Scholar, 28Kohrmann M. Luo M. Kaether C. DesGroseillers L. Dotti C.G. Kiebler M.A. Mol. Biol. Cell. 1999; 10: 2945-2953Crossref PubMed Scopus (257) Google Scholar, 29Mallardo M. Deitinghoff A. Muller J. Goetze B. Macchi P. Peters C. Kiebler M.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2100-2105Crossref PubMed Scopus (145) Google Scholar, 30Roegiers F. Jan Y.N. Trends Cell Biol. 2000; 10: 220-224Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 31Tang S.J. Meulemans D. Vazquez L. Colaco N. Schuman E. Neuron. 2001; 32: 463-475Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 32Wickham L. Duchaine T. Luo M. Nabi I.R. DesGroseillers L. Mol. Cell. Biol. 1999; 19: 2220-2230Crossref PubMed Scopus (209) Google Scholar, 33Villace P. Marion R.M. Ortin J. Nucleic Acids Res. 2004; 32: 2411-2420Crossref PubMed Scopus (129) Google Scholar, 34Krichevsky A.M. Kosik K.S. Neuron. 2001; 32: 683-696Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar, 35Kanai Y. Dohmae N. Hirokawa N. Neuron. 2004; 43: 513-525Abstract Full Text Full Text PDF PubMed Scopus (856) Google Scholar), as well as with poly(A) and ribosomal RNA. Another NXF-interacting factor identified in this study is Unrip/STRAP, which belongs to a family of WD-repeat proteins (for review, see Ref. 36Smith T.F. Gaitatzes C. Saxena K. Neer E.J. Trends Biochem. Sci. 1999; 24: 181-185Abstract Full Text Full Text PDF PubMed Scopus (1019) Google Scholar). Human Unrip participates in the inhibition of transforming growth factor β signaling by direct binding to its receptor (37Matsuda S. Katsumata R. Okuda T. Yamamoto T. Miyazaki K. Senga T. Machida K. Thant A.A. Nakatsugawa S. Hamaguchi M. Cancer Res. 2000; 60: 13-17PubMed Google Scholar, 38Datta P.K. Chytil A. Gorska A.E. Moses H.L. J. Biol. Chem. 1998; 273: 34671-34674Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 39Datta P.K. Moses H.L. Mol. Cell. Biol. 2000; 20: 3157-3167Crossref PubMed Scopus (148) Google Scholar). Unrip also associates with an RNA-binding protein, Unr, which has been implicated in translation regulation and mRNA turnover (40Hunt S.L. Hsuan J.J. Totty N. Jackson R.J. Genes Dev. 1999; 13: 437-448Crossref PubMed Scopus (220) Google Scholar, 41Grosset C. Chen C.Y. Xu N. Sonenberg N. Jacquemin-Sablon H. Shyu A.B. Cell. 2000; 103: 29-40Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). Because WD-repeat proteins are thought to act as scaffolds for the assembly of multiprotein complexes, it is possible that Unrip may facilitate the recruitment of soluble factors onto the NXF-containing mRNP complexes or that it may act by anchoring such complexes to specific subcellular locations. Cell Culture, Fractionation, and Immunofluorescence—Transfections in human 293 or HeLa-derived HLtat cells, luciferase and GFP measurements, DM128 export assays, and chloramphenicol acetyltransferase (CAT) activity measurements were performed as described previously (42Bear J. Tan W. Zolotukhin A.S. Tabernero C. Hudson E.A. Felber B.K. Mol. Cell. Biol. 1999; 19: 6306-6317Crossref PubMed Scopus (105) Google Scholar). Mouse neuroblastoma Neuro2a (N2a) cells (ATCC number CCL-131) were transfected using calcium phosphate or the LT1 (Mirus, Madison, WI) protocol. Primary chicken forebrain neurons were isolated, cultured, and transfected by electroporation as described previously (43Martinez C.Y. Hollenbeck P.J. Methods Cell Biol. 2003; 71: 339-351Crossref PubMed Google Scholar, 44Heidemann S.R. Reynolds M. Ngo K. Lamoureux P. Methods Cell Biol. 2003; 71: 51-65Crossref PubMed Google Scholar). Live cell treatments with colchicine (1 μg/ml), taxol (20 μm), and cytochalasin B (10 μm) were performed for 30-60 min at room temperature. The cytoplasmic and nuclear extracts of 293 cells were prepared as described previously (5Zolotukhin A.S. Tan W. Bear J. Smulevitch S. Felber B.K. J. Biol. Chem. 2002; 277: 3935-3942Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). For indirect immunofluorescence, cells were fixed with 3.7% formaldehyde in PBS for 10 min and permeabilized with 0.2% Triton X-100/PBS for 10 min. If cells were permeabilized prior to fixation, they were treated with 0.004% digitonin in PBS followed by a 2-min fixation in 3.7% formaldehyde/PBS at room temperature. The Alexa 590-, Alexa 488-, or Alexa 320-conjugated anti-mouse or anti-rabbit IgG (Molecular Probes, Eugene, OR) were used as secondary antibodies. In Situ Hybridization—Cells were grown and transfected in 35-mm glass-bottomed polylysine-coated plates (MatTek, Ashland, MA) fixed in 3.7% formaldehyde in PBS for 10 min and stored in 70% ethanol at 4 °C. After rehydration in PBS, cells were permeabilized in 0.2% Triton X-100/PBS, equilibrated in 2× SSC/40% formamide for 5 min, and hybridized overnight at 37 °C with 5′-biotinylated oligodeoxyribonucleotide probes. Hybridization mixes contained 1 μg/ml probe in 2× SSC, 40% formamide, 1× Denhardt's solution, 10% dextran sulfate, and 100 μg/ml denatured herring sperm DNA. Cells were washed twice with 2× SSC and once with 0.2× SSC at room temperature, incubated in blocking solution (DAKO, Carpinteria, CA), and stained with Alexa 594-conjugated streptavidin. Recombinant DNA and Proteins—mNXF2 (GenBank™ accession numbers AY017476 and AF490577) and mNXF7 (GenBank™ accession numbers AY266683 and AY260550) are described elsewhere (21Tan W. Zolotukhin A.S. Tretyakova I. Bear J. Lindtner S. Smulevitch S. Felber B.K. Nucleic Acids Res. 2005; 33: 3855-3865Crossref PubMed Scopus (30) Google Scholar). The mammalian expression plasmids for untagged, HA-, and GFP-tagged mNXF2 and mNXF7 and for GFP-tagged Unrip were constructed as described previously (42Bear J. Tan W. Zolotukhin A.S. Tabernero C. Hudson E.A. Felber B.K. Mol. Cell. Biol. 1999; 19: 6306-6317Crossref PubMed Scopus (105) Google Scholar). The plasmids expressing GFP-TAP/NXF1 and its mutants, GFP-U2AF35 and GFP-U2AF65, were described previously (5Zolotukhin A.S. Tan W. Bear J. Smulevitch S. Felber B.K. J. Biol. Chem. 2002; 277: 3935-3942Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 42Bear J. Tan W. Zolotukhin A.S. Tabernero C. Hudson E.A. Felber B.K. Mol. Cell. Biol. 1999; 19: 6306-6317Crossref PubMed Scopus (105) Google Scholar). CFP-mNXF7 plasmid was constructed by inserting the mNXF7 coding region in pECFP (BD Clontech, Palo Alto, CA). YFP and HA-Stau1 expression plasmids were a generous gift from M. Kiebler. The MAP1B LC-3XFLAG expression plasmid contains a region encoding the light chain of MAP1B that was inserted into p3XFLAG-CMV-14 (Sigma). The expression plasmids for the Ref and p15-1 proteins were provided by E. Izaurralde. For expression in Escherichia coli, MAP1B L chain and its portions were inserted in the pGEX-2T plasmid in-frame with GST, and the proteins were purified following the standard protocols. The plasmid DsRed2-Mito expresses a fluorescently tagged mitochondrial marker protein (BD Clontech). Microscopy—For microscopy, cells were grown in 35-mm glass-bottomed plates. The wide-field epifluorescence images were captured using an inverted microscope (Axiovert 135TV) with PlanApo X40 or X100 objective, equipped with an Axiocam MRM charge-coupled device camera, appropriate filter sets, and Axiovision software (Carl Zeiss, Thornwood, NY). Confocal microscopy was performed as described previously (42Bear J. Tan W. Zolotukhin A.S. Tabernero C. Hudson E.A. Felber B.K. Mol. Cell. Biol. 1999; 19: 6306-6317Crossref PubMed Scopus (105) Google Scholar, 45Stauber R.H. Horie K. Carney P. Hudson E.A. Tarasova N.I. Gaitanaris G.A. Pavlakis G.N. BioTechniques. 1998; 24 (468-471): 462-466Crossref PubMed Scopus (141) Google Scholar). For some live cell experiments, plates were maintained at 37 °C in a closed chamber. The dual color experiments were performed using appropriate controls to exclude leakage between the channels. Some images of N2a neurites and 293 processes were acquired with parameters that maximized the pixel intensity while maintaining signal linearity in these compartments. Under these conditions, the cell body fluorescence intensity was saturated. Image refinement, digital deconvolution, and colocalization were performed using the AutoDeblur and Imaris software (Bitplane, Saint Paul, MN). In Vitro Protein Binding Assays—Reticulocyte-produced proteins were synthesized and metabolically labeled in a coupled transcription/translation system (TnT T7 Quick, Promega, Madison, WI), using T7 promoter-containing PCR fragments as templates and were adjusted with unprogrammed extract to equal molar concentrations. Equimolar amounts of these proteins were used in binding reactions that contained 1-2 μg of E. coli-produced GST-tagged proteins that were immobilized on glutathione-Sepharose beads (Amersham Biosciences). The binding was performed in 200 μl of RBB buffer (15 mm HEPES, pH 7.9, 50 mm KCl, 0.1 mm EDTA, and 0.2% Triton X-100) supplemented with 200 or 400 mm NaCl. Following incubation for 15 min at room temperature, the beads were pelleted and washed three times with binding buffer. Bound proteins were eluted by boiling in SDS-PAGE sample buffer, separated by SDS-PAGE, and detected using a phosphorimaging device. Immunoprecipitations—Complexes of epitope-tagged proteins were immunopurified from transiently transfected 293 cells. Typically, about 3 × 106 cells were extracted with 200 μl of RBB-200 buffer (15 mm HEPES, pH 7.9, 50 mm KCl, 200 mm NaCl, 0.1 mm EDTA, and 0.2% Triton X-100), which was supplemented with RNase and protease inhibitors for 10 min at 4 °C. The extracts were cleared by centrifugation at 10,000 × g for 15 min at 4 °C and, in some experiments, were further fractionated by gel filtration on Chromaspin-200 columns (BD Clontech) to enrich for high molecular mass complexes. Immunoprecipitations were performed at 4 °C for 10 min in 200 μl of RBB-200 buffer, using covalently attached antibodies (anti-HA and anti-GFP agarose, VectorLabs, Burlingame, CA) or anti-FLAG M2-agarose (Sigma). The precipitates were analyzed on immunoblots using horseradish peroxidase-conjugated epitope tag antibodies. Yeast Two-hybrid Interactions—The mNXF2 cDNAs were inserted into the bait plasmid pGBKT7. Screens of a pretransformed mouse brain cDNA library in S. cerevisiae AH 109 (BD Clontech MY4008AH) were performed using the MATCHMAKER two-hybrid system 3 (BD Clontech) with medium stringency selection conditions according to the manufacturer's instructions. The prey plasmids were recovered from the primary colonies, and MAP1A, MAP1B, and Unrip cDNAs were identified by sequencing. After retransformation into AH109, the MAP1A, MAP1B, and Unrip clones were subjected to mating assays against the full-length and the 1-400 amino acid portions of NXF1 and NXF2 that were inserted into the pGBKT7 plasmid and transformed into S. cerevisiae Y187. As control, the empty pGBKT7 plasmid was used. In mating assays, the interactions were rated positive when all selection/indicator criteria were fulfilled, according to the manufacturer's instructions. mNXF2 but Not mNXF7 Exhibits mRNA Export Activity—In cultured cells, exogenous TAP/NXF1 stimulates the expression of DM128 mRNA, a reporter transcript that contains an intron-encoded CAT reporter gene embedded within a portion of HIV-1 env (46McDonald D. Hope T.J. Parslow T.G. J. Virol. 1992; 66: 7232-7238Crossref PubMed Google Scholar, 47Huang X.J. Hope T.J. Bond B.L. McDonald D. Grahl K. Parslow T.G. J. Virol. 1991; 65: 2131-2134Crossref PubMed Google Scholar), and is normally retained in the nucleus. The DM128 mRNA also contains a HIV-1 Rev-responsive element element that, in the presence of the HIV-1 Rev protein, leads to strong activation of CAT expression. Although TAP/NXF1 alone leads to a small stimulation, coexpression of its cofactor p15 augments this effect (48Braun I.C. Herold A. Rode M. Conti E. Izaurralde E. J. Biol. Chem. 2001; 276: 20536-20543Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). We have shown previously that the exogenous TAP/NXF1-p15 as well as the export cofactor of TAP/NXF1, U2AF65, acts by increasing the nuclear export of cat mRNA (5Zolotukhin A.S. Tan W. Bear J. Smulevitch S. Felber B.K. J. Biol. Chem. 2002; 277: 3935-3942Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), and therefore CAT activation provides a measure of nuclear export stimulation in this reporter system. We examined the activity of mNXF2 and mNXF7 in this assay and compared it with that of TAP/NXF1. Mouse neuronal N2a cells were cotransfected with the pDM128 plasmid and vectors encoding untagged TAP/NXF1, mNXF2, or mNXF7 in the presence or in the absence of the p15-1 expression plasmid. The cloning and characterization of mouse NXF cDNAs are described in detail elsewhere (21Tan W. Zolotukhin A.S. Tretyakova I. Bear J. Lindtner S. Smulevitch S. Felber B.K. Nucleic Acids Res. 2005; 33: 3855-3865Crossref PubMed Scopus (30) Google Scholar). All transfections contained a GFP expression plasmid as an internal reference. As positive control, pDM128 was cotransfected with the rev expression plasmid. We found that coexpression of Rev activated the DM128 mRNA expression 16-fold (Fig. 1), as expected (46McDonald D. Hope T.J. Parslow T.G. J. Virol. 1992; 66: 7232-7238Crossref PubMed Google Scholar, 47Huang X.J. Hope T.J. Bond B.L. McDonald D. Grahl K. Parslow T.G. J. Virol. 1991; 65: 2131-2134Crossref PubMed Google Scholar), confirming the validity of our assay conditions. In the absence of p15-1, TAP/NXF-1 activated the DM128 expression about 2-fold, whereas cotransfection of TAP/NXF1 and p15-1 led to an at least 20-fold activation, in agreement with previous data (48Braun I.C. Herold A. Rode M. Conti E. Izaurralde E. J. Biol. Chem. 2001; 276: 20536-20543Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Similarly, we found that mNXF2 alone led to 7-fold activation, and cotransfection of p15-1 resulted in 14-fold effect. In contrast, mNXF7 was inactive both in the absence and in the presence of p15-1 (Fig. 1). Neither of the coexpressed proteins had significant effects on GFP expression (Fig. 1), and similar results were obtained when using mouse PA317 or human 293 cells (21Tan W. Zolotukhin A.S. Tretyakova I. Bear J. Lindtner S. Smulevitch S. Felber B.K. Nucleic Acids Res. 2005; 33: 3855-3865Crossref PubMed Scopus (30) Google Scholar). Because mNXF7 was localized exclusively to the cytoplasm of N2a cells (see Fig. 2A), the lack of export activity could be due to its absence from the nucleus. The lack of an effect by mNXF7 is likely not because of its lower levels but rather its intrinsic properties, because mNXF7 and NXF1 are always expressed to higher levels compared with mNXF2 (see e.g. Fig. 5A). Together, our results show that the mouse mNXF2 is an active export receptor, whereas mNXF7 is inactive in this assay. However, we cannot exclude the possibility that mNXF7 has a more specialized export role that was not revealed using the DM128 reporter mRNA and N2a cells.Fig. 2Subcellular localization of mNXF2 and mNXF7. GFP- or HA-tagged NXF proteins (A) or GFP-mNXF7 and DsRed2-mito (B) were transiently expressed in N2a cells and visualized by confocal (A-C) and wide-field (D and E) microscopy. A, localization of NXF factors within the cell body. GFP-tagged proteins (green) were detected in live cells, and HA-mNXF7 (blue) was detected in fixed cells using indirect immunofluorescence with HA antibody. Raw images are shown, representing midsections through the nuclei. B, images of live cells coexpressing GFP-mNXF7 and DsRed2-mito. Right, merge of GFP-mNXF7 and DsRed2 (mitochondria) signals. Left, differential interference contrast (DIC) image of the same field. Bar, 20 μm. C, time-lapse images of GFP-mNXF7 in N2a neurites. Arrows indicate the particles undergoing directional movement. Top row, fields shown in B by rectangles, elapsed time is shown in minutes, and bar is 20 μm. Bottom row, time is in seconds, and bar is 4 μm. D, live primary chicken forebrain neurons expressing GFP-mNXF7. Cells were electroporated with GFP-mNXF7 expression plasmid immediately after isolation, and the GFP and bright field images (top two panels) were captured at day 2. Bottom panel, time-lapse images of GFP-mNXF7 in the field indicated by rectangles in top panels. Bar, 10 μm. E, in situ colchicine treatments of N2a cells expressing GFP-mNXF7. Time-lapse GFP images of the same neurite were acquired over a period of 30 s, before and after treatment. Data are presented as pseudo-three-dimensional rendering, where the elapsed time is plotted on the vertical axis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5Association of NXF factors with MAP1B L chain and Unrip in vivo. All indicated proteins were transiently expressed in 293 cells. A, FLAG-tagged MAP1B L chain and GFP-NXFs were coexpressed, and MAP1B L chain-containing complexes were precipitated under native conditions using FLAG antibodies. The immunoprecipitates (IP, αFLAG) and 1:100 aliquots of input extracts (Load) were analyzed on Western blots using GFP and FLAG antibodies. Positions of GFP-NXF factors (black arrowheads) and GFP protein (gray arrow-heads) are indicated. B, FLAG-tagged MAP1B L chain was detected with FLAG antibodies using indirect immunofluorescence and confocal microscopy. C, GFP-Unrip and HA-tagged NXFs were coexpressed, and NXFs were analyzed in immunoprecipitated Unrip-containing complexes (IP, αGFP) and 1:100 aliquots of input extracts (Load). D, Unrip-GFP was detected by confocal microscopy as in Fig. 2A.View Large Image Figure ViewerDownload Hi-res image Download (PPT) mNXF7 Colocalizes with Stau1 in RNA Transport Granules—Subcellular localization of mouse NXF factors was studied using GFP- or HA-tagged proteins that were transiently expressed in neuronal N2a cells. GFP-TAP/NXF1 localized to the nucleoplasm and nuclear membrane, whereas GFP-mNXF2 was found both in the cytoplasm and nucleus and was most prominent at the nuclear membrane (Fig. 2A). Hence, the localization of these proteins was that of typical NXF factors, in agreement with their export activity in N2a cells (Fig. 1). In contrast, GFP-mNXF7 was found uniquely in small cytoplasmic foci, both in the cell body and in the neurites, and it was excluded from the nucleus (Fig. 2, A and B). The observed localization did not depend on the tag because the HA-tagged mNXF7 was localized similarly (Fig. 2A) and was not sensitive to actinomycin D or leptomycin B treatments (data not shown). To assess the motility of mNXF7-containing particles in neurites, we used time-lapse microscopy of live N2a cells coexpressing GFP-mNXF7 and, as internal control, a mitochondria-tagged DsRed2-Mito protein. Fig. 2B shows that mNXF7 and mitochondria were segregated into discrete, non-colocalizing particles and were present abundantly in neurites. The majority of mNXF7 particles oscillated along neurites with amplitudes of several micrometers (see also Fig. 2E), and the movements of mNXF7 granules were distinct from those of mitochondria at all times (data not shown). Som" @default.
• W2085248380 created "2016-06-24" @default.
• W2085248380 creator A5006284974 @default.
• W2085248380 creator A5020816891 @default.
• W2085248380 creator A5026212411 @default.
• W2085248380 creator A5060271353 @default.
• W2085248380 creator A5069687616 @default.
• W2085248380 creator A5077914338 @default.
• W2085248380 creator A5086630782 @default.
• W2085248380 date "2005-09-01" @default.
• W2085248380 modified "2023-10-12" @default.
• W2085248380 title "Nuclear Export Factor Family Protein Participates in Cytoplasmic mRNA Trafficking" @default.
• W2085248380 cites W1494985759 @default.
• W2085248380 cites W1529954546 @default.
• W2085248380 cites W1541126279 @default.
• W2085248380 cites W1575786351 @default.
• W2085248380 cites W1636792448 @default.
• W2085248380 cites W1675934496 @default.
• W2085248380 cites W1975669874 @default.
• W2085248380 cites W1978316536 @default.
• W2085248380 cites W1978547782 @default.
• W2085248380 cites W1978610247 @default.
• W2085248380 cites W1979021029 @default.
• W2085248380 cites W1986461283 @default.
• W2085248380 cites W1994852911 @default.
• W2085248380 cites W1996322041 @default.
• W2085248380 cites W1997521460 @default.
• W2085248380 cites W1998206036 @default.
• W2085248380 cites W1998728023 @default.
• W2085248380 cites W2000999799 @default.
• W2085248380 cites W2001243881 @default.
• W2085248380 cites W2005100774 @default.
• W2085248380 cites W2011162362 @default.
• W2085248380 cites W2011991138 @default.
• W2085248380 cites W2014620401 @default.
• W2085248380 cites W2016867961 @default.
• W2085248380 cites W2019178696 @default.
• W2085248380 cites W2027286299 @default.
• W2085248380 cites W2032527184 @default.
• W2085248380 cites W2046721181 @default.
• W2085248380 cites W2051022441 @default.
• W2085248380 cites W2057073231 @default.
• W2085248380 cites W2062564388 @default.
• W2085248380 cites W2066043643 @default.
• W2085248380 cites W2069181654 @default.
• W2085248380 cites W2074497182 @default.
• W2085248380 cites W2074596019 @default.
• W2085248380 cites W2076347860 @default.
• W2085248380 cites W2079979082 @default.
• W2085248380 cites W2081882866 @default.
• W2085248380 cites W2085769384 @default.
• W2085248380 cites W2087672655 @default.
• W2085248380 cites W2094367541 @default.
• W2085248380 cites W2095109616 @default.
• W2085248380 cites W2095651361 @default.
• W2085248380 cites W2098441269 @default.
• W2085248380 cites W2099571103 @default.
• W2085248380 cites W2101062908 @default.
• W2085248380 cites W2106095098 @default.
• W2085248380 cites W2107777962 @default.
• W2085248380 cites W2116438422 @default.
• W2085248380 cites W2128026951 @default.
• W2085248380 cites W2131835564 @default.
• W2085248380 cites W2137990405 @default.
• W2085248380 cites W2140044545 @default.
• W2085248380 cites W2143718571 @default.
• W2085248380 cites W2144564747 @default.
• W2085248380 cites W2149715294 @default.
• W2085248380 cites W2154437245 @default.
• W2085248380 cites W2158346186 @default.
• W2085248380 cites W2160263647 @default.
• W2085248380 cites W2160337186 @default.
• W2085248380 cites W2161728447 @default.
• W2085248380 cites W2162592749 @default.
• W2085248380 cites W2163765191 @default.
• W2085248380 cites W2163833112 @default.
• W2085248380 cites W2165589515 @default.
• W2085248380 cites W2168907321 @default.
• W2085248380 cites W2281559027 @default.
• W2085248380 cites W27872685 @default.
• W2085248380 doi "https://doi.org/10.1074/jbc.m502736200" @default.
• W2085248380 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16014633" @default.
• W2085248380 hasPublicationYear "2005" @default.
• W2085248380 type Work @default.
• W2085248380 sameAs 2085248380 @default.
• W2085248380 citedByCount "48" @default.
• W2085248380 countsByYear W20852483802012 @default.
• W2085248380 countsByYear W20852483802013 @default.
• W2085248380 countsByYear W20852483802014 @default.
• W2085248380 countsByYear W20852483802015 @default.
• W2085248380 countsByYear W20852483802016 @default.
• W2085248380 countsByYear W20852483802017 @default.
• W2085248380 countsByYear W20852483802018 @default.
• W2085248380 countsByYear W20852483802022 @default.
• W2085248380 crossrefType "journal-article" @default.
• W2085248380 hasAuthorship W2085248380A5006284974 @default.
• W2085248380 hasAuthorship W2085248380A5020816891 @default.
• W2085248380 hasAuthorship W2085248380A5026212411 @default.

• next