RNA helicase p54 (DDX6) is a shuttling protein involved in nuclear assembly of stored mRNP particles

2002 ◽  
Vol 115 (2) ◽  
pp. 395-407 ◽  
Author(s):  
David A. Smillie ◽  
John Sommerville
Keyword(s):  
Nuclear Export ◽  
De Novo ◽  
Nuclear Export Signal ◽  
Specific Inhibitor ◽  
Rna Helicase ◽  
Embryonic Cell ◽  
Leptomycin B ◽  
Nuclear Assembly ◽  
Culture Cells ◽  
Nascent Transcripts

Previously, we showed that an integral component of stored mRNP particles in Xenopus oocytes, Xp54, is a DEAD-box RNA helicase with ATP-dependent RNA-unwinding activity. Xp54 belongs to small family of helicases (DDX6) that associate with mRNA molecules encoding proteins required for progress through meiosis. Here we describe the nucleocytoplasmic translocation of recombinant Xp54 in microinjected oocytes and in transfected culture cells. We demonstrate that Xp54 is present in oocyte nuclei, its occurrence in both soluble and particle-bound forms and its ability to shuttle between nucleus and cytoplasm. Translocation of Xp54 from the nucleus to the cytoplasm appears to be dependent on the presence of a leucine-rich nuclear export signal (NES) and is blocked by leptomycin B, a specific inhibitor of the CRM1 receptor pathway. However, the C-terminal region of Xp54 can act to retain the protein in the cytoplasm of full-grown oocytes and culture cells. Cytoplasmic retention of Xp54 is overcome by activation of transcription. That Xp54 interacts directly with nascent transcripts is shown by immunostaining of the RNP matrix of lampbrush chromosome loops and co-immunoprecipitation with de novo-synthesized RNA. However, we are unable to show that nuclear export of this RNA is affected by either treatment with leptomycin B or mutation of the NES. We propose that newly synthesized Xp54 is regulated in its nucleocytoplasmic distribution: in transcriptionally quiescent oocytes it is largely restricted to the cytoplasm and, if imported into the nucleus, it is rapidly exported again by the CRM1 pathway. In transcriptionally active oocytes, it binds to a major set of nascent transcripts, accompanies mRNA sequences to the cytoplasm by an alternative export pathway and remains associated with masked mRNA until the time of translation activation at meiotic maturation and early embryonic cell division.

Developmentally regulated activity of CRM1/XPO1 during early Xenopus embryogenesis

2000 ◽  
Vol 113 (3) ◽  
pp. 451-459 ◽  
Author(s):  
M. Callanan ◽  
N. Kudo ◽  
S. Gout ◽  
M. Brocard ◽  
M. Yoshida ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Intracellular Localization ◽  
Nuclear Export Signal ◽  
Specific Inhibitor ◽  
Amino Acid Identity ◽  
Early Embryogenesis ◽  
Leptomycin B ◽  
Developmentally Regulated ◽  
Neurula Stage

In this work, we have investigated the role of CRM1/XPO1, a protein involved in specific export of proteins and RNA from the nucleus, in early Xenopus embryogenesis. The cloning of the Xenopus laevis CRM1, XCRM1, revealed remarkable conservation of the protein during evolution (96.7% amino acid identity between Xenopus and human). The protein and mRNA are maternally expressed and are present during early embryogenesis. However, our data show that the activity of the protein is developmentally regulated. Embryonic development is insensitive to leptomycin B, a specific inhibitor of CRM1, until the neurula stage. Moreover, the nuclear localization of CRM1 changes concomitantly with the appearance of the leptomycin B sensitivity. These data suggest that CRM1, present initially in an inactive form, becomes functional before the initiation of the neurula stage during gastrula-neurula transition, a period known to correspond to a critical transition in the pattern of gene expression. Finally, we confirmed the gastrula-neurula transition-dependent activation of CRM1 by pull-down experiments as well as by the study of the intracellular localization of a green fluorescent protein tagged with a nuclear export signal motif during early development. This work showed that the regulated activity of CRM1 controls specific transitions during normal development and thus might be a key regulator of early embryogenesis.

MALT1 contains nuclear export signals and regulates cytoplasmic localization of BCL10

Blood ◽  
2005 ◽  
Vol 106 (13) ◽  
pp. 4210-4216 ◽  
Author(s):  
Masao Nakagawa ◽  
Yoshitaka Hosokawa ◽  
Masakatsu Yonezumi ◽  
Koh Izumiyama ◽  
Ritsuro Suzuki ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Specific Inhibitor ◽  
B Cell Lymphoma ◽  
Nuclear Factor Κb ◽  
Dependent Manner ◽  
Nucleocytoplasmic Shuttling ◽  
Leptomycin B ◽  
Apoptosis Inhibitor ◽  
Export Signal

MALT1, BCL10 (B-cell lymphoma 10), and API2 (apoptosis inhibitor 2)-MALT1 are key molecules in mucosa-associated lymphoid tissue (MALT) lymphomagenesis. We previously reported that MALT1 and API2-MALT1 were localized only in cytoplasm, where we suggested that both molecules were likely to be active. In the study presented here, we further examined the localization-determining region by generating various mutants and were able to demonstrate that there were nuclear export signal (NES)-containing domains in the MALT1 C-terminal region. The use of leptomycin B, an NES-specific inhibitor, demonstrated that both MALT1 and API2-MALT1 were predominantly retained in the nuclei, indicating that these molecules were shuttling between nucleus and cytoplasm in an NES-dependent manner. It was also found that MALT1 was involved in the nuclear export of BCL10, which is originally localized in both nucleus and cytoplasm. These results correlate well with the nuclear BCL10 expression pattern in both t(1;14) and t(11;18) MALT lymphomas. The nucleocytoplasmic shuttling of MALT1 and BCL10 complex may indicate that these molecules are involved not only in the nuclear factor κB (NF-κB) pathway but also in other biologic functions in lymphocytes.

Subcellular localisation of human inositol 1,4,5-trisphosphate 3-kinase C: species-specific use of alternative export sites for nucleo-cytoplasmic shuttling indicates divergent roles of the catalytic and N-terminal domains

2006 ◽  
Vol 387 (5) ◽  
pp. 583-593 ◽  
Author(s):  
Marcus M. Nalaskowski ◽  
Sabine Windhorst ◽  
Malte C. Stockebrand ◽  
Georg W. Mayr
Keyword(s):  
Nuclear Export ◽  
Catalytic Domain ◽  
De Novo ◽  
Nuclear Export Signal ◽  
Subcellular Localisation ◽  
Leptomycin B ◽  
Intracellular Targeting ◽  
Mammalian Cell Lines ◽  
Species Specific ◽  
Export Signal

Abstract The three isoforms of human Ins(1,4,5)P3 3-kinase (IP3K) show remarkable differences in their intracellular targeting. Whereas predominant targeting to the cytoskeleton and endoplasmic reticulum has been shown for IP3K-A and IP3K-B, rat IP3K-C shuttles actively between the nucleus and cytoplasm. In the present study we examined the expression and intracellular localisation of endogenous IP3K-C in different mammalian cell lines using an isoform-specific antibody. In addition, human IP3K-C, showing remarkable differences to its rat homologue in the N-terminal targeting domain, was tagged with EGFP and used to examine active transport mechanisms into and out of the nucleus. We found both a nuclear import activity residing in its N-terminal domain and a nuclear export activity sensitive to treatment with leptomycin B. Different from the rat isoform, an exportin 1-dependent nuclear export site of the human enzyme resides outside the N-terminal targeting domain in the catalytic enzyme domain. A phylogenetic survey of vertebrate IP3K sequences indicates that in each of the three isoforms a nuclear export signal has evolved in the catalytic domain either de novo (IP3K-A) or as a substitute for an earlier evolved corresponding N-terminal signal (IP3K-B and IP3K-C). In higher vertebrates, and in particular in primates, re-export of nuclear IP3K activity may be guaranteed by the mechanism discovered.

scholarly journals Exportin 1 Mediates Nuclear Export of the Kinetoplastid Spliced Leader RNA

2003 ◽  
Vol 2 (2) ◽  
pp. 222-230 ◽  
Author(s):  
Gusti M. Zeiner ◽  
Nancy R. Sturm ◽  
David A. Campbell
Keyword(s):  
Nuclear Export ◽  
Ectopic Expression ◽  
Specific Inhibitor ◽  
Leptomycin B ◽  
Spliced Leader ◽  
Small Nuclear Rnas ◽  
Rna Biogenesis ◽  
Common Substrate ◽  
The Common

ABSTRACT The kinetoplastid protozoan spliced leader (SL) RNA is the common substrate pre-mRNA utilized in all trans-splicing reactions. Here we show by fluorescence in situ hybridization that the SL RNA is present in the cytoplasm of Leishmania tarentolae and Trypanosoma brucei. Treatment with the karyopherin-specific inhibitor leptomycin B was toxic to T. brucei and eliminated the cytoplasmic SL RNA, suggesting that cytoplasmic SL RNA was dependent on the nuclear exporter exportin 1 (XPO1). Ectopic expression of xpo1 with a C506S mutation in T. brucei conferred resistance to leptomycin B. A reduction in SL RNA 3′ extension removal and 5′ methylation of nucleotide U4 was observed in wild-type T. brucei treated with leptomycin B, suggesting that the cytoplasmic stage is necessary for SL RNA biogenesis. This study demonstrates spatial and mechanistic similarities between the posttranscriptional trafficking of the kinetoplastid protozoan SL RNA and the metazoan cis-spliceosomal small nuclear RNAs.

scholarly journals Characterization of a Novel Transferable CRM-1-Independent Nuclear Export Signal in a Herpesvirus Tegument Protein That Shuttles between the Nucleus and Cytoplasm

2006 ◽  
Vol 80 (20) ◽  
pp. 10021-10035 ◽  
Author(s):  
Janneke Verhagen ◽  
Michelle Donnelly ◽  
Gillian Elliott
Keyword(s):  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Nuclear Targeting ◽  
Leptomycin B ◽  
Herpesvirus Type ◽  
Close Proximity ◽  
Bovine Herpesvirus Type 1 ◽  
Import And Export ◽  
Export Signal

ABSTRACT A new group of nucleocytoplasmic shuttling proteins has recently been identified in the structural proteins encoded by several alphaherpesvirus UL47 genes. Nuclear import and export signals for the bovine herpesvirus type 1 UL47 protein (VP8 or bUL47) have been described previously. Here, we study the trafficking of bUL47 in detail and identify an import signal different from that shown before. It comprises a 20-residue N-terminal peptide that is fully transferable and targets a large, normally cytosolic protein to the nucleus. A conserved RRPRRS motif within this peptide was shown to be essential but not sufficient for nuclear targeting. Using interspecies heterokaryon assays, we further demonstrate that the export activity of the published leucine-rich nuclear export signal (NES) is also transferable to a large protein but is functionally weak compared to the activity of the HIV-1 Rev NES. We show that nuclear export dictated by this bUL47 NES is sensitive to leptomycin B (LMB) and therefore dependent on the export receptor CRM-1. However, nuclear export of full-length bUL47 is fully resistant to LMB, suggesting the presence of an additional NES. We go on to identify a second NES in bUL47 within a 28-residue peptide that is in close proximity to but entirely separable from the N-terminal import signal, and we use fluorescence loss in photobleaching to confirm its activity. This NES is resistant to leptomycin B, and therefore utilizes an export receptor other than CRM-1. As this new sequence bears little similarity to other export signals so far defined, we suggest it may be involved in bUL47 export from the nucleus via a novel cellular receptor.

scholarly journals Reconstitution of nuclear protein export in isolated nuclear envelopes

2002 ◽  
Vol 158 (5) ◽  
pp. 849-854 ◽  
Author(s):  
Jan Peter Siebrasse ◽  
Elias Coutavas ◽  
Reiner Peters
Keyword(s):  
Protein Kinase ◽  
Nuclear Export ◽  
Xenopus Oocytes ◽  
Nuclear Protein ◽  
Nuclear Export Signal ◽  
Protein Export ◽  
Leptomycin B ◽  
Permeabilized Cells ◽  
Export Signal ◽  
Kinase A

Signal-dependent nuclear protein export was studied in perforated nuclei and isolated nuclear envelopes of Xenopus oocytes by optical single transporter recording. Manually isolated and purified oocyte nuclei were attached to isoporous filters and made permeable for macromolecules by perforation. Export of a recombinant protein (GG-NES) containing the nuclear export signal (NES) of the protein kinase A inhibitor through nuclear envelope patches spanning filter pores could be induced by the addition of GTP alone. Export continued against a concentration gradient, and was NES dependent and inhibited by leptomycin B and GTPγS, a nonhydrolyzable GTP analogue. Addition of recombinant RanBP3, a potential cofactor of CRM1-dependent export, did not promote GG-NES export at stoichiometric concentration but gradually inhibited export at higher concentrations. In isolated filter-attached nuclear envelopes, export of GG-NES was virtually abolished in the presence of GTP alone. However, a preformed export complex consisting of GG-NES, recombinant human CRM1, and RanGTP was rapidly exported. Unexpectedly, export was strongly reduced when the export complex contained RanGTPγS or RanG19V/Q69L-GTP, a GTPase-deficient Ran mutant. This paper shows that nuclear transport, previously studied in intact and permeabilized cells only, can be quantitatively analyzed in perforated nuclei and isolated nuclear envelopes.

scholarly journals Control of NF–κB transcriptional activation by signal induced proteolysis of IκBα

1999 ◽  
Vol 354 (1389) ◽  
pp. 1601-1609 ◽  
Author(s):  
R. T. Hay ◽  
L. Vuillard ◽  
J. M. P. Desterro ◽  
M. S. Rodriguez
Keyword(s):  
Nuclear Localization ◽  
Transcriptional Activation ◽  
Nuclear Localization Signal ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Cytoplasmic Process ◽  
Leptomycin B ◽  
Rapid Degradation ◽  
Localization Signal ◽  
Export Signal

In unstimulated cells the transcription factor NF–κB is held in the cytoplasm in an inactive state by IκB inhibitor proteins. Ultimately activation of NF–κB is achieved by ubiquitination and proteasome–mediated degradation of IκBα and we have therefore investigated factors which control this proteolysis. Signal–induced degradation of IκBα exposes the nuclear localization signal of NF–κB, thus allowing it to translocate into the nucleus and activate transcription from responsive genes. An autoregulatory loop is established when NF–κB induces expression of the IκBα gene and newly synthesized IκBα accumulates in the nucleus where it negatively regulates NF–κB–dependent transcription. As part of this post–induction repression, the nuclear export signal on IκBα mediates transport of NF–κB–IκBα complexes from the nucleus to the cytoplasm. As nuclear export of IκBα is blocked by leptomycin B this drug was used to examine the effect of cellular location on susceptibility of IκBα to signal–induced degradation. In the presence of leptomycin B, IκBα is accumulated in the nucleus and in this compartment is resistant to signal–induced degradation. Thus signal–induced degradation of IκBα is mainly, if not exclusively a cytoplasmic process. An efficient nuclear export of IκBα is therefore essential for maintaining a low level of IκBα in the nucleus and allowing NF–κB to be transcriptionally active upon cell stimulation. We have detected a modified form of IκBα, conjugated to the small ubiquitin–like protein SUMO–1, which is resistant to signal–induced degradation. SUMO–1 modified IκBα remains associated with NF–κB and thus overexpression of SUMO–1 inhibits the signal–induced activation of NF–κB–dependent transcription. Reconstitution of the conjugation reaction with highly purified proteins demonstrated that in the presence of a novel E1 SUMO–1 activating enzyme, Ubch9 directly conjugated SUMO–1 to IκBα on residues K21 and K22, which are also used for ubiquitin modification. Thus, while ubiquitination targets proteins for rapid degradation, SUMO–1 modification acts antagonistically to generate proteins resistant to degradation.

scholarly journals A Carboxyl Leucine-Rich Region of Parathyroid Hormone-Related Protein Is Critical for Nuclear Export

Endocrinology ◽  
2006 ◽  
Vol 147 (2) ◽  
pp. 990-998 ◽  
Author(s):  
Jared C. Pache ◽  
Douglas W. Burton ◽  
Leonard J. Deftos ◽  
Randolph H. Hastings
Keyword(s):  
Amino Acid ◽  
Nuclear Export ◽  
Chromosome Region ◽  
Nuclear Export Signal ◽  
Wild Type ◽  
Leptomycin B ◽  
Intact Cells ◽  
Parathyroid Hormone Related Protein ◽  
Truncation Mutant ◽  
Chromosome Region Maintenance

PTHrP is an oncofetal protein with distinct proliferative and antiapoptotic roles that are affected by nucleocytoplasmic shuttling. The protein’s nuclear export is sensitive to leptomycin B, consistent with a chromosome region maintenance protein 1-dependent pathway. We determined that the 109–139 region of PTHrP was involved in its nuclear export by demonstrating that a C-terminal truncation mutant, residues 1–108, exports at a reduced rate, compared with the wild-type 139 amino acid isoform. We searched for potential nuclear export sequences within the 109–139 region, which is leucine rich. Comparisons with established nuclear export sequences identified a putative consensus signal at residues 126–136. Deletion of this region resulted in nuclear export characteristics that closely matched those of the C-terminal truncation mutant. Confocal microscopic analyses of transfected 293, COS-1, and HeLa cells showed that steady-state nuclear levels of the truncated and deletion mutants were significantly greater than levels of wild-type PTHrP and were unaffected by leptomycin B, unlike the wild-type protein. In addition, both mutants demonstrated greatly reduced nuclear export with assays using nuclear preparations and intact cells. Based on these results, we conclude that the 126–136 amino acid sequence closely approximates the structure of a chromosome region maintenance protein 1-dependent leucine-rich nuclear export signal and is critical for nuclear export of PTHrP.

scholarly journals The Mobile FG Nucleoporin Nup98 Is a Cofactor for Crm1-dependent Protein Export

2010 ◽  
Vol 21 (11) ◽  
pp. 1885-1896 ◽  
Author(s):  
Masahiro Oka ◽  
Munehiro Asally ◽  
Yoshinari Yasuda ◽  
Yutaka Ogawa ◽  
Taro Tachibana ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Nuclear Protein ◽  
Mutational Analysis ◽  
Specific Inhibitor ◽  
Protein Export ◽  
Dependent Manner ◽  
Repeat Region ◽  
Leptomycin B ◽  
Dependent Protein ◽  
Nuclear Export Receptor

Nup98 is a mobile nucleoporin that forms distinct dots in the nucleus, and, although a role for Nup98 in nuclear transport has been suggested, its precise function remains unclear. Here, we show that Nup98 plays an important role in Crm1-mediated nuclear protein export. Nuclear, but not cytoplasmic, dots of EGFP-tagged Nup98 disappeared rapidly after cell treatment with leptomycin B, a specific inhibitor of the nuclear export receptor, Crm1. Mutational analysis demonstrated that Nup98 physically and functionally interacts with Crm1 in a RanGTP-dependent manner through its N-terminal phenylalanine-glycine (FG) repeat region. Moreover, the activity of the Nup98-Crm1 complex was modulated by RanBP3, a known cofactor for Crm1-mediated nuclear export. Finally, cytoplasmic microinjection of anti-Nup98 inhibited the Crm1-dependent nuclear export of proteins, concomitant with the accumulation of anti-Nup98 in the nucleus. These results clearly demonstrate that Nup98 functions as a novel shuttling cofactor for Crm1-mediated nuclear export in conjunction with RanBP3.

scholarly journals Shuttling Imbalance of MLF1 Results in p53 Instability and Increases Susceptibility to Oncogenic Transformation

2007 ◽  
Vol 28 (1) ◽  
pp. 422-434 ◽  
Author(s):  
Noriko Yoneda-Kato ◽  
Jun-ya Kato
Keyword(s):  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Chromosomal Translocation ◽  
Nuclear Accumulation ◽  
Tumor Suppressor P53 ◽  
Oncogenic Transformation ◽  
Early Passage ◽  
Leptomycin B ◽  
Nuclear Shuttling ◽  
Export Signal

ABSTRACT Myeloid leukemia factor 1 (MLF1) stabilizes the activity of the tumor suppressor p53 by suppressing its E3 ubiquitin ligase, COP1, through a third component of the COP9 signalosome (CSN3). However, little is known about how MLF1 functions upstream of the CSN3-COP1-p53 pathway and how its deregulation by the formation of the fusion protein nucleophosmin (NPM)-MLF1, generated by t(3;5)(q25.1;q34) chromosomal translocation, leads to leukemogenesis. Here we show that MLF1 is a cytoplasmic-nuclear-shuttling protein and that its nucleolar localization on fusing with NPM prevents the full induction of p53 by both genotoxic and oncogenic cellular stress. The majority of MLF1 was located in the cytoplasm, but the treatment of cells with leptomycin B rapidly induced a nuclear accumulation of MLF1. A mutation of the nuclear export signal (NES) motif identified in the MLF1 sequence enhanced the antiproliferative activity of MLF1. The fusion of MLF1 with NPM translocated MLF1 to the nucleolus and abolished the growth-suppressing activity. The introduction of NPM-MLF1 into early-passage murine embryonic fibroblasts allowed the cells to escape from cellular senescence at a markedly earlier stage and induced neoplastic transformation in collaboration with the oncogenic form of Ras. Interestingly, disruption of the MLF1-derived NES sequence completely abolished the growth-promoting activity of NPM-MLF1 in murine fibroblasts and hematopoietic cells. Thus, our results provide important evidence that the shuttling of MLF1 is critical for the regulation of cell proliferation and a disturbance in the shuttling balance increases the cell's susceptibility to oncogenic transformation.

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