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.

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.

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.

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.

Nuclear localization of cyclin B1 regulates DNA damage–induced apoptosis

Blood ◽  
2003 ◽  
Vol 101 (5) ◽  
pp. 1928-1933 ◽  
Author(s):  
Lisa A. Porter ◽  
I. Howard Cukier ◽  
Jonathan M. Lee
Keyword(s):  
Dna Damage ◽  
Nuclear Export ◽  
Intracellular Localization ◽  
Ectopic Expression ◽  
Cell Types ◽  
Cyclin B1 ◽  
Nuclear Accumulation ◽  
Leptomycin B ◽  
Radiation Induced ◽  
Induced Apoptosis

Some cells undergo apoptosis in response to DNA damage, whereas others do not. To understand the biochemical pathways controlling this differential response, we have studied the intracellular localization of cyclin B1 in cell types sensitive or resistant to apoptosis induced by DNA damage. We found that cyclin B1 protein accumulates in the nucleus of cells that are sensitive to γ radiation–induced apoptosis (thymocytes, lymphoid cell lines), but remains cytoplasmic in apoptosis-resistant cells (primary and transformed fibroblasts). Treatment of both cell types with leptomycin B, an inhibitor of CRM1-dependent cyclin B1 nuclear export, induces apoptosis. Furthermore, ectopic expression of cyclin B1-5xE, a protein that preferentially localizes to the nucleus, is sufficient to trigger apoptosis. Conversely, expression of cyclin B1-5xA, a predominantly cytoplasmic protein, fails to induce apoptosis. This suggests that nuclear accumulation is necessary for cyclin B1–dependent apoptosis. Our observations are consistent with the idea that localization of cyclin B1 is among the factors determining the cellular decision to undergo apoptosis in response to DNA damage.

scholarly journals Nmd3p Is a Crm1p-Dependent Adapter Protein for Nuclear Export of the Large Ribosomal Subunit

2000 ◽  
Vol 151 (5) ◽  
pp. 1057-1066 ◽  
Author(s):  
Jennifer Hei-Ngam Ho ◽  
George Kallstrom ◽  
Arlen W. Johnson
Keyword(s):  
Nuclear Export ◽  
Ribosomal Subunit ◽  
Specific Inhibitor ◽  
Small Gtpase ◽  
Nuclear Accumulation ◽  
Nuclear Pore ◽  
Adapter Protein ◽  
Leptomycin B ◽  
Ribosomal Subunits ◽  
Late Step

In eukaryotic cells, nuclear export of nascent ribosomal subunits through the nuclear pore complex depends on the small GTPase Ran. However, neither the nuclear export signals (NESs) for the ribosomal subunits nor the receptor proteins, which recognize the NESs and mediate export of the subunits, have been identified. We showed previously that Nmd3p is an essential protein from yeast that is required for a late step in biogenesis of the large (60S) ribosomal subunit. Here, we show that Nmd3p shuttles and that deletion of the NES from Nmd3p leads to nuclear accumulation of the mutant protein, inhibition of the 60S subunit biogenesis, and inhibition of the nuclear export of 60S subunits. Moreover, the 60S subunits that accumulate in the nucleus can be coimmunoprecipitated with the NES-deficient Nmd3p. 60S subunit biogenesis and export of truncated Nmd3p were restored by the addition of an exogenous NES. To identify the export receptor for Nmd3p we show that Nmd3p shuttling and 60S export is blocked by the Crm1p-specific inhibitor leptomycin B. These results identify Crm1p as the receptor for Nmd3p export. Thus, export of the 60S subunit is mediated by the adapter protein Nmd3p in a Crm1p-dependent pathway.

scholarly journals The Final Step in 5.8S rRNA Processing Is Cytoplasmic in Saccharomyces cerevisiae

2009 ◽  
Vol 30 (4) ◽  
pp. 976-984 ◽  
Author(s):  
Emma Thomson ◽  
David Tollervey
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Export ◽  
18S Rrna ◽  
Rrna Processing ◽  
Leptomycin B ◽  
5.8S Rrna ◽  
Final Maturation ◽  
Rrna Degradation

ABSTRACT The 18S rRNA component of yeast (Saccharomyces cerevisiae) 40S ribosomes undergoes cytoplasmic 3′ cleavage following nuclear export, whereas exported pre-60S subunits were believed to contain only mature 5.8S and 25S rRNAs. However, in situ hybridization detected 3′-extended forms of 5.8S rRNA in the cytoplasm, which were lost when Crm1-dependent preribosome export was blocked by treatment with leptomycin B (LMB). LMB treatment rapidly blocked processing of 6S pre-rRNA to 5.8S rRNA, leading to TRAMP-dependent pre-rRNA degradation. The 6S pre-rRNA was coprecipitated with the 60S export adapter Nmd3 and cytoplasmic 60S synthesis factor Lsg1. The longer 5.8S+30 pre-rRNA (a form of 5.8S rRNA 3′ extended by ∼30 nucleotides) is processed to 6S by the nuclear exonuclease Rrp6, and nuclear pre-rRNA accumulated in the absence of Rrp6. In contrast, 6S to 5.8S processing requires the cytoplasmic exonuclease Ngl2, and cytoplasmic pre-rRNA accumulated in strains lacking Ngl2. We conclude that nuclear pre-60S particles containing the 6S pre-rRNA bind Nmd3 and Crm1 and are exported to the cytoplasm prior to final maturation by Ngl2.

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.

scholarly journals Loss of IκBα-Mediated Control over Nuclear Import and DNA Binding Enables Oncogenic Activation of c-Rel

1998 ◽  
Vol 18 (9) ◽  
pp. 5445-5456 ◽  
Author(s):  
Shrikesh Sachdev ◽  
Mark Hannink
Keyword(s):  
Dna Binding ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Specific Inhibitor ◽  
Nuclear Localization Sequence ◽  
Treatment Results ◽  
Leptomycin B ◽  
Localization Sequence ◽  
Nuclear Shuttling

ABSTRACT The IκBα protein is able both to inhibit nuclear import of Rel/NF-κB proteins and to mediate the export of Rel/NF-κB proteins from the nucleus. We now demonstrate that the c-Rel–IκBα complex is stably retained in the cytoplasm in the presence of leptomycin B, a specific inhibitor of Crm1-mediated nuclear export. In contrast, leptomycin B treatment results in the rapid and complete relocalization of the v-Rel–IκBα complex from the cytoplasm to the nucleus. IκBα also mediates the rapid nuclear shuttling of v-Rel in an interspecies heterokaryon assay. Thus, continuous nuclear export is required for cytoplasmic retention of the v-Rel–IκBα complex. Furthermore, although IκBα is able to mask the c-Rel-derived nuclear localization sequence (NLS), IκBα is unable to mask the v-Rel-derived NLS in the context of the v-Rel–IκBα complex. Taken together, our results demonstrate that IκBα is unable to inhibit nuclear import of v-Rel. We have identified two amino acid differences between c-Rel and v-Rel (Y286S and L302P) which link the failure of IκBα to inhibit nuclear import and DNA binding of a mutant c-Rel protein to oncogenesis. Our results support a model in which loss of IκBα-mediated control over c-Rel leads to oncogenic activation of c-Rel.

Role of small nuclear RNAs in eukaryotic gene expression

2013 ◽  
Vol 54 ◽  
pp. 79-90 ◽  
Author(s):  
Saba Valadkhan ◽  
Lalith S. Gunawardane
Keyword(s):  
Gene Expression ◽  
Protein Interactions ◽  
Rna Stability ◽  
Splice Sites ◽  
Branch Site ◽  
Small Nuclear Rnas ◽  
Eukaryotic Gene Expression ◽  
Eukaryotic Gene ◽  
Rna Biogenesis

Eukaryotic cells contain small, highly abundant, nuclear-localized non-coding RNAs [snRNAs (small nuclear RNAs)] which play important roles in splicing of introns from primary genomic transcripts. Through a combination of RNA–RNA and RNA–protein interactions, two of the snRNPs, U1 and U2, recognize the splice sites and the branch site of introns. A complex remodelling of RNA–RNA and protein-based interactions follows, resulting in the assembly of catalytically competent spliceosomes, in which the snRNAs and their bound proteins play central roles. This process involves formation of extensive base-pairing interactions between U2 and U6, U6 and the 5′ splice site, and U5 and the exonic sequences immediately adjacent to the 5′ and 3′ splice sites. Thus RNA–RNA interactions involving U2, U5 and U6 help position the reacting groups of the first and second steps of splicing. In addition, U6 is also thought to participate in formation of the spliceosomal active site. Furthermore, emerging evidence suggests additional roles for snRNAs in regulation of various aspects of RNA biogenesis, from transcription to polyadenylation and RNA stability. These snRNP-mediated regulatory roles probably serve to ensure the co-ordination of the different processes involved in biogenesis of RNAs and point to the central importance of snRNAs in eukaryotic gene expression.

scholarly journals Genetic factors influencing a neurobiological substrate for psychiatric disorders

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Till F. M. Andlauer ◽  
Thomas W. Mühleisen ◽  
Felix Hoffstaedter ◽  
Alexander Teumer ◽  
Katharina Wittfeld ◽  
...  
Keyword(s):  
Psychiatric Disorders ◽  
Genetic Risk ◽  
Meta Analysis ◽  
Anterior Cingulate ◽  
Psychiatric Disease ◽  
Salience Network ◽  
Genome Wide Association Studies ◽  
Genome Wide ◽  
Common Substrate ◽  
The Common

AbstractA retrospective meta-analysis of magnetic resonance imaging voxel-based morphometry studies proposed that reduced gray matter volumes in the dorsal anterior cingulate and the left and right anterior insular cortex—areas that constitute hub nodes of the salience network—represent a common substrate for major psychiatric disorders. Here, we investigated the hypothesis that the common substrate serves as an intermediate phenotype to detect genetic risk variants relevant for psychiatric disease. To this end, after a data reduction step, we conducted genome-wide association studies of a combined common substrate measure in four population-based cohorts (n = 2271), followed by meta-analysis and replication in a fifth cohort (n = 865). After correction for covariates, the heritability of the common substrate was estimated at 0.50 (standard error 0.18). The top single-nucleotide polymorphism (SNP) rs17076061 was associated with the common substrate at genome-wide significance and replicated, explaining 1.2% of the common substrate variance. This SNP mapped to a locus on chromosome 5q35.2 harboring genes involved in neuronal development and regeneration. In follow-up analyses, rs17076061 was not robustly associated with psychiatric disease, and no overlap was found between the broader genetic architecture of the common substrate and genetic risk for major depressive disorder, bipolar disorder, or schizophrenia. In conclusion, our study identified that common genetic variation indeed influences the common substrate, but that these variants do not directly translate to increased disease risk. Future studies should investigate gene-by-environment interactions and employ functional imaging to understand how salience network structure translates to psychiatric disorder risk.

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