scholarly journals Formation of a Tap/NXF1 Homotypic Complex Is Mediated through the Amino-Terminal Domain of Tap and Enhances Interaction with Nucleoporins

2008 ◽  
Vol 19 (1) ◽  
pp. 327-338 ◽  
Author(s):  
Leah H. Matzat ◽  
Stephen Berberoglu ◽  
Lyne Lévesque
Keyword(s):  
Nuclear Export ◽  
Rna Binding ◽  
Direct Interaction ◽  
Nuclear Pore ◽  
Amino Terminal ◽  
Multimeric Complex ◽  
Rna Export ◽  
Amino Terminal Domain

Nuclear export of mRNAs is mediated by the Tap/Nxt1 pathway. Tap moves its RNA cargo through the nuclear pore complex by direct interaction with nucleoporin phenylalanine-glycine repeats. This interaction is strengthened by the formation of a Tap/Nxt1 heterodimer. We now present evidence that Tap can form a multimeric complex with itself and with other members of the NXF family. We also show that the homotypic Tap complex can interact with both Nxt1 and nucleoporins in vitro. The region mediating this oligomerization is localized to the first 187 amino acids of Tap, which overlaps with its RNA-binding domain. Removal of this domain greatly reduces the ability of Tap to bind nucleoporins in vitro and in vivo. This is the first report showing that the Tap amino terminus modulates the interaction of Tap with nucleoporins. We speculate that this mechanism has a regulatory role for RNA export independent of RNA binding.

scholarly journals Analysis of Cellular Factors That Mediate Nuclear Export of RNAs Bearing the Mason-Pfizer Monkey Virus Constitutive Transport Element

2000 ◽  
Vol 74 (13) ◽  
pp. 5863-5871 ◽  
Author(s):  
Yibin Kang ◽  
Hal P. Bogerd ◽  
Bryan R. Cullen
Keyword(s):  
Nuclear Export ◽  
Biological Activities ◽  
Critical Role ◽  
Nuclear Pore ◽  
Nucleocytoplasmic Shuttling ◽  
Rna Molecules ◽  
Constitutive Transport Element ◽  
Rna Export

ABSTRACT There is now convincing evidence that the human Tap protein plays a critical role in mediating the nuclear export of mRNAs that contain the Mason-Pfizer monkey virus constitutive transport element (CTE) and significant evidence that Tap also participates in global poly(A)+ RNA export. Previously, we had mapped carboxy-terminal sequences in Tap that serve as an essential nucleocytoplasmic shuttling domain, while others had defined an overlapping Tap sequence that can bind to the FG repeat domains of certain nucleoporins. Here, we demonstrate that these two biological activities are functionally correlated. Specifically, mutations in Tap that block nucleoporin binding also block both nucleocytoplasmic shuttling and the Tap-dependent nuclear export of CTE-containing RNAs. In contrast, mutations that do not inhibit nucleoporin binding also fail to affect Tap shuttling. Together, these data indicate that Tap belongs to a novel class of RNA export factors that can target bound RNA molecules directly to the nuclear pore without the assistance of an importin β-like cofactor. In addition to nucleoporins, Tap has also been proposed to interact with a cellular cofactor termed p15. Although we were able to confirm that Tap can indeed bind p15 specifically both in vivo and in vitro, a mutation in Tap that blocked p15 binding only modestly inhibited CTE-dependent nuclear RNA export. However, p15 did significantly enhance the affinity of Tap for the CTE in vitro and readily formed a ternary complex with Tap on the CTE. This result suggests that p15 may play a significant role in the recruitment of the Tap nuclear export factor to target RNA molecules in vivo.

scholarly journals Selective nuclear export of mRNAs is promoted by DRBD18 in Trypanosoma brucei

2021 ◽  
Author(s):  
Amartya Mishra ◽  
Jan Naseer Kaur ◽  
Daniel I. McSkimming ◽  
Eva Hegedusova ◽  
Ashutosh P. Dubey ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Nuclear Export ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Direct Interaction ◽  
Nuclear Pore ◽  
Control Gene Expression ◽  
Export Receptors ◽  
Posttranscriptional Level

Kinetoplastids, including Trypanosoma brucei, control gene expression primarily at the posttranscriptional level. Nuclear mRNA export is an important, but understudied, step in this process. The general heterodimeric export factors, Mex67/Mtr2, function in the export of mRNAs and tRNAs in T. brucei, but RNA binding proteins (RBPs) that regulate export processes by controlling the dynamics of Mex67/Mtr2 ribonucleoprotein formation or transport have not been identified. Here, we report that DRBD18, an essential and abundant T. brucei RBP, associates with Mex67/Mtr2 in vivo, likely through its direct interaction with Mtr2. DRBD18 downregulation results in partial accumulation of poly(A)+ mRNA in the nucleus, but has no effect on localization of intron-containing or mature tRNAs. Comprehensive analysis of transcriptomes from whole cell and cytosol in DRBD18 knockdown parasites demonstrates that depletion of DRBD18 leads to impairment of nuclear export of a subset of mRNAs. CLIP experiments reveal association of DRBD18 with several of these mRNAs. Moreover, DRBD18 knockdown leads to a partial accumulation of the Mex67/Mtr2 export receptors in the nucleus. Taken together, the current study supports a model in which DRBD18 regulates the selective nuclear export of mRNAs by promoting the mobilization of export competent mRNPs to the cytosol through the nuclear pore complex.  

scholarly journals Localization matters: nuclear-trapped Survivin sensitizes glioblastoma cells to temozolomide by elevating cellular senescence and impairing homologous recombination

2021 ◽  
Author(s):  
Thomas R. Reich ◽  
Christian Schwarzenbach ◽  
Juliana Brandstetter Vilar ◽  
Sven Unger ◽  
Fabian Mühlhäusler ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Nuclear Export ◽  
Cell Model ◽  
Chromosome Instability ◽  
Direct Interaction ◽  
High Grade Glioma ◽  
Strand Break ◽  
Γh2ax Foci

AbstractTo clarify whether differential compartmentalization of Survivin impacts temozolomide (TMZ)-triggered end points, we established a well-defined glioblastoma cell model in vitro (LN229 and A172) and in vivo, distinguishing between its nuclear and cytoplasmic localization. Expression of nuclear export sequence (NES)-mutated Survivin (SurvNESmut-GFP) led to impaired colony formation upon TMZ. This was not due to enhanced cell death but rather due to increased senescence. Nuclear-trapped Survivin reduced homologous recombination (HR)-mediated double-strand break (DSB) repair, as evaluated by γH2AX foci formation and qPCR-based HR assay leading to pronounced induction of chromosome aberrations. Opposite, clones, expressing free-shuttling cytoplasmic but not nuclear-trapped Survivin, could repair TMZ-induced DSBs and evaded senescence. Mass spectrometry-based interactomics revealed, however, no direct interaction of Survivin with any of the repair factors. The improved TMZ-triggered HR activity in Surv-GFP was associated with enhanced mRNA and stabilized RAD51 protein expression, opposite to diminished RAD51 expression in SurvNESmut cells. Notably, cytoplasmic Survivin could significantly compensate for the viability under RAD51 knockdown. Differential Survivin localization also resulted in distinctive TMZ-triggered transcriptional pathways, associated with senescence and chromosome instability as shown by global transcriptome analysis. Orthotopic LN229 xenografts, expressing SurvNESmut exhibited diminished growth and increased DNA damage upon TMZ, as manifested by PCNA and γH2AX foci expression, respectively, in brain tissue sections. Consequently, those mice lived longer. Although tumors of high-grade glioma patients expressed majorly nuclear Survivin, they exhibited rarely NES mutations which did not correlate with survival. Based on our in vitro and xenograft data, Survivin nuclear trapping would facilitate glioma response to TMZ.

scholarly journals Nuclear Import of IκBα Is Accomplished by a Ran-Independent Transport Pathway

2000 ◽  
Vol 20 (5) ◽  
pp. 1571-1582 ◽  
Author(s):  
Shrikesh Sachdev ◽  
Sriparna Bagchi ◽  
Donna D. Zhang ◽  
Angela C. Mings ◽  
Mark Hannink
Keyword(s):  
Nuclear Import ◽  
Nuclear Export ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Nuclear Pore ◽  
Transport Pathway ◽  
Repeat Domain ◽  
Nuclear Export Receptor

ABSTRACT The inhibitor of kappa B alpha (IκBα) protein is able to shuttle between the cytoplasm and the nucleus. We have utilized a combination of in vivo and in vitro approaches to provide mechanistic insight into nucleocytoplasmic shuttling by IκBα. IκBα contains multiple functional domains that contribute to shuttling of IκBα between the cytoplasm and the nucleus. Nuclear import of IκBα is mediated by the central ankyrin repeat domain. Similar to previously described nuclear import pathways, nuclear import of IκBα is temperature and ATP dependent and is blocked by a dominant-negative mutant of importin β. However, in contrast to classical nuclear import pathways, nuclear import of IκBα is independent of soluble cytosolic factors and is not blocked by the dominant-negative RanQ69L protein. Nuclear export of IκBα is mediated by an N-terminal nuclear export sequence. Nuclear export of IκBα requires the CRM1 nuclear export receptor and is blocked by the dominant-negative RanQ69L protein. Our results are consistent with a model in which nuclear import of IκBα is mediated through direct interactions with components of the nuclear pore complex, while nuclear export of IκBα is mediated via a CRM1-dependent pathway.

scholarly journals Mutations in Tap Uncouple RNA Export Activity from Translocation through the Nuclear Pore Complex

2006 ◽  
Vol 17 (2) ◽  
pp. 931-943 ◽  
Author(s):  
Lyne Lévesque ◽  
Yeou-Cherng Bor ◽  
Leah H. Matzat ◽  
Li Jin ◽  
Stephen Berberoglu ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Point Mutations ◽  
Nuclear Pore ◽  
Nucleocytoplasmic Shuttling ◽  
Transport Receptors ◽  
Cellular Mrnas ◽  
Rna Export ◽  
Rna Complex ◽  
Transport Receptor

Interactions between transport receptors and phenylalanine-glycine (FG) repeats on nucleoporins drive the translocation of receptor-cargo complexes through nuclear pores. Tap, a transport receptor that mediates nuclear export of cellular mRNAs, contains a UBA-like and NTF2-like folds that can associate directly with FG repeats. In addition, two nuclear export sequences (NESs) within the NTF2-like region can also interact with nucleoporins. The Tap-RNA complex was shown to bind to three nucleoporins, Nup98, p62, and RanBP2, and these interactions were enhanced by Nxt1. Mutations in the Tap-UBA region abolished interactions with all three nucleoporins, whereas the effect of point mutations within the NTF2-like domain of Tap known to disrupt Nxt1 binding or nucleoporin binding were nucleoporin dependent. A mutation in any of these Tap domains was sufficient to reduce RNA export but was not sufficient to disrupt Tap interaction with the NPC in vivo or its nucleocytoplasmic shuttling. However, shuttling activity was reduced or abolished by combined mutations within the UBA and either the Nxt1-binding domain or NESs. These data suggest that Tap requires both the UBA- and NTF2-like domains to mediate the export of RNA cargo, but can move through the pores independently of these domains when free of RNA cargo.

Arabidopsis At2g40730 encodes a cytoplasmic protein involved in nuclear tRNA export

Botany ◽  
2011 ◽  
Vol 89 (3) ◽  
pp. 175-190 ◽  
Author(s):  
Aaron D. Johnstone ◽  
Robert T. Mullen ◽  
Dev Mangroo
Keyword(s):  
Nuclear Export ◽  
Cell Cycle Progression ◽  
Nuclear Pore ◽  
Cytoplasmic Protein ◽  
Cycle Progression ◽  
Cellular Processes ◽  
Gtpase Ran ◽  
Cytoplasmic Side

Nuclear tRNA export plays an essential role in several key cellular processes, such as regulation of protein synthesis, cell cycle progression, response to nutrient availability and DNA damage, and development. While the overall mechanism of nuclear tRNA export is, in general, poorly understood, the details of specific steps are emerging from studies conducted in different organisms aimed at identifying and characterizing components involved in the process. Here, we report that Arabidopsis thaliana (L.) Heynh At2g40730 encodes CTEXP, a cytoplasmic protein component of the nuclear tRNA export process. CTEXP bound tRNA directly and saturably, and like the nuclear tRNA export receptor PAUSED, overexpression of CTEXP restored export of a nuclear export-defective lysine amber suppressor tRNA in tobacco cells. CTEXP was also found to associate with nucleoporins of the nuclear pore complex (NPC), PAUSED, and the GTPase Ran in vivo. CTEXP interacted directly with PAUSED in vitro and RanGTP, but not RanGDP. Furthermore, a portion of CTEXP appeared to associate with the NPC. Taken together, the data suggest that CTEXP assists with unloading of tRNAs from PAUSED at the cytoplasmic side of the NPC in plant cells.

scholarly journals The GLFG repetitive region of the nucleoporin Nup116p interacts with Kap95p, an essential yeast nuclear import factor.

1995 ◽  
Vol 131 (6) ◽  
pp. 1699-1713 ◽  
Author(s):  
M K Iovine ◽  
J L Watkins ◽  
S R Wente
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Structural Basis ◽  
Nuclear Pore ◽  
Temperature Sensitive ◽  
Repetitive Region ◽  
Amino Terminal ◽  
Section Electron ◽  
Rna Export

Nup116p is a member of a family of five yeast nuclear pore complex (NPC) proteins that share an amino terminal region of repetitive tetrapeptide "GLFG" motifs. Previous experiments characterized the unique morphological perturbations that occur in a nup116 null mutant: temperature-sensitive formation of nuclear envelope seals over the cytoplasmic face of the NPC (Wente, S. R., and G. Blobel. 1993. J. Cell Biol. 123:275-284). Three approaches have been taken to dissect the structural basis for Nup116p's role in NPC function. First, deletion mutagenesis analysis of NUP116 revealed that the GLFG region was required for NPC function. This was not true for the other four yeast GLFG family members (Nup49p, Nup57p, Nup100p, and Nup145p). Moreover, deletion of either half of Nup116p's GLFG repeats or replacement of Nup116p's GLFG region with either Nup100p's GLFG region or Nsp1p's FXFG repetitive region abolishes the function of Nup116p. At a semipermissive growth temperature, the cells lacking Nup116p's GLFG region displayed a diminished capacity for nuclear import. Second, overexpression of Nup116p's GLFG region severely inhibited cell growth, rapidly blocked polyadenylated-RNA export, and fragmented the nucleolus. Although it inhibited nuclear export, the overexpressed GLFG region appeared predominantly localized in the cytoplasm and NPC/nuclear envelope structure was not perturbed in thin section electron micrographs. Finally, using biochemical and two-hybrid analysis, an interaction was characterized between Nup116p's GLFG region and Kap95p, an essential yeast homologue of the vertebrate nuclear import factor p97/Imp90/karopherin beta. These data show that Nup116p's GLFG region has an essential role in mediating nuclear transport.

scholarly journals The amino-terminal domain of pyrrolysyl-tRNA synthetase is dispensable in vitro but required for in vivo activity

FEBS Letters ◽  
2007 ◽  
Vol 581 (17) ◽  
pp. 3197-3203 ◽  
Author(s):  
Stephanie Herring ◽  
Alexandre Ambrogelly ◽  
Sarath Gundllapalli ◽  
Patrick O'Donoghue ◽  
Carla R. Polycarpo ◽  
...  
Keyword(s):  
Trna Synthetase ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Amino Terminal Domain

scholarly journals Dynamics of Nuclear Pore Distribution in Nucleoporin Mutant Yeast Cells

1997 ◽  
Vol 136 (4) ◽  
pp. 747-759 ◽  
Author(s):  
Naïma Belgareh ◽  
Valérie Doye
Keyword(s):  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Pore Distribution ◽  
Yeast Cells ◽  
Nuclear Pore ◽  
Amino Terminal ◽  
Dividing Cells ◽  
Green Fluorescent ◽  
Amino Terminal Domain

To follow the dynamics of nuclear pore distribution in living yeast cells, we have generated fusion proteins between the green fluorescent protein (GFP) and the yeast nucleoporins Nup49p and Nup133p. In nup133− dividing cells that display a constitutive nuclear pore clustering, in vivo analysis of GFP-Nup49p localization revealed changes in the distribution of nuclear pore complex (NPC) clusters. Furthermore, upon induction of Nup133p expression in a GAL-nup133 strain, a progressive fragmentation of the NPC aggregates was observed that in turn led to a wild-type nuclear pore distribution. To try to uncouple Nup133p- induced NPC redistribution from successive nuclear divisions and nuclear pore biogenesis, we devised an assay based on the formation of heterokaryons between nup133− mutants and cells either expressing or overexpressing Nup133p. Under these conditions, the use of GFP-Nup133p and GFP-Nup49p fusion proteins revealed that Nup133p can be rapidly targeted to the clustered nuclear pores, where its amino-terminal domain is required to promote the redistribution of preexisting NPCs.

Circ-0000284 arouses malignant phenotype of cholangiocarcinoma cells and regulates the biological functions of peripheral cells through cellular communication

2019 ◽  
Vol 133 (18) ◽  
pp. 1935-1953 ◽  
Author(s):  
Shuming Wang ◽  
Yilin Hu ◽  
Xiurui Lv ◽  
Bin Li ◽  
Dianhua Gu ◽  
...  
Keyword(s):  
Cell Lines ◽  
Rna Binding ◽  
Direct Interaction ◽  
Luciferase Reporter ◽  
Circular Rnas ◽  
Biological Functions ◽  
Normal Cells ◽  
Cholangiocarcinoma Cell

Abstract Circular RNAs (circRNAs) play a vital role in cancers. Accumulated evidences showed that the physiological condition of cells can be reflected by the circRNAs in the exosomes they secrete, and these exosomal circRNAs can be captured by the receptor cells, thereby inducing a series of cellular responses. We performed qRT-PCR to detect the expression level of circ-0000284 in cholangiocarcinoma cell lines, tissues and plasma exosomes. Then the direct interaction between circ-0000284 and miR-637 was investigated through dual-luciferase reporter assay, RNA binding protein immunoprecipitation (RIP) assay and Fluorescent in situ hybridization (FISH) assay. Subsequently, EdU (5-ethynyl-2′-deoxyuridine), migration, invasion assay, flow cytometry and nude mouse tumorigenicity assay were adopted to evaluate the effect of circ-0000284 on migration, invasion, proliferation and apoptosis of cholangiocarcinoma cells. Additionally, TEM was conducted to investigate the shape and size of exosomes from cholangiocarcioma and 293T cell lines. Circ-0000284 was evidently elevated in cholangiocarcinoma cell lines, tumor tissues and plasma exosomes. Meanwhile, the high expression of circ-0000284 enhanced the migration, invasion and proliferation abilities of cholangiocarcinoma cells in vivo and in vitro. Besides, the levels of circ-0000284 were increased in cholangiocarcinoma cells and exosomes from them. Moreover, exosomes from cholangiocarcinoma cells enhanced circ-0000284 expression and stimulated migration and proliferation of the surrounding normal cells. Our findings suggest that on the one hand circ-0000284 functions as a competitive endogenous RNA to promote cholangiocarcinoma progression, and on the other hand, circ-0000284 can be directly transferred from cholangiocarcinoma cells to surrounding normal cells via exosomes and in this way regulate the biological functions of surrounding normal cells.

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