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1.
J Biol Chem ; 285(53): 42097-104, 2010 Dec 31.
Article in English | MEDLINE | ID: mdl-20978285

ABSTRACT

Retrovirus replication requires specialized transport mechanisms to export genomic mRNA from the nucleus to the cytoplasm of the infected cell. This regulation is mediated by a combination of viral and/or cellular factors that interact with cis-acting RNA export elements linking the viral RNA to the cellular CRM1 or NXF1 nuclear export pathways. Endogenous type D murine LTR retrotransposons (musD) were reported to contain an RNA export element located upstream of the 3'-LTR. Although functionally equivalent, the musD export element, termed the musD transport element, is distinct from the other retroviral RNA export elements, such as the constitutive transport element of simian/Mason-Pfizer monkey retroviruses and the RNA transport element found in rodent intracisternal A-particle LTR retrotransposons. We demonstrate here that the minimal RNA transport element (musD transport element) of musD comprises multiple secondary structure elements that presumably serve as recognition signals for the cellular export machinery. We identified two classes of tertiary interactions, namely kissing loops and a pseudoknot. This work constitutes the first example of an RNA transport element requiring such structural motifs to mediate nuclear export.


Subject(s)
RNA/metabolism , 3' Untranslated Regions , Animals , Biological Transport , HeLa Cells , Humans , Mice , Models, Genetic , Mutagenesis, Site-Directed , Nucleic Acid Conformation , Oligonucleotides, Antisense/genetics , Promoter Regions, Genetic , Retroelements/genetics , Retroviridae/genetics , Terminal Repeat Sequences
2.
Nucleic Acids Res ; 37(21): 7151-62, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19786495

ABSTRACT

The conserved mRNA export receptor NXF1 (Mex67 in yeast) assembles with messenger ribonucleoproteins (mRNP) in the nucleus and guides them through the nuclear pore complex into the cytoplasm. The DEAD family RNA helicase Dbp5 is essential for nuclear export of mRNA and is thought to dissociate Mex67 from mRNP upon translocation, thereby generating directional passage. However, the molecular mechanism by which Dbp5 recognizes Mex67-containing mRNP is not clear. Here we report that the human NXF1-binding protein RBM15 binds specifically to human DBP5 and facilitates its direct contact with mRNA in vivo. We found that RBM15 is targeted to the nuclear envelope, where it colocalizes extensively with DBP5 and NXF1. Gene silencing of RBM15 leads to cytoplasmic depletion and nuclear accumulation of general mRNA as well as individual endogenous transcripts, indicating that RBM15 is required for efficient mRNA export. We propose a model in which RBM15 acts locally at the nuclear pore complex, by facilitating the recognition of NXF1-mRNP complexes by DBP5 during translocation, thereby contributing to efficient mRNA export.


Subject(s)
DEAD-box RNA Helicases/metabolism , Nucleocytoplasmic Transport Proteins/metabolism , RNA Transport , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Cell Line , Humans , Nuclear Envelope/metabolism , RNA Interference , RNA Splicing , RNA-Binding Proteins/antagonists & inhibitors , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , Ribonucleoproteins/metabolism
3.
J Immunol ; 183(5): 3064-72, 2009 Sep 01.
Article in English | MEDLINE | ID: mdl-19696432

ABSTRACT

The two known isoforms of IL-15 contain either a long signal peptide (LSP) or a short signal peptide (SSP), and are produced by alternatively spliced transcripts. It has been proposed that SSP IL-15 remains exclusively intracellular, and its function is unclear. In this study, we show that, similar to LSP IL-15, the SSP IL-15 is stabilized and secreted efficiently upon coexpression of IL-15Ralpha. Coinjection of SSP IL-15- and IL-15Ralpha-expressing plasmids into mice resulted in increased plasma levels of bioactive heterodimeric IL-15 and mobilization and expansion of NK and T cells. Therefore, SSP IL-15 is secreted and bioactive when produced as a heterodimer with IL-15Ralpha in the same cell. The apparent t(1/2) of this heterodimer is lower compared with LSP IL-15/IL-15Ralpha, due to different intracellular processing. Coexpression of both LSP IL-15 and SSP IL-15 in the presence of IL-15Ralpha results in lower levels of bioactive IL-15, indicating that LSP and SSP IL-15 compete for the binding to IL-15Ralpha when expressed in the same cell. Because the SSP IL-15 interaction to IL-15Ralpha leads to a complex with lower apparent stability, SSP IL-15 functions as competitive inhibitor of LSP IL-15. The data suggest that usage of alternative splicing is an additional level of control of IL-15 activity. Expression of both SSP and LSP forms of IL-15 appears to be conserved in many mammals, suggesting that SSP may be important for expressing a form of IL-15 with lower magnitude or duration of biological effects.


Subject(s)
Interleukin-15 Receptor alpha Subunit/metabolism , Interleukin-15/metabolism , Molecular Chaperones/metabolism , Peptide Fragments/metabolism , Protein Sorting Signals , Alternative Splicing , Amino Acid Sequence , Animals , Cell Line , Female , Humans , Intracellular Fluid/metabolism , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Protein Isoforms/metabolism , Protein Stability , RNA, Messenger/metabolism , Transfection
4.
J Biol Chem ; 284(38): 26106-16, 2009 Sep 18.
Article in English | MEDLINE | ID: mdl-19586903

ABSTRACT

The human SPEN family proteins SHARP, RBM15/OTT1, and RBM15B/OTT3 share the structural domain architecture but show distinct functional properties. Here, we examined the function of OTT3 and compared it with its paralogues RBM15 and SHARP. We found that OTT3, like RBM15, has post-transcriptional regulatory activity, whereas SHARP does not, supporting a divergent role of RBM15 and OTT3. OTT3 shares with RBM15 the association with the splicing factor compartment and the nuclear envelope as well as the binding to mRNA export factors NXF1 and Aly/REF. Mutational analysis revealed direct interaction of OTT3 and RBM15 with NXF1 via their C-terminal regions. Biochemical and subcellular localization studies showed that OTT3 and RBM15 also interact with each other in vivo, further supporting a shared function. Genetic knockdown of RBM15 in mouse is embryonically lethal, indicating that OTT3 cannot compensate for the RBM15 loss, which supports the notion that these proteins, in addition to sharing similar activities, likely have distinct biological roles.


Subject(s)
Nuclear Envelope/metabolism , Nucleocytoplasmic Transport Proteins/metabolism , RNA-Binding Proteins/metabolism , Active Transport, Cell Nucleus/physiology , Amino Acid Motifs/physiology , Animals , HeLa Cells , Humans , Mice , Nuclear Envelope/genetics , Nucleocytoplasmic Transport Proteins/genetics , RNA-Binding Proteins/genetics , Structural Homology, Protein
5.
Virology ; 377(1): 88-99, 2008 Jul 20.
Article in English | MEDLINE | ID: mdl-18485438

ABSTRACT

We previously identified an RNA transport element (RTE) present at a high copy number in the mouse genome. Here, we show that a related element, RTE-D, is part of a mobile LTR-retrotransposon, which belongs to a family of intracisternal A-particle related elements (IAP). We demonstrate that RTE-D is essential for the mobility of the retrotransposon and it can be substituted by other known RNA export signals. RTE-deficient IAP transcripts are retained in the nucleus, while the RTE-containing transcripts accumulate in the cytoplasm allowing Gag protein expression. RTE-D acts as a posttranscriptional control element in a heterologous reporter mRNA and is activated by the cellular RNA binding protein 15 (RBM15), as reported for the previously described RTE. We identified a complex family of RTE-containing IAPs in mouse and mapped the active RTE-D-containing IAPs to the Mmr10 group of LTR-retrotransposons. These data reveal that, despite a complex evolutionary history, retroelements and retroviruses share the dependency on posttranscriptional regulation.


Subject(s)
Genes, Intracisternal A-Particle , RNA Transport/genetics , Terminal Repeat Sequences , Animals , Base Sequence , Gene Expression , Genes, Reporter , HeLa Cells , Humans , Mice , Molecular Sequence Data , Nucleic Acid Conformation , RNA Processing, Post-Transcriptional , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Homology, Nucleic Acid , Transfection
7.
J Biol Chem ; 281(48): 36915-28, 2006 Dec 01.
Article in English | MEDLINE | ID: mdl-17001072

ABSTRACT

Retroviruses/retroelements provide tools enabling the identification and dissection of basic steps for post-transcriptional regulation of cellular mRNAs. The RNA transport element (RTE) identified in mouse retrotransposons is functionally equivalent to constitutive transport element of Type D retroviruses, yet does not bind directly to the mRNA export receptor NXF1. Here, we report that the RNA-binding motif protein 15 (RBM15) recognizes RTE directly and specifically in vitro and stimulates export and expression of RTE-containing reporter mRNAs in vivo. Tethering of RBM15 to a reporter mRNA showed that RBM15 acts by promoting mRNA export from the nucleus. We also found that RBM15 binds to NXF1 and the two proteins cooperate in stimulating RTE-mediated mRNA export and expression. Thus, RBM15 is a novel mRNA export factor and is part of the NXF1 pathway. We propose that RTE evolved as a high affinity RBM15 ligand to provide a splicing-independent link to NXF1, thereby ensuring efficient nuclear export and expression of retrotransposon transcripts.


Subject(s)
Nucleocytoplasmic Transport Proteins/physiology , RNA-Binding Proteins/physiology , RNA/chemistry , Alternative Splicing , Amino Acid Motifs , Amino Acid Sequence , Animals , Base Sequence , HeLa Cells , Humans , Ligands , Mice , Models, Biological , Molecular Sequence Data , Nucleocytoplasmic Transport Proteins/metabolism , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Retroviridae/metabolism
8.
Retrovirology ; 3: 6, 2006 Jan 13.
Article in English | MEDLINE | ID: mdl-16412225

ABSTRACT

Studies of retroviral mRNA export identified two distinct RNA export elements utilizing conserved eukaryotic mRNA export mechanism(s), namely the Constitutive Transport Element (CTE) and the RNA Transport Element (RTE). Although RTE and CTE are potent in nucleocytoplasmic mRNA transport and expression, neither element is as powerful as the Rev-RRE posttranscriptional control. Here, we found that whereas CTE and the up-regulatory mutant RTEm26 alone increase expression from a subgenomic gag and env clones, the combination of these elements led to a several hundred-fold, synergistic increase. The use of the RTEm26-CTE combination is a simple way to increase expression of poorly expressed retroviral genes to levels otherwise only achieved via more cumbersome RNA optimization. The potent RTEm26-CTE element could be useful in lentiviral gene therapy vectors, DNA-based vaccine vectors, and gene transfer studies of other poorly expressed genes.


Subject(s)
Gene Products, rev/metabolism , HIV/genetics , RNA, Messenger/metabolism , RNA, Viral/metabolism , Simian Immunodeficiency Virus/genetics , Animals , Biological Transport , Cattle , Cell Line , Genes, env , Growth Hormone/genetics , HeLa Cells , Humans , Kidney , Protein Processing, Post-Translational , RNA Processing, Post-Transcriptional , RNA Transport/genetics , RNA, Messenger/genetics , RNA, Viral/genetics , Restriction Mapping , Terminal Repeat Sequences , Transfection , rev Gene Products, Human Immunodeficiency Virus
9.
J Biol Chem ; 280(36): 31981-90, 2005 Sep 09.
Article in English | MEDLINE | ID: mdl-16014633

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.


Subject(s)
Cytoplasm/metabolism , Nucleocytoplasmic Transport Proteins/physiology , RNA, Messenger/metabolism , RNA-Binding Proteins/physiology , Active Transport, Cell Nucleus/genetics , Animals , Carrier Proteins/metabolism , Cell Nucleus/metabolism , Cytoplasm/genetics , Genes, Reporter , Humans , Lipoproteins/metabolism , Mice , Microtubule-Associated Proteins/metabolism , Neoplasm Proteins/metabolism , Nucleocytoplasmic Transport Proteins/genetics , RNA, Ribosomal/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Trans-Activators/metabolism , Tumor Cells, Cultured , Two-Hybrid System Techniques
10.
Nucleic Acids Res ; 33(12): 3855-65, 2005.
Article in English | MEDLINE | ID: mdl-16027110

ABSTRACT

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


Subject(s)
Cell Nucleus/metabolism , Nucleocytoplasmic Transport Proteins/physiology , RNA, Messenger/metabolism , RNA-Binding Proteins/physiology , Active Transport, Cell Nucleus , Amino Acid Sequence , Animals , Brain/metabolism , Cytoplasmic Granules/chemistry , Humans , Mice , Molecular Sequence Data , Nuclear Envelope/chemistry , Nuclear Localization Signals , Nucleocytoplasmic Transport Proteins/analysis , Nucleocytoplasmic Transport Proteins/genetics , Protein Structure, Tertiary , RNA-Binding Proteins/analysis , RNA-Binding Proteins/genetics
11.
J Virol ; 79(4): 2356-65, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15681436

ABSTRACT

We previously identified an RNA transport element (RTE), present in a subclass of rodent intracisternal A particle retroelements (F. Nappi, R. Schneider, A. Zolotukhin, S. Smulevitch, D. Michalowski, J. Bear, B. Felber, and G. Pavlakis, J. Virol. 75:4558-4569, 2001), that is able to replace Rev-responsive element regulation in human immunodeficiency virus type 1. RTE-directed mRNA export is mediated by a still-unknown cellular factor(s), is independent of the CRM1 nuclear export receptor, and is conserved among vertebrates. Here we show that this RTE folds into an extended RNA secondary structure and thus does not resemble any known RTEs. Computer searches revealed the presence of 105 identical elements and more than 3,000 related elements which share at least 70% sequence identity with the RTE and which are found on all mouse chromosomes. These related elements are predicted to fold into RTE-like structures. Comparison of the sequences and structures revealed that the RTE and related elements can be divided into four groups. Mutagenesis of the RTE revealed that the minimal element contains four internal stem-loops, which are indispensable for function in mammalian cells. In contrast, only part of the element is essential to mediate RNA transport in microinjected Xenopus laevis oocyte nuclei. Importantly, the minimal RTE able to promote RNA transport has key structural features which are preserved in all the RTE-related elements, further supporting their functional importance. Therefore, RTE function depends on a complex secondary structure that is important for the interaction with the cellular export factor(s).


Subject(s)
Genome , RNA Transport/genetics , RNA/chemistry , Animals , Base Sequence , Humans , Mice , Molecular Sequence Data , Nucleic Acid Conformation , Oocytes , RNA/analysis , Sequence Homology, Nucleic Acid , Xenopus laevis
12.
Mol Cell Biol ; 23(18): 6618-30, 2003 Sep.
Article in English | MEDLINE | ID: mdl-12944487

ABSTRACT

Human immunodeficiency virus type 1 (HIV) gag/pol and env mRNAs contain cis-acting regulatory elements (INS) that impair stability, nucleocytoplasmic transport, and translation by unknown mechanisms. This downregulation can be counteracted by the viral Rev protein, resulting in efficient export and expression of these mRNAs. Here, we show that the INS region in HIV-1 gag mRNA is a high-affinity ligand of p54nrb/PSF, a heterodimeric transcription/splicing factor. Both subunits bound INS RNA in vitro with similar affinity and specificity. Using an INS-containing subgenomic gag mRNA, we show that it specifically associated with p54nrb in vivo and that PSF inhibited its expression, acting via INS. Studying the authentic HIV-1 mRNAs produced from an infectious molecular clone, we found that PSF affected specifically the INS-containing, Rev-dependent transcripts encoding Gag-Pol and Env. Both subunits contained nuclear export and nuclear retention signals, whereas p54nrb was continuously exported from the nucleus and associated with INS-containing mRNA in the cytoplasm, suggesting its additional role at late steps of mRNA metabolism. Thus, p54nrb and PSF have properties of key factors mediating INS function and likely define a novel mRNA regulatory pathway that is hijacked by HIV-1.


Subject(s)
Gene Expression Regulation, Viral , HIV-1/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Regulatory Sequences, Ribonucleic Acid , Cells, Cultured/virology , DNA-Binding Proteins , Fusion Proteins, gag-pol/genetics , Fusion Proteins, gag-pol/metabolism , Gene Products, env/genetics , Gene Products, env/metabolism , Gene Products, gag/genetics , Gene Products, gag/metabolism , Gene Products, rev/genetics , Gene Products, rev/metabolism , HIV-1/drug effects , Humans , Nuclear Matrix-Associated Proteins/genetics , Nuclear Matrix-Associated Proteins/metabolism , Octamer Transcription Factors , PTB-Associated Splicing Factor , Proviruses/genetics , RNA Processing, Post-Transcriptional , RNA Stability , RNA, Viral/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/pharmacology , rev Gene Products, Human Immunodeficiency Virus
13.
J Biol Chem ; 277(6): 3935-42, 2002 Feb 08.
Article in English | MEDLINE | ID: mdl-11724776

ABSTRACT

TAP/NXF1 is a conserved mRNA export receptor serving as a link between messenger ribonucleoproteins (mRNPs) and the nuclear pore complex. The mechanism by which TAP recognizes its export substrate is unclear. We show here that TAP is added to spliced mRNP in human cells. We identified a distinct region of TAP that targets it to mRNP. Using yeast two-hybrid screens and in vitro binding studies, we found that this region coincides with a direct binding site for U2AF35, the small subunit of the splicing factor U2AF. This interaction is evolutionarily conserved across metazoa, indicating its significance. We further found in human cells that the exogenously expressed large U2AF subunit, U2AF65, accumulates in spliced mRNP, leading to the recruitment of U2AF35 and TAP. Similarly to TAP, U2AF65 stimulated directly the nuclear export and expression of an mRNA that is otherwise retained in the nucleus. Together with our finding that U2AF is continuously exported from the nucleus, these data suggest that U2AF participates in nuclear export, by facilitating TAP's addition to its mRNA substrates.


Subject(s)
Nuclear Proteins/metabolism , Nucleocytoplasmic Transport Proteins , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Ribonucleoproteins/metabolism , Binding Sites , HeLa Cells , Humans , Protein Binding , RNA Splicing , Splicing Factor U2AF
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