Jenifer Bear

Identification of Novel Import and Export Signals of Human TAP, the Protein That Binds to the Constitutive Transport Element of the Type D Retrovirus mRNAs

ABSTRACT The nuclear export of the unspliced type D retrovirus mRNA depends on the cis-acting constitutive transport RNA element (CTE) that has been shown to interact with the human TAP (hTAP) protein promoting the export of the CTE-containing mRNAs. We report here that hTAP is a 619-amino-acid protein extending the previously identified protein by another 60 residues at the N terminus and that hTAP shares high homology with the predicted rat and mouse TAP proteins. We found that hTAP is a nuclear protein that accumulates in the nuclear rim and the nucleoplasm. We further demonstrated that hTAP is able to shuttle between the nucleus and the cytoplasm. Identification of the signals responsible for nuclear import (NLS) and export (NES) revealed that they are distinct but partially overlapping. NLS and NES of hTAP are active transferable signals that do not share similarities with known elements. The C-terminal portion contributes further to hTAP’s nuclear retention and contains a signal(s) for nuclear rim association. Taken together, our data show that hTAP is a dynamic protein capable of bidirectional trafficking across the nuclear envelope. These data further support hTAP’s role as an export factor of the CTE-containing mRNAs.

PSF acts through the human immunodeficiency virus type 1 mRNA instability elements to regulate virus expression.

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.

The mRNA export in Caenorhabditis elegans is mediated by Ce-NXF-1, an ortholog of human TAP/NXF and Saccharomyces cerevisiae Mex67p.

Human TAP and Saccharomyces cerevisiae Mex67p belong to a family of proteins that mediate mRNA export. Computer searches identified previously two Caenorhabditis elegans genes, C15H11.3 and C115H11.6, that encode putative homologs of hTAP and Mex67p (Segref et al., EMBO J, 1997, 16:3256-3271). Using RNA interference experiments in C. elegans, we found that functional knockout of C15H11.3 resulted in nuclear accumulation of poly(A)-containing RNAs and was lethal for both embryos and adult nematodes. No embryonic or progeny abnormality was observed in functional knockout of C15H11.6. Taken together, these data established that the C15H11.3 gene product is an ortholog of hTAP and Mex67p; thus, it was named Ce-NXF-1. Ce-NXF-1 binds RNA directly and is a nucleocytoplasmic shuttle protein accumulating in the nucleoplasm and at the nuclear rim. The rim association is mediated via unique signals present in the C-terminal portion of all TAP/NXF and Mex67p proteins. This region was shown to interact with the FG-repeat domains of nucleoporins Nup98, Nup153, and Nup214, indicating that the rim association occurs through components of the nuclear pore complex. In summary, Ce-NXF-1 belongs together with hTAP and Mex67p to a family of proteins that participate in mRNA export and can provide a direct molecular link between mRNAs and components of the nuclear pore complex. Therefore, despite differences in mRNA metabolism between these species, they utilize a conserved mRNA transport mechanism.

Circulating IL-15 exists as heterodimeric complex with soluble IL-15Rα in human and mouse serum

IL-15 is an important cytokine for the function of the immune system, but the form(s) of IL-15 produced in the human body are not fully characterized. Coexpression of the single-chain IL-15 and the IL-15 receptor alpha (IL-15Rα) in the same cell allows for efficient production, surface display, and eventual cleavage and secretion of the bioactive IL-15/IL-15Rα heterodimer in vivo, whereas the single-chain IL-15 is poorly secreted and unstable. This observation led to the hypothesis that IL-15 is produced and secreted only as a heterodimer with IL-15Rα. We purified human IL-15/IL-15Rα complexes from overproducing human cell lines and developed an ELISA specifically measuring the heterodimeric form of IL-15. Analysis of sera from melanoma patients after lymphodepletion revealed the presence of circulating IL-15/IL-15Rα complexes in amounts similar to the total IL-15 quantified by a commercial IL-15 ELISA that detects both the single-chain and the heterodimeric forms of the cytokine. Therefore, in lymphodepleted cancer patients, the serum IL-15 is exclusively present in its heterodimeric form. Analysis of the form of IL-15 present in either normal or lymphodepleted mice agrees with the human data. These results have important implications for development of assays and materials for clinical applications of IL-15.

Identification of a Novel Posttranscriptional Regulatory Element by Using a rev- and RRE-Mutated Human Immunodeficiency Virus Type 1 DNA Proviral Clone as a Molecular Trap

ABSTRACT Human immunodeficiency virus (HIV) and all other lentiviruses utilize the essential viral protein Rev, which binds to RRE RNA, to export their unspliced and partially spliced mRNAs from the nucleus. We used a rev- and RRE-defective HIV type 1 (HIV-1) molecular clone in complementation experiments to establish a method for the rapid isolation of posttranscriptional regulatory elements from the mammalian genome by selecting for rescue of virus replication. Viruses rescued by this method contained a novel element with homology to rodent intracisternal A-particle (IAP) retroelements. A functional element was contained within a 247-nucleotide fragment named RNA transport element (RTE), which was able to promote replication of the Rev- and RRE-defective HIV-1 in both human lymphoid cell lines and primary lymphocytes, demonstrating its potent posttranscriptional function. RTE was functional in many cell types, indicating that the cellular factors that recognize RTE are widely expressed and evolutionarily conserved. RTE also promoted RNA export fromXenopus oocyte nuclei. RTE-mediated RNA transport was CRM1 independent, and RTE did not show high affinity for binding to mRNA export factor TAP/NXF1. Since CRM1 and TAP/NXF1 are critical export receptors associated with the two recognized mRNA export pathways, these results suggest that RTE functions via a distinct export mechanism. Taken together, our results identify a novel posttranscriptional control element that uses a conserved cellular export mechanism.

RNA-binding Motif Protein 15 Binds to the RNA Transport Element RTE and Provides a Direct Link to the NXF1 Export Pathway

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.

Long-Lasting Decrease in Viremia in Macaques Chronically Infected with Simian Immunodeficiency Virus SIVmac251 after Therapeutic DNA Immunization

ABSTRACT Rhesus macaques chronically infected with highly pathogenic simian immunodeficiency virus (SIV) SIVmac251 were treated with antiretroviral drugs and vaccinated with combinations of DNA vectors expressing SIV antigens. Vaccination during therapy increased cellular immune responses. After the animals were released from therapy, the virus levels of 12 immunized animals were significantly lower (P = 0.001) compared to those of 11 animals treated with only antiretroviral drugs. Vaccinated animals showed a persistent increase in immune responses, thus indicating both a virological and an immunological benefit following DNA therapeutic vaccination. Several animals show a long-lasting decrease in viremia, suggesting that therapeutic vaccination may provide an additional benefit to antiretroviral therapy.

Characterization and favorable in vivo properties of heterodimeric soluble IL-15/IL-15Rα cytokine compared to IL-15 monomer

Background: IL-15 acts in vivo in association with IL-15Rα, as a heterodimeric cytokine. Results: Authentically processed and glycosylated IL-15·sIL-15Rα heterodimer was purified from human cells and characterized by sequencing and functional studies. Conclusion: IL-15 heterodimer shows favorable in vivo properties compared with monomeric IL-15. Significance: Characterization and availability of the IL-15 heterodimer is important for functional studies and clinical applications. Interleukin-15 (IL-15), a 114-amino acid cytokine related to IL-2, regulates immune homeostasis and the fate of many lymphocyte subsets. We reported that, in the blood of mice and humans, IL-15 is present as a heterodimer associated with soluble IL-15 receptor α (sIL-15Rα). Here, we show efficient production of this noncovalently linked but stable heterodimer in clonal human HEK293 cells and release of the processed IL-15·sIL-15Rα heterodimer in the medium. Purification of the IL-15 and sIL-15Rα polypeptides allowed identification of the proteolytic cleavage site of IL-15Rα and characterization of multiple glycosylation sites. Administration of the IL-15·sIL-15Rα heterodimer reconstituted from purified subunits resulted in sustained plasma IL-15 levels and in robust expansion of NK and T cells in mice, demonstrating pharmacokinetics and in vivo bioactivity superior to single chain IL-15. These identified properties of heterodimeric IL-15 provide a strong rationale for the evaluation of this molecule for clinical applications.

Kaposi's Sarcoma-Associated Herpesvirus ORF57 Interacts with Cellular RNA Export Cofactors RBM15 and OTT3 To Promote Expression of Viral ORF59

ABSTRACT Kaposis sarcoma-associated herpesvirus (KSHV) encodes ORF57, which promotes the accumulation of specific KSHV mRNA targets, including ORF59 mRNA. We report that the cellular export NXF1 cofactors RBM15 and OTT3 participate in ORF57-enhanced expression of KSHV ORF59. We also found that ectopic expression of RBM15 or OTT3 augments ORF59 production in the absence of ORF57. While RBM15 promotes the accumulation of ORF59 RNA predominantly in the nucleus compared to the levels in the cytoplasm, we found that ORF57 shifted the nucleocytoplasmic balance by increasing ORF59 RNA accumulation in the cytoplasm more than in the nucleus. By promoting the accumulation of cytoplasmic ORF59 RNA, ORF57 offsets the nuclear RNA accumulation mediated by RBM15 by preventing nuclear ORF59 RNA from hyperpolyadenylation. ORF57 interacts directly with the RBM15 C-terminal portion containing the SPOC domain to reduce RBM15 binding to ORF59 RNA. Although ORF57 homologs Epstein-Barr virus (EBV) EB2, herpes simplex virus (HSV) ICP27, varicella-zoster virus (VZV) IE4/ORF4, and cytomegalovirus (CMV) UL69 also interact with RBM15 and OTT3, EBV EB2, which also promotes ORF59 expression, does not function like KSHV ORF57 to efficiently prevent RBM15-mediated nuclear accumulation of ORF59 RNA and RBM15s association with polyadenylated RNAs. Collectively, our data provide novel insight elucidating a molecular mechanism by which ORF57 promotes the expression of viral intronless genes.

Nuclear export factor RBM15 facilitates the access of DBP5 to mRNA

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.