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 , TransfectionABSTRACT
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/metabolismABSTRACT
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 VirusABSTRACT
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/geneticsABSTRACT
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 laevisABSTRACT
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 VirusABSTRACT
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.
