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.Many eukaryotic mRNAs are subject to regulated turnover via cis-acting signals which are recognized by trans-acting factors. The best-studied example is the cytoplasmic mRNA decay mediated by cis-acting AU-rich RNA elements and trans-acting ARE-binding proteins. Another important mechanism is nonsense-mediated mRNA decay, which serves to eliminate the transcripts that contain premature stop codons (for recent reviews, see references 17, 34, and 40).In contrast to cytoplasmic pathways, nuclear mRNA turnover is less well understood. Among the mRNAs that are subject to nuclear downregulation are the Rev-responsive element (RRE)-containing mRNAs of human immunodeficiency virus type 1 (HIV-1) (11,22,25,26,32,33,42,48,54; reviewed in references 13, 21, 27, and 47). These unspliced and nonterminally spliced transcripts need to be exported from the nucleus before completion of splicing to produce the Gag-Pol and Env proteins. This regulatory step is accomplished by the viral Rev protein, which binds to the RRE and links these transcripts to the CRM1 export receptor. In the absence of Rev, these mRNAs are further spliced to completion or degraded. However, even when devoid of splice sites, the unspliced or nonterminally spliced mRNAs are poorly expressed due to the presence of cis-acting instability elements (INS/ CRS) that are scattered throughout the gag/pol and env mRNAs (11,25,32,42,50,51,54). These elements act at several steps, impairing mRNA stability, nucleocytoplasmic transport, and translation (5,15,22,29,33,51,54), whereas Rev counteracts these defects, resulting in efficient expression. However, Rev is unable to export nonterminally spliced mRNAs that do not contain a functional INS (51), and hence, INS are an integral part of Rev regula...

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.Posttranscriptional regulation is an essential regulatory step of many retroviruses and is necessary for virus production. This key regulatory step mediates the export of the unspliced, fulllength viral RNA, which requires the interaction of viral and/or cellular factors. This controlled export of the viral RNA to the cytoplasm ensures the availability of the genomic RNA for packaging into the progeny virions and the production of the Gag/Pol polyproteins. Among the best-studied export systems are those used by the simian type D retroviruses (SRV/D) and the lentiviruses, such as human immunodeficiency virus type 1 (HIV-1) (for reviews see references 6, 14, and 28).SRV/D expression is controlled by the essential cis-acting constitutive transport element (CTE) (5,12,54,63). The SRV/D CTE (11, 55) and a related CTE-like element in a murine intracisternal A-particle retroelement (54) fold into an extended RNA stem-loop structure containing two conserved internal loops and an AAGA bulge. These loops and the bulge, the spacing of the loops within the RNA element, as well as the overall secondary structure of the element, have been shown to be essential features for CTE function (11,55). Recently, we showed that the human TAP protein (hTAP) binds specifically to these internal loops and promotes nucleocytoplasmic transport of the CTE-containing intron lariat from the Xenopus oocyte nucleus (22). TAP had previously been identified as a factor binding to Tip, a herpesvirus saimiri protein responsible for cell transformation (60). The role of hTAP interaction with Tip is still unclear.Whereas the SRV/D retroviruses have been proposed to utilize the cellular hTAP protein to export their unspliced mRNA (22), HIV-1 uses the viral Rev protein to promote the transport of the Rev responsive element (RRE)-containing...

Molecular clones of human immunodeficiency virus type 1 that contained either 37 point mutations in the Rev-responsive element (RRE) that did not affect the overlapping env reading frame or both a mutated RRE and two mutations that eliminated Rev were constructed. The mutations in the RRE were shown to remove both negative and Rev-inducible positive elfects of the RRE on gene expression (G. Nasioulas, A

, 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.

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.General mRNA export in eukaryotes is mediated by NXF1 protein orthologues that are conserved from yeast to humans and bind to the export-ready mRNP, targeting them to the nuclear pore complex (NPC) 2 (1-6). NXF1 acts as part of a stable heterodimer with its cofactor p15/NXT1 (7-9). Splicing changes the mRNP protein composition, allowing NXF1-p15 to bind and export to occur, whereas the pre-mRNPs are normally retained in the nucleus until completely spliced (10 -13). In particular, a set of proteins known as exon junction complex (EJC) is deposited onto mRNP as a result of splicing (14), providing critical determinants for the subsequent metabolic steps, including nuclear export, quality control, cytoplasmic trafficking, and translation (15). EJC consists of a stably bound core composed of eIF4AIII, Y14-Magoh, and MLN51/Barentsz that serves as a platform for a multitude of other EJC and EJC-associated factors that are bound more transiently. EJC is thought to commit the spliced mRNPs to nuclear export by providing binding sites for the NXF1-p15 export receptor. In one scenario, the EJC factor UAP56 recruits Aly/REF proteins, which bind directly to NXF1-p15, which in turn tethers the export substrate to the NPC (16 -21). Alternatively, NXF1 may assemble with the spliced mRNP via interactions with non-EJC factors such as SR proteins: SRp20 and 9G8 (22, 23), ASF/SF2 (24), and U2AF (25). Thus, it appears that several pathways lead to the binding of NXF1-p15 with the export-ready mRNP. Upon NXF1-p15-dependent targeting to NPC, such complexes are translocated to the cytoplasm by a yet unknown mechanism. NXF1 is a conserved export receptor for cellular mRNAs (1-6). Proteins of the NXF family can act on nuclear as well as on cytoplasmic mRNA trafficking (26 -28).According to the current model, general mRNA metabolism requires the acquisition of an export signal as a result of splicing, whereas pre-mRNA is generally retained in the nucleus due both to the lack of active export and ...

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.

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.TAP (also called NXF1)/Mex67p proteins define a conserved mRNA export pathway in eukaryotes (1-4). The Saccharomyces cerevisiae Mex67p and Caenorhabditis elegans Ce-NXF-1 are essential for mRNA export (4, 5), and the vertebrate TAP mediates the export of mRNA, but not the other classes of RNA (3, 6, 7). The Schizosaccharomyces pombe spMex67p is nonessential, but its mRNA export function is required in the absence of the essential Rae1p (8,9). TAP belongs to the NXF family of proteins that share the ability to bind both RNA and the components of the nuclear pore complex (10). The proposed substrate binding domain of TAP (aa 1 1-372) comprises an RNA binding domain and leucine-rich repeat domain and exhibits general RNA binding and high affinity binding to the viral CTE RNA (2, 3, 11). This region is also required for the direct binding to REF (also called Aly) proteins (1, 12), which are the metazoan homologues of yeast mRNA export factor Yra1p (13).At all metabolic steps, mRNA is part of ribonucleoprotein complexes (mRNP). The primary transcripts associate with a subset of heterogeneous nuclear ribonucleoproteins (hnRNPs) (reviewed in Ref. 14) and with the splicing machinery (15, 16), leading to their nuclear retention before splicing. Splicing changes the mRNP composition by promoting the assembly of export-competent mRNP (17, 18) and by depositing "splicing signature" proteins onto ligated exons (19). This protein complex is composed of the splicing-associated factors SRm160, DEK, and RNP S1, as well as REF and the RNA-binding protein Y14 (19, 20). Hence, REF was proposed to provide a link to export, possibly by recruiting TAP (18,21,22). As shown in human cells, TAP is added efficiently to the endogenous postsplicing mRNP complexes (23). In contrast, TAP is not added to the complexes produced by splicing in vitro (18, 19) or in microinjected oocyte nuclei (2). In this work, we addressed the assembly of TAP with the endogenous mR...

Human immunodeficiency virus type 1 (HIV-1) Rev contains a leucine-rich nuclear export signal that is essential for its nucleocytoplasmic export mediated by hCRM1. We examined the role of selected nucleoporins, which are located in peripheral structures of the nuclear pore complex and are thought to be involved in export, in Rev function in human cells. First, we found that upon actinomycin D treatment, Nup98, but not Nup214 or Nup153, is able to translocate to the cytoplasm of HeLa cells, demonstrating that Nup98 may act as a soluble factor. We further showed that Rev can recruit Nup98 and Nup214, but not Nup153, to the nucleolus. We also found that the isolated FG-containing repeat domains of Nup98 and Nup214, but not those of Nup153, competitively inhibit the Rev/RRE-mediated expression of HIV. Taken together, the recruitment of Nup98 and Nup214 by Rev and the competitive inhibition exhibited by their NP domains demonstrate direct participation of Nup98 and Nup214 in the Rev-hCRM1-mediated export.