An NXF1 mRNA with a retained intron is expressed in hippocampal and neocortical neurons and is translated into a protein that functions as an Nxf1 cofactor.

The Nxf1 protein is a major nuclear export receptor for the transport of mRNA, and it also is essential for export of retroviral mRNAs with retained introns. In the latter case, it binds to RNA elements known as constitutive transport elements (CTEs) and functions in conjunction with a cofactor known as Nxt1. The NXF1 gene also regulates expression of its own intron-containing RNA through the use of a functional CTE within intron 10. mRNA containing this intron is exported to the cytoplasm, where it can be translated into the 356-amino acid short Nxf1(sNxf1) protein, despite the fact that it is a prime candidate for nonsense-mediated decay (NMD). Here we demonstrate that sNxf1 is highly expressed in nuclei and dendrites of hippocampal and neocortical neurons in rodent brain. Additionally, we show that sNxf1 localizes in RNA granules in neurites of differentiated N2a mouse neuroblastoma cells, where it shows partial colocalization with Staufen2 isoform SS, a protein known to play a role in dendritic mRNA trafficking. We also show that sNxf1 forms heterodimers in conjunction with the full-length Nxf1 and that sNxf1 can replace Nxt1 to enhance the expression of CTE-containing mRNA and promote its association with polyribosomes.

The Tpr protein regulates export of mRNAs with retained introns that traffic through the Nxf1 pathway.

Post-transcriptional regulation of mRNA includes restriction mechanisms to prevent export and expression of mRNAs that are incompletely spliced. Here we present evidence that the mammalian protein Tpr is involved in this restriction. To study the role of Tpr in export of mRNA with retained introns, we used reporters in which the mRNA was exported either via the Nxf1/Nxt1 pathway using a CTE or via the Crm1 pathway using Rev/RRE. Our data show that even modest knockdown of Tpr using RNAi leads to a significant increase in export and translation from the mRNA containing the CTE. In contrast, Tpr perturbation has no effect on export of mRNA containing the RRE, either in the absence or presence of Rev. Also, no effects were observed on export of a completely spliced mRNA. Taken together, our results indicate that Tpr plays an important role in quality control of mRNA trafficked on the Nxf1 pathway.

The shuttling SR protein 9G8 plays a role in translation of unspliced mRNA containing a constitutive transport element.

The splicing regulatory SR protein, 9G8, has recently been proposed to function in mRNA export in conjunction with the export protein, Tap/NXF1. Tap interacts directly with the Mason-Pfizer monkey virus constitutive transport element (CTE), an element that enables export of unspliced, intron-containing mRNA. Based on our previous finding that Tap can promote polysome association and translation of CTE-RNA, we investigated the effect of 9G8 on cytoplasmic RNA fate. 9G8 was shown to enhance expression of unspliced RNA containing either the Mason-Pfizer monkey virus-CTE or the recently discovered Tap-CTE. 9G8 also enhanced polyribosome association of unspliced RNA containing a CTE. Hyperphosphorylated 9G8 was present in monosomes and small polyribosomes, whereas soluble fractions contained only hypophosphorylated protein. Our results are consistent with a model in which hypophosphorylated SR proteins remain stably associated with messenger ribonucleoprotein (mRNP) complexes during export and are released during translation initiation concomitant with increased phosphorylation. These results provide further evidence for crucial links between RNA splicing, export and translation.

The envelope gene is a cytopathic determinant of CCR5 tropic HIV-1.

Late stage AIDS associated CCR5 tropic HIV-1 clones (R5-AIDS HIV-1) exhibit greater cytopathic effects (CPE) than earlier isolates from the same patients. In this study, envelopes from a series of three biological clones derived from the same patient were evaluated as a cytopathic determinant of R5-AIDS HIV-1 for thymocytes. In a single round of replication in thymocytes, the AIDS associated clone mediated greater initiation of reverse transcription. This enhancement was not due to broadened coreceptor tropism, as all clones studied were exclusively R5 tropic. The full-length R5-AIDS env mediated greater infectivity than R5 pre-AIDS env when used to pseudotype a reporter virus. R5-AIDS env pseudotypes were more resistant to TAK-779 and showed more rapid infection kinetics but similar resistance to a CD4 blocking mAb. We conclude that the enhanced thymic replication and CPE shown by the R5-AIDS clone is due to enhanced efficiency of Env-mediated entry via CCR5.

An intron with a constitutive transport element is retained in a Tap messenger RNA.

Alternative splicing is a key factor contributing to genetic diversity and evolution. Intron retention, one form of alternative splicing, is common in plants but rare in higher eukaryotes, because messenger RNAs with retained introns are subject to cellular restriction at the level of cytoplasmic export and expression. Often, retention of internal introns restricts the export of these mRNAs and makes them the targets for degradation by the cellular nonsense-mediated decay machinery if they contain premature stop codons. In fact, many of the database entries for complementary DNAs with retained introns represent them as artefacts that would not affect the proteome. Retroviruses are important model systems in studies of regulation of RNAs with retained introns, because their genomic and mRNAs contain one or more unspliced introns. For example, Mason-Pfizer monkey virus overcomes cellular restrictions by using a cis-acting RNA element known as the constitutive transport element (CTE). The CTE interacts directly with the Tap protein (also known as nuclear RNA export factor 1, encoded by NXF1), which is thought to be a principal export receptor for cellular mRNA, leading to the hypothesis that cellular mRNAs with retained introns use cellular CTE equivalents to overcome restrictions to their expression. Here we show that the Tap gene contains a functional CTE in its alternatively spliced intron 10. Tap mRNA containing this intron is exported to the cytoplasm and is present in polyribosomes. A small Tap protein is encoded by this mRNA and can be detected in human and monkey cells. Our results indicate that Tap regulates expression of its own intron-containing RNA through a CTE-mediated mechanism. Thus, CTEs are likely to be important elements that facilitate efficient expression of mammalian mRNAs with retained introns.

The Wilms' tumor 1 (WT1) gene (+KTS isoform) functions with a CTE to enhance translation from an unspliced RNA with a retained intron.

The Wilms' tumor 1 (WT1) gene plays an important role in mammalian urogenital development, and dysregulation of this gene is observed in many human cancers. Alternative splicing of WT1 RNA leads to the expression of two major protein isoforms, WT1(+KTS) and WT1(-KTS). Whereas WT1(-KTS) acts as a transcriptional regulator, no clear function has been ascribed to WT1(+KTS), despite the fact that this protein is crucial for normal development. Here we show that WT1(+KTS) functions to enhance expression from RNA possessing a retained intron and containing either a cellular or viral constitutive transport element (CTE). WT1(+KTS) expression increases the levels of unspliced RNA containing a CTE and specifically promotes the association of this RNA with polyribosomes. These studies provide further support for links between different steps in RNA metabolism and for the existence of post-transcriptional operons.

Mutations in tap uncouple RNA export activity from translocation through the nuclear pore complex.

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.

Human immunodeficiency virus type 1 Nef associates with lipid rafts to downmodulate cell surface CD4 and class I major histocompatibility complex expression and to increase viral infectivity.

Lipid rafts are membrane microdomains that are functionally distinct from other membrane regions. We have shown that 10% of human immunodeficiency virus type 1 (HIV-1) Nef expressed in SupT1 cells is present in lipid rafts and that this represents virtually all of the membrane-associated Nef. To determine whether raft targeting, rather than simply membrane localization, has functional significance, we created a Nef fusion protein (LAT-Nef) containing the N-terminal 35 amino acids from LAT, a protein that is exclusively localized to rafts. Greater than 90% of the LAT-Nef protein was found in the raft fraction. In contrast, a mutated form, lacking two cysteine palmitoylation sites, showed less than 5% raft localization. Both proteins were equally expressed and targeted nearly exclusively to membranes. The LAT-Nef protein was more efficient than its nonraft mutant counterpart at downmodulating both cell surface CD4 and class I major histocompatibility complex (MHC) expression, as well as in enhancing first-round infectivity and being incorporated into virus particles. This demonstrates that targeting of Nef to lipid rafts is mechanistically important for all of these functions. Compared to wild-type Nef, LAT-Nef downmodulated class I MHC nearly as effectively as the wild-type Nef protein, but was only about 60% as effective for CD4 downmodulation and 30% as effective for infectivity enhancement. Since the LAT-Nef protein was found entirely in rafts while the wild-type Nef protein was distributed 10% in rafts and 90% in the soluble fraction, our results suggest that class I MHC downmodulation by Nef may be performed exclusively by raft-bound Nef. In contrast, CD4 downmodulation and infectivity enhancement may require a non-membrane-bound Nef component as well as the membrane-bound form.

Tap and NXT promote translation of unspliced mRNA.

Tap has been proposed to play a role in general mRNA export and also functions in expression of RNA with retained introns that contain the MPMV CTE (constitutive transport element). Tap forms a functional heterodimer with NXT/p15. We have previously demonstrated that unspliced intron-containing CTE RNA is efficiently exported to the cytoplasm in mammalian cells. Here we show that Tap and NXT proteins function together to enhance translation of proteins from the exported CTE RNA. Pulse chase experiments show that Tap/NXT significantly increases the rate of protein synthesis. Sucrose gradient analysis demonstrates that Tap and NXT efficiently shift the unspliced RNA into polyribosomal fractions. Furthermore, Tap, but not NXT is detected in polyribosomes. Taken together, our results indicate that Tap and NXT serve a role in translational regulation of RNA after export to the cytoplasm. They further suggest that Tap/NXT may play a role in remodeling of cytoplasmic RNP complexes, providing a link between export pathways and cytoplasmic fate.

Sam68 enhances the cytoplasmic utilization of intron-containing RNA and is functionally regulated by the nuclear kinase Sik/BRK.

Cells normally restrict the nuclear export and expression of intron-containing mRNA. In many cell lines, this restriction can be overcome by inclusion of cis-acting elements, such as the Mason-Pfizer monkey virus constitutive transport element (CTE), in the RNA. In contrast, we observed that CTE-mediated expression from human immunodeficiency virus Gag-Pol reporters was very inefficient in 293 and 293T cells. However, addition of Sam68 led to a dramatic increase in the amount of Gag-Pol proteins produced in these cells. Enhancement of CTE function was not seen when a Sam68 point mutant (G178E) that is defective for RNA binding was used. Additionally, the effect of Sam68 was inhibited in a dose-dependent manner by coexpression of an activated form of the nuclear kinase Sik/BRK that hyperphosphorylated Sam68. RNA analysis showed that cytoplasmic Gag-Pol-CTE RNA levels were only slightly enhanced by the addition of Sam68, compared to a 60- to 70-fold increase in the levels of Gag-Pol protein expression. Thus, in this system, Sam68 functioned to enhance the cytoplasmic utilization of RNA containing the CTE. These results suggest that Sam68 may interact with specific RNAs in the nucleus to provide a "mark" that affects their cytoplasmic fate. They also provide further evidence of links between signal transduction and RNA utilization.