Export of hepatitis B virus RNA on a Rev-like pathway: inhibition by the regenerating liver inhibitory factor IkappaB alpha.

Nuclear export of hepatitis B virus (HBV) RNA is mediated by a specific RNA element but, in contrast to lentivirus genomic RNA, does not depend on viral proteins. We show that nonetheless, the export of HBV RNA can be blocked by competitive inhibitors of Rev-mediated lentivirus RNA export, suggesting that the export pathways of both viral species share components and might be driven by the same nuclear export machinery. HBV RNA export is also inhibited by overexpression of IkappaB alpha, as reported previously for the export of human immunodeficiency virus RNA. Since IkappaB alpha is strongly overexpressed during liver regeneration, its inhibition of HBV RNA export might contribute to elimination or silent persistence of HBV.     

The interactions with Ro60 and La differentially affect nuclear export of hY1 RNA.

Ro RNPs are evolutionarily conserved ribonucleoprotein particles that consist of a small RNA, known as Y RNA, associated with several proteins, such as La, Ro60, and Ro52. The Y RNAs (Y1-Y5), which are transcribed by RNA polymerase III, have been shown to reside almost exclusively in the cytoplasm as Ro RNPs. To obtain more insight into the nuclear export pathway of Y RNAs, hY1 RNA export was studied in Xenopus laevis oocytes. Injection of various hY1 RNA mutants showed that an intact Ro60 binding site is a prerequisite for nuclear export, whereas the presence of an intact La binding site resulted in strong nuclear retention of hY1 RNA. Competition studies with various classes of RNAs indicated that, in addition to Ro60, another titratable factor was necessary for nuclear export of hY1 RNA. This factor appears also to be involved in nuclear export of tRNA. Because export of hY1 RNA could not be blocked by a synthetic peptide containing the recently identified nuclear export signal of the HIV-1 Rev protein, nuclear export of hY1 RNA does not seem to be dependent on a Rev-like nuclear export signal.     

Regulation of the export of RNA from the nucleus

Transport of macromolecules across the nuclear envelope is an essential activity in eukaryotic cells. RNA molecules within cells are found complexed with proteins and the bound proteins likely contain signals for RNA export. RNAs microinjected into Xenopus oocyte nuclei are readily exported, and their export can be competed by self RNA but not by RNAs of other classes. This indicates that the rate-limiting step in RNA export is the interaction of RNAs with class-specific proteins, at least when substrate RNAs are present at saturating levels. Export of host mRNAs is inhibited following infection by some animal viruses, while the export of viral RNAs occurs. The HIV-1 RNA-binding protein, Rev, mediates the export of intron-containing viral RNAs that would normally be retained in nuclei. This requires a nuclear export signal (NES) within Rev and an element within the RNA to which Rev binds. In yeast, heat shock causes accumulation of poly(A)(+)RNA within nuclei but heat-shock mRNAs are transcribed and exported efficiently. This requires elements within heat shock mRNA that probably interact with a cellular protein to facilitate RNA export. In these cases, the proteins that recognize critical sequences in the RNAs probably direct the RNAs to an RNA export pathway not generally used for mRNA export. This would circumvent the general retention of most poly(A)(+)mRNAs following heat shock in yeast and the need for complete splicing of viral mRNAs that travel through the normal mRNA export pathway.     

The RNA transport element of the murine musD retrotransposon requires long-range intramolecular interactions for function

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.     

The constitutive transport element (CTE) of Mason-Pfizer monkey virus (MPMV) accesses a cellular mRNA export pathway.

The constitutive transport elements (CTEs) of type D retroviruses are cis-acting elements that promote nuclear export of incompletely spliced mRNAs. Unlike the Rev response element (RRE) of human immunodeficiency virus type 1 (HIV-1), CTEs depend entirely on factors encoded by the host cell genome. We show that an RNA comprised almost entirely of the CTE of Mason-Pfizer monkey virus (CTE RNA) is exported efficiently from Xenopus oocyte nuclei. The CTE RNA and an RNA containing the RRE of HIV-1 (plus Rev) have little effect on export of one another, demonstrating differences in host cell requirements of these two viral mRNA export pathways. Surprisingly, even very low amounts of CTE RNA block export of normal mRNAs, apparently through the sequestration of cellular mRNA export factors. Export of a CTE-containing lariat occurs when wild-type CTE, but not a mutant form, is inserted into the pre-mRNA. The CTE has two symmetric structures, either of which supports export and the titration of mRNA export factors, but both of which are required for maximal inhibition of mRNA export. Two host proteins bind specifically to the CTE but not to non-functional variants, making these proteins candidates for the sequestered mRNA export factors.     

The nucleoporin nup153 plays a critical role in multiple types of nuclear export

The fundamental process of nucleocytoplasmic transport takes place through the nuclear pore. Peripheral pore structures are presumably poised to interact with transport receptors and their cargo as these receptor complexes first encounter the pore. One such peripheral structure likely to play an important role in nuclear export is the basket structure located on the nuclear side of the pore. At present, Nup153 is the only nucleoporin known to localize to the surface of this basket, suggesting that Nup153 is potentially one of the first pore components an RNA or protein encounters during export. In this study, anti-Nup153 antibodies were used to probe the role of Nup153 in nuclear export in Xenopus oocytes. We found that Nup153 antibodies block three major classes of RNA export, that of snRNA, mRNA, and 5S rRNA. Nup153 antibodies also block the NES protein export pathway, specifically the export of the HIV Rev protein, as well as Rev-dependent RNA export. Not all export was blocked; Nup153 antibodies did not impede the export of tRNA or the recycling of importin beta to the cytoplasm. The specific antibodies used here also did not affect nuclear import, whether mediated by importin alpha/beta or by transportin. Overall, the results indicate that Nup153 is crucial to multiple classes of RNA and protein export, being involved at a vital juncture point in their export pathways. This juncture point appears to be one that is bypassed by tRNA during its export. We asked whether a physical interaction between RNA and Nup153 could be observed, using homoribopolymers as sequence-independent probes for interaction. Nup153, unlike four other nucleoporins including Nup98, associated strongly with poly(G) and significantly with poly(U). Thus, Nup153 is unique among the nucleoporins tested in its ability to interact with RNA and must do so either directly or indirectly through an adaptor protein. These results suggest a unique mechanistic role for Nup153 in the export of multiple cargos.     

The molecular mechanisms of messenger RNA nuclear export

In eukaryotic cells, the nuclear membrane creates a barrier between the nucleus and the cytoplasm. Whereas RNA synthesis occurs in the nucleus, they mostly function in the cytoplasm; thus export of RNA molecules from the nucleus to the cytoplasm is indispensable for normal function of the cells. The molecular mechanisms involved in each kind of cellular RNA export is gradually understood. The focus of this review will be mRNA export. mRNAs are multiformed. In order to ensure that this variety of mRNA molecules are all exported, cells are probably equipped with multiple export pathways. A number of proteins is predicted to be involved in mRNA export. Ascertaining which proteins play crucial roles in the pathways is the key point in the study of mRNA export.     

PHAX, a mediator of U snRNA nuclear export whose activity is regulated by phosphorylation

In metazoa, assembly of spliceosomal U snRNPs requires nuclear export of U snRNA precursors. Export depends upon the RNA cap structure, nuclear cap-binding complex (CBC), the export receptor CRM1/Xpo1, and RanGTP. These components are however insufficient to support U snRNA export. We identify PHAX (phosphorylated adaptor for RNA export) as the additional factor required for U snRNA export complex assembly in vitro. In vivo, PHAX is required for U snRNA export but not for CRM1-mediated export in general. PHAX is phosphorylated in the nucleus and then exported with RNA to the cytoplasm, where it is dephosphorylated. PHAX phosphorylation is essential for export complex assembly while its dephosphorylation causes export complex disassembly. The compartmentalized PHAX phosphorylation cycle can contribute to the directionality of export.     

HIV-1 rev promotes the nuclear export of unspliced and singly spliced RNAs in a mammalian cell-free export system

Rev has been shown to promote the export of HIV-1 RNAs fromXenopus oocyte nuclei, but a system to examine the direct effect of Rev on HIV-1 RNA export in mammalian somatic cells does not exist. In this report, the development of a cell-free RNA export system using COS cells is described. This system is capable of examining the movement of RNA from nuclei of COS cells transfected with an HIV-1 proviral construct into reconstituted cytosol from nontransfected cells. A reproducible preparation of nuclei free of residual cytoplasmic RNA is demonstrated. Export of RNA from these nuclei into reconstituted cell-free extracts was saturable and dependent on temperature and energy. Further validation of the system was obtained by confirming that the nuclear export of HIV-1-unspliced and partially spliced RNAs was dependent upon the expression of HIV-1 Rev and that the presence of Rev appeared to decrease the export of an HIV-1-spliced RNA. The system was also able to demonstrate that Rev did not appear to significantly enhance the export of an HIV-1 protease-containing RNA that has been shown to be dependent upon Rev for maximal expression. Consequently, the system appears useful for the examination of parameters of nuclear export of HIV-1 and cellular RNAs.     

Coupling of nuclear RNA export and protein import in vertebrate cells

Nuclear RNA export is a highly dynamic process in which factors that carry the RNAs out of the nucleus must be re-imported. These RNA export factors are bifunctional molecules that recognize specific RNA sequences or structures and interact with shared nuclear proteins, including components of the nuclear pore complex. Inhibition of protein import by inactivation of the GTPase Ran, or its associated activating and recycling factors, blocks RNA export. However a few classes of RNAs escape this inhibition, perhaps because they do not use shuttling export factors or their factors have been stockpiled in the nucleus. Because of its critical role in gene expression, RNA export is a target for control by both cells and viruses.     

A role for nucleoporin FG repeat domains in export of human immunodeficiency virus type 1 Rev protein and RNA from the nucleus.

The human immunodeficiency virus type 1 Rev protein contains a nuclear export signal (NES) that is required for Rev-mediated RNA export in mammals as well as in the yeast Saccharomyces cerevisiae. The Rev NES has been shown to specifically interact with a human (hRIP/RAB1) and a yeast (yRip1p) protein in the two-hybrid assay. Both of these interacting proteins are related to FG nucleoporins on the basis of the presence of typical repeat motifs. This paper shows that Rev is able to interact with multiple FG repeat-containing nucleoporins from both S. cerevisiae and mammals; moreover, the ability of Rev NES mutants to interact with these FG nucleoporins parallels the ability of the mutants to promote RNA export in yeast and mammalian cells. The data also show that, after Xenopus oocyte nuclear injection, several FG nucleoporin repeat domains inhibit the export of both Rev protein and U small nuclear RNAs, suggesting that these nucleoporins participate in Rev-mediated and cellular RNA export. Interestingly, not all FG nucleoporin repeat domains produced the same pattern of RNA export inhibition. The results suggest that Rev and cellular mediators of RNA export can interact with multiple components of the nuclear pore complex during transport, analogous to the proposed mode of action of the nuclear protein import receptor.     

Identification of a novel cis-acting RNA element involved in nuclear export of hY RNAs

Ro RNPs are small cytoplasmic RNA-protein complexes of unknown function that have been found in all metazoan cells studied so far. In human cells, Ro RNPs consist of one of four small RNA molecules, termed hY RNAs and at least two well-characterized proteins, Ro60 and La. In previous Xenopus laevis oocyte microinjection studies, we showed that an intact Ro60 binding site (Stem-loop 1) is a prerequisite for efficient nuclear export of hY1 RNA, whereas an intact La-binding site promotes nuclear retention (Simons et al. RNA, 1996, 2:264-273). Here we present evidence that the distal half (Stem 2) of the conserved base-paired stem structure found in all hY RNAs also plays a critical role in the export process. A minimal RNA molecule containing this region, L1S2 RNA, competes effectively for the export of full-length hY1 RNAs and is itself exported very rapidly in a Ro60-independent and RanGTP-dependent manner. Mutational analyses of this RNA shows that a 5'/3' terminal double-stranded stem structure (>10 bp) of no specific nucleotide sequence constitutes a novel nuclear export element (NEE). Cross-competition studies indicate that this type of NEE may also be involved in export of other classes of RNAs. Like full-length hY1 RNA, L1S2 RNA also competes for export of ET-202 RNA, an RNA that was selected for its efficient nuclear export in the presence of the nuclear transport inhibitor, VSV Matrix protein (Grimm et al. Proc Natl Acad Sci USA, 1997, 94:10122-10127). However, export of L1S2 RNA is strongly inhibited by VSV-M protein, showing that these RNAs use partially overlapping, but not identical export pathways. We propose that export of Y RNAs is mediated by two contiguous cis-acting elements in the 5'/3' double-stranded stem region that is conserved between different Y RNAs.     

Monomethylated cap structures facilitate RNA export from the nucleus

RNA export from the nucleus has been analyzed in Xenopus oocytes. U1 snRNAs made by RNA polymerase II were exported into the cytoplasm, while U1 snRNAs synthesized by RNA polymerase III, and therefore with a different cap structure, remained in the nucleus. Export of the polymerase II-transcribed RNAs was inhibited by the cap analog m7GpppG. Spliced mRNAs carrying monomethylguanosine cap structures were rapidly exported, while hypermethylated cap structures delayed mRNA export. The export of a mutant precursor mRNA unable to form detectable splicing complexes was also significantly delayed by incorporation of a hypermethylated cap structure. The results suggest that the m7GpppN cap structure is likely to be a signal for RNA export from the nucleus.     

RNA-binding of the human cytomegalovirus transactivator protein UL69, mediated by arginine-rich motifs, is not required for nuclear export of unspliced RNA

The human cytomegalovirus protein pUL69 belongs to a family of regulatory factors that is conserved within the Herpesviridae and includes the proteins ICP27 of herpes simplex virus type 1 and EB2 of Epstein–Barr virus. ICP27 and EB2 have been shown to facilitate the nuclear export of viral mRNAs via interacting with the cellular mRNA export factor REF. Furthermore, direct RNA-binding of these proteins was found to be essential for their stimulating effects on mRNA export. Recently, we demonstrated that pUL69 shares common features with ICP27 and EB2 such as (i) nucleocytoplasmic shuttling and (ii) stimulation of nuclear RNA export via binding to the cellular mRNA export machinery. Here, we demonstrate that pUL69 can also interact with RNA both in vivo and in vitro via a complex N-terminal RNA-binding domain consisting of three arginine-rich motifs. Interestingly, the RNA-binding domain of pUL69 overlaps with both the NLS and the binding site of the cellular mRNA export factors UAP56 and URH49. While the deletion of the UAP56/URH49-binding site abolished pUL69-mediated RNA export, an RNA-binding deficient pUL69 mutant which still interacts with UAP56/URH49 retained its RNA export activity. This surprising finding suggests that, in contrast to its homologues, RNA-binding is not a prerequisite for pUL69-mediated nuclear RNA export.