Protein and RNA export from the nucleus

The presence of the nuclear envelope necessitates the movement of proteins and RNAs between the nucleus and the cytoplasm. Elaborate cellular machinery exists to promote the nuclear transport of macromolecules. Recent advances in the field have illuminated our comprehension of both nuclear import and export as powerful means of gene regulation. As our appreciation of the importance of the process has grown, its study has matured, moving beyond the single cell to the entire organism. This review discusses basic mechanisms and regulation of protein, mRNA, and ribosome export with an emphasis on developmental examples.     

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.     

Regulation of retroviral RNA export

Viruses are obligate intracellular parasites and have to use the host cell machinery for their replication. Many viruses are able to divert different parts of this machinery to preferentially enhance virus replication at the expense of the cell. The mechanisms by which different viruses do this have, over the years, given us great insight into many cellular processes. Although we still know relatively little about how RNA is exported from the nucleus to the cytoplasm and how this process is regulated, retroviruses have already emerged as one of the most important model systems for these studies. This review will attempt to summarize what we have learnt from these viruses to date and what we hope to achieve in the near future.     

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.     

The origin of nucleus: rebuild from the prokaryotic ancestors of ribosome export factors

Various hypotheses have been proposed on the evolutionary origin of eukaryotic nucleus. Because one of the major cargoes in the nucleocytoplasmic export in the eukaryotic cell is the ribosome, its stimulating proteins called Ribosome Export Factors (REFs) might have an evolutionary history of inscribing the origin of eukaryotic nucleus. With the aim of understanding the evolutionary origin of the nucleus, here we employed the yeast REFs and searched for their evolutionary origin in more than 500 genomes of archaea and eubacteria by the PSI-BLAST search. Our results showed that the non-membranous REFs (non-mREFs) originated exclusively from eubacterial proteins, whereas the membranous REFs (mREFs) are from both archaeal and eubacterial proteins. Since the non-mREFs just work inside the nucleus while the mREFs shuttle between the nucleus and the cytoplasm, these results suggest that the extant REFs working inside the nucleus have derived exclusively from eubacterial proteins, implying that the nucleus arose in a cell that contained chromosomes possessing a substantial fraction of eubacterial genes, in line with the predictions of several models entailing endosymbiosis at eukaryote origins.     

A compartmentalized phosphorylation/dephosphorylation system that regulates U snRNA export from the nucleus

PHAX (phosphorylated adaptor for RNA export) is the key regulator of U snRNA nuclear export in metazoa. Our previous work revealed that PHAX is phosphorylated in the nucleus and is exported as a component of the U snRNA export complex to the cytoplasm, where it is dephosphorylated (M. Ohno, A. Segref, A. Bachi, M. Wilm, and I. W. Mattaj, Cell 101:187-198, 2000). PHAX phosphorylation is essential for export complex assembly, whereas its dephosphorylation causes export complex disassembly. Thus, PHAX is subject to a compartmentalized phosphorylation/dephosphorylation cycle that contributes to transport directionality. However, neither essential PHAX phosphorylation sites nor the modifying enzymes that contribute to the compartmentalized system have been identified. Here, we identify PHAX phosphorylation sites that are necessary and sufficient for U snRNA export. Mutation of the phosphorylation sites inhibited U snRNA export in a dominant-negative way. We also show, by both biochemical and RNA interference knockdown experiments, that the nuclear kinase and the cytoplasmic phosphatase for PHAX are CK2 kinase and protein phosphatase 2A, respectively. Our results reveal the composition of the compartmentalized phosphorylation/dephosphorylation system that regulates U snRNA export. This finding was surprising in that such a specific system for U snRNA export regulation is composed of two such universal regulators, suggesting that this compartmentalized system is used more broadly for gene expression regulation.     

Export of adenoviral late mRNA from the nucleus requires the Nxf1/Tap export receptor

One important function of the human adenovirus E1B 55-kDa protein is induction of selective nuclear export of viral late mRNAs. This protein interacts with the viral E4 Orf6 and four cellular proteins to form an infected-cell-specific E3 ubiquitin ligase. The assembly of this enzyme is required for efficient viral late mRNA export, but neither the relevant substrates nor the cellular pathway that exports viral late mRNAs has been identified. We therefore examined the effects on viral late gene expression of inhibition of the synthesis or activity of the mRNA export receptor Nxf1, which was observed to colocalize with the E1B 55-kDa protein in infected cells. When production of Nxf1 was impaired by using RNA interference, the efficiency of viral late mRNA export was reduced to a corresponding degree. Furthermore, synthesis of a dominant-negative derivative of Nxf1 during the late phase of infection interfered with production of a late structural protein. These observations indicate that the Nxf1 pathway is responsible for export of viral late mRNAs. As the infected-cell-specific E3 ubiquitin ligase targets its known substrates for proteasomal degradation, we compared the concentrations of several components of this pathway (Nxf1, Thox1, and Thoc4) in infected cells that did or did not contain this enzyme. Although the concentration of a well-established substrate, Mre11, decreased significantly in cells infected by adenovirus type 5 (Ad5), but not in those infected by the E1B 55-kDa protein-null mutant Hr6, no E1B 55-kDa protein-dependent degradation of the Nxf1 pathway proteins was observed.     

Nuclear RNA export in yeast.

Eukaryotic cells massively exchange macromolecules (proteins and RNAs) between the nucleus and cytoplasm through the nuclear pore complexes. Whereas a mechanistic picture emerges of how proteins are imported into and exported from the nucleus, less is known about nuclear exit of the different classes of RNAs. However, the yeast Saccharomyces cerevisiae offers an experimental system to study nuclear RNA export in vivo and thus to genetically dissect the different RNA export machineries. In this review, we summarize our current knowledge and recent progress in identifying components involved in nuclear RNA export in yeast.