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.     

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.     

Protein export from the mitochondrial matrix

Assembly of a functional mitochondrion requires import of proteins from the cytosol and export of proteins from the matrix. Most previous studies have focused on the import pathway followed by nucleus-encoded proteins. However, it is now clear that proteins encoded in the nucleus as well as those encoded in the mitochondrion also move from the matrix into and across the inner membrane, a process defined here as export. These exported proteins are found in at least three cellular locations: the inner mitochondrial membrane, the intermembrane space and the cell surface. Here, we consider the pathways for export and the relationships between import and export.     

Protein export elements from Lactococcus lactis

Summary Broad-host-range plasmids carrying -amylase or -lactamase reporter genes lacking a signal sequence were used to select export elements from Lactococcus lactis chromosomal DNA that could function as signal sequences. Fragments containing such elements were identified by their ability to direct the export of the reporter proteins in Escherichia coli. Several of the selected export elements were also active in Bacillus subtilis and L. lactis, although the efficiencies depended strongly on the host organism and reporter gene used. The export elements AL9 and BL1 were highly efficient in L. lactis in the expression and secretion of at least two heterologous proteins (Bacillus licheniformis -amylase and E. coli TEM--lactamase). AL9 even permitted growth of this organism on starch as the sole carbon source. Nucleotide sequence analysis of five selected fragments indicated that these encode oligopeptides with the major characteristics of typical signal peptides. The putative expression signals had a limited similarity to previously described expression signals for E. coli, B. subtilis and L. lactis. Differences in both expression and export efficiency are likely to underlie the host-specific effects.     

Protein export through the bacterial flagellar type III export pathway

For construction of the bacterial flagellum, which is responsible for bacterial motility, the flagellar type III export apparatus utilizes both ATP and proton motive force across the cytoplasmic membrane and exports flagellar proteins from the cytoplasm to the distal end of the nascent structure. The export apparatus consists of a membrane-embedded export gate made of FlhA, FlhB, FliO, FliP, FliQ, and FliR and a water-soluble ATPase ring complex consisting of FliH, FliI, and FliJ. FlgN, FliS, and FliT act as substrate-specific chaperones that do not only protect their cognate substrates from degradation and aggregation in the cytoplasm but also efficiently transfer the substrates to the export apparatus. The ATPase ring complex facilitates the initial entry of the substrates into the narrow pore of the export gate. The export gate by itself is a proton-protein antiporter that uses the two components of proton motive force, the electric potential difference and the proton concentration difference, for different steps of the export process. A specific interaction of FlhA with FliJ located in the center of the ATPase ring complex allows the export gate to efficiently use proton motive force to drive protein export. The ATPase ring complex couples ATP binding and hydrolysis to its assembly–disassembly cycle for rapid and efficient protein export cycle. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

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.     

Protein kinase Czeta-dependent LKB1 serine 428 phosphorylation increases LKB1 nucleus export and apoptosis in endothelial cells

LKB1 is a serine-threonine protein kinase that, when inhibited, may result in unregulated cell growth and tumor formation. However, how LKB1 is regulated remains poorly understood. The aim of the present study was to define the upstream signaling events responsible for peroxynitrite (ONOO(-))-induced LKB1 activation. Exposure of cultured human umbilical vein endothelial cells to a low concentration of ONOO(-) (5 microM) significantly increased the phosphorylation of LKB1 at Ser(428) and protein kinase Czeta (PKCzeta) at Thr(410). These effects were accompanied by increased activity of the lipid phosphatase PTEN, decreased activity and phosphorylation (Ser(473)) of Akt, and induction of apoptosis. ONOO(-) enhanced Akt-Ser(473) phosphorylation in LKB1-deficient HeLa S3 cells or in HeLa S3 cells transfected with kinase-dead LKB1. Conversely, ONOO(-) inhibited Akt Ser(473) phosphorylation when wild type LKB1 were reintroduced in HeLa S3 cells. Further analysis revealed that PKCzeta directly phosphorylated LKB1 at Ser(428) in vitro and in intact cells, resulting in increased PTEN phosphorylation at Ser(380)/Thr(382/383). Finally, ONOO(-) enhanced PKCzeta nuclear import and LKB1 nuclear export. We conclude that PKCzeta mediates LKB1-dependent Akt inhibition in response to ONOO(-), resulting in endothelial apoptosis.     

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.     

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.     

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.