ZAP is a CRM1-dependent nucleocytoplasmic shuttling protein

The zinc finger antiviral protein (ZAP) is a recently isolated host antiviral factor. It specifically inhibits the replication of Moloney murine leukemia virus (MMLV) and Sindbis virus (SIN) by preventing the accumulation of viral RNA in the cytoplasm. In this report, we demonstrate that ZAP is predominantly localized in the cytoplasm at steady state but shuttles between the nucleus and the cytoplasm in a CRM1-dependent manner. Two nuclear localization sequences (NLS) and one nuclear export sequence (NES) were identified. One NLS was mapped to amino acids 68-RARVCRRK-75 and the other mapped to a region including amino acids K405 and K406. The NES was mapped to amino acids 284-LEDVSVDV-291. These findings help to understand why ZAP specifically prevents the accumulation of viral RNA in the cytoplasm. These findings also suggest possible functions of ZAP in the nucleus.     

A novel transferable nuclear export signal mediates CRM1-independent nucleocytoplasmic shuttling of the human cytomegalovirus transactivator protein pUL69.

The best studied nuclear export processes are mediated by classical leucine-rich nuclear export signals that specify recognition by the CRM1 export receptor. However, details concerning alternative nuclear export signals and pathways are beginning to emerge. Within the family of Herpesviridae, a set of homologous regulatory proteins that are exemplified by the ICP27 of herpes simplex virus were described recently as nucleocytoplasmic shuttling proteins. Here we report that pUL69 of the beta-herpesvirus human cytomegalovirus is a nuclear protein that is able to shuttle between the nucleus and the cytoplasm independently of virus-encoded cofactors. In contrast to proteins containing a leucine-rich export signal, the shuttling activity of pUL69 was not affected by leptomycin B, indicating that pUL69 trafficking is not mediated by the export receptor CRM1. Importantly, we identified and characterized a novel type of transferable, leptomycin B-insensitive export signal that is distinct from other export signals described previously and is required for pUL69-mediated activation of gene expression. These data suggest that pUL69 is exported via a novel nuclear export pathway, based on a so far unique nuclear export signal of 28 amino acids.     

The cellular DExD/H-box RNA-helicases UAP56 and URH49 exhibit a CRM1-independent nucleocytoplasmic shuttling activity

Cellular DExD/H-box RNA-helicases perform essential functions during mRNA biogenesis. The closely related human proteins UAP56 and URH49 are members of this protein family and play an essential role for cellular mRNA export by recruiting the adaptor protein REF to spliced and unspliced mRNAs. In order to gain insight into their mode of action, we aimed to characterize these RNA-helicases in more detail. Here, we demonstrate that UAP56 and URH49 exhibit an intrinsic CRM1-independent nucleocytoplasmic shuttling activity. Extensive mapping studies identified distinct regions within UAP56 or URH49 required for (i) intranuclear localization (UAP56 aa81-381) and (ii) interaction with REF (UAP56 aa51-428). Moreover, the region conferring nucleocytoplasmic shuttling activity was mapped to the C-terminus of UAP56, comprising the amino acids 195-428. Interestingly, this region coincides with a domain within Uap56p of S. pombe that has been reported to be required for both Rae1p-interaction and nucleocytoplasmic shuttling. However, in contrast to this finding we report that human UAP56 shuttles independently from Rae1. In summary, our results reveal nucleocytoplasmic shuttling as a conserved feature of yeast and human UAP56, while their export receptor seems to have diverged during evolution.     

The fission yeast meiotic regulator Mei2p undergoes nucleocytoplasmic shuttling.

Schizosaccharomyces pombe Mei2p is an RNA-binding protein that switches the cell cycle from mitotic to meiotic. Mei2p forms a unique dot in the nucleus prior to meiosis I, aided by a non-coding RNA molecule termed meiRNA. Here we show that Mei2p intrinsically undergoes nucleocytoplasmic shuttling. Artificial acceleration of nuclear migration of Mei2p advances nuclear dot formation, but meiRNA does not appear to promote the dot formation by modulating the migration rate of Mei2p into the nucleus. Rather, this RNA is likely to facilitate the assembly of Mei2p into a dot structure and trap the protein as such in the nucleus.     

Characterization of RNA aptamer binding by the Wilms' tumor suppressor protein WT1

The interaction of the zinc finger protein WT1 with RNA aptamers has been investigated using a quantitative binding assay, and the results have been compared to those from a previous study of the DNA binding properties of this protein. A recombinant peptide containing the four zinc fingers of WT1 (WT1-ZFP) binds to representatives of three specific families of RNA aptamers with apparent dissociation constants ranging from 13.8 +/- 1.1 to 87.4 +/- 10.4 nM, somewhat higher than the dissociation constant of 4.12 +/- 0.4 nM for binding to DNA. An isoform that contains an insertion of three amino acids between the third and fourth zinc fingers (WT1[+KTS]-ZFP) also binds to these RNAs with slightly reduced affinity (the apparent dissociation constants ranging from 22.8 to 69.8 nM) but does not bind to DNA. The equilibrium binding of WT1-ZFP to the highest-affinity RNA molecule was compared to the equilibrium binding to a consensus DNA molecule as a function of temperature, pH, monovalent salt concentration, and divalent salt concentration. The interaction of WT1-ZFP with both nucleic acids is an entropy-driven process. Binding of WT1-ZFP to RNA has a pH optimum that is narrower than that observed for binding to DNA. Binding of WT1-ZFP to DNA is optimal at 5 mM MgCl(2), while the highest affinity for RNA was observed in the absence of MgCl(2). Binding of WT1 to both nucleic acid ligands is sensitive to increasing monovalent salt concentration, with a greater effect observed for DNA than for RNA. Point mutations in the zinc fingers associated with Denys-Drash syndrome have dramatically different effects on the interaction of WT1-ZFP with DNA, but a consistent and modest effect on the interaction with RNA. The role of RNA sequence and secondary structure in the binding of WT1-ZFP was probed by site-directed mutagenesis. Results indicate that a hairpin loop is a critical structural feature required for protein binding, and that some consensus nucleotides can be substituted provided proper base pairing of the stem of the hairpin loop is maintained.     

Facilitated nucleocytoplasmic shuttling of the Ran binding protein RanBP1

The Ran binding protein RanBP1 is localized to the cytosol of interphase cells. A leucine-rich nuclear export signal (NES) near the C terminus of RanBP1 is essential to maintain this distribution. We now show that RanBP1 accumulates in nuclei of cells treated with the export inhibitor, leptomycin B, and collapse of the nucleocytoplasmic Ran:GTP gradient leads to equilibration of RanBP1 across the nuclear envelope. Low temperature prevents nuclear accumulation of RanBP1, suggesting that import does not occur via simple diffusion. Glutathione S-transferase (GST)-RanBP1(1-161), which lacks the NES, accumulates in the nucleus after cytoplasmic microinjection. In permeabilized cells, nuclear accumulation of GST-RanBP1(1-161) requires nuclear Ran:GTP but is not inhibited by a dominant interfering G19V mutant of Ran. Nuclear accumulation is enhanced by addition of exogenous karyopherins/importins or RCC1, both of which also enhance nuclear Ran accumulation. Import correlates with Ran concentration. Remarkably, an E37K mutant of RanBP1 does not import into the nuclei under any conditions tested despite the fact that it can form a ternary complex with Ran and importin beta. These data indicate that RanBP1 translocates through the pores by an active, nonclassical mechanism and requires Ran:GTP for nuclear accumulation. Shuttling of RanBP1 may function to clear nuclear pores of Ran:GTP, to prevent premature release of import cargo from transport receptors.     

Mouse Disabled1 (DAB1) is a nucleocytoplasmic shuttling protein

Disabled1 (DAB1) is an intracellular mediator of the Reelin-signaling pathway and essential for correct neuronal positioning during brain development. So far, DAB1 has been considered a cytoplasmic protein. Here, we show that DAB1 is subject to nucleocytoplasmic shuttling. In its steady state, DAB1 is mainly located in the cytoplasm. However, treatment with leptomycine B, a specific inhibitor of the CRM1 (chromosomal region maintenance 1)-RanGTP-dependent nuclear export, resulted in nuclear accumulation of DAB1. By using deletion or substitutional mutants of DAB1 fused with enhanced green fluorescent protein, we have mapped a bipartite nuclear localization signal and two CRM1-dependent nuclear export signals. These targeting signals were functional in both Neuro2a cells and primary cerebral cortical neurons. Using purified recombinant proteins, we have shown that CRM1 binds to DAB1 directly in a RanGTP-dependent manner. We also show that tyrosine phosphorylation of DAB1, which is indispensable for the layer formation of the brain, by Fyn tyrosine kinase or Reelin stimulation did not affect the subcellular localization of DAB1 in vitro. These results suggest that DAB1 is a nucleocytoplasmic shuttling protein and raise the possibility that DAB1 plays a role in the nucleus as well as in the cytoplasm.     

Nephropathy and defective spermatogenesis in mice transgenic for a single isoform of the Wilms' tumour suppressor protein, WT1-KTS, together with one disrupted Wt1 allele

The Wilms' tumour suppressor protein, WT1, is a zinc finger protein essential for the development of several organs, including the kidney and gonads. In each of these tissues WT1 is required at multiple stages of development and its persistent expression in podocytes and Sertoli cells suggests WT1 may also have a role in the maintenance of kidney and testis function throughout adult life. Naturally occurring isoforms of WT1 are generated by alternative mRNA splicing. An altered ratio of the splice isoforms WT1-KTS and WT1 + KTS appears to be sufficient to account for the developmental abnormalities (pseudohermaphroditism and nephropathy) characteristic of Frasier syndrome. We show that mice with a transgene encoding WT1-KTS do not differ from their wild-type littermates unless they are also heterozygous for a null mutation at the endogenous Wt1 locus. Animals with both genetic modifications develop proteinuria, together with multiple glomerular cysts, and male infertility. These pathologic changes may be explained as a consequence of altering the WT1 isoform ratio in tissues that express WT1 during adulthood. The results suggest WT1 misexpression could contribute to human glomerulocystic kidney disease.     

Adapter protein SH2-B beta undergoes nucleocytoplasmic shuttling: implications for nerve growth factor induction of neuronal differentiation

The adapter protein SH2-B has been shown to bind to activated nerve growth factor (NGF) receptor TrkA and has been implicated in NGF-induced neuronal differentiation and the survival of sympathetic neurons. However, the mechanism by which SH2-B enhances and maintains neurite outgrowth is unclear. We examined the ability of truncation mutants to regulate neuronal differentiation and observed that certain truncation mutants localized in the nucleus rather than in the cytoplasm or at the plasma membrane as reported for wild-type SH2-B beta. Addition of the nuclear export inhibitor leptomycin B caused both overexpressed wild-type and endogenous SH2-B beta to accumulate in the nucleus of both PC12 cells and COS-7 cells as did deletion of a putative nuclear export sequence (amino acids 224 to 233) or mutation of two critical lysines in that sequence. Deleting or mutating the nuclear export signal caused SH2-B beta to lose its ability to enhance NGF-induced differentiation of PC12 cells. Neither the NGF-induced phosphorylation of ERKs 1 and 2 nor their subcellular distribution was altered in PC12 cells stably expressing the nuclear export-defective SH2-B beta(L231A, L233A). These data provide strong evidence that SH2-B beta shuttles constitutively between the nucleus and cytoplasm. However, SH2-B beta needs continuous access to the cytoplasm and/or plasma membrane to participate in NGF-induced neurite outgrowth. These data also suggest that the stimulatory effect of SH2-B beta on NGF-induced neurite outgrowth of PC12 cells is either downstream of ERKs or via some other pathway yet to be identified.     

Physical and functional interaction between the HCMV IE2 protein and the Wilms' tumor suppressor WT1

Human cytomegalovirus (HCMV) is a major renal pathogen in congenitally infected infants and renal allograft recipients. It has been shown that human kidney cells of glomerular, tubular, and vascular origin were all infected by HCMV in vitro. It has previously been demonstrated that the IE2 protein of HCMV directly associates with the zinc finger domain of Egr-1. The zinc finger region of WT1 is a sequence-specific DNA-binding domain which also recognizes the consensus DNA binding site (5'-CGCCCCCGC-3') of Egr-1, thus suggesting a possible interaction between WT1 and IE2. Here we demonstrate that HCMV IE2 binds to the C-terminal region of WT1 containing zinc finger domain in vivo as well as in vitro and that WT1 can inhibit IE2-driven transactivation of the responsive promoter. Our results suggest that WT1 may be able to regulate the functional activity of HCMV IE2. Furthermore, these data may provide new insights into the possible involvement of HCMV in WT1-related pathogeneses.     

Members of the tristetraprolin family of tandem CCCH zinc finger proteins exhibit CRM1-dependent nucleocytoplasmic shuttling.

Members of the tristetraprolin (TTP) family of CCCH tandem zinc finger proteins can bind directly to certain types of AU-rich elements (AREs) in mRNA. Experiments in TTP-deficient mice have shown that TTP is involved in the physiological destabilization of at least two cytokine mRNAs, those encoding tumor necrosis factor alpha and granulocyte-macrophage colony-stimulating factor. The two other known mammalian members of the TTP family, CMG1 and TIS11D, also contain ARE-binding CCCH tandem zinc finger domains and can also destabilize ARE-containing mRNAs. To investigate the effects of primary sequence on the subcellular localization of these proteins, we constructed green fluorescent protein fusions with TTP, CMG1, and TIS11D; these were predominantly cytoplasmic when expressed in 293 or HeLa cells. Deletion and mutation analyses revealed functional nuclear export signals in the amino terminus of TTP and in the carboxyl termini of CMG1 and TIS11D. This type of leucine-rich nuclear export signal interacts with the nuclear export receptor CRM1; abrogation of CRM1 activity resulted in nuclear accumulation of TTP, CMG1, and TIS11D. These proteins are thus nucleocytoplasmic shuttling proteins and rely on CRM1 for their export from the nucleus. Although TTP, CMG1, and TIS11D lack known nuclear import sequences, mapping experiments revealed that their nuclear accumulation required an intact tandem zinc finger domain but did not require RNA binding ability. These findings suggest possible roles for nuclear import and export in the regulation of cellular TTP, CMG1, and TIS11D activity.