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Results for “Nuclear Localization Signals”

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PubMed2004

Phospholipid scramblase 1 is imported into the nucleus by a receptor-mediated pathway and interacts with DNA.

Phospholipid scramblase 1 (PLSCR1) is a multiply palmitoylated, Ca(2+)-binding, endofacial plasma membrane protein originally identified by its capacity to accelerate transbilayer movement of membrane phospholipids. We recently reported that when palmitoylation of PLSCR1 does not occur, it is localized to the nucleus rather than the plasma membrane. Nuclear localization of PLSCR1 was also observed upon induction of its de novo synthesis by cytokines such as interferon alpha that activate the PLSCR1 gene. Despite its capacity to enter the nucleus, its sequence does not predict a nuclear localization signal. To gain insight into the mechanism and potential significance of nuclear PLSCR1, we investigated the conditions required for its import and retention in the nucleus. We show that nuclear localization of PLSCR1 is dependent on cytosolic factors and energy. Furthermore, we show that PLSCR1 is specifically transported into the nucleus by the importin alpha/beta import pathway, and binds directly and with high affinity to importin alpha. Analysis of deletion mutants suggested that the NLS of PLSCR1 is between residues 242 and 290 and, furthermore, that a peptide within this region encompassing residues (257)GKISKHWTGI(266) is sufficient for nuclear import when conjugated to BSA. In addition, in intact cells, mutation of positively charged amino acids within this putative NLS in the full-length protein completely blocked its entry into the nucleus, consistent with its role in targeting PLSCR1 to the nucleus. Release of PLSCR1 from the nucleus was only observed after treatment of cells with both detergent and an elevated NaCl concentration, or following DNase treatment of the nucleus, suggesting ionic interactions of PLSCR1 with a nuclear component bound to genomic DNA or directly with genomic DNA. Purified PLSCR1 was also found to bind directly to a genomic DNA-cellulose conjugate, and its elution from DNA also required an elevated NaCl concentration. These data support a mechanism of receptor-mediated nuclear import of PLSCR1 and suggest a potential nuclear function for this plasma membrane protein.

Active Transport, Cell Nucleus↗

PubMed2001

A nucleus-localization-deficient mutant serves as a dominant-negative inhibitor of gut-enriched Krüppel-like factor function.

Cancer cells differ from normal cells in many aspects, including loss of differentiation and uninhibited cell proliferation. Recent studies have suggested that gut-enriched Krüppel-like factor (GKLF) played an important role in the regulation of cell growth in the colon. Studies from this laboratory have shown that GKLF protein predominantly expressed in the cytoplasm but not the nucleus of colon cancer cells, suggesting that impaired nuclear translocation of GKLF might contribute to cancer formation. In this report, a region containing putative nuclear localization signal (NLS) of GKLF (PKRGRR; amino acids 385-390) was investigated. Mutation of KR to WT had no effect on the inhibitory properties of GKLF on cyclin D1 promoter activity and [(3)H]thymidine uptake in HT-29 cells, whereas mutation of RR to GL abolished GKLF function completely. Additional mutation analyses demonstrated that Arg(390) is the most critical moiety within this region that mediated GKLF function and its nucleus localization. Cotransfection of Arg(390) mutant (RR/RS) completely inhibited wild-type GKLF function, and GFP-RR/RS GKLF fusion proteins failed to translocate to the nucleus. The results from this study demonstrate that Arg(390) confers the NLS of GKLF and that the nucleus-localization-deficient mutant serves as dominant-negative inhibitor of GKLF function.

Active Transport, Cell Nucleus↗

PubMed2000

Identification of domains of the HPV11 E1 protein required for DNA replication in vitro.

The HPV E1 and E2 proteins along with cellular factors, are required for replication of the viral genome. In this study we show that in vitro synthesized HPV11 E1 can support DNA replication in a cell-free system and is able to cooperate with E2 to recruit the host polymerase alpha primase to the HPV origin in vitro. Deletion analysis revealed that the N-terminal 166 amino acids of E1, which encompass a nuclear localization signal and a cyclin E-binding motif, are dispensable for E1-dependent DNA replication and for recruitment of pol alpha primase to the origin in vitro. A shorter E1 protein lacking the N-terminal 190 amino acids supported cell-free DNA replication at less than 25% the efficiency of wild-type E1 and was active in the pol alpha primase recruitment assay. An even shorter E1 protein lacking a functional DNA-binding domain due to a truncation of the N-terminal 352 amino acids was inactive in both assays despite the fact that it retains the ability to associate with E2 or pol alpha primase in the absence of ori DNA. We provide additional functional evidence that E1 interacts with pol alpha primase through the p70 subunit of the complex by showing that p70 can be recruited to the HPV origin by E1 and E2 in vitro, that the domain of E1 (amino acids 353-649) that binds to pol alpha primase in vitro is the same as that needed for interaction with p70 in the yeast two-hybrid system, and that exogenously added p70 competes with the interaction between E1 and pol alpha primase and inhibits E1-dependent cell-free DNA replication. On the basis of these results and the observation that pol alpha primase competes with the interaction between E1 and E2 in solution, we propose that these three proteins assemble at the origin in a stepwise process during which E1, following its interaction with E2, must bind to DNA prior to interacting with pol alpha primase.

Animals↗

PubMed2000

Dihydrotestosterone as a selective cellular/nuclear localization vector for anti-gene peptide nucleic acid in prostatic carcinoma cells.

Peptide nucleic acids (PNAs) are synthetic structural analogues of DNA and RNA that, if allowed to enter the cell, bind to the complementary polynucleotide sequence and inhibit DNA transcription and mRNA translation. Although PNAs have a very limited ability in penetrating nuclei of living cells, there are indications that covalent linkage of the PNA to appropriate vectors, e.g., a nuclear localization signal, permits access to the genome. Here we test the ability of dihydrotestosterone (T) covalently linked to PNA to act as a vector for targeting c-myc DNA to prostatic cancer cell nuclei. LNCaP cells, which express the androgen receptor gene, and DU145 cells, in which the androgen receptor gene is silent, offer a model to test this biologically active hormone as a cell-specific vector. T vector was covalently linked to the NH2-terminal position of a PNA complementary to a unique sequence of c-myc oncogene (PNAmyc-T). To localize PNAmyc-T and vector-free PNA within the cells, a rhodamine (R) group was attached at the COOH-terminal position (PNAmyc-R, PNAmyc-TR); cellular uptake was monitored by confocal fluorescence microscopy. PNAmyc-R was detected only in the cytoplasm of both prostatic cell lines, whereas PNAmyc-TR was localized in nuclei as well as in cytoplasm of LNCaP cells. In contrast, PNAmyc-TR uptake in DU145 cells was minimal and exclusively cytoplasmic. In LNCaP cells, MYC protein remained unchanged by exposure to vector-free PNAmyc, whereas a significant and persistent decrease was induced by PNAmyc-T. In DU145 cells, MYC expression was unaltered by PNAmyc with or without the T vector. Our data show that the T vector facilitates cell-selective nuclear localization of PNA and its consequent inhibition of c-myc expression. These findings suggest a strategy for targeting of cell-specific anti-gene therapy in prostatic carcinoma.

Antisense Elements (Genetics)↗

PubMed2001

Human cell lines expressing hormone regulated T7 RNA polymerase localized at distinct intranuclear sites.

Although several systems are now available for the controlled expression of eukaryotic genes transcribed by RNA polymerase II, regulated expression has been more difficult to achieve in the case of genes transcribed by RNA polymerase III. In the present study the gene for bacteriophage T7 RNA polymerase, implanted with a eukaryotic nuclear localization signal, was linked to a 5'-flanking ecdysone-responsive promoter and stably transformed human cell lines were constructed in which the ecdysone promoter-T7 RNA polymerase gene had been integrated intact, as demonstrated by a polymerase chain reaction assay. Exposure of these cells to the ecdysone analog ponasterone A resulted in the appearance of a single protein having the expected size of T7 RNA polymerase in immunoblots of cell extracts probed with an affinity purified antibody raised against the C-terminus of T7 RNA polymerase. The induced T7 RNA polymerase was exclusively localized in the nucleus of induced cells and was undetectable in uninduced cells either by immunoblotting or immunofluorescence. The induced T7 RNA polymerase was present at numerous punctate foci dispersed throughout the nucleoplasmic regions of the nucleus and was also present in the nucleoli. Both of these observed intranuclear localizations have relevance to the potential applications of this system.

Bacteriophage T7↗

PubMed1998

Targeting to transcriptionally active loci by the hydrophilic N-terminal domain of Drosophila DNA topoisomerase I.

DNA topoisomerase I (topo I) from Drosophila melanogaster contains a nonconserved, hydrophilic N-terminal domain of about 430 residues upstream of the conserved core domains. Deletion of this N terminus did not affect the catalytic activity of topo I, while further removal of sequences into the conserved regions inactivated its enzymatic activity. We have investigated the cellular function of the Drosophila topo I N-terminal domain with top1-lacZ transgenes. There was at least one putative nuclear localization signal within the first 315 residues of the N-terminal domain that allows efficient import of the large chimeric proteins into Drosophila nuclei. The top1-lacZ fusion proteins colocalized with RNA polymerase II (pol II) at developmental puffs on the polytene chromosomes. Either topo I or the top1-lacZ fusion protein was colocalized with RNA pol II in some but not all of the nonpuff, interband loci. However, the fusion proteins as well as RNA pol II were recruited to heat shock puffs during heat treatment, and they returned to the developmental puffs after recovery from heat shock. By immunoprecipitation, we showed that two of the largest subunits of RNA pol II coprecipitated with the N-terminal 315-residue fusion protein by using antibodies against beta-galactosidase. These data suggest that the topo I fusion protein can be localized to the transcriptional complex on chromatin and that the N-terminal 315 residues were sufficient to respond to cellular processes, especially during the reprogramming of gene expression.

Animals↗

PubMed2005

Nuclear/nucleolar localization properties of C-terminal nucleocapsid protein of SARS coronavirus.

A novel coronavirus (CoV) has recently been identified as the aetiological agent of severe acute respiratory syndrome (SARS). Nucleocapsid (N) proteins of the Coronaviridae family have no discernable homology, but they share a common nucleolar-cytoplasmic distribution pattern. There are three putative nuclear localization signal (NLS) motifs present in the N. To determine the role of these putative NLSs in the intracellular localization of the SARS-CoV N, we performed a confocal microscopy analysis using rabbit anti-N antisera. In this report, we show that the wild type N was distributed mainly in the cytoplasm. The N-terminal of the N, which contains the NLS1 (aa38-44), was localized to the nucleus. The C-terminus of the N, which contains both NLS2 (aa257-265) and NLS3 (aa369-390) was localized to the cytoplasm and the nucleolus. Results derived from analysis of various deletion mutations show that the region containing amino acids 226-289 is able to mediate nucleolar localization. The deletion of two hydrophobic regions that flanked the NLS3 recovered its activity and localized to the nucleus. Furthermore, deletion of leucine rich region (220-LALLLLDRLNRL) resulted in the accumulation of N to the cytoplasm and nucleolus, and when fusing this peptide to EGFP localization was cytoplasmic, suggesting that the N may act as a shuttle protein. Differences in nuclear/nucleolar localization properties of N from other members of coronavirus family suggest a unique function for N, which may play an important role in the pathogenesis of SARS.

Animals↗

PubMed2002

Functional analysis and the molecular dissection of zinc-fingers and homeoboxes 1 (ZHX1).

Zinc-fingers and homeoboxes 1 (ZHX1) is a protein that interacts with the activation domain of the A subunit of nuclear factor-Y. The function of ZHX1, as a transcription factor, was characterized and their domains were mapped. To determine the nuclear localization signal, expression vectors, in which various truncated forms of ZHX1 were fused to the C-terminal of green fluorescence protein (GFP), were transfected into human embryonic kidney (HEK) 293 cells. All GFP-ZHX1 fusion proteins including an arginine-rich region that corresponds to the amino acid sequence between 734 and 768 were localized in the nuclei. A dimerization domain of the ZHX1 was also mapped using protein-protein interaction assays. The homeodomain (HD) 1 consisting of the amino acid sequence between 272 and 432 of ZHX1 was necessary and sufficient for dimerization. Lastly, the transcriptional activity of ZHX1 was examined using a mammalian one-hybrid system. ZHX1, fused to the C-terminal of the GAL4 DNA-binding domain, was co-transfected with luciferase reporter plasmids with or without five copies of the GAL4-binding site into HEK293 cells. The luciferase activity was decreased in both concentration- and GAL4-binding site-dependent manner. The acidic region corresponding to the amino acid sequence between 831 and 873 was a repressor domain and dimerization was prerequisited for full repressor activity.

Active Transport, Cell Nucleus↗

PubMed2003

Sla1, a Schizosaccharomyces pombe homolog of the human La protein, induces ectopic meiosis when its C terminus is truncated.

Sla1 is a Schizosaccharomyces pombe homolog of the human La protein. La proteins are known to be RNA-binding proteins that bear conserved RNA recognition motifs (La and RRMs), but their biological functions still have not been fully resolved. In this study, we show that the S. pombe La homolog (Sla1) is involved in regulating sexual development. Sla1 truncated in the C terminus (Sla1DeltaC) induced ectopic sporulation in the ras1Delta strain and several other sporulation-deficient mutants. The C terminus contains a nuclear localization signal. While full-length Sla1 localizes in the nucleus, Sla1DeltaC is found throughout the cell, suggesting the cytoplasmic localization of Sla1DeltaC is involved in its sporulation-inducing activity. Further deletion analysis of Sla1 indicated that a small region (35 amino acids) that includes a portion of RRM2 is sufficient to induce sporulation. The La motif (RRM1) is not involved in this activity. Strikingly, Sla1DeltaC induced haploid meiosis in a heterothallic strain, similar to the pat1-114 or mei2-SATA mutation. Sla1DeltaC induced sporulation in a mei3 disruptant but not in a mei2 disruptant, indicating that Sla1DeltaC requires Mei2 to induce haploid meiosis. Deletion of the chromosomal sla1 gene lowered the temperature sensitivity of the pat1-114 mutant. Two-hybrid analysis indicated that Pat1 interacts with Sla1DeltaC but not full-length Sla1. Thus, Sla1DeltaC may block Pat1 activity. This block would remove the inhibition on Mei2, which would then drive the cell into haploid meiosis. Finally, Sla1 was degraded prior to the start of meiosis when we monitored Sla1 in cells in which meiosis was synchronously induced. The ability of truncated Sla1 to induce ectopic meiosis represents a very novel function that has hitherto not been suspected for the La family of proteins.

Amino Acid Motifs↗

PubMed1999

Crystal structure of the nuclear matrix targeting signal of the transcription factor acute myelogenous leukemia-1/polyoma enhancer-binding protein 2alphaB/core binding factor alpha2.

Transcription factors of the acute myelogenous leukemia (AML)/polyoma enhancer-binding protein (PEBP2alpha)/core-binding factor alpha (CBFA) class are key transactivators of tissue-specific genes of the hematopoietic and bone lineages. AML-1/PEBP2alphaB/CBFA2 proteins participating in transcription are associated with the nuclear matrix. This association is solely dependent on a highly conserved C-terminal protein segment, designated the nuclear matrix targeting signal (NMTS). The NMTS of AML-1 is physically distinct from the nuclear localization signal, operates autonomously, and supports transactivation. Our data indicate that the related AML-3 and AML-2 proteins are also targeted to the nuclear matrix in situ by analogous C-terminal domains. Here we report the first crystal structure of an NMTS in an AML-1 segment fused to glutathione S-transferase. The model of the NMTS consists of two loops connected by a flexible U-shaped peptide chain.

Amino Acid Sequence↗

PubMed1999

Nuclear localization of Schizosaccharomyces pombe Mcm2/Cdc19p requires MCM complex assembly.

The minichromosome maintenance (MCM) proteins MCM2-MCM7 are conserved eukaryotic replication factors that assemble in a heterohexameric complex. In fission yeast, these proteins are nuclear throughout the cell cycle. In studying the mechanism that regulates assembly of the MCM complex, we analyzed the cis and trans elements required for nuclear localization of a single subunit, Mcm2p. Mutation of any single mcm gene leads to redistribution of wild-type MCM subunits to the cytoplasm, and this redistribution depends on an active nuclear export system. We identified the nuclear localization signal sequences of Mcm2p and showed that these are required for nuclear targeting of other MCM subunits. In turn, Mcm2p must associate with other MCM proteins for its proper localization; nuclear localization of MCM proteins thus requires assembly of MCM proteins in a complex. We suggest that coupling complex assembly to nuclear targeting and retention ensures that only intact heterohexameric MCM complexes remain nuclear.

Cell Cycle Proteins↗

PubMed2000

A novel binding factor facilitates nuclear translocation and transcriptional activation function of the pituitary tumor-transforming gene product.

Pituitary tumor-transforming gene (PTTG) is a recently characterized oncogene whose expression product contains a transcriptional activation domain at the C terminus. To understand the mechanisms involved in PTTG biological functions, we used yeast two-hybrid screening to identify proteins that interact with PTTG. This study reports the isolation and characterization of a novel PTTG-binding factor (PBF). PBF contains an open reading frame of 179 amino acids with a predicted molecular mass of 22 kDa. In Northern blot analyses, PBF mRNA was ubiquitously expressed in human tissues. Glutathione S-transferase pull-down and co-immunoprecipitation assays demonstrate that PBF interacts specifically with PTTG under both in vitro and in vivo conditions. The PTTG binding domain in PBF was located within the C-terminal 30-amino acid region that contain a nuclear localization signal. Immunofluorescence and subcellular fractionation studies showed that PTTG is predominantly expressed in the cytoplasm with partial nuclear localization, whereas PBF is localized both in the cytoplasm and the nucleus. The interaction between PBF and PTTG facilitated PTTG translocation from the cytoplasm to the nucleus. Furthermore, PBF is required for transcriptional activation of basic fibroblast growth factor by PTTG. In summary, we have characterized a novel PTTG-binding protein that facilitates PTTG nuclear translocation and potentiates its transcriptional activation function.

Amino Acid Sequence↗

PubMed1995

Inhibition of HIV-1 Tat-mediated LTR transactivation and HIV-1 infection by anti-Tat single chain intrabodies.

Genes encoding the rearranged immunoglobulin heavy and light chain variable regions of anti-HIV-1 Tat, exon 1 or exon 2 specific monoclonal antibodies have been used to construct single chain intracellular antibodies 'intrabodies' for expression in the cytoplasm of mammalian cells. These anti-Tat single chain intrabodies (anti-Tat sFvs) are additionally modified with a C-terminal human C kappa domain to increase cytoplasmic stability and/or the C-terminal SV40 nuclear localization signal to direct the nascent intrabody to the nuclear compartment, respectively. The anti-Tat sFvs with specific binding activity against the N-terminal activation domain of Tat, block Tat-mediated transactivation of HIV-1 LTR as well as intracellular trafficking of Tat in mammalian cells. As a result, the transformed lymphocytes expressing anti-Tat sFvs are resistant to HIV-1 infection. Thus, these studies demonstrate that stably expressed single chain intrabodies and their modified forms can effectively target molecules in the cytoplasm and nuclear compartments of eukaryotic cells. Furthermore, these studies suggest that anti-Tat sFvs used either alone or in combination with other genetically based strategies may be useful for the gene therapy of HIV-1 infection and AIDS.

Amino Acid Sequence↗

PubMed2004

A novel chimeric ribozyme vector produces potent inhibition of ICAM-1 expression on ischemic vascular endothelium.

BACKGROUND: Inhibition of intercellular adhesion molecule-1 (ICAM-1) expression can ameliorate the inflammation induced by ischemia-reperfusion injury (IRI) in animal models. However, current strategies to reduce ICAM-1 expression have been limited by the lack of stability, poor specificity, and the transient nature of synthesized regulatory molecules (antisense/ribozyme). METHODS: A chimeric expression vector was generated by fusing a ribozyme targeting sequence against ICAM-1 to stabilizing stem-loop structures and nuclear localization signals that are components of endogenous U1 small nuclear RNA. Oligonucleotide scanning was used to predict accessible sites for targeting within the rat ICAM-1 transcript. Efficacy of the chimeric ribozyme vector was tested by transfection of rat aortic endothelial (RAE) cells (in vitro) and intraportal delivery in a rat hepatic IRI model (in vivo). RESULTS: Transfection of RAE cells with the chimeric ribozyme vector produced potent and specific inhibition of ICAM-1 mRNA and protein levels by >65%. This reduction in ICAM-1 expression was accompanied by a proportional decrease in neutrophil adhesion to RAE cells. In vivo intraportal delivery of the chimeric targeting vector to rats sustaining hepatic IRI produced a marked reduction in ICAM-1 expression on liver endothelium after reperfusion. CONCLUSIONS: A chimeric ribozyme vector effectively inhibited ICAM-1 expression in vascular endothelial cells and in rat liver following IRI, demonstrating a novel gene targeting technique that may be ideally suited to clinical applications aimed at ameliorating IRI.

Animals↗

PubMed2004

Identification and cloning of a novel chromatin-associated protein partner of Epstein-Barr nuclear protein 2.

In a screen for binding partners of the Epstein-Barr virus transformation-related protein EBNA2, we cloned a novel, evolutionarily conserved protein showing similarity to the Drosophila Parallel Sister Chromatids Protein (PASC). We have named this protein "Friend of EBNA2" (FOE). Human FOE encodes a protein of 1227 amino acids with a functional bipartite nuclear localization signal, an arginine-rich motif, a putative nuclear export signal as well as with three highly acidic regions and a predicted coiled-coil domain. FOE and EBNA2 coimmunoprecipitate from lymphocyte nuclear extracts. RNA and protein blots show that FOE is expressed in all human tissues. FOE is a nuclear protein with the bulk of the protein associated with the insoluble nuclear fraction biochemically defined as the nuclear matrix. Indirect immunofluorescence and dynamic imaging studies suggest that FOE associates with transcriptionally active nuclear subregions in interphase cells and concentrates at the ends of formed chromosomes during mitosis.

Amino Acid Sequence↗

PubMed1995

Implication of mammalian ribosomal protein S3 in the processing of DNA damage.

A human apurinic/apyrimidinic endonuclease activity, called AP endonuclease I, is missing from or altered specifically in cells cultured from Xeroderma pigmentosum group-D individuals (XP-D cells) (Kuhnlein, U., Lee, B., Penhoet, E. E., and Linn, S. (1978) Nucleic Acids Res. 5,951-960). We have now observed that another nuclease activity, UV endonuclease III, is similarly not detected in XP-D cells and is inseparable from the AP endonuclease I activity. This activity preferentially cleaves the phosphodiester backbone of heavily ultraviolet-irradiated DNA at unknown lesions as well as at one of the phosphodiester bonds within a cyclobutane pyrimidine dimer. The nuclease activities have been purified from mouse cells to yield a peptide of M(r) = 32,000, whose sequence indicates identity with ribosomal protein S3. The nuclease activities all cross-react with immunopurified antibody directed against authentic rat ribosomal protein S3, and, upon expression in Escherichia coli of a cloned rat cDNA for ribosomal protein S3, each of the activities was recovered and was indistinguishable from those of the mammalian UV endonuclease III. Moreover, the protein expressed in E. coli and its activities cross-react with the rat protein antibody. Ribosomal protein S3 contains a potential nuclear localization signal, and the protein isolated as a nuclease also has a glycosylation pattern consistent with a nuclear localization as determined by lectin binding. The unexpected role of a ribosomal protein in DNA damage processing and the unexplained inability to detect the nuclease activities in extracts from XP-D cells are discussed.

Amino Acid Sequence↗

PubMed1996

The KRAB zinc finger gene ZNF74 encodes an RNA-binding protein tightly associated with the nuclear matrix.

We previously cloned ZNF74, a developmentally expressed zinc finger gene commonly deleted in DiGeorge syndrome. Here, the intron/exon organization of the human gene and the functional properties of the expressed protein are presented. This zinc finger gene from the transcription factor IIIA/Kruppel family contains three exons. A truncated Kruppel-associated box (KRAB) located at the N terminus of the predicted 64-kDa zinc finger protein is encoded by exon 2. The remainder of the protein including the zinc finger domain as well as the 3'-untranslated region (UTR) is encoded by exon 3. Both 5'-UTR (exon 1) and 3'-UTR contain repetitive Alu elements. In vitro translation of a cDNA encoding the entire ZNF74 coding region produced a 63-kDa protein as determined on sodium dodecyl sulfate-polyacrylamide gel. A bacterially expressed fusion protein shown to bind tightly to 65zinc was used to test the nucleic acid binding properties of ZNF74. By RNA binding assays, ZNF74 was found to bind specifically to poly(U) and poly(G) RNA homopolymers. The restricted binding to these homopolymers and not to poly(A) and poly(C) suggested that ZNF74 displays RNA sequence preferences. RNA binding was mediated by the zinc finger domain. Immunofluorescence studies on transfected cells revealed ZNF74 nuclear localization. The labeling pattern observed in the nuclei clearly excluded the nucleoli. The zinc finger region lacks a classical nuclear localization signal but was found to be responsible for nuclear targeting. Subcellular and in situ sequential fractionations further showed that ZNF74 is associated with the nuclear matrix. The RNA binding properties of this protein and its tight association with the nuclear matrix, a subnuclear compartment involved in DNA replication as well as RNA synthesis and processing, suggest a role for ZNF74 in RNA metabolism.

Amino Acid Sequence↗

PubMed2000

Gene activation by Varicella-zoster virus IE4 protein requires its dimerization and involves both the arginine-rich sequence, the central part, and the carboxyl-terminal cysteine-rich region.

Varicella-zoster virus (VZV) open reading frame 4-encoded protein (IE4) possesses transactivating properties for VZV genes as well as for those of heterologous viruses. Since most transcription factors act as dimers, IE4 dimerization was studied using the mammalian two-hybrid system. Introduction of mutations in the IE4 open reading frame demonstrated that both the central region and the carboxyl-terminal cysteine-rich domain were important for efficient dimerization. Within the carboxyl-terminal domain, substitution of amino acids encompassing residues 443-447 totally abolished dimerization. Gene activation by IE4 was studied by transient transfection with an IE4 expression plasmid and a reporter gene under the control of either the human immunodeficiency virus, type 1, long terminal repeat or the VZV thymidine kinase promoter. Regions of IE4 important for dimerization were also shown to be crucial for transactivation. In addition, the arginine-rich domains Rb and Rc of the amino-terminal region were also demonstrated to be important for transactivation, whereas the Ra domain as well as an acidic and bZIP-containing regions were shown to be dispensable for gene transactivation. A nucleocytoplasmic shuttling of IE4 has also been characterized, involving a nuclear localization signal identified within the Rb domain and a nuclear export mechanism partially depending on Crm-1.

Amino Acid Sequence↗