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Results for “Nuclear Factor 45 Protein”

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At least 19 recordsLinked to original sources

PubMed2003

Selective regulation of gene expression by nuclear factor 110, a member of the NF90 family of double-stranded RNA-binding proteins.

Members of the nuclear factor 90 (NF90) family of double-stranded RNA (dsRNA)-binding proteins have been implicated in several biological processes including the regulation of gene expression. cDNA sequences predict that the proteins have a functional nuclear localization signal and two dsRNA-binding motifs (dsRBMs), and are identical at their N termini. Isoforms are predicted to diverge at their C termini as well as by the insertion of four amino acid residues (NVKQ) between the two dsRBMs. In this study, we verified the expression of four of the isoforms by cDNA cloning and mass spectrometric analysis of proteins isolated from human cells. Cell fractionation studies showed that NF90 and its heteromeric partner, NF45, are predominantly nuclear and largely chromatin-associated. The C-terminally extended NF90 species, NF110, are almost exclusively chromatin-bound. Both NF110 isoforms are more active than NF90 isoforms in stimulating transcription from the proliferating cell nuclear antigen reporter in a transient expression system. NF110b, which carries the NVKQ insert, was identified as the strongest activator. It stimulated transcription of some, but not all, promoters in a fashion that suggested that it functions in concert with other transcription factors. Finally, we demonstrate that NF110b associates with the dsRBM-containing transcriptional co-activator, RNA helicase A, independently of RNA binding.

Amino Acid Sequence↗

PubMed1998

Activities of adenovirus virus-associated RNAs: purification and characterization of RNA binding proteins.

Most human adenoviruses encode two virus-associated (VA) RNAs, VA RNAI and VA RNAII, that accumulate to high levels in the cytoplasm of infected cells. The function of VA RNAI in blocking the activation of the cellular kinase PKR is well known, but the role of VA RNAII is obscure. Herein we characterize and purify several human proteins that interact preferentially with VA RNAII in Northwestern blot assays. Two of these proteins were identified as RNA helicase A and NF90, a component of the heterodimeric nuclear factor of activated T cells (NFAT). They copurified with the smaller NFAT subunit, NF45, which did not bind VA RNAII, and with an unidentified protein, p97, which did bind VA RNAII. Both RNA helicase A and NF90 contain two copies of a double-stranded (ds) RNA binding motif and bind strongly to dsRNA. NF90 interacts with RNAs in the following order of affinity: dsRNA > VA RNAII > VA RNAI > single-stranded RNA. Furthermore, VA RNAII is more effective than VA RNAI as an inhibitor of RNA helicase activity. These data identify RNA helicase A and NF90 as cellular proteins with an affinity for dsRNA and other structured RNA molecules and suggest that their functions are subject to regulation by RNA ligands including VA RNAII.

Adenoviridae↗

PubMed1994

Cloning and expression of cyclosporin A- and FK506-sensitive nuclear factor of activated T-cells: NF45 and NF90.

Nuclear Factor of Activated T-cells (NF-AT) is a crucial transcription factor required for T-cell expression of interleukin 2. Purified NF-AT contains 45-kDa and 90-kDa subunits (Corthésy, B., and Kao, P. N. (1994) J. Biol. Chem. 269, 20682-20690). Partial internal amino acid sequences derived from each subunit indicate that these proteins are novel. The amino acid sequences were used to clone the cDNAs encoding each subunit. The cDNAs predict proteins of novel structures: NF45 has limited similarity to prokaryotic transcription factor sigma-54 and to human DNA topoisomerase II; NF90 has limited similarity to Drosophila Staufen in a domain predicted to bind double-stranded RNA. RNA encoding NF45 and NF90 exists in nonstimulated Jurkat T-cells and in all other cell types examined (HeLa, HepG2, K562). Immunofluorescence microscopy was used to demonstrate that both proteins are located in the nucleus of Jurkat T-cells. Clones NF45 and NF90 with a polyhistidine fusion tag were transiently expressed and processed in the native environment of Jurkat T-cells. Histidine-tagged NF45 and NF90 proteins, affinity-purified on nickel chelate columns, encode a NF-AT DNA-binding activity that is enhanced following T-cell stimulation, and this enhancement is blocked when T-cells are stimulated in the presence of cyclosporin A or FK506.

Amino Acid Sequence↗

PubMed2002

Host cell proteins binding to the encapsidation signal epsilon in hepatitis B virus RNA.

The highly conserved encapsidation signal (epsilon) of hepatitis B viral (HBV) pregenomic RNA has been reported as an essential component for encapsidation and protein priming of HBV polymerase. Here, we report that two HBV epsilon RNA-binding host proteins (80 and 43 kDa) and a copurifying protein (100 kDa) were purified and characterized by the combined methods of UV cross-linking analysis with the epsilon RNA and column chromatography. Amino-terminal microsequencing showed that 80- and 43-kDa proteins were identified as the heterodimeric nuclear factor of activated T cells (NF90/NF45) and 100 kDa as a molecular chaperone, the GRP94. The heterodimeric factor interacted preferentially with the upper-bulge region of HBV epsilon RNA helping the HBV polymerase bind the lower-bulge region. Using in vitro protein priming analysis, the initial oligonucleotide of the protein-priming product was deduced as 5'-GAAC-3', which is the complementary sequence of both regions of DR1 and epsilon in the pregenomic RNA. Previously, we also proposed that the GRP94 was associated with HBV polymerase in the human liver cell HepG2. These results suggest that the heterodimeric factor plays an important role in the priming activity of HBV polymerase.

Amino Acid Sequence↗

PubMed1996

Mapping interleukin enhancer binding factor 2 gene (ILF2) to human chromosome 1 (1q11-qter and 1p11-p12) by polymerase chain reaction amplification of human-rodent somatic cell hybrid DNA templates.

Interleukin-2 (IL-2) is the first lymphokine secreted following T cell activation. Several transcription factors regulate IL-2 gene expression, including the nuclear factor of activated T cells (NFAT). NFAT acts at the antigen receptor response element-2 (ARRE-2) sequence in the IL-2 enhancer and is the nuclear target of T cell stimulation signals and the immunosuppressant drugs cyclosporine and FK506, which are potent inhibitors of IL-2 gene transcription. NFAT has been cloned and found to consist of two subunits, NF45 (ILF2) and NF90 (ILF3). This communication reports the assignment of NF45, interleukin enhancer binding factor 2 gene (ILF2), to human chromosome 1 (1q11-qter and 1p11-p12) by polymerase chain reaction (PCR) amplification of ILF2-specific DNA sequences from well-characterized human-rodent somatic cell hybrid DNA.

Animals↗

PubMed1998

Mapping interleukin enhancer binding factor 3 gene (ILF3) to human chromosome 19 (19q11-qter and 19p11-p13.1) by polymerase chain reaction amplification of human-rodent somatic cell hybrid DNA templates.

Interleukin-2 (IL-2) is the first lymphokine secreted following T cell activation. Several transcription factors regulate IL-2 gene expression, including the nuclear factor of activated T cells (NFAT). NFAT acts at the antigen receptor response element-2 (ARRE-2) sequence in the IL-2 enhancer and is the nuclear target of T cell stimulation signals and the immunosuppressant drugs cyclosporine (Sandimmune) and FK-506 (tacrolimus), which are potent inhibitors of IL-2 gene transcription. NFAT has been cloned and found to consist of two subunits, NF45 (ILF2) and NF90 (ILF3). We have recently assigned the gene encoding the small NFAT subunit, NF45 (ILF3) to human chromosome 1 (1q11-qter and 1p11-p12). This communication reports the assignment of the gene encoding the large NFAT subunit, NF90 or interleukin enhancer binding factor 3 gene (ILF3), to human chromosome 19 (19q11-qter and 19p11-p13.1) by polymerase chain reaction (PCR) amplification of ILF3-specific DNA sequences from well-characterized human-rodent somatic cell hybrid DNAs.

Animals↗

PubMed1998

DNA-dependent protein kinase interacts with antigen receptor response element binding proteins NF90 and NF45.

The DNA-dependent protein kinase (DNA-PK) is composed of a large catalytic subunit of approximately 470 kDa (DNA-PKcs) and the DNA-binding protein, Ku. Absence of DNA-PK activity confers sensitivity to x-rays and defects in both DNA double-strand break repair and V(D)J recombination. However the precise function of DNA-PK in DNA double-strand break repair is not known. Here we show, using electrophoretic mobility shift assays, that polypeptides in a fraction purified from human cells interact with DNA-PK and stabilize the formation of a complex containing DNA-PKcs-Ku and DNA. Five polypeptides in this fraction have been identified by amino-terminal sequence analysis and/or immunoblotting. These proteins are NF90 and NF45, which are the 90- and 45-kDa subunits of a protein known to bind specifically to the antigen receptor response element of the interleukin 2 promoter, and the alpha, beta, and gamma subunits of eukaryotic translation initiation factor eIF-2. We also show that NF90, NF45, and eIF-2 beta are substrates for DNA-PK in vitro. In addition, recombinant NF90 promotes formation of a complex between DNA-PKcs, Ku, and DNA, and antibodies to recombinant NF90 or recombinant NF45 immunoprecipitate DNA-PKcs in vitro. Together, our data suggest that NF90, in complex with NF45, interacts with DNA-PKcs and Ku on DNA and that NF90 and NF45 may be important for the function of DNA-PK.

Antigens, Nuclear↗

PubMed2005

NF45/ILF2 tissue expression, promoter analysis, and interleukin-2 transactivating function.

NF45/ILF2 associates with NF90/ILF3 in the nucleus and regulates IL-2 gene transcription at the antigen receptor response element (ARRE)/NF-AT DNA target sequence (P.N. Kao, L. Chen, G. Brock, J. Ng, A.J. Smith, B. Corthesy, J. Biol. Chem. 269 (1994) 20691-20699). NF45 is widely expressed in normal tissues, especially testis, brain, and kidney, with a predominantly nuclear distribution. NF45 mRNA expression is increased in lymphoma and leukemia cell lines. The human and murine NF45 proteins differ only by substitution of valine by isoleucine at amino acid 142. Fluorescence in situ hybridization localized the human NF45 gene to chromosome 1q21.3, and mouse NF45 gene to chromosome 3F1. Promoter analysis of 2.5 kB of the murine NF45 gene reveals that significant activation is conferred by factors, possible including NF-Y, that bind to the CCAAT-box sequence. The function of human NF45 in regulating IL-2 gene expression was characterized in Jurkat T-cells stably transfected with plasmids directing expression of NF45 cDNA in sense or antisense orientations. NF45 sense expression increased IL-2 luciferase reporter gene activity 120-fold, and IL-2 protein expression 2-fold compared to control cells. NF45 is a highly conserved, regulated transcriptional activator, and one target gene is IL-2.

Amino Acid Sequence↗

PubMed2003

Regulation of IL-2 gene expression and nuclear factor-90 translocation in vaccinia virus-infected cells.

Nuclear factor-90 (NF-90) has been described as a regulatory subunit of a complex containing DNA-dependent protein kinase (DNA-PK), Ku, and NF-45, which are capable of binding the interleukin-2 (IL-2) enhancer region and stimulating IL-2 gene expression. Vaccinia virus (VV) infection of Jurkat cells induced a nuclear factor that bound specifically to the IL-2 promoter sequence and led to the expression of the IL-2 transcript. Induction of this IL-2 promoter binding factor occurred concomitantly with the induction of NF-90 and translocation of NF-90 to the nucleus. Electrophoretic mobility supershift analysis using specific anti-NF-90 serum suggested the presence of NF-90 in the IL-2 promoter binding complex. As NF-90 can bind to double-stranded RNA (dsRNA) and be phosphorylated by the dsRNA-dependent protein kinase, PKR, we investigated whether accumulation of dsRNA in VV-infected cells could regulate IL-2 gene expression. Infection of Jurkat cells with a VV mutant that produces free dsRNA led to similar levels of induced NF-90 within the cell, but the protein remained localized within the cytosol. This mutant did not lead to the accumulation of an IL-2 promoter binding complex or to the synthesis of IL-2 mRNA. Other VV mutants that produced excess dsRNA also inhibited protein binding to the IL-2 enhancer, suggesting that the presence of viral dsRNA has a role in retaining NF-90 in the cytosol and regulating IL-2 gene expression.

DNA-Binding Proteins↗

PubMed1998

CsA-sensitive purine-box transcriptional regulator in bronchial epithelial cells contains NF45, NF90, and Ku.

Human bronchial epithelial (HBE) cells express interleukin (IL)-2 [Y. Aoki, D. Qiu, A. Uyei, and P. N. Kao. Am. J. Physiol. 272 (Lung Cell. Mol. Physiol. 16): L276-L286, 1997]. 16HBE-transformed cells contain constitutive and inducible nuclear DNA-binding activity for the purine-box/nuclear factor (NF) of activated T cell (NFAT) target DNA sequence in the human IL-2 enhancer. Transcriptional activation through the purine-box DNA sequence requires stimulation with phorbol 12-myristate 13-acetate + ionomycin, and this activation is inhibited by cyclosporin A. Immunohistochemical staining of 16HBE cells demonstrates nuclear expression of the purine-box DNA-binding proteins NF45 and NF90 and no expression of NFATp or NFATc. NF90 and NF45 associate with the DNA-dependent protein kinase catalytic subunit and the DNA-targeting subunits Ku80 and Ku70 (N. S. Ting, P. N. Kao, D. W. Chan, L. G. Lintott, and S. P. Lees-Miller. J. Biol. Chem. 273: 2136-2145, 1998). Antibodies to Ku potently inhibit the purine-box DNA-binding complex. The purine-box transcriptional regulator in 16HBE cells likely comprises NF45, NF90, Ku80, Ku70, and the DNA-dependent protein kinase catalytic subunit.

Antigens, Nuclear↗

PubMed2002

The RNA binding protein nuclear factor 90 functions as both a positive and negative regulator of gene expression in mammalian cells.

Nuclear factor 90 (NF90) was originally isolated in a complex that binds to the antigen recognition response element (ARRE-2) present in the interleukin-2 promoter. To characterize the transcriptional properties of NF90 in mammalian cells, we examined its ability to modulate promoter function in cellular transfection assays. NF90-Gal4 fusion proteins inhibited transcription from the adenovirus major late promoter in a fashion that was dependent on Gal4 targeting. Conversely, NF90 activated the cytomegalovirus immediate-early promoter, to which it was not targeted. These effects required distinct but overlapping domains in the C terminus of NF90, which contains a functional nuclear localization signal and two double-stranded-RNA binding motifs. NF90 is present in cellular complexes together with the NF45 protein. Transfection assays showed that NF45 binds NF90 strongly and stimulates its ability to activate but not to inhibit gene expression. This report characterizes NF90 as both a positive and negative regulator of gene expression, depending on the promoter context, and suggests a role for NF45 as a regulator of NF90.

Adenoviridae↗

PubMed2001

Nuclear factor 90 is a substrate and regulator of the eukaryotic initiation factor 2 kinase double-stranded RNA-activated protein kinase.

Nuclear factor 90 (NF90) is a member of an expanding family of double-stranded (ds) RNA-binding proteins thought to be involved in gene expression. Originally identified in complex with nuclear factor 45 (NF45) as a sequence-specific DNA-binding protein, NF90 contains two double stranded RNA-binding motifs (dsRBMs) and interacts with highly structured RNAs as well as the dsRNA-activated protein kinase, PKR. In this report, we characterize the biochemical interactions between these two dsRBM containing proteins. NF90 binds to PKR through two independent mechanisms: an RNA-independent interaction occurs between the N terminus of NF90 and the C-terminal region of PKR, and an RNA-dependent interaction is mediated by the dsRBMs of the two proteins. Co-immunoprecipitation analysis demonstrates that NF90, NF45, and PKR form a complex in both nuclear and cytosolic extracts, and both proteins serve as substrates for PKR in vitro. NF90 is phosphorylated by PKR in its RNA-binding domain, and this reaction is partially blocked by the NF90 N-terminal region. The C-terminal region also inhibits PKR function, probably through competitive binding to dsRNA. A model for NF90-PKR interactions is proposed.

Animals↗

PubMed1999

Nuclear factor-90 of activated T-cells: A double-stranded RNA-binding protein and substrate for the double-stranded RNA-dependent protein kinase, PKR.

NFAT transcription factors play a central role in initiating T-cell activation through the induction of immediate-early T-cell specific genes including interleukin-2 (IL-2). NFAT transcription factors bind to a sequence in the IL-2 enhancer known as the antigen receptor response element 2 (ARRE-2). Multiple proteins exhibiting ARRE-2 binding activity have been isolated, including a heterodimer from stimulated T-cell nuclear extracts consisting of Mr = 90 000 (NF90) and Mr = 45 000 (NF45) subunits. The subunits of this heterodimer have been cloned, and NF90 was found to encode a protein containing two domains that are predicted to form motifs capable of binding to double-stranded RNA. Using in vitro translated polypeptides, we have demonstrated that NF90 specifically binds to double-stranded RNA. Furthermore, NF90 was phosphorylated in a double-stranded RNA-dependent manner likely by the interferon-induced, double-stranded RNA-dependent protein kinase, PKR. The NF90 protein was found to be expressed not only in T-cells, but also in nonimmune HeLa cells. In HeLa cells, the protein was almost exclusively localized to the ribosome salt wash fraction of cell lysates.

Amino Acid Sequence↗

PubMed2002

Ilf2 is regulated during meiosis and associated to transcriptionally active chromatin.

Analysis of gene expression during testis development demonstrated accumulation of Ilf2 mRNA in pachytene spermatocytes. In these cells, the protein was localized in the nucleus, but it was absent from chromatin of the XY pachytene bivalent, in which there is no transcriptional activity. Nucleolar signal is inmmunolocalized in spermatogonia, Sertoli cells and oocytes. By in situ hybridisation, Ilf2 expression is detected in proliferative cells of adult ovary and a defined pattern is also exhibited in different tissues of embryos. The presence of ILF2 in active chromatin is corroborated in NIH3T3 cultured cells after transfection with Ilf2-EGFP constructs.

Amino Acid Sequence↗

PubMed2006

The double-stranded RNA binding protein 76:NF45 heterodimer inhibits translation initiation at the rhinovirus type 2 internal ribosome entry site.

Poliovirus (PV) plus-strand RNA genomes initiate translation in a cap-independent manner via an internal ribosome entry site (IRES) in their 5' untranslated region. Viral translation is codetermined by cellular IRES trans-acting factors, which can influence viral propagation in a cell-type-specific manner. Engineering of a poliovirus recombinant devoid of neuropathogenic properties but highly lytic in malignant glioma cells was accomplished by exchange of the cognate poliovirus IRES with its counterpart from human rhinovirus type 2 (HRV2), generating PV-RIPO. Neuroblast:glioma heterokaryon analyses revealed that loss of neurovirulence is due to trans-dominant repression of PV-RIPO propagation in neuronal cells. The double-stranded RNA binding protein 76 (DRBP76) was previously identified to bind to the HRV2 IRES in neuronal cells and to inhibit PV-RIPO translation and propagation (M. Merrill, E. Dobrikova, and M. Gromeier, J. Virol. 80:3347-3356, 2006). The results of size exclusion chromatography indicate that DRBP76 heterodimerizes with nuclear factor of activated T cells, 45 kDa (NF45), in neuronal but not in glioma cells. The DRBP76:NF45 heterodimer binds to the HRV2 IRES in neuronal but not in glioma cells. Ribosomal profile analyses show that the heterodimer preferentially associates with the translation apparatus in neuronal cells and arrests translation at the HRV2 IRES, preventing PV-RIPO RNA assembly into polysomes. Results of this study suggest that the DRBP76:NF45 heterodimer selectively blocks HRV2 IRES-driven translation initiation in neuron-derived cells.

5' Untranslated Regions↗

PubMed1998

Tex261, a novel gene presumably related but distinct from steroidogenic acute regulatory (StAR) gene, is regulated during the development of germ cells.

Tex261 is a new gene cloned from a subtractive cDNA library from 10-day postnatal mouse testis. Tex261 transcribes three mRNAs of 3.5, 1.6 and 1.4 kb. The 3.5 kb and 1.4 kb transcripts are expressed in different gonadal and somatic tissues analyzed. However, the 1.6 Kb transcript is only detected in testis and differentially regulated during development. This 1.6 kb mRNA is highly expressed in adult testis, with detection beginning at 15 days of postnatal life, which coincides with the presence of pachytene cells in prepuberal mouse. This expression was confirmed in pachytene cells by run-off transcription assay and by in situ hybridization. A region of 86 amino acids from the predicted Tex261 was recently reported as a part of the steroidogenic acute regulatory protein StAR gene by its sequence identity to a rat StAR cloned cDNA. We demonstrate her that, in the mouse, StAR and Tex261 are two different genes with different expected functions, yet, a high identity (43%) at amino acid level is detected in a region of 153 amino acids corresponding to a transmembrane protein.

Amino Acid Sequence↗

PubMed2006

Molecular cloning, characterization and expression analysis of grass carp (Ctenopharyngodon idellus) NF45 (ILF2) cDNA, a subunit of the nuclear factor of activated T-cells (NF-AT).

NF45 (ILF2) and NF90 (ILF3) regulate the IL-2 gene transcription via interaction with the antigen receptor response element. Much work on NF45 has been done in human and mammals while little in fish. In the present study, we have cloned and characterized the full-length cDNA of NF45 in grass carp (Ctenopharyngodon idellus). The grass carp NF45 cDNA of 1563bp contains a short 5'UTR of 24bp, a 3'UTR of 375bp and an open reading frame of 1164bp coding for a protein of 387 aa with a predicted molecular mass of 42.8kDa. The encoded protein shares 86.3-96.7% identities to other homologues. RT-PCR was optimized to estimate the expression level of NF45 in grass carp. The results showed that NF45 is constitutively expressed in most selected tissues, including head kidney, spleen, heart, brain, liver, and gill, although low levels were observed in spleen, liver and gill. The ubiquitous expression of NF45 is consistent with a postulated role in gene regulation at the level of transcription. Stimulating the fish with PHA significantly up-regulated the expression of NF45 in most tissues examined, which potentially indicated that NF45 was involved in the immune responses triggered by PHA.

Amino Acid Sequence↗

PubMed2006

Molecular cloning, characterization and expression analysis of an ILF2 homologue from Tetraodon nigroviridis.

Interleukin-2 enhancer binding factor 2 (ILF2) was reported to regulate transcription of interleukin-2 (IL-2), a central cytokine in the regulation of T-cell responses. This property of ILF2 was well characterized in human and mammals, but little is known in bony fish. In this paper, an ILF2 homologue was cloned and well characterized from Tetraodon nigrovirid is for the further investigation of the function of ILF2 in bony fish. The full-length Tetraodon ILF2 cDNA was 1380 bp in size and contained an open reading frame (ORF) of 1164 bp that translates into a 387 amino-acid peptide with a molecular weight of 42.9 kDa, a 5' untranslated region (UTR) of 57 bp, and a 3' UTR of 159 bp containing a poly A tail. The deduced peptide of Tetraodon ILF2 shared an overall identity of 58%~93% with other known ILF2 sequences, and contained two Nglycosylation sites, two N-myristoylation sites, one RGD cell attachment sequence, six protein kinase C phosphorylation sites, one amino-terminal RGG-rich single-stranded RNAbinding domain, and a DZF zinc-finger nucleic acid binding domain, most of which were highly conserved through species compared. Constitutive expression of Tetraodon ILF2 was observed in all tissues examined, including gill, gut, head kidney, spleen, liver, brain and heart. The highest expression was detected in heart, followed by liver, head kidney and brain. Stimulation with LPS did not significantly alter the expression of Tetraodon ILF2. Gene organization analysis showed that the Tetraodon ILF2 gene have fifteen exons, one more than other known ILF2 genes in human and mouse. Genes upand down-stream from the Tetraodon ILF2 were Rpa12, Peroxin-11b, Smad4, Snapap and Txnip homologue, which were different from that in human and mouse.

Amino Acid Sequence↗