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PubMed1988

Effects of anti-C23 (nucleolin) antibody on transcription of ribosomal DNA in Chironomus salivary gland cells.

Protein C23 (also called nucleolin or 100-kDa nucleolar protein) is a major nucleolar phosphoprotein involved in ribosome biogenesis. To determine the effects of protein C23 on preribosomal RNA (pre-rRNA) synthesis anti-C23 antiserum was microinjected into nuclei of Chironomus tentans salivary glands. Transcription was measured by incubation of the glands with 32P-labeled RNA precorsors followed by microdissection of nucleoli, RNA extraction, and electrophoretic analyses. Injection of the anti-C23 antibody caused a 2- to 3.5-fold stimulation of 32P incorporation into 38S pre-rRNA. No stimulation was observed in salivary glands injected with preimmune serum or antiserum preabsorbed with protein C23. The stimulatory effect was selective for pre-rRNA as indicated by the lack of stimulation of 32P incorporation into extranucleolar RNA. Injection of the antiserum produced little or no effect on pre-RNA processing as measured by the relative amounts of 32P-labeled intermediate cleavage products of pre-rRNA in stimulated versus control glands. When protein extracts of Chironomus tentans salivary gland nuclei were probed on Western blots with anti-C23 antibody the predominant cross-reacting species was a 110-kDa polypeptide which had an electrophoretic mobility similar to that of protein C23. These results suggest that protein C23 not only is involved in ribosome assembly but also plays a role in regulating the transcription of the preribosomal RNA.

Animals↗
PubMed1990

Nucleolin gene organization in rodents: highly conserved sequences within three of the 13 introns.

The complete nucleotide (nt) sequence of the rat nuc gene encoding nucleolin, the major nucleolar-specific protein in eukaryotic exponentially growing cells, is compared with the corresponding locus recently characterized in mouse. [Bourbon et al., J. Mol. Biol. 200 (1988) 627-638]. In both murine species the genomic organization has been strikingly conserved during evolution, i.e., the coding region extends over 9 kb and is split into 14 exons, encoding a 712-amino acid protein. Moreover, all the exon-intron junction positions were strictly maintained during evolution. More unexpectedly, this analysis revealed that several introns contain highly conserved sequence elements of about 120 nt. The nt sequence of the homologous locus isolated from a Chinese hamster genomic clone established that these regions were under unusually high selective constraints (84-96% identity between the hamster and murine nuc genes) and, although they do not contain open reading frames, they surprisingly appear to be more conserved than most of the exons, suggesting that they play an important role. Such an element of 130 nt presents features of known genes transcribed by RNA polymerase III. Furthermore, in the rat nuc pre-mRNA the 5'- and 3'-end regions of the last intron are fully complementary over 16 nt, and so are predicted to be included in a prominent stem structure. Moreover, an homologous RNA stem structure can be derived from the mouse sequence, including two compensatory nt changes. As the secondary structure would occlude the canonical sequences required for the proper excision of this intron in both murine species, this remarkable finding could be relevant to the regulation of the nuc gene expression at the RNA processing level.

Amino Acid Sequence↗
PubMed1991

Structure of an intronless mouse nucleolin pseudogene: insights into mechanisms enabling the conversion of RNA to DNA.

The complete nucleotide (nt) sequence of a mouse nucleolin (Nuc) pseudogene (termed E4) is reported. The E4 element displays the hallmarks of retroposons generated from processed RNA messages, i.e., lack of introns (out of 14 in the active nuc gene), presence of a poly(dA) tail at the 3' end, and is a short directly repeated sequence (TTCTAATATTTGA) at both ends. In addition, the genetic information corresponding to the 5' part of the nuc mRNA is missing. The most salient feature is the presence in the 3' half of the terminal direct repeats of a motif (TTTGA) corresponding precisely to the nuc messenger sequence at the truncation site. This unusual structure suggests that the E4 pseudogene, which appeared in the mouse genome about 15 Myr ago, arose from an insertion event involving a weak hybridization between a recipient DNA and a nuc mRNA-like intermediate. This hypothesis is supported by the fact that the chromosomal integration site is enriched in A and T residues and contains sequences capable of adopting a Z-DNA conformation. On the basis of these data, an RNA-mediated transposition model involving a strand switch during the retrotranscription process is proposed.

Animals↗
PubMed1995

Human DNA helicase IV is nucleolin, an RNA helicase modulated by phosphorylation.

The cDNA encoding human DNA helicase IV (HDH IV), a 100-kDa protein which unwinds DNA in the 5' to 3' direction with respect to the bound strand, was cloned and sequenced. It was found to be identical to the human cDNA encoding nucleolin, a ubiquitous eukaryotic protein essential for pre-ribosome assembly. HDH IV/nucleolin can unwind RNA-RNA duplexes, as well as DNA-DNA and DNA-RNA duplexes. Phosphorylation of HDH IV/nucleolin by cdc2 kinase and casein kinase II enhanced its unwinding activity in an additive way. The Gly-rich C-terminal domain possesses a limited ATP-dependent duplex-unwinding activity which contributes to the helicase activity of HDH IV/nucleolin.

Adenosine Triphosphatases↗
PubMed2005

Role of nucleolin in posttranscriptional control of MMP-9 expression.

Matrix-metalloproteinases (MMPs), which are able to degrade extra cellular matrix (ECM) components, are crucial in ECM-remodeling, under physiological (e.g., embryogenesis, wound healing, angiogenesis) or pathophysiological conditions (e.g., arthritis, cancer progression and metastasis, fibrosis). Treating HT1080 cells, a human fibrosarcoma cell line, with the iron chelator 2,2-Dipyridyl, which mimics certain aspects of hypoxia, leads to a 3-fold elevated Matrix-metalloproteinase-9 (MMP-9) protein level. This elevation occurs within 3 h, without any change of mRNA-concentration. The rapid increase in MMP-9 expression is caused by an enhancement of translational efficiency characterized by a recruitment of translationally inactive MMP-9 mRNP-complexes into the rough endoplasmatic reticulum (rER). Reporter gene assays, which depend on the untranslated regions (UTR) of MMP-9 mRNA, reveal that the posttranscriptional regulation is mainly attributed to the 3'UTR. RNA/protein interaction studies indicate that the elevated binding of nucleolin ( approximately 64 kDa form) to the 3'UTR may be of major importance for the increased efficiency of MMP-9 translation. The results show that MMP-9 expression can be regulated posttranscriptionally, affecting the efficiency of translation and localization of the mRNA.

2,2'-Dipyridyl↗
PubMed2026

Nucleolin promotes neuropathic pain by increasing chromatin accessibility at the Ccl2 promoter in primary sensory neurons.

Nerve injury-induced transcriptional alterations in primary sensory neurons of the dorsal root ganglion (DRG) constitute a key molecular basis for the development of neuropathic pain. Nucleolin (NCL), a highly conserved multifunctional nucleolar protein, regulates gene transcription. Here, we identify that NCL is expressed exclusively in the nuclei of DRG neurons. Peripheral nerve injury time-dependently upregulates Ncl mRNA and NCL protein levels in injured DRG neurons. Blocking this upregulation through DRG microinjection of the adeno-associated virus 9 (AAV9) expressing an shRNA targeting Ncl attenuates nerve injury-induced increases of C-C motif chemokine ligand 2 (CCL2) mRNA and its protein in injured DRG and alleviates the development and maintenance of mechanical, heat and cold hypersensitivities. Conversely, mimicking DRG NCL upregulation through DRG microinjection of AAV9 carrying the full-length Ncl coding sequence increases Ccl2 mRNA and CCL2 protein levels in microinjected DRGs and produces neuropathic pain-like symptoms in the absence of nerve injury. Mechanistically, peripheral nerve injury increases NCL occupancy at the Ccl2 promoter and enhances chromatin accessibility at this locus, resulting in elevated CCL2 expression in injured DRG neurons, which is reversed by NCL knockdown. Given that Ncl mRNA is co-expressed with Ccl2 mRNA in individual DRG neurons, our findings suggest that NCL upregulation in the DRG contributes to neuropathic pain likely by increasing chromatin accessibility at the Ccl2 promoter in primary sensory neurons.

Animals↗
PubMed2004

Phagocytosis of M. paratuberculosis fails to activate expression of NADH dehydrogenase and nucleolin-related protein in bovine macrophages.

Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is a facultative intracellular bacterium and causal agent of Johne's disease in cattle. Following phagocytosis, M. paratuberculosis resides and replicates in macrophage phagosomes that fail to mature. Differential display reverse transcription polymerase chain reaction (DDRT-PCR) was used as a high throughput initial screen to begin to test the hypothesis that macrophage gene expression patterns would be differentially affected by M. paratuberculosis when compared to readily degraded bacteria or non-degradable latex beads. Gene expression profiles from immortalized bovine macrophage cells (BOMAC) exposed to M. paratuberculosis were compared to gene expression profiles for BOMAC cells exposed to Escherichia coli, latex beads or PBS. Amplicons representing genes specifically activated or repressed during M. paratuberculosis phagocytosis were cloned for further investigation. Northern blot hybridizations preformed using DDRT-PCR-derived amplicons 3-1-4, 5-2-10, 5-4-2 and 4-1-6 confirmed stimuli dependent differential gene expression. Expression pattern observed for amplicon 3-1-4 represents genes that are up-regulated following phagocytosis of E. coli or latex beads, but not M. paratuberculosis. Amplicon 5-2-10 exhibited a pattern of expression representative of genes that are up-regulated strongly following phagocytosis of E. coli or latex beads but only moderately following M. paratuberculosis phagocytosis. Expression pattern of the gene for amplicon 5-4-2 was representative of genes that are specifically suppressed following M. paratuberculosis phagocytosis, while the amplicon 4-1-6 gene expression pattern represented genes that are generally suppressed following phagocytosis of any of the three stimuli. DNA sequencing and Genbank database analysis of these amplicons revealed that amplicon 3-1-4, whose expression failed to activate following M. paratuberculosis phagocytosis, had high levels of similarity to a Rattus norvegicus nucleolin-related protein (NRP). Amplicon 5-2-10, which increased expression moderately following M. paratuberculosis phagocytosis, was a near perfect match to bovine nicotinamide adenine dinucleotide dehydrogenase (FNADH dehydrogenase) subunit 1 (ND1). Failure to activate these two genes at levels observed following phagocytosis of either E. coli or latex beads may uncover new mechanisms for the survival of M. paratuberculosis within bovine macrophage cells.

Animals↗
PubMedDate not supplied

Proteomic evidence for roles for nucleolin and poly[ADP-ribosyl] transferase in drug resistance.

One-hundred twenty-four proteins have been identified in the soluble nuclear protein mixture from MCF-7 human breast cancer cells, of which more than 90% are classically categorized as nuclear proteins. Proteins were also studied from three drug resistant MDF-7 lines, selected previously from the same parent line by exposure to etoposide, to mitoxantrone, or to adriamycin in the presence of verapamil. Both quantitative gel comparisons and stable isotope labeling were used to identify a total of fourteen proteins whose abundances are altered by more than 2-fold in the three resistant lines. Several cytoskeleton proteins, cytokeratin 8, cytokeratin 19, septin 2, and alpha tropomyosin, are decreased in common across the three resistant cell lines. PARP-l (poly[ADP-ribosyl]transefrase or connexion) is found to be less abundant in all three resistant lines. Nucleolin is more abundant in lines resistant to etoposide and mitoxantrone, while the mitotic checkpoint protein BUB 3 is more abundant in the line resistant to adriamycin/verapamil.

Antibiotics, Antineoplastic↗
PubMed2001

Identification of nucleolin as a new L-selectin ligand.

Apart from leucocyte-endothelial interactions, the adhesion molecule L-selectin mediates the homotypic adhesion of leucocytes during recruitment at sites of acute inflammation, as well as intercellular adhesion of haematopoietic progenitor cells during haematopoiesis. There is evidence that, in addition to P-selectin glycoprotein ligand-1, other as-yet-unidentified proteins function as L-selectin ligands on human leucocytes and haematopoietic progenitor cells. In the present study, we show: (i) by affinity chromatography on L-selectin-agarose; (ii) by protein identification using MS; and (iii) by covalent cell-surface labelling with sulphosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate that the multifunctional nuclear protein nucleolin is partly exposed on the cell surface, and is a ligand of L-selectin in human leucocytes and haematopoietic progenitor cells.

Amino Acid Sequence↗
PubMed2001

A polymorphic dinucleotide repeat in the rat nucleolin gene forms Z-DNA and inhibits promoter activity.

Many sequences in eukaryotic genomes have the potential to adopt a left-handed Z-DNA conformation. We used a previously described assay based on the binding of a mAb to Z-DNA to inquire whether Z-DNA is formed in the rat nucleolin (Ncl) gene in metabolically active, permeabilized nuclei. Using real-time PCR to measure Z-DNA formation, the potential Z-DNA sequence element Z1 [(CA)(10)(CG)(8)] in the promoter region was found to be enriched 571- to 4,040-fold in different cell lines, whereas Z2 [AC(GC)(5)CCGT(CG)(2)] in the first intron was enriched 12- to 34-fold. Ncl promoter activity was 1.5- to 16-fold stronger than that of the simian virus 40 promoter and enhancer. This activity was further increased 36-54% when Z1 was deleted. The inhibitory effect of Z1 on Ncl promoter activity was independent of location and orientation. The Ncl Z1 element is identical to the genetic marker D9Arb5. Five allelic variants of Z1 were identified by sequence analysis of genomic DNA from various rats. The two most common alleles differed significantly (up to 27%) in their capacity to inhibit Ncl promoter activity. This finding suggests that differences in Z-DNA formation by polymorphic dinucleotide repeats may be one of the factors contributing to genetic variation.

Alleles↗
PubMed1999

G4 DNA binding by LR1 and its subunits, nucleolin and hnRNP D, A role for G-G pairing in immunoglobulin switch recombination.

The immunoglobulin heavy chain switch regions contain multiple runs of guanines on the top (nontemplate) DNA strand. Here we show that LR1, a B cell-specific, duplex DNA binding factor, binds tightly and specifically to synthetic oligonucleotides containing G-G base pairs (KD </= 0.25 nM). LR1 also binds to single-stranded G-rich sequences (KD approximately 10 nM). The two subunits of LR1, nucleolin and hnRNP D, bind with high affinity to G4 DNA (KD = 0.4 and 0.5 nM, respectively). LR1 therefore contains two independent G4 DNA binding domains. We propose that LR1 binds with G-G-paired structures that form during the transcription of the S regions that is prerequisite to recombination in vivo. Interactions of donor and acceptor S regions with subunits of the LR1 could then juxtapose the switch regions for recombination.

Antibodies↗
PubMed2001

N(omega)-arginine dimethylation modulates the interaction between a Gly/Arg-rich peptide from human nucleolin and nucleic acids.

We studied the interaction between a synthetic peptide (sequence Ac-GXGGFGGXGGFXGGXGG-NH(2), where X = arginine, N(omega),N(omega)-dimethylarginine, DMA, or lysine) corresponding to residues 676-692 of human nucleolin and several DNA and RNA substrates using double filter binding, melting curve analysis and circular dichroism spectroscopy. We found that despite the reduced capability of DMA in forming hydrogen bonds, N(omega),N(omega)-dimethylation does not affect the strength of the binding to nucleic acids nor does it have any effect on stabilization of a double-stranded DNA substrate. However, circular dichroism studies show that unmethylated peptide can perturb the helical structure, especially in RNA, to a much larger extent than the DMA peptide.

Amino Acid Sequence↗
PubMed2004

Altered expression of protein p42IP4/centaurin-alpha 1 in Alzheimer's disease brains and possible interaction of p42IP4 with nucleolin.

The protein p42IP4 (centaurin-alpha1) is a brain-specific InsP4/PtdIns3 (PIP)-binding protein, whish is localized in neurons of the human brain. In Alzheimer's disease (AD) the intraneuronal expression of the protein was shown to be elevated. In addition, p42IP4 immunostaining decorated neuritic plaques. Attempting to explain the putative role of the protein in AD, we have concentrated on its well-known interactions with casein kinase I, which is known to be prominently involved in AD pathophysiology. Meanwhile, specific interaction of p42IP4 with nucleolin, another player in AD pathology, has been revealed. Based on these data, we propose alternative concepts of how p42IP4 might act in AD pathomechanisms.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Infection of macrophage-like THP-1 cells with Mycobacterium avium results in a decrease in their ability to phosphorylate nucleolin.

This study of the phosphorylation ability of macrophage-like cells upon infection with Mycobacterium avium was undertaken to establish potential targets of the interference with host response mechanisms. Cytosolic and membrane fractions from noninfected and infected cells were incubated with [gamma-(32)P]ATP, in the presence of Mg(2+) and the absence of Ca(2+), and the patterns of phosphoproteins synthesized were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Lower levels of a 110-kDa phosphoprotein were observed in association with cytosolic fractions from mycobacterium-infected cells compared to noninfected cells or cells treated with lipopolysaccharide or having ingested Escherichia coli or killed M. avium. The 110-kDa phosphoprotein was present in the soluble fraction (230,000 x g supernatant) after the kinase incubation, from where it was partially purified and identified as phosphonucleolin by amino acid sequencing. The decrease in nucleolin phosphorylation observed was not related to changes in the cytosolic or membrane levels of this protein, and was detected also in the cytosolic fraction of (32)P-labeled intact cells.

Cell Membrane↗
PubMedDate not supplied

Simultaneous measurement of nucleolin and estrogen receptor in breast cancer cells by laser scanning cytometry.

BACKGROUND: The purpose of the study was to test the feasibility of laser scanning cytometry (LSC) to simultaneously measure estrogen receptor (ER) and nucleolin (NU) expression in the nuclei of the same individual breast cancer cells. MATERIALS AND METHODS: Cancer cells from 64 breast tumors were labeled with anti-NU and biotinylated anti-ER antibodies, then with secondary FITC-conjugated antibody and streptavidin-APC conjugate, respectively, and measured by LSC. The expression of NU in the nucleus and NU aggregates (NUA), number of NUA, nuclear and NUA areas and ER expression were assessed for aspiration each cell. RESULTS: ER-bound APC fluorescence correlated with nuclear NU (r=0.65; p<0.001) and NUA-bound FITC fluorescence (r=0.59; p<0.001). Good correlation was found between percentages of ER-positive cells in LSC and by image analysis in paraffin-embedded sections (r=0.59, p<0.001). CONCLUSION: ER and NU expression can be measured simultaneously in the same nuclei of breast cancer cells.

Adult↗
PubMed1997

Ultrastructural changes in the Schizosaccharomyces pombe nucleolus following the disruption of the gar2+ gene, which encodes a nucleolar protein structurally related to nucleolin.

The nucleolar protein gar2, from the fission yeast Schizosaccharomyces pombe, is the functional homolog of NSR1 from Saccharomyces cerevisiae, and is structurally related to nucleolin from vertebrates. By immunocytochemistry at the electron microscope level, we show that gar2 co-localizes with RNA polymerase I and the gar1 protein along the dense fibrillar component of the nucleolus in a wild-type strain of S. pombe, suggesting that gar2 is involved in the transcription and/or in the early steps of maturation of the ribosomal RNAs. Since the effects of disruption of the gar2+ gene might also shed light on the role of the gar2 protein, we analyzed the ultrastructure of the nucleolus of a gar2-disruption mutant. The nucleolus of the gar2- mutant is dramatically reorganized when compared with that of the wild-type gar2+ strain: a truncated protein containing the NH2-terminus of the gar2 protein is accumulated in an unusual nucleolar "dense body". Our results also suggest that the NH2-terminus might be sufficient for nucleolar localization via interaction with specific nucleolar components and support the hypothesis that gar2 in wild-type S. pombe interacts with nascent pre-rRNA via its two RNA-binding domains in combination with the glycine/arginine-rich domain. We also report that disruption of the gar2+ gene results in a mutant that is defective in cytokinesis and nuclear division.

Cell Nucleolus↗