Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “RNA-binding”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 613 records · Page 34Linked to original sources
PubMed1991

Selection of the same major T cell determinants of influenza nucleoprotein after vaccination or exposure to infectious virus.

In the present study, we have compared the T cell antigenic determinants on nucleoprotein (NP) of influenza A/NT/60/68 virus recognized by BALB/c mice (H-2d) after vaccination using several different vehicles with the determinants recognized after exposure to infectious virus. Mice were immunized s.c. with: 1) purified recombinant NP with three different adjuvants--alum, saponin, or CFA; 2) whole inactivated A/Okuda virus in PBS or saponin; or 3) live attenuated Salmonella typhimurium AroA- vector expressing NP. A series of overlapping synthetic peptides that cover more than 90% of the amino acid sequence of NP were used to map the Th cell epitopes. The results showed that the same limited number of major epitopes were recognized after each of the different immunization regimes. Secondary in vivo boosting using the same vehicles as for the primary immunization did not increase the number of different T cell sites recognized. The T cell responses after intranasal infection with infectious A/NT/60/68 or A/PR/8/34 virus also showed a similar pattern of recognition of the major CD4-positive T cell epitopes. The only exception was that the region corresponding to residues 401-419 was only recognized after exposure to NP from A/NT/60/68 but not A/PR/8/34. This is probably because the two viruses differ in amino acid sequence at positions 408 and 411 within this part of the NP molecule. In contrast to the results observed with CD4-positive T cell epitopes, the major determinant recognized by CD8-positive T cells was only presented after live viral infection. The results in this study have important implications for vaccine design, inasmuch as they indicate that the same dominant CD4 T cell determinants on NP presented by vaccination with NP are also recognized by T cells from mice exposed to infectious virus.

Animals↗
PubMed1991

A frameshift mutation at the NH2 terminus of the nucleoprotein gene does not affect generation of cytotoxic T lymphocyte epitopes.

BALB/3T3 cells infected with a retroviral vector encoding the influenza virus nucleoprotein (NP) gene are efficiently lysed by CTL generated in BALB/c mice (H-2d background). Cells transduced with a mutant form of NP which contains a frameshift mutation at its NH2 terminus (NPm) do not express biochemically detectable levels of protein but nevertheless present Ag to CTL with high efficiency. Cold target inhibition studies indicate that the same CTL epitope(s) are recognized in cells harboring NP or NPm. L929 cells transduced with the NPm gene also present Ag efficiently to CTL raised in C3H mice (H-2k background). Cells engineered to express 5- to 15-fold lower levels of wild-type NP were not capable of presenting Ag to CTL, arguing against the notion that CTL are able to lyse cells expressing very low levels of Ag which might have resulted from suppression of the frameshift mutation in NPm. Implications to the mechanism of epitope generation in class I MHC-restricted immune responses are considered.

Animals↗
PubMed1991

Characterization of Escherichia coli SecA protein binding to a site on its mRNA involved in autoregulation.

In order to understand further the autogenous regulation of Escherichia coli secA translation, we have set up a purified system to study the binding of SecA protein to portions of its mRNA. Specific SecA protein-RNA binding was demonstrated by UV cross-linking, filter binding, and gel shift assays. Use of the filter binding assay allowed optimization of binding, which was influenced by Mg2+ and ATP concentrations, and a measurement of the affinity of this interaction. A nested series of RNAs lacking either 5' or 3' portions of geneX-secA sequences were used to localize the SecA protein binding site to sequences around the geneX-secA intergenic region. These studies imply that SecA protein directly regulates its own translation by a specific RNA binding activity that presumably blocks translational initiation.

Adenosine Triphosphatases↗
PubMed1991

Antigen presentation and the association of class-I molecules.

We have identified two mutant cell lines which are not able to present epitopes of influenza virus synthesized in the cytoplasm but can present the same epitope when exposed to it as a peptide in the extracellular medium. The cell lines also have a defect in class-I assembly, with reduced expression of assembled alpha chain: beta 2M heterodimers at their cell surface. This led to the suggestion that the two traits were the result of the same mutation and that stable assembly of class-I molecules is dependent on peptide binding. Consistent with this idea was the finding that exposure to specific peptides in the extracellular fluid promotes stable association of class-I heavy chains with beta 2M and restores expression of class-I at the cell surface. We have gone on to show that stable assembly of class-I molecules can be supported in detergent extracts of the mutant cells when specific peptides are added. Peptides stabilized a conformational change in the class-I heavy chain and association with beta 2M by binding to the complexes. This effect is apparent at peptide concentrations around 100-fold lower than required in "peptide feeding" experiments with whole cells. We have also demonstrated that the conformational change induced in heavy chain is influenced by the concentration of beta 2M, and consequently have been able to demonstrate the formation of empty class-I molecules.

Animals↗
PubMed1992

The glycine-rich domain of nucleolin has an unusual supersecondary structure responsible for its RNA-helix-destabilizing properties.

Nucleolin, a major nucleolar protein implicated in preribosome assembly and transcriptional regulation, possesses a C-terminal domain unusually rich in glycine, arginine, and phenylalanine residues. A polypeptide (p10), corresponding to this domain, has been synthesized by means of an Escherichia coli expression system and purified to homogeneity. Nitrocellulose binding assays have clearly shown that this domain of nucleolin is capable of interacting with RNA, and indeed all nucleic acids tested, in an efficient but nonspecific manner. A combination of circular dichroism and infrared spectroscopy provide strong evidence that repeated beta-turns are a major structural component of this polypeptide, which is entirely consistent with its amino acid composition and above all the presence of repeat motifs such as RGGF. Circular dichroism technique also shows that the interaction of p10 with RNA involves an unstacking of the nucleotide bases and an unfolding of the RNA secondary structure. While the role of the C-terminal domain of nucleolin in vivo has yet to be established, our findings suggest that it may act to unfold regions of ribosomal RNA so that a second domain of nucleolin has access to its specific binding site.

Amino Acid Sequence↗
PubMed1991

High expression of era gene.

For high production of Era protein, lambda Ea8.5 gene has been chosen from Gene Bank because the Ea8.5 protein contains a sequence of several amino acids at its N-terminus which is identical to that of Era and also because it can be highly expressed in E. coli. The 5'-end sequence of era gene was substituted by a synthetic oligonucleotide which was identical to that of the 5'-end of Ea8.5 gene, so that the transcripts of era gene was provided with a strong translational initiation signal without changing the amino acid sequence of its translational product. Plasmid pCE31, which harbored the recombined era gene under the control of PL promoter, could express very high levels of Era protein, while synthesis of other cellular proteins was nearly shut off during the period of induction. As a result, Era constituted over 80% of the total cellular protein. Electrophoretic pure Era protein with specific guanine nucleotide-binding activity was obtained by a simple procedure including lyzing the cells and washing the pellet of the lysate.

Bacterial Proteins↗
PubMed1991

Comparison of the activities of variant forms of eIF-4D. The requirement for hypusine or deoxyhypusine.

Eukaryotic protein synthesis initiation factor 4D (eIF-4D) (current nomenclature, eIF-5A) contains the unique amino acid hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine). The first step in hypusine biosynthesis, i.e. the formation of the intermediate, deoxyhypusine (N epsilon-(4-aminobutyl)lysine), was carried out in vitro using spermidine, deoxyhypusine synthase, and ec-eIF-4D(Lys), an eIF-4D precursor prepared by over-expression of human eIF-4D cDNA in Escherichia coli. In a parallel reaction, using N-(3-aminopropyl)cadaverine in place of spermidine, a variant form of eIF-4D containing homodeoxyhypusine (N epsilon-(5-aminopentyl)lysine) was prepared. Evidence that N-(3-aminopropyl)cadaverine can also act as the amine substrate for deoxyhypusine synthase in intact cells was obtained by incubating putrescine- and spermidine-depleted Chinese hamster ovary cells with [3H]cadaverine. In these cells, in which [3H]cadaverine is readily converted to N-(3-aminopropyl) [3H]cadaverine, small amounts of [3H]homodeoxyhypusine and another 3H-labeled compound, presumed to be N epsilon-(5-amino-2-hydroxy[3H]pentyl)lysine, were found. eIF-4D stimulates methionyl-puromycin synthesis, an in vitro model assay for translation initiation. Whereas the unmodified precursor ec-eIF-4D(Lys) appeared inactive, the deoxyhypusine-containing form provided a significant degree of stimulation. The variant form containing homodeoxyhypusine, on the other hand, showed little or no activity. These findings emphasize the importance of hypusine or deoxyhypusine for the biological activity of eIF-4D and demonstrate the influence of both the length and chemical nature of its amino alkyl side chain.

Animals↗
PubMed1991

[Detection of influenza virus nucleoprotein on the surface of infected cells and in free nonvirion form].

The inner viral nucleoprotein synthesized de novo is shown to be exposed on the surface of the chicken embryo infected with influenza virus. The kinetics of the nucleoprotein located on the surface does not correlate with the kinetics of cell destruction. In culture or allantois virus containing liquids the large number of extracellular viral nucleoprotein prone to antinucleoprotein monoclonal antibodies was found. The accumulation of this nucleoprotein occurs in the period when cell destruction is absent, it is eliminated by the adsorption of the virus on erythrocytes or centrifugation at 70,000 g for 2 hours (20%) or by centrifugation at 10,000 g for 4 hours (50%).

Animals↗
PubMed1991

Granzyme A binding to target cell proteins. Granzyme A binds to and cleaves nucleolin in vitro.

The physiologic substrates of cytotoxic T lymphocyte granule-associated serine esterases (referred to hereafter as proteases or "granzymes"), and the role of these enzymes in cell-mediated activity remain unclear. We have developed an assay for possible ligands of the trypsin-like dimeric serine protease granzyme A based on Western immunoblotting techniques. This protein-binding assay demonstrates the selective binding of granzyme A to several proteins present in the target cell P815. The binding specificity is preserved when enzyme binding is performed in the presence of excess competing proteins, including such cationic species as lysozyme and RNase. Enzyme binding is inhibited, however, by heat or detergent inactivation of granzyme A. Subcellular fractionation of target cells shows that the nuclear fraction contains most granzyme A binding reactivity, which is recovered in the nuclear salt wash fraction. A protein with Mr = 100,000 and two closely migrating proteins with Mr = 35,000 and 38,000 are the predominant reactive moieties, and the N-terminal sequence of the 100-kDa protein confirmed that this protein was murine nucleolin. Incubation of granzyme A with nucleolin generates a discrete proteolytic cleavage product of Mr = 88,000. Since nucleolin is known to shuttle between nucleus and cytoplasm, the interaction of granzyme A and nucleolin may be important in the process of apoptosis which accompanies cytotoxic T lymphocyte-mediated lysis of target cells.

Amino Acid Sequence↗
PubMed1991

Identification of an amino acid-regulated mRNA from rat liver as the mammalian equivalent of bacterial ribosomal protein L22.

Amino acid deprivation of rat hepatoma cells induced the levels of a 612-base pair mRNA termed ASI (Shay, N. F., Nick, H. S., and Kilberg, M. S. (1990) J. Biol. Chem. 265, 17844-17848). The ASI mRNA was present at levels equal to or greater than actin in every rat tissue tested. The corresponding full-length cDNA was cloned, and the present report demonstrates that the deduced 184-residue amino acid sequence shares greater than 30% identity to a number of bacterial and chloroplast L22 ribosomal proteins, including those from Escherichia coli and Halobacterium halobium. A monospecific anti-peptide antibody was produced that upon immunochemical analysis of subcellular fractions of rat liver recognized a band in the microsomal fraction and, more specifically, reacted with a single polypeptide in the ribosomal large subunit fraction. The antibody did not react with any proteins of the mitochondrial large subunit, but did recognize a protein in human liver homogenate at the same relative mobility (23 kDa) as that observed for rat liver.

Amino Acid Sequence↗
PubMed1990

[Regulation of intestinal cholesterol metabolism].

The small bowel epithelium is of major importance in the cholesterol homeostasis of the organism. The morphological and functional heterogeneity of the gut, complicates studies on intestinal cholesterol metabolism. Cholesterol from diet, de novo synthesis and lipoproteins is strictly compartmentalized intracellularly and supports different metabolic needs. Interactions of cholesterol and lipoproteins were studied in cultured intestinal cells (IEC-6, CaCo-2). Newly synthesized cholesterol is mainly utilized for local purposes like membrane synthesis and is essential for cell growth. In contrast to other cell systems it cannot be replaced by LDL cholesterol in case of blocked synthesis. LDL is specifically bound, internalized and degraded. HDL3 also displays specific binding, internalisation and retroendocytosis, but is not degraded. The observed induction of HMG-CoA reductase, suppression of ACAT, as well as cholesterol efflux after contact of HDL3 with lipid droplets argue for intracellular cholesterol transfer to intact HDL3 and finally resecretion into the medium. HDL3 therefore appears as a mediator of reverse cholesterol transport also in the small intestinal epithelial cell.

Carrier Proteins↗
PubMed1991

Autoantibodies to nucleolin in systemic lupus erythematosus and other diseases.

The 110-kDa intracellular phosphoprotein (110K) described previously by this laboratory as a common IgM autoantigen in SLE and certain other systemic autoimmune disorders and viral infections is identified as nucleolin in the present investigation. Using rabbit antiserum to rat nucleolin as a probe, IgM autoantibody-reactive 110K co-migrated with human lymphocyte nucleolin in one- and two-dimensional immunoblots. Rabbit anti-nucleolin also specifically depleted autoreactive 110K from detergent lysates of human cells. Because nucleolin shares amino acid sequence similarity and/or forms dynamic particles with other prominent autoantigens, the present observation raises the possibility that the nucleolin/anti-nucleolin system may be of special significance for the development of humoral autoreactivity to nuclear Ag.

Antibody Specificity↗
PubMed1991

Increased stability of nucleolin in proliferating cells by inhibition of its self-cleaving activity.

Nucleolin is the major nucleolar phosphoprotein of exponentially growing eukaryotic cells and is presumably involved in pre-rRNA transcription and ribosome biogenesis. Monoclonal antibodies against nucleolin were selected by a differential dot-immunobinding assay. Nucleolin expression during T lymphocyte activation was monitored by the specific antibody. Results showed that nucleolin fluctuated in parallel to DNA synthesis. The intact 105-kDa nucleolin molecule was the major species in actively dividing cells, whereas the degraded forms were relatively abundant in nondividing cells. These results imply that stability of nucleolin molecule is cell proliferation-dependent. When affinity purified nucleolin containing undetectable contaminants was incubated at 37 degrees C, the majority of 105-kDa nucleolin was cleaved by 6 h and completely degraded within 24 h. This purified nucleolin was further separated from possible copurified protease, if any, on a reducing sodium dodecyl sulfate-polyacrylamide gel. After renaturation, the 105-kDa nucleolin immobilized in the gel was also cleaved at 37 degrees C. These data have confirmed that nucleolin protein autocatalyzes its own degradation. The self-cleaving activity of nucleolin was inhibited by nuclear extracts prepared from proliferating cells. Apparently, a proteolytic inhibitor(s) in the nuclei of proliferating cells stabilized the nucleolin molecule. It provides an unique regulatory mechanism for nucleolin expression.

Antibodies, Monoclonal↗
PubMed1991

Molecular cloning of matrin 3. A 125-kilodalton protein of the nuclear matrix contains an extensive acidic domain.

We report here the cloning and sequencing of matrin 3, an acidic internal matrix protein, from a rat insuloma cDNA library. The nucleotide sequence has a single open reading frame encoding a polypeptide of 845 amino acids. The Genbank and National Biomedical Research Foundation databases did not contain any sequences similar to that of matrin 3. The primary structure consists of 33% charged residues and is generally hydrophilic. The amino-terminal region (residues 1-120) is positively charged and contains a large number of amino acids with free hydroxyl groups (26 of the first 100 residues) as in the lamins and several non-lamin intermediate filament proteins. A highly acidic domain (approximately 170 amino acids) near the carboxyl terminus, in which 32% of the amino acid residues are acidic (Glu or Asp), is a characteristic found in other nuclear proteins (Earnshaw, W. C. (1987) J. Cell Biol. 105, 1479-1482). A putative nuclear targeting signal sequence (Ser-Lys-Lys-Lys-Leu-Lys-Lys-Val-Glu) is located in the middle of the highly acidic domain. The corresponding human deduced partial amino acid sequence is 96% identical to the rat sequence, indicating that matrin 3 is a highly conserved protein.

Amino Acid Sequence↗