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At least 19 recordsLinked to original sources
PubMed1990

T-cell epitopes on the 70-kDa protein of the (U1)RNP complex in autoimmune rheumatologic disorders.

High-titre IgG antibodies against the immunodominant 70-kDa protein of the (U1)ribonucleoprotein (RNP) complex are present in virtually 100% of patients with mixed connective tissue disease (MCTD), and less commonly in a variety of other autoimmune rheumatic diseases. As T-cell 'help' is assumed to be required for this potentially pathogenic form of immune response, investigations to define T-cell epitopes on the 70-kDa protein were undertaken. In prior studies we expressed the 70-kDa protein and a number of its fragments, spanning most of the molecule, as recombinant fusion proteins using the pGEX expression-vector system. These fusion proteins were used as antigens in the epitope mapping studies reported here. PBMC were isolated from patients with (U1)RNP-positive rheumatic diseases and from both normal controls and rheumatologic patients with other autoantibody reactivities, including those to Ro, La and dsDNA. Reactivity to the purified 70-kDa protein was assayed by thymidine incorporation and was evident only in anti-(U1)RNP positive patients but was not restricted to MCTD patients, being present also in patients with SLE and rheumatoid arthritis. The stimulation indices (SIs) observed were in the two- to five-fold range. Using the 70-kDa protein fragments, a T-cell stimulatory epitope was localized to the C-terminal 63 amino acids of the autoantigen. A T-cell line, derived from PBMC of a (U1)RNP positive patient with MCTD, also reacted predominantly with this C-terminal fragment but with an SI of approximately 15-fold. Thus, we have demonstrated the presence and specificity of autoreactive T lymphocytes to a defined peptide epitope in systemic rheumatic disease.

Arthritis, Rheumatoid↗
PubMed1976

Cytochemical studies on RNP complexes produced by puff 2-48BC in Drosophila hydei: uranyl acetate and phosphotungstic acid staining.

Differential staining of the core and RNP particles of RNP complexes in puff 2--48 BC in salivary gland chromosomes of Drosophila hydei was achieved with aqueous uranyl acetate (UA) at low pH, with UA in acetone, with phosphotungstic acid (PTA) in organic solvents, and with aqueous PTA at pH 5 And 6. A comparison of the results of UA and PTA staining under various conditions indicate that the proteins in the core region and in the RNP particles connected to it differ with respect to their amino-acid composition (arginine and lysine residues).--The staining mechanism of PTA and UA is discussed.

Acetates↗
PubMed1978

[Messenger RNA and RNP complexes of normal mouse liver cells and of hepatomas].

The messenger RNA spectra of highly malignant ascites hepatoma 22A and hepatoma 48, belonging to the group of hepatomas with minimum deviations, examined by a sedimentation method in the sacchrose concentration gradient, are characterized by higher heterogenecity as compared with that of the normal liver. The nuclear RNA contents of these hepatomas differ but insignificantly. Whereas in cytoplasm of hepatoma 22A and, to a less extent, of hepatoma 48 some deviations were found in quantitative correlations between polyribosomes and messenger somatic cells (free cytosol particles) in favour of the latter.

Animals↗
PubMed1992

Yeast 5S rRNA binding to ribosomal protein L1a alters the fluorescence of tryptophan residues lying outside the binding site.

The yeast ribosomal protein L1a contains two tryptophan residues located at positions 95 and 183. Spectrofluorometric analysis showed that the average tryptophan environment is moderately polar. Quenching studies of the yeast 5S rRNA-L1a protein complex (RNP) with acrylamide and iodide revealed tryptophan heterogeneity. The two tryptophan residues are located in the non-RNA-binding region of the L1a molecule. However, dissociation of the yeast 5S rRNA-L1a protein RNP complex to its components resulted in a decline of tryptophan fluorescence. The observation implied that the environment of the tryptophan-containing L1a regions which were not known to be involved in RNA binding was influenced by association with the 5S rRNA molecule.

Acrylamide↗
PubMed1979

Further characterization of the replicative complex of vesicular stomatitis virus.

Replicating vesicular stomatitis virus ribonucleoprotein (RNP) complexes were isolated in nonequilibrium Renografin density gradients. These nascent RNPs had the same buoyant density as virion nucleocapsids in both isopycnic Renografin and CsCl gradients. Both transcribing and replicating RNP complexes were shown to be stable in sucrose gradients, whereas only replicating RNP complexes were stable in Renografin gradients. Size analysis of the 5-min-pulse-labeled RNA species from the replicating RNPs using methylmercury gels revealed that the nascent strands were primarily less than full-length molecules. Longer times of radiolabeling demonstrated that the nascent RNA accumulated as 42S RNA, which was primarily of the same sense as the virion strand when it was radiolabeled at 5 h postinfection. The percentage of this radiolabeled RNA which was plus stranded was higher at 2.5 h postinfection, reflective of the shift in plus- to minus-stranded full-length 42S RNA synthesis which occurs in the cell. Addition of cycloheximide to the infected cells before the addition of the radiolabel prevented the formation of these RNP complexes. Both the change in the percentage of minus strands found in the RNP complexes at the different times postinfection and the sensitivity to cycloheximide indicate that the RNP complex which was isolated was indeed the replicative complex.

Cycloheximide↗
PubMed1992

Purification of ribonucleoproteins by a novel approach: isolation of the SSB1 ribonucleoprotein from yeast and demonstration that it has no role in mRNA splicing.

A novel approach is described to purify potential ribonucleoproteins (RNP) of yeast. The method assays a yeast RNP complex, assembled in vitro on actin pre-mRNA, by low-ionic strength acrylamide gel electrophoresis. The minimal protein components of this RNP complex were three proteins, one of 30 kDa and two at 42-44 kDa, defined by formation of the complex on biotinylated-RNA, binding of this complex to avidin-agarose, and salt elution of the protein in the biotinylated-RNP complex. Using the assay for RNP complex formation, an RNP protein was purified to homogeneity on the basis of its affinity towards single-stranded DNA and RNA. This RNP protein turned out to be identical to a known RNP protein, the single-stranded binding protein 1 (ssb1) of yeast, on the basis of identical gel electrophoretic migration, antibody cross-reactivity, and identical properties on the gel complex formation assay. In vitro mRNA splicing was normal in extracts made from a yeast strain missing ssb1 (ssb1- strain). Addition of anti-ssb1 antibody to splicing extracts made from a wild type strain did not inhibit or diminish splicing. Instead, mRNA splicing was reproducibly stimulated several fold, indicating competition between ssb1 and splicing factors for binding to single-stranded RNA in the extracts. RNP complexes still formed in the ssb1- strain, demonstrating that it would be possible to purify other RNP proteins from this strain using the gel complex formation assay.

Actins↗
PubMed1978

[Ribonucleoprotein mRNA-containing cytoplasmic particles from mouse lymphocytes before and after injection of antigen].

In mouse spleen lymphocytes mRNA was discovered not only in poliribosomes but also in lighter RNP complexes having sedimentation coefficients of 80--120S and buyoant density of 1,40--1,52 g/cm3. The incorporation of 14C-pulse aminoacid lable has shown that the most active polypeptide synthesis takes place in light RNP-complexes and not in polyribosomes. The pattern and the contents of labeled mRNA-containing particules in spleen cytoplasm changed within different periods after the antigen injection. The greatest contents of these particles have been discovered on the second day after the antign injection, 3H- and 14C-radioactivity of RNP-complexes with higher sedimentation coefficients, and that of polyribosomes was increased; the optical density of polyribosomes peak was also increased. On the 4th day after the antigen injection contents of light RNP-complexes with higher sedimentation coefficients were decreased. On the 5th day the decrease of RNP-complexes contents was more pronounced. The changes of radioactive RNP-complexes contents in spleen cytoplasm at different periods of the immune response, which can be discovered during determined period of labling, are probably due to acceleration and then to retardation of the transition of light RNP complexes into polyribosomes. It, probably, reflects the existens in spleen lymphocytes mechanisms of translation regulation, irrespective of mRNA stability which are realized during immune reaction. Periods of the translation increasing or inhibiting in immune lymphocytes don't correspond to periods of increasing or decreasing of number of antibody synthesising cells in spleen.

Animals↗
PubMed1975

Nuclear ribonucleoprotein complexes containing U1 and U2 RNA.

Nuclear ribonucleoprotein (RNP) complexes that contain the U1 and U2 RNA of chromatin of Novikoff hepatoma cells were extracted with 0.01 M Tris-HCl (pH 8.0) after the nuclei were initially washed with 0.075 M NaCl and 0.025 M EDTA (pH 8.0). These RNP complexes were purified by chromatography on Sepharose 6B columns and centrifugation on sucrose density gradients. The identity of the U1 and U2 RNA in these particles was established by their electrophoretic mobility in polyacrylamide gels and their T1 RNase fingerprints which were identical with those of authentic U1 and U2 RNA (R. Reddy et al. (1974), J. Biol. Chem.249, 6486-6494; H. Shibata et al. (1974), Mol. Cell. Biochem. 4, 3-19). The nuclear riboncleoproteins had a buoyant density of 1.47 g/ml in CsCl gradients. Two-dimensional polyacrylamide gel electrophoresis of their proteins showed these RNP complexes contain 10 polypeptide spots, of which two are phosphorylated in vivo.

Animals↗
PubMed1978

Isolation and characterization of a virus-specific ribonucleoprotein complex from reticuloendotheliosis virus-transformed chicken bone marrow cells.

Chicken bone marrow cells transformed by reticuloendotheliosis virus (REV) produce in the cytoplasm a ribonucleoprotein (RNP) complex which has a sedimentation value of approximately 80 to 100S and a density of 1.23 g/cm3. This RNP complex is not derived from the mature virion. An endogenous RNA-directed DNA polymerase activity is associated with the RNP complex. The enzyme activity was completely neutralized by anti-REV DNA polymerase antibody but not by anti-avian myeloblastosis virus DNA polymerase antibody. The DNA product from the endogenous RNA-directed DNA polymerase reaction of the RNP complex hybridized to REV RNA but not to avian leukosis virus RNA. The RNA extracted from the RNP hybridized only to REV-specific complementary DNA synthesized from an endogenous DNA polymerase reaction of purified REV. The size of the RNA in the RNP is 30 to 35S, which represents the subunit size of the genomic RNA. No 60S mature genomic RNA was found within the RNP complex. The significance of finding the endogenous DNA polymerase activity in the viral RNP in infected cells and the maturation process of 60S virion RNA of REV are discussed.

Animals↗
PubMed1992

Purification of a glucose-binding protein from rat liver nuclei. Evidence for a role in targeting of nuclear mRNP to nuclear pore complex.

A nuclear carbohydrate-binding protein with a molecular mass of 67 kDa (CBP67), which is specific for glucose residues, was purified to essential homogeneity from rat liver nuclear extracts. This protein could also be isolated from nuclear ribonucleoprotein (RNP) complexes by extraction in the presence of 0.6 M or 2 M NaCl, but it was absent in polysomal RNP complex. The binding of the purified protein, which has an isoelectric point of 7.3, to glucose-containing glycoconjugates depends on the presence of Ca2+ and Mg2+. Using closed nuclear envelope vesicles as a system to study nuclear transport of RNA, it was shown that both entrapped polysomal mRNA and nuclear RNA precursors are readily exported from the vesicles in an ATP-dependent manner. The transport was unidirectional and strongly promoted by the poly(A) segment attached to these RNAs. In contrast, nuclear RNP complexes entrapped into the vesicles together with glucose-conjugated bovine serum albumin or nucleoplasmin, or bird nest glycoprotein, were not exported into the extravesicular space. However, transport of nuclear RNP complexes could be achieved in the presence of glucose or after co-addition of a glucose-recognizing lectin from Pellina semitubulosa. In Western blots, radioiodinated CBP67 binds to an 80-kDa polypeptide both in isolated rat liver nuclear envelopes and pore-complex laminae. From these results we postulate that CBP67 may direct nuclear RNP complexes to the nuclear pore.

Animals↗
PubMed1978

[Nuclear and cytoplasmic ribonucleoprotein complexes of normal mouse liver cells, Guelstein hepatomas 22A and 48 and of liver of tumor-bearing animals].

Nuclear and cytoplasmic RNP complexes obtained from normal mouse liver cells, Guelstein hepatomas of different degrees of malignancy (22A and 48) as well as from liver of tumor-bearing mice were subjected to chromatography on a celite column (NPC--chromatography). In addition cytoplasmic RNP complexes were centrifuged in sucrose and CsCl density gradients. The results of the NPC-chromatography indicate that nuclear rapidly labelled RNA species of all tissues under study are constituents of the two main types of RNP particles differing from each other by the tightness of RNA-protein bonds. No precursor-product relationship could be revealed between the above types of RNP-particles of nucleus, labelled under conditions of a partial Actinomycin D block. Rapidly labelled nonribosomal cytoplasmic RNAs represent constituents of RNP-particles resembling nuclear ones in their degree of heterogeneity and chromatographic position. Sedimentation analysis of cytoplasmic RNP-particles from tumours showed an increase in relative proportion of monoribosomes and informosomes (free non-ribosomal cytosol RNP-complexes) at the expense of polyribosomes and mRNP complexes. Thus, the liver cells of experimental tumour-bearing animals undergo changes (although not very well-defined), typical for tumour cells.

Animals↗
PubMed1991

A novel 40S multi-snRNP complex isolated from rat liver nuclei.

Two structurally distinct RNP complexes (MI and MII), each with a sedimentation value of approx. 40S, were isolated from rat liver nuclear extracts by sucrose gradient centrifugation and subsequent native gel electrophoresis of the 40S hnRNP-containing fractions. MII RNP contained the bulk of hnRNA and hnRNP proteins (i.e. the 32-45KD core proteins and polypeptides of 60-80 and 110-130KD). MI RNP was characterized by the exclusive presence of U-snRNAs (U1, U2, U4, U5 and U6), their well known snRNP polypeptides and a number of Sm-associated proteins in the range of 50-210KD. Immunoselection experiments employing a monoclonal antibody with an established specificity for the U2-snRNP-specific B" polypeptide proved that the RNA and protein components characteristic of MI were part of a single multi-snRNP unit. The prominent 200/210KD protein doublet of MI was identified immunochemically as the rat homologue of the yeast PRP8 protein, a known U5-associated splicing component. Based on the major biochemical and immunochemical features of MI and MII RNP complexes, we conclude that MII represents the monomeric 40S hnRNP structure, whereas MI defines a novel multi-snRNP entity.

Animals↗
PubMed1975

Induced RNP production in different cell types of Drosophila.

The typical RNP complexes produced in puff 2-48BC in the salivary gland chromosomes as a consequence of treatments interfering with the cellular respiratory metabolism have been found in various other cell types after treatment with 5-10- minus 2 M vitamin B6 for 4 hr. In Drosophila hydei the typical RNP complexes were present in puff 2-48BC in the nuclei of gastric caeca cells, in nuclei of cells of the imaginal (wing) discs and embryonic cells in primary culture. Similar RNP complexes were observed in a puff in D. virilis salivary gland nuclei, but not in salivary gland nuclei of D. melanogaster.

Animals↗
PubMed1976

Detection of mRNA sequences in nuclear 30S ribonucleoprotein subcomplexes.

RNA from nuclear 30S ribonucleoprotein (RNP) complexes of mouse ascites cells has been shows to contain sequences homologous to poly(A) + mRNA by its ability to hybridize with complementary DNA prepared from poly(A) + mRNA template. Analysis of the hybridization kinetics of poly(A) + mRNA with its own complementary DNA revealed several abundancy classes. The total complexity of poly(A) + mRNA from ascites cells was estimated to be approximately 30,000 sequences of average molecular weight (6 X 10(5)). When the hybridization reaction of 30S RNP-RNA with mRNA-specific cDNA was compared to the homologous reaction the majority, and most probably all, of the poly(A) + mRNA sequences were found to be present in the RNA. The kinetics of hybridization suggest that 10-15% of the RNA in this RNP complex is homologous to poly(A) + mRNA. The 30S RNP subcomplexes therefore contain nuclear poly(A) + mRNA sequences as well as the bulk of heterogeneous RNA.

Base Sequence↗
PubMed1992

Ro ribonucleoprotein assembly in vitro. Identification of RNA-protein and protein-protein interactions.

The human Y RNAs, small RNAs with an unknown function, are complexed with at least three proteins: the 60,000 M(r) Ro protein (Ro60), the 52,000 M(r) Ro protein (Ro52) and the La protein (La). In this study we examined the intermolecular interactions between the components of these so-called Ro ribonucleoprotein (Ro RNP) complexes. Incubation of 32P-labelled hY1 RNA in HeLa S100 extract allows the reconstitution of Ro RNP complexes, which were analysed by immunoprecipitation with monospecific antisera. By immunodepletion of HeLa S100 extracts for either Ro60, Ro52 or La, followed by supplementation with recombinant Ro60 or La, it was demonstrated that both Ro60 and La bind to hY1 RNA directly without being influenced by one of the other proteins. However, binding of Ro52 to hY1 RNA required the presence of Ro60, which strongly suggests that the association of Ro52 with Ro RNPs is mediated by protein-protein interactions between Ro60 and Ro52.

Autoantigens↗
PubMed1978

Ribonucleoprotein-like structures from coronavirus particles.

The structure of the ribonucleoprotein (RNP) complex of three coronaviruses was investigated. A single-stranded helix of diam. 14 to 16 nm and up to 320 nm in length was released from disrupted particles of human coronavirus strain 229E and mouse hepatitis virus strain 3 after incubation in mild conditions. The helical complexes appeared to be composed of globular subunits with long axes of 5 to 7 nm surrounding a hollow core of diam. 3 to 4 nm. The complexes were shown to be sensitive to both pancreatic RNase and to pronase. No undegraded internal component was obtained from disrupted avian infectious bronchitis virus particles. We conclude that these structures are RNP complexes. The similarity between these RNPs and those of other large lipid containing RNA viruses is discussed.

Coronaviridae↗
PubMed1978

Rat liver nuclear skeleton and small molecular weight RNA species.

Small molecular weight RNA species (smwRNAs) were studied in rat liver nuclei with and without chromatin as well as with and without nuclear envelope and nucleoplasm. From all the species identified, only two, N5 and 5Sb, were related to ribosomes. The others were localized exclusively in the nuclear skeleton or the spongelike network that was described in the preceding communication. This network or protein matrix contains a less abundant but exclusive set of molecules designated 5Sa, N1, and 4.5S, as well as other more abundant molecules which also exist in rat liver endoplasmic reticulum but not in polysomes or postribosomal RNP complexes. The smwRNAs behave like HnRNA; they remain located in the nuclear skeleton when nuclei are deprived of nucleoplasm and chromatin. With the information presently available, it is not possible to know whetherer both species are in the same or different RNP complexes and whether some of the smwRNAs contribute to the architecture of the nuclear skeleton. Distinct from any other nuclear RNA species, smwRNAs have two unique properties: facility of extraction, and resistance to nuclear ribonuclease digestion.

Animals↗