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PubMed1975

Effects of actinomycin D on the association of newly formed ribonucleoproteins with the cistrons of ribosomal RNA in Triturus oocytes.

The effect of actinomycin D(AMD) on the association of the nascent ribonucleo-protein (RNP) fibrils containing the precursors of ribosomal RNA (pre-rRNA) with their template deoxyribonucleoprotein (rDNP) strands has been studied in lampbrush stage oocytes from Triturus alpestris. Ovary pieces were incubated in vitro either in media containing radioactive ribonucleosides and then, for various times, in solutions containing 25 mug/ml AMD, or were directly exposed to the drug. The ultrastructure of the nucleoli and the nuclear periphery was studied by electron microscopy of thin sections and positively stained spread preparations of isolated nuclear contents, and by light and electron microscope autoradiography. The fate of the labeled pre-rRNA was followed by gel electrophoresis of RNA extracted from manually isolated nuclei. Our results show that the growing fibrils which contain the nascent pre-rRNA progressively detach from the DNP strands, the majority being released between 45 and 180 min after application of the drug. The release pattern seems to be random and does not show preference for regions close to the initiator or terminator sites of the transcribed rDNP units. There is a pronounced tendency to removal of groups of adjacent mascent fibrils. The effect of the drug is very heterogeneous. Even after 3 h of treatment with AMD the nucleoli exhibit several individual transcriptional units which appear almost completely covered with lateral fibrils. Autoradiography revealed that most of this released RNP remains within the confinements of the nucleoli which show some foci of aggregation and condensation of fibrillar components but no clear "segregation" phenomenon. In the gel-electrophoretic analysis, a significant but moderate decrease of labeled pre-rRNA was noted only in the first stable pre-rRNA component, whereas pre-rRNA classes of lower molecular weight are very stable under these conditions. The results are discussed in relation to the stability of rDNA transcription complexes and as a basis for an explanation of the ultrastructural changes which are generally observed in nucleoli of AMD-treated cells. It is postulated that inhibition of transcription results in a slow but progressive release of the arrested incomplete RNP fibrils from the template.

Animals↗

PubMed1989

In vitro reconstitution of hnRNP particles.

The assembly of hnRNP-like particles was studied by in vitro reconstitution, UV-crosslinking and CsCl-equilibrium centrifugation. Using total nuclear protein and RNA extracts from HeLa cells for RNP reconstitution, RNP particles sedimenting with the same buoyant density of p = 1.4 g/cm3 as 'native' 40 S core hnRNPs were obtained. Under the stringent reconstitution conditions used, hnRNP complexes containing only the Cl-core hnRNP protein could be identified.

Cell Nucleus↗

PubMed2000

MSSP, a protein binding to an origin of replication in the c-myc gene, interacts with a catalytic subunit of DNA polymerase alpha and stimulates its polymerase activity.

MSSP has been identified as a protein that binds to both single- and double-stranded sequences of a putative DNA replication origin sequence in the human c-myc gene. MSSP possesses versatile functions, including stimulation of DNA replication, transcriptional regulation, apoptosis induction, and cell transformation coordinated by c-Myc. MSSP contains two RNP domains, RNP1-A and RNP1-B, both of which are necessary for all of the functions of MSSP. In this study, we found that MSSP binds to the N-terminal region of a catalytic subunit of a human DNA polymerase alpha via its RNP domains both in vitro and in human cells. Furthermore, MSSP was released from the putative DNA replication origin of the c-myc gene after it complexed with DNA polymerase alpha, and MSSP stimulated DNA polymerase activity in vitro.

Cell Line↗

PubMed1999

Key residues revealed in a major conformational epitope of the U1-70K protein.

Epitopes depending on three-dimensional folding of proteins have during recent years been acknowledged to be main targets for many autoantibodies. However, a detailed resolution of conformation-dependent epitopes has to date not been achieved in spite of its importance for understanding the complex interaction between an autoantigen and the immune system. In analysis of immunodominant epitopes of the U1-70K protein, the major autoantigen recognized by human ribonucleoprotein (RNP)-positive sera, we have used diversely mutated recombinant Drosophila melanogaster 70K proteins as antigens in assays for human anti-RNP antibodies. Thus, the contribution of individual amino acids to antigenicity could be assayed with the overall structure of the major antigenic domain preserved, and analysis of how antigenicity can be reconstituted rather than obliterated was enabled. Our results reveal that amino acid residue 125 is situated at a crucial position for recognition by human anti-RNP autoantibodies and that flanking residues at positions 119-126 also appear to be of utmost importance for recognition. These results are discussed in relation to structural models of RNA-binding domains, and tertiary structure modeling indicates that the residues 119-126 are situated at easily accessible positions in the end of an alpha-helix in the RNA binding region. This study identifies a major conformation-dependent epitope of the U1-70K protein and demonstrates the significance of individual amino acids in conformational epitopes. Using this model, we believe it will be possible to analyze other immunodominant regions in which protein conformation has a strong impact.

Amino Acid Sequence↗

PubMed1992

Identification of proteins associating with poly(A)-binding-protein mRNA.

Synthesis of poly(A)-binding protein is regulated at the translational level. We have investigated the binding of proteins to this mRNA on the premise that the protein(s) of the mRNP complex may be involved in regulating the expression of the mRNA. We found the first 243 nucleotides of the 5' untranslated region to contain sequences essential for RNP formation. A large, single-stranded bulge structure encompassing stretches rich in adenine nucleotides and a potential stem-loop domain appear to be the primary sites for protein binding. Removal of the 243-nucleotide segment results in a drastic reduction in protein binding and a concomitant increase in translational efficiency in vitro. We suggest that proteins binding to this region, including poly(A)-binding protein itself, may be essential for regulating translation of this mRNA.

Base Sequence↗

PubMed2002

Immune responses to small nuclear ribonucleoproteins: antigen-dependent distinct B cell epitope spreading patterns in mice immunized with recombinant polypeptides of small nuclear ribonucleoproteins.

Complex patterns of autoantibody reactivities with the small nuclear ribonucleoproteins (snRNPs) are observed in systemic lupus erythematosus. To investigate the role of individual snRNP components in the initiation and diversification of anti-snRNP Ab responses, we immunized A/J mice with recombinant Smith D (SmD), Smith B (SmB), and A ribonucleoprotein (A-RNP) with alum as adjuvant. Sera at different time points after initial immunizations were analyzed by Western blot and immunoprecipitation assays. In SmD-immunized mice, specific Abs to A-RNP and SmB were generated by 2 mo postimmunization, in addition to the detection of cross-reactive Abs between the immunogen and other snRNPs. Whereas Abs reactive with the immunogen decreased by 5 mo, Abs capable of immunoprecipitating A-RNP and SmB increased. In SmB-immunized mice, specific Abs to A-RNP were readily detectable, in addition to cross-reactive Abs. In contrast, A-RNP-immunized mice had only cross-reactive Abs to SmB without detectable Abs to SmD. However, in these mice, specific Abs to the 70-kDa protein were generated. Abs, which precipitated the native snRNP particle, were generated in all three groups of the immunized mice. Our results show that different initiating Ags from the same multiprotein antigenic complex induce distinct patterns of epitope spreading to proteins within that complex. These data have significant implications for the mechanisms of autoantibody diversification in systemic lupus erythematosus.

Animals↗

PubMed1996

The use of antibody to C5b-9 in the subclassification of lupus erythematosus.

Fifty-five patients with biopsy-proven cutaneous lupus erythematosus (LE) were identified in whom a prospective and retrospective review of the clinical and laboratory data allowed subclassification into systemic (SLE), subacute (SCLE), or discoid (DLE) variants. In addition to conventional direct immunofluorescence, an indirect immunofluorescent technique, using a monoclonal antibody, was employed to assess deposition of the membranolytic attack complex (C5b-9) in skin lesions. Deposition of C5b-9 within the epidermis correlated with a diagnosis of SCLE with or without antibodies to Ro and was seen in SLE patients with antibodies to extractable nuclear antigens Ro, La, Sm, and RNP, and in DLE patients with positive antinuclear antibodies and/or extracutaneous manifestations. In the SLE group, vascular C5b-9 deposition was present in six patients. Of these, four had circulating lupus anticoagulant, one had lymphocytic vasculitis, and two had antibodies to Ro. In two patients with SLE there was keratinocyte decoration for immunoglobulin G but not for C5b-9, in the absence of seropositivity for antibodies to Ro, La, Sm, and ribonucleoprotein (RNP). The immunohistological examination of skin lesions using a monoclonal antibody to C5b-9 is a valuable adjunct in the subclassification of LE. The presence of C5b-9 within skin lesions of patiens with LE implies a pathogenic role for complement-mediated pore formation.

Biomarkers↗

PubMed1982

Age- and ploidy-related changes in rat liver nuclear proteins as assessed by one- and two-dimensional gel electrophoresis.

Hepatocyte nuclei from young (3--5 week), mature (8--12 week), and aged (over 32 weeks) rats were isolated and characterized by flow cytometry. Nuclei were bulk separated into diploid (2C), tetraploid (4C), and octoploid (8C) enriched fractions on sucrose gradients. Total, 0.35 M NaCl soluble, and residual proteins were prepared from all nuclear stages and examined by one- and two-dimensional gel electrophoresis. Within limits of sensitivity of these techniques, the following general features emerged. (a) A majority of proteins visualized were common to and present in similar relative quantities in nuclei from all age and ploidy groups. (b) A relatively higher proportion of nonhistone proteins (NHP) were saline-soluble in 2C nuclei from young rats than at any subsequent stage of development. (c) Several age-related and to a lesser extent ploidy-related fluctuations in pattern among the NHP were evident. These reflected primarily differences in solubility rather than major quantitative changes among individual proteins. (d) Exceptions to the foregoing included a group of high molecular weight components (greater than 100 000), a major and a minor component between 45 000 and 50 000, and a heterogeneous group of proteins in 2C nuclei from very young animals. There were no obvious differences among the histones, although these proteins were not examined in detail. The complex pattern of changes observed are discussed in terms of known aspects of hepatocyte differentiation and are related to possible changes in nucleoplasmic, nuclear matrix and Hn-RNP associated proteins.

Aging↗

PubMed1979

Non-nucleolar transcription complexes of rat liver as revealed by spreading isolated nuclei.

Miller's technique was applied to isolated nuclei of rat liver. Both the usual nucleolar and non-nucleolar transcription complexes were visualized. In addition, an unusual type of putative non-ribosomal transcription unit was revealed. It was charcaterized by a high density of the lateral ribonucleoprotein (RNP) fibrils. Although these particular units exhibited a regular increase of fibril lengths, the length of the transcript-covered deoxyribonucleoprotein (DNP) fibres and the morphological aspect of the RNP fibrils distinguished them from the nucleolar 'Christmas-tree'-like figures. The linear and granular configuration of the transcripts and the absence of terminal knobs made them similar to non-nucleolar nascent RNP fibrils.

Animals↗

PubMed1985

Isofocusing of antigenic small nuclear ribonucleoproteins. I. Compositional analysis.

This report describes the behavior of antigenic small nuclear ribonucleoproteins (snRNPs) in native isofocusing gels. These RNA-protein complexes exhibited true isofocusing characteristics only when in the complexed form. Deproteinized snRNAs migrated to pH ranges which varied according to the pH of the application site. Immunological assays using lupus sera which recognized the La, Sm, and RNP determinants on these snRNPs established that the La and the Sm/RNP antigens segregated to pH 4.7-4.9 and 5.5-7.5, respectively. RNase digestion of these snRNPs did not alter the isofocusing migration of either the Sm or the La determinants. These antigenically active fractions contained the appropriate protein and RNA species shown by immunoprecipitation studies to associate with these antigenic determinants. The isofocusing fractions containing the uridylic acid-snRNPs were fully immunoprecipitable by anti-Sm sera, confirming their particulate integrity after isofocusing.

Animals↗

PubMedDate not supplied

The SS-A/SS-B autoantigenic complex: localization and assembly.

The subcellular distribution of Ro ribonucleoprotein particles (Ro RNPs) and their constituents was determined using mammalian tissue culture cells and Xenopus laevis oocytes. Cell fractionation revealed that Ro RNPs are restricted to the cytoplasm, while the Ro RNP-specific proteins were also present in the nucleus. Transport studies indicated that the protein components of Ro RNPs are actively imported into the nucleus while the RNA component accumulates in the cytoplasm. These results suggest a model for the assembly of Ro RNPs.

Animals↗

PubMedDate not supplied

[Morphometric study of nucleolus-organizing regions of chromosomes from swine embryo kidney cells during Go-, G2-period and mitosis].

Quantitative characteristics of nucleolus-organizing regions of chromosomes (NORs, or fibrillar centers, FCs) and some other nucleolar components have been studied with the aid of complete series of ultrathin sections of PK-cells. It has been found that: 1) the number of FCs per cell in the G0-period, in the G2-period and at metaphase is equal to 7.0, 33.7 and 8.0, respectively; 2) volumes of individual FCs in the G0-period (0.033 micron 3), G2-period (0.014 micron 3) and at metaphase (0.025 micron 3) are different; 3) the total volume of FCs, calculated for a haploid set of chromosomes, do not differ in the G0 (0.105 micron 3) and G2 (0.107 micron 3) periods, but exceed twice the FCs volume at metaphase (0.04-0.05 micron 3). These data show that the activation and inactivation of ribosomal genes in interphase PK-cells are not accompanied by a change in the total volumes of FCs and are probably connected with the "fragmentation" and fusion of FCs. Complete inactivation of ribosomal genes at mitosis leads to a decrease in the total volumes of FCs; 4) the nucleolus volume is proportional to the volume of the dense fibrillar RNP-component; in the G2-period the nucleolus volume also correlates with the number of FCs (r = 0.99); 5) the volume of the dense fibrillar component within individual fibrillar complexes--the structures corresponding to one nucleolus-organizing region--is not constant. This is an indirect evidence for the differences in the functional activity of NORs of different chromosomes.

Animals↗

PubMed2001

Influenza virus propagation is impaired by inhibition of the Raf/MEK/ERK signalling cascade.

Influenza A viruses are important worldwide pathogens in humans and different animal species. The functions of most of the ten different viral proteins of this negative-strand RNA virus have been well elucidated. However, little is known about the virus-induced intracellular signalling events that support viral replication. The Raf/MEK/ERK cascade is the prototype of mitogen-activated protein (MAP) kinase cascades and has an important role in cell growth, differentiation and survival. Investigation of the function of this pathway has been facilitated by the identification of specific inhibitors such as U0126, which blocks the cascade at the level of MAPK/ERK kinase (MEK). Here we show that infection of cells with influenza A virus leads to biphasic activation of the Raf/MEK/ERK cascade. Inhibition of Raf signalling results in nuclear retention of viral ribonucleoprotein complexes (RNPs), impaired function of the nuclear-export protein (NEP/NS2) and concomitant inhibition of virus production. Thus, signalling through the mitogenic cascade seems to be essential for virus production and RNP export from the nucleus during the viral life cycle.

Active Transport, Cell Nucleus↗

PubMed1983

The purification, characterization and amino acid analysis of nuclear ribonuclear protein and Sm antigens reacting with human autoimmune sera.

'Monospecific' antibodies directed against nuclear ribonucleoprotein (nRNP) and Smith (Sm) antigens from the sera of patients with mixed connective tissue disease (MCTD) and systemic lupus erythematosus (SLE) were used to isolate and purify the respective antigens from calf thymus nuclear extracts. The antibodies were allowed to interact with the nuclear antigens and the resulting complexes were purified by Protein-A-Sepharose and poly-U-Sepharose chromatography. When the Protein-A-Sepharose antigen-antibody complex was sequentially eluted with 20% and 60% ethylene glycol, 2 fractions could be identified, one containing antigenically active Sm and the other antigenically active Sm and nRNP. The nRNP fraction contained 5 major proteins with mol. wts ranging from 38,000 to 150,000. The Sm antigen had a mol. wt of 68,000 and was noted to 'co-purify' with the nRNP antigen. Amino acid analysis of the purified proteins demonstrated a high content of glycine, serine and acidic amino acid residues similar to previously described core proteins of heterogeneous nuclear RNP (hnRNP), a precursor to mammalian messenger RNA. Analysis of the RNA moiety following nuclease phosphorylase reactions demonstrated the presence of a RNA polynucleotide with a 'capped' structure at the 5' terminus, a feature consistent with the concept that the nRNP antigen is part of the hnRNP complex.

Amino Acids↗

PubMed1996

Direct analysis of nematode cis- and trans-spliceosomes: a functional role for U5 snRNA in spliced leader addition trans-splicing and the identification of novel Sm snRNPs.

Most nuclear pre-mRNAs in nematodes are processed by both cis- and trans-splicing. In trans-splicing, the 5' terminal exon, the spliced leader sequence (SL), is derived from a trans-splicing specific Sm snRNP, the SL RNP. Because U snRNPs are required cofactors for trans-splicing, and because this processing reaction proceeds via a two-step reaction pathway identical to that of cis-splicing, it has long been assumed that trans-splicing is catalyzed in a complex analogous to the cis-spliceosome. However, similarities or differences between cis- and trans-spliceosomes have not been established. In particular, the role of U5 snRNP in trans-splicing has been unclear. Here, we have used affinity selection to analyze the U snRNA constituents of nematode cis- and trans-spliceosomes. We find that U5 snRNP is an integral component of the trans-spliceosome and, using site-specific crosslinking, we show that U5 snRNP establishes specific Interactions with the SL RNA exon. We also identify two novel Sm snRNPs that are enriched in both cis- and trans-spliceosomes. Finally, we provide evidence that a SL RNP-containing multi-snRNP (SL, U4, U5, and U6 RNPs) may be a functional precursor in trans-spliceosome assembly.

Animals↗

PubMed2004

Functional domains of the influenza A virus PB2 protein: identification of NP- and PB1-binding sites.

Influenza virus genomic RNA segments are packaged into ribonucleoprotein (RNP) structures by the PB1, PB2, and PA subunits of an RNA polymerase and a single-strand RNA-binding nucleoprotein (NP). Assembly and function of these ribonucleoproteins depend on a complex set of protein-protein and protein-RNA interactions. Here, we identify new functional domains of PB2. We show that PB2 contains two regions that bind NP and also identify a novel PB1 binding site. The regions of PB2 responsible for binding NP and PB1 show considerable overlap, and binding of NP to the PB2 fragments could be outcompeted by PB1. The binding domains of PB2 acted as trans-dominant inhibitors of viral gene expression, and consistent with the in vitro binding data, their inhibitory activity depended on the concentration of wild-type PB2, NP, and PB1. This provides evidence for functionally significant and potentially regulatory interactions between PB2 and NP.

Binding Sites↗

PubMed2003

Clinical significance of antibodies to TS1-RNA in patients with mixed connective tissue disease.

OBJECTIVE: To investigate the clinical significance of anti-TS1-RNA antibodies in patients with mixed connective tissue disease (MCTD). METHODS: Anti-TS1-RNA antibodies were detected by immunoprecipitation using 32P-UTP labeled TS1-RNA as the antigen source. In total, 104 patients with MCTD, 30 with Sjögren's syndrome, 30 with systemic lupus erythematosus (SLE), 25 with systemic sclerosis, 23 with polymyositis or dermatomyositis, and 10 with rheumatoid arthritis were examined. Specificity of anti-TS1-RNA antibodies was analyzed by immunoprecipitation using HeLa cell extracts. RESULTS: The frequency of anti-TS1-RNA antibodies was 31.7% in patients with MCTD, significantly higher than in SLE (p < 0.05). In anti-TS1-RNA positive patients, the incidence of hypertension and proteinuria and the frequency of anti-Sm and anti-dsDNA antibodies associated with SLE were higher than those of anti-TS1-RNA negative patients. Clinical features of SS such as sicca complex, the serum level of IgA, and anti-SSA antibodies were also elevated. The frequency of anti-TS1-RNA antibodies was significantly higher in SLE patients with anti-U1-RNP antibodies (p < 0.01); however, anti-TS1-RNA positive sera did not precipitate the specific RNA including U1 RNA in immunoprecipitation using HeLa cell extracts. In longitudinal studies, the level of anti-TS1-RNA antibodies changed in parallel with disease activity. CONCLUSION: We found that the level of anti-TS1-RNA antibodies was possibly correlated with the disease activity of lupus-like clinical features in patients with MCTD.

Adult↗

PubMed2004

Effect of mutated transporters associated with antigen-processing 2 on characteristic major histocompatibility complex binding peptides: analysis using electrospray ionization tandem mass spectrometry.

A novel allele of transporters associated with the antigen-processing (TAP) 2 gene, TAP2*Bky2 (Val(577)), is significantly increased in Japanese patients with Sjögren's syndrome (SS), and has a strong association with SS-A/Ro autoantibody production in SS and autoantibody including anti-SS-A/Ro and anti-U1 RNP antibody in systemic lupus erythematosus (SLE). To determine the influence of this natural mutated TAP on peptides loaded onto MHC class I, we analyzed the repertoire of peptides loaded onto MHC class I on transfectants with TAP1 and TAP2 or mutated TAP2 by electrospray ionization tandem mass spectrometry (ESI-MS/MS). After comparison of the peptide profiles we identified three peptides from only mutated TAP transfectants. Moreover, one of these peptides is derived from snRNP A, which is a target for anti-U1 RNP antibody. To our knowledge this is the first report to show that the natural mutation of TAP2 changes the peptide profile loaded onto MHC class I molecules.

ATP Binding Cassette Transporter, Subfamily B, Mem↗