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PubMed1994

Patterns of nuclear immunofluorescence and reactivities to recombinant nuclear antigens in autoimmune hepatitis.

BACKGROUND/AIMS: Antibodies to nuclear antigens have not been fully characterized in autoimmune hepatitis. The aims of this study were to determine the different patterns of immunofluorescence associated with antinuclear antibodies and to correlate these patterns with reactivities to recombinant nuclear antigens and clinical manifestations of autoimmune hepatitis. METHODS: Sera from 65 patients were tested by indirect immunofluorescence and by enzyme immunoassay for antibodies to recombinant centromere, ribonucleoproteins, and ribonucleoprotein complexes. RESULTS: Homogeneous and speckled patterns of immunofluorescence occurred with similar frequencies (34% vs. 38%) in the majority of patients (72%). Patients with speckled patterns were younger and had higher serum aspartate aminotransferase levels at presentation than counterparts with other patterns. Antibodies to centromere (42%) and 52K ribonucleoprotein complex (23%) were the most common reactivities. Of the 35 patients with antibodies, 16 (46%) had multiple specificities. Individual reactivities were not associated with specific immunofluorescent patterns, and they did not identify patients with distinctive features. CONCLUSIONS: Antinuclear antibodies produce mainly homogeneous and speckled patterns of immunofluorescence in patients with autoimmune hepatitis. Speckled patterns are associated with a younger age and greater aminotransferase activity. Multiple autoantibodies are frequently associated with each immunofluorescent pattern. Antibodies to centromere and the 52K ribonucleoprotein complex have a previously unrecognized high frequency and uncertain significance.

Adult↗
PubMed1989

Evidence that the interferon-induced Daudi cell human lupus inclusions are de novo synthesized complexes of ribonucleoprotein and membrane.

Quantitative electron microscope autoradiography has been used to define the macromolecular composition of the interferon-induced human lupus-type inclusions (LI) in the human B lymphoblastoid cell line, Daudi. LI were first apparent in Daudi cell cultures 12 h after the addition of 100 units/ml of the purified recombinant human leukocyte interferon, IFLrA. Radiolabels were added at this time and allowed to incorporate over the following 12 h during which an estimated greater than 99% of the LI material present at 24 h was formed. The LI-incorporated radiolabels were present only during this discrete 12-h period after the interferon activation of LI cell pathways in order to detect LIs de novo synthesized macromolecular components. The estimate relative specific activities of the LI-incorporated radiolabels were: choline at 4.042, mannose at 2.631, uridine at 0.664, glucosamine at 0.578, and amino acids at 0.477. With thymidine the estimated LI specific activity was 0.000. LI isolated from whole cells retained the tubular elements and the interwoven membrane network. These results provide direct evidence that the interferon-induced Daudi cell LI are de novo synthesized complexes of ribonucleoprotein and membrane.

Amino Acids↗
PubMed1986

Antibodies against small nuclear ribonucleoproteins immunoprecipitate complexes containing ts110 Moloney murine sarcoma virus genomic and messenger RNAs.

Small nuclear ribonucleoproteins (snRNPs) are believed to play a role in processing premessenger RNAs. In this study, snRNPs were immunoprecipitated from extracts of cells infected with ts110 Moloney murine sarcoma virus (ts110 MoMuSV). Both the unspliced 4.0 kb and the spliced 3.5-kb ts110 MoMuSV specific RNA species were found in the immunoprecipitates obtained with monoclonal antibody anti-Sm and polyclonal anti-Sm, anti-(U1) RNP and anti-La sera. Although only a portion of the total ts110 RNAs was present in these immunoprecipitates, immune recognition by the anti-snRNPs was specific and not due to contaminating anti-RNA (at least for the anti-Sm sera) or, to anti-viral protein activities. Genomic 8.3-kb RNA and subgenomic 3.0-kb spliced env mRNA from Moloney murine leukemia virus (MoMuLV) infected cells as well as the cellular actin mRNA were also detected in immunoprecipitates obtained with the same antisera. The fact that pre-mRNAs and mature mRNAs of different origin can be recovered from immunoprecipitates formed with anti-snRNP sera establishes their tight association and confirms the role of snRNPs in mRNA processing.

Actins↗
PubMed1997

Interaction of rice and human SRP19 polypeptides with signal recognition particle RNA.

The signal recognition particle (SRP) controls the transport of secretory proteins into and across lipid bilayers. SRP-like ribonucleoprotein complexes exist in all organisms, including plants. We characterized the rice SRP RNA and its primary RNA binding protein, SRP19. The secondary structure of the rice SRP RNA was similar to that found in other eukaryotes; however, as in other plant SRP RNAs, a GUUUCA hexamer sequence replaced the highly conserved GNRA-tetranucleotide loop motif at the apex of helix 8. The small domain of the rice SRP RNA was reduced considerably. Structurally, rice SRP19 lacked two small region that can be present in other SRP19 homologues. Conservative structure prediction and site-directed mutagenesis of rice and human SRP19 polypeptides indicated that binding to the SRP RNAs occurred via a loop that is present in the N-domain of both proteins. Rice SRP19 protein was able to form a stable complex with the rice SRP RNA in vitro. Furthermore, heterologous ribonucleoprotein complexes with components of the human SRP were assembled, thus confirming a high degree of structural and functional conservation between plant and mammalian SRP components.

Amino Acid Sequence↗
PubMed2005

Small non-coding RNAs in Archaea.

Biochemical and informatics analyses conducted over the past few years have revealed the presence of a plethora of small non-coding RNAs in various species of Archaea. A large proportion of these RNAs contain a common structural motif called the RNA kink turn (K-turn). The best-characterized are the C/D box and the H/ACA box guide small (s)RNAs. Both contain the K-turn fold and require the binding of the L7Ae protein to stabilize the structure of this crucial motif. These sRNAs assemble with L7Ae and several other proteins into complex and dynamic ribonucleoprotein machines that mediate guide-directed ribose methylation or pseudouridylation to specific locations in ribosomal or transfer RNA. Analyses of new archaeal sRNA libraries have identified additional classes of novel sRNAs; many of these contain the RNA K-turn motif and suggest that the RNAs might function as ribonucleoprotein complexes. Some have characteristics of small interfering RNAs or of micro RNAs that have been implicated in the post-transcriptional control of gene expression, whereas others appear to be involved in protein translocation or in ribosomal RNA processing and ribosome assembly. A complete understanding of the structure of the K-turn motif and its contribution to various RNA-RNA and RNA-protein interactions will be absolutely essential to fully elucidate the biological organization, activity and function of these novel archaeal ribonucleoprotein machines.

Archaea↗
PubMed1994

Characterization of single-stranded DNA binding proteins in rat glial-enriched nuclei.

Single-stranded DNA binding proteins (SSBs) are those proteins which preferentially bind single-stranded DNA as opposed to double-stranded DNA and are known to be involved in recombination, amplification, and repair of DNA. To characterize single-stranded DNA binding proteins of glial cells and to examine their potential involvement in induction of neurogenic tumors in rats, nuclei were isolated from target glia and non-target liver of carcinogenically sensitive Sprague-Dawley (SD) and resistant Berlin-Druckrey-IV (BD-IV) rats of various ages and rapidly proliferating glioma cells. Nuclei were fractionated into chromatin, a preribosomal RNA protein complex, heterogeneous nuclear ribonucleoprotein complex (hnRNP), and nucleoplasm. SSBs were isolated, quantitated, and characterized by electrophoresis. A comparison of the contents of SSBs relative to RNA and their electrophoretic profiles between chromatin and hnRNP revealed that SSBs of liver chromatin were mainly associated with RNA. However, it was found that glial chromatin, particularly that of juvenile rats, was enriched with a heterogeneous series of SSBs which were not found in liver chromatin and presumably associated with chromosomal DNA. Some of these SSBs were enriched in glial chromatin of sensitive SD rats compared with that of resistant BD-IV rats. High mobility group proteins (HMG) 1 and 2 constituted major SSB components in the nucleoplasm and a greater amount of these HMGs were found in juvenile glia, compared to adult glia and juvenile and adult liver. Fractionation of glial SSBs and determination of their biological functions may contribute to the further understanding of the role these proteins may play in the processes of carcinogenesis.

Animals↗
PubMed1972

Transcription of the influenza ribonucleic acid genome by a virion polymerase. 3. Completeness of the transcription process.

The virion ribonucleic acid (RNA) polymerase of influenza strain A(0)/WS transcribed at least 81% of the viral genome in vitro. The polymerase is shown to be associated with each of the major size classes of the virion RNA-ribonucleoprotein complexes. Under optimal in vitro conditions, at least 45% of the RNA contained in a population of influenza virions was involved in a repetitive transcription process. The detectable proteins associated with enzymatically active complexes containing RNA, ribonucleoprotein, and polymerase have been identified by polyacrylamide gel electrophoresis.

Centrifugation, Density Gradient↗
PubMed1998

Identification of a hormonally regulated luteinizing hormone/human chorionic gonadotropin receptor mRNA binding protein. Increased mrna binding during receptor down-regulation.

To elucidate the molecular events associated with the regulation of luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor mRNA stability during hCG-induced receptor down-regulation, we have identified an LH/hCG receptor-specific mRNA binding protein. Proteins were isolated from control and down-regulated rat ovary and were incubated with in vitro transcribed RNAs corresponding to the full-length LH/hCG receptor, as well as 5'- and 3'-truncated receptor forms. Resultant ribonucleoprotein complexes were analyzed by RNA gel mobility shift. A prominent Mr 50,000 ribonucleoprotein complex was identified with the following characteristics: 1) specificity for LH/hCG receptor open reading frame sequences located between nucleotides 102 and 282; 2) lack of competition by nonspecific RNAs; 3) a 3-fold increase in RNA binding activity during hCG-induced receptor down-regulation; and 4) limited tissue expression. This report describes the first evidence of an LH/hCG receptor mRNA binding protein, which we term LRBP-1, for luteinizing hormone receptor RNA binding protein-1. This protein is a candidate for a trans-acting factor involved in the hormonal regulation of LH/hCG receptor mRNA stability in rat ovary.

Animals↗
PubMed1979

The 5-S RNA binding protein from yeast (Saccharomyces cerevisiae) ribosomes. Evolution of the eukaryotic 5-S RNA binding protein.

The ribonucleoprotein complex between 5-S RNA and its binding protein (5-S RNA . protein complex) of yeast ribosomes was released from 60-S subunits with 25 mM EDTA and the protein component was purified by chromatography on DEAE-cellulose. This protein, designated YL3 (Mr = 36000 on dodecylsulfate gels), was relatively insoluble in neutral solutions (pH 4--9) and migrated as one of four acidic 60-S subunit proteins when analyzed by the Kaltschmidt and Wittman two-dimensional gel system. Amino acid analyses indicated lower amounts of lysine and arginine than most ribosomal proteins. Sequence homology was observed in the N terminus of YL3, and two prokaryotic 5-S RNA binding proteins, EL18 from Escherichia coli and HL13 from Halobacterium cutirubrum: Ala1-Phe2-Gln3-Lys4-Asp5-Ala6-Lys7-Ser8-Ser9-Ala10-Tyr11-Ser12-Ser13-Arg14-Phe15-Gln16-Tyr17-Pro18-Phe19-Arg20-Arg21-Arg22-Arg23-Glu24-Gly25-Lys26-Thr27-Asp28-Tyr29-Tyr35; of particular interest was homology in the cluster of basic residues (18--23). Since the protein contained one methionine residue it could be split into two fragments, CN1 (Mr = 24700) and CN2 (Mr = 11300) by CNBr treatment; the larger fragment originated from the N terminus. The N-terminal amino acid sequence of CN2 shared a limited sequence homology with an internal portion of a second 5-S RNA binding protein from E. coli, EL5, and, based also on the molecular weights of the proteins and studies on the protein binding sites in 5-S RNAs, a model for the evolution of the eukaryotic 5-S RNA binding protein is suggested in which a fusion of the prokaryotic sequences may have occurred. Unlike the native 5-S RNA . protein complex, a variety of RNAs interacted with the smaller CN2 fragment to form homogeneous ribonucleoprotein complexes; the results suggest that the CN1 fragment may confer specificity on the natural 5-S RNA-protein interaction.

Amino Acid Sequence↗
PubMed2005

Regulation of luteinizing hormone receptor expression: evidence of translational suppression in vitro by a hormonally regulated mRNA-binding protein and its endogenous association with luteinizing hormone receptor mRNA in the ovary.

Our previous studies have identified a luteinizing hormone receptor (LHR) mRNA-binding protein (LRBP) that binds to the coding region (LBS) of rat LHR mRNA. The identity of LRBP was later established as mevalonate kinase (MVK). The present study examined if LRBP binding to LHR mRNA impairs translation. A full-length FLAG-tagged rat LHR mRNA was synthesized and translated in vitro. The translation product was immunoprecipitated and analyzed on SDS-PAGE. The addition of LRBP inhibited LHR mRNA translation. This inhibitory effect was reversed by an excess of wild type (wt) LBS. To determine whether this reversal of the inhibitory effect of LRBP was indeed due to the sequestration of LRBP by the wtLBS, a translation reaction was performed in the presence of mutated LBS in which all the cytidine in the wtLBS was mutated to uridine. This mutation of LBS has been shown to render it incapable of interacting with LRBP. Unlike wtLBS, the mutated LBS was unable to reverse the inhibitory effect of LRBP on LHR mRNA translation. The addition of mevalonate, which has been shown to compete for LHR mRNA binding to LRBP, also reduced the extent of translation inhibition by LRBP. Endogenous association of LHR mRNA with MVK was assessed by immunoprecipitation of the ribonucleoprotein complex with MVK antibody followed by reverse transcription-PCR of the RNA associated with the immune complex. Amplification of LHR mRNA, if any, associated with the immunoprecipitate obtained from ovarian ribonucleoprotein complex with gene-specific primers confirmed the association of LHR mRNA with MVK. Collectively, the present data support the novel function of LRBP as a translational inhibitor of LHR mRNA in the ovary.

Animals↗
PubMed1982

The binding of transfer ribonucleic acids to 5 S and 5.8 S eukaryotic ribosomal ribonucleic acid-protein complexes.

Rat liver 5 S and 5.8 S rRNAs were oxidized with periodate and the 3' termini were coupled to Sepharose 4B through an adipic acid dihydrazide spacer. Ribosomal proteins were passed through the nucleic acid affinity columns to form ribonucleoprotein complexes containing the nucleic acid and the proteins that bind to it (5 S . L6, L7, L19; and 5.8 S . L6, L19, S9, S13). Pure isoaccepting species of yeast initiator-tRNA (tRNAfMet) and elongator-tRNAs (tRNAmMet, and tRNAPhe) were chromatographed on the ribosomal ribonucleoprotein affinity columns. The three rRNAs were bound to the 5 S and 5.8 S ribosomal ribonucleoprotein complexes. The elongation and initiation ternary complexes, EF-1 alpha . GTP . Phe-tRNAPhe, and eIF-2 . GTP . Met-tRNAfMet, also were bound to both ribosomal ribonucleoprotein affinity columns, whereas the binary complex EF-1 alpha . GTP and puromycin were not.

Animals↗
PubMed2006

Translational repression is sufficient for gene silencing by bacterial small noncoding RNAs in the absence of mRNA destruction.

Stress-induced Hfq-binding small RNAs of Escherichia coli, SgrS and RyhB, down-regulate the expression of target mRNAs through base-pairing. These small RNAs form ribonucleoprotein complexes with Hfq and RNase E. The regulatory outcomes of the RNase E/Hfq/small RNA-containing ribonucleoprotein complex (sRNP) are rapid degradation of target mRNAs and translational inhibition. Here, we ask to what extent the sRNP-mediated mRNA destabilization contributes to the overall silencing of target genes by using strains in which the rapid degradation of mRNA no longer occurs. We demonstrate that translational repression occurs in the absence of sRNP-mediated mRNA destabilization. We conclude that translational repression is sufficient for gene silencing by sRNP. One possible physiological role of mRNA degradation mediated by sRNP is to rid the cell of translationally inactive mRNAs, making gene silencing irreversible.

Biological Transport↗
PubMed1984

Characterization of the La (SS-B) antigen from several mammalian sources.

The La or SS-B antigen is associated with rheumatic diseases, systemic lupus erythematosus, and Sjogren's syndrome, and is part of a larger ribonucleoprotein complex. Immunoaffinity chromatography allowed for the efficient separation of the La antigen from the bulk of the cellular proteins, with a minimum of protease exposure. Protein blot analysis of the affinity-isolated material indicated a major immunoreactive polypeptide of 50,000 m.w. A comparison of this antigen in a number of mammalian sources (human, rabbit, and rat) suggested strong conservation of the native polypeptide m.w. Likewise, in a direct comparison of this antigen from Epstein-Barr virus-infected cells in which there are distinct differences in the antigen-associated RNA species, the immunoreactive polypeptide species were of similar size. The La protein is readily susceptible to endogenous proteolysis, with the resulting generation of smaller, discrete polypeptides that still retain antigenicity. By using the La protein to monitor potential degradation, we have developed a simple two-step procedure to isolate the La-associated snRNP complex. The complexes thus isolated provide material suitable as a source of both the active antigen and of the functional ribonucleoprotein complex.

Animals↗
PubMed1983

Isolation and characterization of nuclear hnRNP complexes from Drosophila melanogaster tissue culture cells.

Fractionation on sucrose gradients of nuclear described extracts prepared from cultured Drosophila melanogaster cells by sonication of the nuclei in the presence of rat liver cytosol RNAase inhibitor revealed a complex polysome-like pattern of nuclear ribonucleoprotein complexes. The bulk of these heterogeneous ribonucleoprotein (hnRNP) complexes sedimented in the 30S to 80S zone of the sucrose gradient. According to biochemical and morphological data, the monomer particle proved to be the 45S hnRNP and its average diameter was found by electron microscopy to be 24-26 nm. The buoyant density of both the mono and polyparticles was about 1.4 g/cm3, with a slight degree of heterogeneity. The proteins from different zones of the sucrose gradient were composed primarily of similar polypeptides of 47 000, 56 000, 64 000, 96 000 and 130 000 daltons. Complete dissociation of nuclear hnRNP complexes was observed by resedimentation of the particles in the presence of 0.7 M NaCl or 4 M urea. RNAase A digestion (0.1 microgram/ml at 0 degree C for 10 min) resulted in the solubilization of part of the hnRNP and aggregation of some particles. The bulk of the RNA isolated from the different sized hnRNP complexes sedimented in the 7 to 11S region in the sucrose gradient. The large hnRNP complexes contained hnRNA strands larger than 15S, up to 28S. The base composition of the RNA from the 45S monoparticles proved to be AU type: A + U/G + C = 1.7. The RNAs from the 60-75S and 90- 100S polyparticles were also AU type, with an A + U/G + C ratio of 1.46 and 1.21, respectively. The hnRNP complexes exhibited marked heterogeneity in the electron microscope. Our biochemical and morphological observation point to a nonrandom organization of hnRNP particles in Drosophila melanogaster nuclei.

Animals↗
PubMed2005

U1 small nuclear ribonucleoprotein immune complexes induce type I interferon in plasmacytoid dendritic cells through TLR7.

Plasmacytoid dendritic cells (PDCs), which produce IFN-alpha in response to autoimmune complexes containing nuclear antigens, are thought to be critically involved in the pathogenesis of systemic lupus erythematosus (SLE). One of the immunostimulatory components of SLE immune complexes (SLE-ICs) is self DNA, which is recognized through Tlr9 in PDCs and B cells. Small nuclear ribonucleoproteins (snRNPs) are another major component of SLE-ICs in 30% to 40% of patients. In this study, we show that murine PDCs are activated by purified U1snRNP/anti-Sm ICs to produce IFN-alpha and proinflammatory cytokines and to up-regulate costimulatory molecules. The induction of IFN-alpha and IL-6 by U1snRNPs in murine bone marrow-derived PDCs required the presence of intact U1RNA and was largely dependent on Tlr7 but independent of Tlr3. Intracellularly delivered isolated U1snRNA and oligoribonucleotides derived from the stem loop regions and the Sm-binding site of U1snRNA efficiently induced IFN-alpha and IL-6 in Flt3L-cultured DCs in a Tlr7-dependent manner. The U1snRNA component of U1snRNP immune complexes, found in patients with SLE, acts as an endogenous "self" ligand for Tlr7 and triggers IFN-alpha and IL-6 production in PDCs.

Animals↗
PubMed2002

Reconstitution of the signal recognition particle of the halophilic archaeon Haloferax volcanii.

The signal recognition particle (SRP) is a ribonucleoprotein complex involved in the recognition and targeting of nascent extracytoplasmic proteins in all three domains of life. In Archaea, SRP contains 7S RNA like its eukaryal counterpart, yet only includes two of the six protein subunits found in the eukaryal complex. To further our understanding of the archaeal SRP, 7S RNA, SRP19 and SRP54 of the halophilic archaeon Haloferax volcanii have been expressed and purified, and used to reconstitute the ternary SRP complex. The availability of SRP components from a haloarchaeon offers insight into the structure, assembly and function of this ribonucleoprotein complex at saturating salt conditions. While the amino acid sequences of H.volcanii SRP19 and SRP54 are modified presumably as an adaptation to their saline surroundings, the interactions between these halophilic SRP components and SRP RNA appear conserved, with the possibility of a few exceptions. Indeed, the H.volcanii SRP can assemble in the absence of high salt. As reported with other archaeal SRPs, the limited binding of H.volcanii SRP54 to SRP RNA is enhanced in the presence of SRP19. Finally, immunolocalization reveals that H.volcanii SRP54 is found in the cytosolic fraction, where it is associated with the ribosomal fraction of the cell.

Amino Acid Sequence↗
PubMed2002

Determinants of the recognition of enteroviral cloverleaf RNA by coxsackievirus B3 proteinase 3C.

The initiation of enteroviral positive-strand RNA synthesis requires the presence of a functional ribonucleoprotein complex containing a cloverleaf-like RNA secondary structure at the 5' end of the viral genome. Other components of the ribonucleoprotein complex are the viral 3CD proteinase (the precursor protein of the 3C proteinase and the 3D polymerase), the viral 3AB protein and the cellular poly(rC)-binding protein 2. For a molecular characterization of the RNA-binding properties of the enteroviral proteinase, the 3C proteinase of coxsackievirus B3 (CVB3) was bacterially expressed and purified. The recombinant protein is proteolytically active and forms a stable complex with in vitro-transcribed cloverleaf RNA of CVB3. The formation of stable complexes is also demonstrated with cloverleaf RNA of poliovirus (PV) 1, the first cloverleaf of bovine enterovirus (BEV) 1, and human rhinovirus (HRV) 2 but not with cloverleaf RNA of HRV14 and the second cloverleaf of BEV1. The apparent dissociation constants of the protein:RNA complexes range from approx. 1.7 to 4.6 microM. An electrophoretic mobility shift assay with subdomain D of the CVB3 cloverleaf demonstrates that this RNA is sufficient to bind the CVB3 3C proteinase. Binding assays using mutated versions of CVB3 and HRV14 cloverleaf RNAs suggest that the presence of structural features rather than a defined sequence motif of loop D are important for 3C proteinase-RNA interaction.

3C Viral Proteases↗