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PubMedDate not supplied

Subacute cutaneous lupus erythematosus--clinical, histopathological and immunophenotypical study of five cases.

Subacute cutaneous lupus erythematosus (SCLE) is a recently described distinct subset of lupus erythematosus (LE) having characteristic clinical, serological and genetic features. The clinicopathological and serological findings of 5 patients are reported. From a clinical point of view, we describe two types of cutaneous lesions: psoriasiform and annular pattern. ANA were present in 80% of the cases, anti-Ro was present in 60%, and anti-La in 20%; anti-RNP was positive in 1 patient. Circulating immune complexes were detected in 2 patients, and low levels of complement factors (C3, C4) in another 2 patients. The B- cell alloantigens HLA-A2, HLA-Bw4 or HLA-DR3 were present in 60% of the cases. The cutaneous histopathology revealed important changes in the epidermis. Our findings are similar to those described by most other authors. Also, we would like to emphasize the low disease activity and benign course of our patients.

Adult↗

PubMed1998

RNA recognition by RNP proteins during RNA processing.

The ribonucleoprotein (RNP) domain is one of the most common eukaryotic protein folds. Proteins containing RNP domains function in important steps of posttranscriptional regulation of gene expression by directing the assembly of multiprotein complexes on primary transcripts, mature mRNAs, and stable ribonucleoprotein components of the RNA processing machinery. The diverse functions performed by these proteins depend on their dual ability to recognize RNA and to interact with other proteins, often utilizing specialized auxiliary domains. Crystallographic and NMR structures of several RNP domains and a handful of structures of RNA-protein complexes have begun to reveal the molecular basis for RNP-RNA recognition.

Alternative Splicing↗

PubMed1986

In vivo splicing of the premRNAs from early region 3 of adenovirus-2: association of precursors, intermediates and products with hnRNP.

To elucidate the function of hnRNP in the splicing of premRNA, we studied the distribution of the transcripts from the early region 3 of adenovirus-2 in hnRNP from infected HeLa cells. In addition to premRNAs and mRNAs, we detected excised IVS 1 and the products of cleavage of premRNA at the 5' splice site of IVS 1 (free leader 1 and IVS 1-exon 2). All these molecules were present in hnRNP and persisted in the salt-resistant complexes after a 400 mM KCl treatment. None of them was exclusively part of the salt dissociable monoparticles. The precursors, intermediates and products of the splicing reaction were associated with both monoparticles and salt resistant complexes. This eliminates the possibility that one of the classes of RNP is exclusively involved in one of the steps of RNA processing. Whereas the size of hnRNP was related to that of its RNA for most molecules, the small molecules free leader 1 and excised IVS 1 were found in the large hnRNP containing premRNA as well as in small size hnRNP. A probable interpretation of these results is that the cleavage products are associated with the premRNP complex immediately after cleavage and are then released in the form of an individual RNP.

Adenoviridae↗

PubMed1992

Translocation of a specific premessenger ribonucleoprotein particle through the nuclear pore studied with electron microscope tomography.

A specific premessenger ribonucleoprotein (RNP) particle in the salivary glands of the dipteran Chironomus tentans was studied with electron microscope tomography during translocation from the cell nucleus to the cytoplasm. The RNP particle consists of a thin RNP fiber tightly folded into a ribbon, which is bent into a ring-like structure. Upon translocation through the pore, the particle is first orientated in a specific manner at the pore entrance, and subsequently the bent ribbon is gradually straightened and transported through the pore with the 5' end of the RNA in the lead. Concomitantly, the elementary RNP fiber constituting the ribbon is gradually unpacked and will appear more or less extended on the cytoplasmic side of the pore complex. The ordered nature of the process suggests a specific recognition of the RNP particle at the nuclear pore.

Animals↗

PubMed1981

Distribution of messenger ribonucleic acid in polysomes and nonpolysomal particles of sea urchin embryos: translational control of actin synthesis.

We have used cell-free translation and two-dimensional gel electrophoresis to examine the complexities of the polysomal and cytoplasmic nonpolysomal [ribonucleo-protein (free RNP)] messenger ribonucleic acid (mRNA) populations of sea urchin eggs and embryos. We show that all species of mRNA detected by this method are represented in both the polysomes and free RNPs; essentially all messages present in polysomes are also in the free RNP fraction. However, the cytoplasmic distribution is clearly nonrandom since some templates are relatively concentrated in the free RNPs and others are predominantly in the polysomes. The polypeptides synthesized under the direction of unfertilized egg mRNA are qualitatively indistinguishable from those made by using embryonic mRNA, indicating that the complexity of the abundant class mRNA remains unchanged from egg through early development. However large changes in the abundancies of specific mRNAs occur, and changes are detected in the polysomal/free RNP distribution of some mRNAs through development. The differences in the realtive abundancies of specific mRNAs between polysomes and free RNPs and the developmental changes that take place indicate significant cytoplasmic selection of mRNA for translation. Three different forms of actin (termed alpha, beta, and gamma) were identified among the translation products. Messages for all three are present in the unfertilized egg and early cleavage embryo, yet the gamma form is preferentially located in the polysomes and the alpha and beta in the free RNPs. The relative concentrations of the three change greatly during development as do their relative distributions into polysomes and free RNPs. Examinations of in vivo labeled proteins largely support the in vitro findings. The results indicate that the synthesis of actin mRNAs increases greatly during development and that the expression of the actin mRNAs is partly controlled at the translation level during early development.

Actins↗

PubMed1999

An unusual chemical reactivity of Sm site adenosines strongly correlates with proper assembly of core U snRNP particles.

The small nuclear ribonucleoprotein particles (snRNP) U1, U2, U4, and U5 contain a common set of eight Sm proteins that bind to the conserved single-stranded 5'-PuAU3-6GPu-3' (Sm binding site) region of their constituent U snRNA (small nuclear RNA), forming the Sm core RNP. Using native and in vitro reconstituted U1 snRNPs, accessibility of the RNA within the Sm core RNP to chemical structure probes was analyzed. Hydroxyl radical footprinting of in vitro reconstituted U1 snRNP demonstrated that riboses within a large continuous RNA region, including the Sm binding site, were protected. This protection was dependent on the binding of the Sm proteins. In contrast with the riboses, the phosphate groups within the Sm core site were accessible to modifying reagents. The invariant adenosine residue at the 5' end, as well as an adenosine two nucleotides downstream of the Sm binding site, showed an unexpected reactivity with dimethyl sulfate. This novel reactivity could be attributed to N7-methylation of the adenosine and was not observed in naked RNA, indicating that it is an intrinsic property of the RNA- protein interactions within the Sm core RNP. Further, this reactivity was observed concomitantly with formation of the Sm subcore intermediate during Sm core RNP assembly. As the Sm subcore can be viewed as the commitment complex in this assembly pathway, these results suggest that the peculiar reactivity of the Sm site adenosine bases may be diagnostic for proper assembly of the Sm core RNP. Consistent with this idea, a strong correlation was found between the unusual N7-A methylation sensitivity of the Sm core RNP and its ability to be imported into the nucleus of Xenopus laevis oocytes.

Adenosine↗

PubMed1980

Nuclear antigens: characterization of the acidic nuclear ribonucleoprotein-Sm-antigen complex by analytical and preparative isoelectric focusing.

The soluble ribonucleoprotein nuclear antigen (RNP), associated with the Sm antigen and present in extracts of rabbit thymus acetone powder, was purified by ion-exchange chromatography. Analytical isoelectric focussing (IEF) showed two bands at pH 5.2 and two bands at pH 5.4. After treatment by RNAse these 4 bands disappeared, and a new band with a pI of 6.1, representing the intact protein moiety, appeared. During preparative IEF in granulated gels the antigenicity of RNP was lost. Only the Sm antigen could be detected by counter-immunoelectrophoresis. Fractions with Sm antigenicity refocussed as two intense bands (pI 6.8 and 7.2) and one or two fainter bands (pI 6.1). On reducing SDS-PAGE the SM antigen revealed two bands of apparent molecular weights of about 110,000 and 28,500, indicating a total molecular weight of 138,500.

Animals↗

PubMed1996

Differential binding of BvgA to two classes of virulence genes of Bordetella pertussis directs promoter selectivity by RNA polymerase.

Transcription of virulence genes of Bordetella pertussis is co-ordinately regulated by the BvgA and BvgS proteins, which are members of the two-component family of bacterial signal-transduction proteins. BvgS is the transmembrane sensor and BvgA the transcriptional regulator. By gel mobility shift assays we demonstrate that phosphorylated BvgA (BvgA approximately P) forms distinct complexes with the filamentous haemagglutinin (PFHA) promoter DNA at different BvgA approximately P: DNA ratios. DNase I protection analyses show that phosphorylation of BvgA not only enhances affinity of the protein for the binding sites of the PFHA and bvgP1 promoters, but it extends significantly the bound region towards position -35 of these promoters. Conversely, a 10-fold higher amount of BvgA approximately P is required for binding to a large DNA region, from -168 to -60, of the pertussis toxin (Ptox) promoter sequence. These findings suggest that the molecular interaction of BvgA approximately P with the Ptox promoter is different from its interaction with the PFHA and bvgP1 promoters. The sigma 70 Escherichia coli RNA polymerase (RNP) does not bind to the bvg-regulated promoters. However, following the formation of a BvgA approximately P-promoter complex, the E. coli RNP specifically recognizes and binds to the bvg-regulated promoters. Thus, BvgA approximately P exerts its action at the level of promoter recognition by directing promoter selectivity by RNP.

Adhesins, Bacterial↗

PubMed1989

Molecular characterization of the SM and RNP nuclear antigens.

Patients with systemic lupus erythematosus (SLE) often possess antibodies against two nuclear antigens, designated "Sm" and "RNP". The exact relationship between Sm and RNP is not clear; the present study was conducted to define these two different nuclear antigens. Rabbit thymus extracts were used to obtain purified Sm/RNP complex and free Sm antigens by using a combination of 25-60% ammonium sulfate precipitation, DEAE-Sephacel and hydroxylapatite chromatography. By using the separated antigens, sera characterized as anti-Sm, anti-Sm/RNP and anti-RNP could be distinguished by enzyme-linked immunosorbent assay (ELISA). Anti-Sm and/or anti-RNP antibodies were detected in 32 (52%) of 62 sera from patients with SLE. Of these 32, 6 contained anti-Sm only, 10 contained anti-RNP only and 16 had both. When HeLa nuclear extracts were used as antigens by immunoblotting, sera with anti-RNP reacted primarily with 2 polypeptides of 68 and 45 KD; sera with anti-Sm recognized mainly on 2 polypeptides of 26 and 14 KD; sera with anti-Sm/RNP recognized both groups. When purified Sm/RNP complex from rabbit thymus extracts was used as antigen by immunoblotting, sera with anti-RNP reacted with 68 KD protein and putative degradation products of the 68 KD protein. (major: 63 KD, 45 KD, 40 KD; minor: 54-47 KD); sera with anti-Sm recognized 14 KD protein; sera with anti-Sm/RNP reacted with both groups. Although Sm and RNP can exist as a Multimolecular complex, the epitopes recognized by anti-Sm and anti-RNP differ greatly. The Sm determinants reside primarily on proteins of 26 KD and 14 KD, whereas the RNP determinants reside mainly on a protein of 68 KD.

Animals↗

PubMed1997

Autoepitope-mapping of the U1-70K protein with human-Drosophila chimeric proteins.

The 70K protein is the major autoantigen for anti-RNP autoantibodies directed against the U1 small nuclear ribonucleoprotein complex particle. The U1-70K protein has been epitope-mapped by various groups, and a major antigenic region of about 70 amino acids has been found which overlaps with the RNA binding motif. Attempts to map the major antigenic region further with smaller cloned fragments or with peptides have been hampered by total loss of, or strongly reduced, antigenicity. Thus the major antigenic region is composed of conformational epitopes and a detailed analysis of particular epitopes has not been possible. In the present work, we examine the antigenicity of chimeric proteins assembled from the highly conserved Drosophila melanogaster 70K proteins grafted with human 70K segments. With this approach, the effects on antigenicity of exchanging particular segments can be assayed with the overall structure of the major antigenic domain kept relatively constant. Our results, supported by depletion experiments, show that residues 99-128 from the human protein are essential for recognition by both human and canine anti-RNP autoantibodies. These residues have to be presented in a manner that allows correct conformational interaction between the different protein domains.

Amino Acid Sequence↗

PubMedDate not supplied

Neuropeptide and monoamine components of the parabrachial pontine complex.

The present investigation examined the distributions of immunoreactive neurotensin (NT), cholecystokinin octapeptide (CCK), substance P (SP), methionine enkephalin (ENK), vasoactive intestinal polypeptide (VIP), somatostatin (SS), rat neurophysin II (RNP II), vasopressin (VP), oxytocin (OXY), tyrosine hydroxylase (TH), and serotonin in the parabrachial nuclear complex (PB) of the rat. All of these substances were localized to the PB and they appeared to be chemoarchitecturally organized within the complex. The lateral subdivision (PBL) was organized medial-lateral and ventral-dorsal. Specifically NT, CCK, and SP immunoreactive fibers were found to be the most dense in the ventral aspect of the PBL. The distribution of NT-containing fibers was similar to the pattern of CCK-containing fibers and these were localized primarily to the central zone of the PBL. Immunoreactive SP fibers and cells were found in the external and internal zones ventrally and surrounding the dorsal and dorsolateral nuclei in the PBL. Somatostatin, ENK and VIP were found to be the most dense in the dorsal PBL. Serotonin- and TH-containing cells and fibers were found in both the PBL and PBM. These results, coupled with the observations of neuronal connections of the PB and the known functions of this region, underscore the potential involvement for these neuropeptides and monoamines in limbic-brainstem mechanisms of autonomic control.

Animals↗

PubMed1982

Anti-RNP monoclonal antibodies derived from a mouse strain with lupus-like autoimmunity.

systemic lupus erythematosus (SLE) and related rheumatic and connective-tissue diseases are often associated with the production of antibodies directed against a variety of specific cellular components. Recent evidence indicates that two such autoantigens, the Sm and RNP antigens recognized by SLE sera, exist in small ribonucleoprotein complexes found in the nuclei of higher eukaryotes. Studies of the structure and function of these autoantigenic particles with human sera used as probes have been limited because of the multiplicity of autoantibodies often found in an individual serum. Through this communication, we report that MRL/Mp-+/+ (MRL/n) mice, which spontaneously develop a disease exhibiting many of the characteristics of human SLE, possess anti-RNP antibodies in addition to anti-Sm and anti-DNA as previously reported. Spleen cells from one such autoimmune mouse were used to produce a stable hybridoma secreting antibodies that react simultaneously with a protein of Mr 40,000 and a doublet of approximately 70,000, a pattern of reactivity identical to and characteristic of human SLE anti-RNP autoantibodies.

Animals↗

PubMed1998

Polyneuropathy with endoneurial immune complex deposition as the first manifestation of systemic lupus erythematosus.

A 72-year-old male presented with progressive sensorimotor polyneuropathy. Later weight loss, proteinuria and deteriorating renal function were noted. The electrophysiological examinations revealed extensive, symmetrical demyelinating and axonal polyneuropathy. Frozen sections obtained from sural nerve biopsy sample showed the presence of immune complexes and complements in the walls of the epi- and endoneurial blood vessels, and perineurium suggestive of systemic lupus erythematous (SLE). IgG and Clq deposits were also present along the basement membranes of Schwann cells. The electron microscopy confirmed the presence of immune complex deposition. Diagnosis of SLE was proven by positive serology (anti-nuclear antibodies, anti-Sm, anti-RNP, anti-double-stranded DNA) and renal biopsy showing membranous lupus nephritis with extensive immune complex deposition in the tubular basement membranes. Despite combined immunosuppressive treatment for 10 months, the patient died of complications of generalized immune complex vasculitis. The manifestation of SLE in elderly patients, especially in males, is very rare. Moreover, the polyneuropathy is an unusual initial symptom of SLE. Immune complex deposition in Schwann cell basement membrane probably plays an important role in the pathomechanism of sensorimotor polyneuropathy in SLE.

Aged↗

PubMed1987

Distribution of snRNP complexes in rat liver nuclear extracts: biochemical and immunochemical analysis.

Rat liver nuclei were extracted with 0.14 M NaCl and the extracts submitted to sucrose gradient fractionation. Aliquots of the nuclear residue remaining after the 0.14 M NaCl extraction were also extracted either with 0.3 M NaCl or 1 M urea, and the extracts similarly submitted to sucrose gradient fractionation. Thereafter, both the presence and relative distribution of individual U-snRNA (U1-U6) species was followed. Results showed an extensive association of all U-snRNAs to RNP structures of greater than or equal to 40 S. However, characteristic differences in the association of mostly U1 and U5--which were the major identifiable species in the extracts--to these structures were observed. Only a small fraction of U1 appeared complexed to less than or equal to 40 S RNP structures, while most of it sedimenting in the greater than 20 S region of the gradient. In contrast, U5-snRNA had a tight and almost exclusive association to 40 S RNP structures. No pool of 10-12 S U5-snRNP complexes was detected. Combined immunoprecipitation and immunoblotting experiments on nuclear 0.14 M NaCl extracts using anti-Sm and/or anti-RNP antisera showed that all snRNA species, whether recovered as 10-12 S complexes or segregated with greater than or equal to 40 S RNP components, existed as snRNP structures bearing at least their Sm-antigenic polypeptides. These and our previous results [Guialis, A., Arvanitopoulou, A, Patrinou-Georgoula, M. and Sekeris, C.E. (1983) FEBS Lett. 151, 127-133], support the existence of snRNP-enriched RNP structures of greater than or equal to 40 S. In such structures the core polypeptides (Mr = 32,000-45,000) of 40 S monoparticles are not obligatory components.

Animals↗

PubMedDate not supplied

Immune complexes in progressive systemic sclerosis and mixed connective tissue disease.

Sera from patients with progressive systemic sclerosis (PSS) and mixed connective tissue disease (MCTD) were studied for the presence of circulating immune complexes (CICs) by Clq precipitins, cryoglobulins and the Raji cell and Clq radioimmunoassays. The Raji cell assay was the most sensitive, detecting ICs in 82% of patients with MCTD and in 55% with PSS. However, the median value in MCTD was significantly higher than in PSS (79 vs 20 microgram equivalent AHG/ml serum), and in MCTD, unlike PSS, the CIC levels appeared to parallel disease activity. Ribonucleoprotein (RNP) antigen could not be demonstrated in the Raji cell bound complexes.

Adult↗

PubMed2003

Concomitant early appearance of anti-ribonucleoprotein and anti-nucleosome antibodies in lupus prone mice.

OBJECTIVE: To gain insights on initial stages of the autoimmune response in lupus prone mice taking advantage of new sensitive and quantitative techniques for the detection of autoantibodies specific for RNA- (ribonucleoproteins) and DNA-protein (chromatin) complexes. METHODS: DNA and nucleosome antibodies were detected by ELISA, antibodies to SmB, U1A-RNP, Ro52, Ro60 and La by a new radioligand assay, using de novo synthesized radio-labeled antigens. RESULTS: Analysis of anti-chromatin (including anti-nucleosome, anti-dsDNA and anti-histone antibodies) and of anti-snRNP antibodies (including anti-U1A-RNP, anti-SmB, anti-Ro52, anti-Ro60, anti-La antibodies) was performed in sequential sera from B/W, MRL+/+, MRL Yaa and MRL lpr/lpr mice. In a cohort of 105 MRL+/+ mice of different ages, 59, 51, and 57 mice were positive for anti-nucleosome, anti-SmB and anti-U1A-RNP, respectively. None of them was positive for anti-dsDNA. Importantly, antibody positivities were not randomly distributed but were significantly clustered in individual mice. Appearance of DNA- and RNA-protein complex antibodies started at approximately 18-20 weeks of age, preceding that of the anti-dsDNA (or anti-histone) antibodies that only started at 30-32 weeks. Anti-nucleosome, anti-SmB and anti-U1A-RNP antibody responses did not display any cross-reactivity as demonstrated by inhibition and adsorption experiments. CONCLUSION: These data indicate that anti-nucleosome and anti-snRNP antibodies appear early and concomitantly in lupus prone mice even though they do not share any cross-reactivity. These results fit with the assumption that their production is triggered by tightly physically associated nucleosomes and snRNP autoantigens contained in the same apoptotic bodies.

Animals↗

PubMed1984

Subcellular location of polypeptides that react with anti-Sm and anti-RNP antibodies.

Whole nuclear and cytoplasmic fractions from HeLa cells were analyzed in protein gel blots probed with either monoclonal anti-Sm or polyclonal anti-(U1)RNP antibodies. The cells were fractionated by a nonaqueous procedure, to minimize proteolysis and artifactual leakage of nuclear components to the cytoplasmic fraction. Unexpectedly, more reactive proteins were detected in the nucleus than shown earlier in partially purified small nuclear ribonucleoprotein particles (snRNPs). In addition, reactive polypeptides were now found in the cytoplasm. These results are discussed in reference to the possibility that the nucleus and cytoplasm of adult somatic human cells may have a more complex than anticipated set of populations of polypeptides bearing Sm or RNP antigenic determinants, including some proteins that might not be in snRNP form.

Antibodies↗

PubMed1994

NMR studies of U1 snRNA recognition by the N-terminal RNP domain of the human U1A protein.

The RNP domain is a very common motif found in hundreds of proteins, including many protein components of the RNA processing machinery. The 70-90 amino acid domain contains two highly conserved stretches of 6-8 amino acids (RNP-1 and RNP-2) in the central strands of a four-stranded antiparallel beta-sheet, packed against two alpha-helices by a conserved hydrophobic core. Using multidimensional heteronuclear NMR, we have mapped intermolecular contacts between the human U1A protein 102 amino acid N-terminal RNP domain and a 31-mer oligonucleotide derived from stem-loop II of U1 snRNA. Chemical shift changes induced on the protein by the RNA define the surface of the beta-sheet as the recognition interface. The reverse face of the protein, with the two alpha-helices, remains exposed to the solvent in the presence of the RNA, and is potentially available for protein-protein contacts in spliceosome assembly or splice site selection. Protein-RNA contacts occur at the single-stranded apical loop of the hairpin, but also in the major groove of the helical stem at neighbouring U.G and U.U non-Watson-Crick base pairs. Examination of a proposed model for the complex in the light of the present results reveals several features of RNA recognition by RNP proteins. The quality of the spectra for this complex of 22 kDa demonstrates the feasibility of NMR investigation of RNA-protein complexes.

Amino Acid Sequence↗