Independent deposition of heterogeneous nuclear ribonucleoproteins and small nuclear ribonucleoprotein particles at sites of transcription.

The major nuclear ribonucleoproteins (RNPs) involved in pre-mRNA processing are classified in broad terms either as small nuclear RNPs (snRNPs), which are major participants in the splicing reaction, or heterogeneous nuclear RNPs (hnRNPs), which traditionally have been thought to function in general pre-mRNA packaging. We obtained antibodies that recognize these two classes of RNP in Drosophila melanogaster. Using a sequential immunostaining technique to compare directly the distribution of these RNPs on Drosophila polytene chromosomes, we found that the two patterns were very similar qualitatively but not quantitatively, arguing for the independent deposition of the two RNP types and supporting a role for hnRNP proteins, but not snRNPs, in general transcript packaging.

Analysis of an immunodominant epitope of topoisomerase I in patients with systemic sclerosis.

In this paper an immunodominant epitope of Topoisomerase I is described. An epitope expression sublibrary was constructed from Topoisomerase I cDNA. The subclones were screened with an antiserum from a patient with systemic sclerosis (SSc). The positive clones defined one immunodominant B cell epitope (epitope III), which was located at the carboxyterminal part of the protein. The epitope, 52 amino acids in length, neither contains the p30gag sequence nor the suggested active site Tyr-723, both presumed antibody recognition sites. More than 70% of our anti-TopoI sera recognize this epitope III, indicating that it is a major recognition site of the anti-TopoI autoantibodies in SSc sera. DNA relaxation experiments show that all sera that recognize epitope III and most sera with antibodies to other epitopes inhibit Topoisomerase I activity.

Zinc finger-like structure in U1-specific protein C is essential for specific binding to U1 snRNP.

The U1 small nuclear ribonucleoprotein (snRNP) contains three specific proteins denoted 70K, A and C, in addition to the common proteins. Specific functions of these proteins are not known although recently protein C was shown to be involved in the binding of U1 snRNP to the 5' splice site of a pre-mRNA. Unlike proteins A and 70K, U1-C lacks an RNA binding domain (RNP-80 motif) and does not appear to bind directly to U1 snRNA. However, at the amino terminal end protein C contains a zinc finger-like structure of the CC-HH type found in transcription factor TF IIIA. Several lines of evidence indicate that the zinc finger-like structure is essential for the binding of protein C to U1 snRNP particles: i) deletion analysis of protein C showed that the N-terminal 45 amino acids are sufficient for binding to U1 snRNPs, ii) modification of the cysteine residues in the N-terminal domain with N-ethylmaleimide and iii) single point mutations of the cysteines and histidines contributing to the putative zinc finger abolished binding of protein C to U1 snRNPs. Interestingly, unlike the proteins U1-A and U1-70K the U1-C protein is unable to bind to naked U1 snRNA. On the other hand it is shown that protein C does not bind to the known protein constituents of the U1 particle without the U1 snRNA being present. These data indicate that the binding of protein C to U1 snRNP is dependent on the presence of both the U1 snRNA and one or more of the U1 snRNP proteins.

Analysis of U1snRNP-specific A protein cross-linked complexes.

The organization of the U1snRNP-specific A protein (34 kDa) has been analyzed by 12 and 16 A thiol-reversible chemical cross-linking and Western blotting. A-containing cross-linked complexes had molecular masses of 43, 47, 56, 62, 67, 105 and 125 kDa. None of these complexes could be cross-linked following ribonuclease digestion, suggesting that UsnRNA may play important roles in the spatial organization of A and other proteins. Moreover, the data suggest that A is proximal to, and may have interactions with, UsnRNP-specific proteins C and 70 kDa as well as with UsnRNP-common proteins B, E and G.

Use of recombinant RNP peptides 70K and A in an ELISA for measurement of antibodies in mixed connective tissue disease: a longitudinal follow up of 18 patients.

In a three year prospective study disease activity variables and levels of antibody against the RNP-peptides 70K and A were measured in 18 patients with mixed connective tissue disease. Antibody measurement entailed use of cloned autoantigens in an enzyme linked immunosorbent assay (ELISA). Fluctuations in antibody levels against 70K and A were most commonly noted in patients who also had changes in disease activity, but these changes in serology and disease activity were synchronous in only a minority of the episodes. Even major disease flares were associated with changes in anti-A levels in only a few, and with changes in anti-70K levels in none of the episodes. The data indicate that measurements of anti-70K and anti-A levels are not useful in monitoring disease activity or response to treatment in mixed connective tissue disease, and suggest that these antibody specificities do not play a direct part in the pathogenesis of disease manifestations.

Epitope patterns of anti-RNP antibodies in rheumatic diseases. Evidence for an antigen-driven autoimmune response.

Autoantibodies against small nuclear ribonucleoproteins (snRNP) are common in systemic lupus erythematosus and related disorders. The 3 categories, anti-(U1)RNP, anti-(U1, U2)RNP, and anti-Sm, all contain a common antibody specificity directed against the U1 snRNP-associated A protein. To determine the specificity of anti-U1 snRNP A protein antibodies for various antigenic sites, we tested 26 different anti-snRNP-positive sera for reactivity with fragments of the U1 snRNP A protein, which was produced using recombinant DNA technology. Several different fragments were shown to contain autoimmune-reactive epitopes, which indicates that the antibody response against the U1 snRNP A protein is polyclonal. Antibodies against a discontinuous or conformational epitope were found in most of the sera tested, regardless of whether they were classified as anti-(U1)RNP, anti-Sm, or anti-(U1, U2) RNP. These results strongly support the hypothesis that the anti-snRNP autoimmune response is antigen driven.

A recombinant topoisomerase I used for autoantibody detection in sera from patients with systemic sclerosis.

We report the expression of a cDNA clone encoding 695 carboxyl-terminal amino acids of human DNA topoisomerase I (topoI) in Escherichia coli. More than 96% of the anti-HeLa topoI-positive sera from patients with a connective tissue disease displayed also an immunoreactivity with this recombinant protein (the HTopoA protein). Sera from patients with a definite diagnosis systemic sclerosis and reacting with HeLa topoI, all reacted with the HTopoA protein as well. Sera from patients with systemic sclerosis that did not contain anti-topoI antibodies (about 30% of the systemic sclerosis sera), as concluded from HeLa immunoblot, displayed also no immunoreactivity with our recombinant antigen. By expressing different fragments of HTopoA, we were able to assign at least three different autoimmune epitope regions on the HTopoA protein and we show that over a period of 5 years the amount of anti-topoI antibodies against these regions may fluctuate.

Mapping of B cell epitopes on small nuclear ribonucleoproteins that react with human autoantibodies as well as with experimentally-induced mouse monoclonal antibodies.

Small nuclear ribonucleoprotein (snRNP) particles are a class of RNA-containing particles in the nucleus of eukaryotic cells. They consist of uridylate-rich small nuclear RNA complexed with several proteins. snRNP particles U1, U2, U4/U6, and U5 all contain a common protein core consisting of proteins B'/B, D, D', E, F, and G. In addition to this core, U1 snRNP particles contain proteins 70K, A, and C, whereas U2 snRNP particles contain proteins A' and B". Almost any of the small nuclear RNA-associated polypeptides is targeted by autoantibodies in the sera from patients with SLE or related connective tissue diseases. We immunized a genetically non-autoimmune mouse with recombinant human B" protein and obtained three mAb reactive with native U2 snRNP particles. Two of these mAb particles cross-reacted with U1 snRNP, 9A9 and 11A1, via epitopes present on the U2 snRNP B" protein as well as on the U1 snRNP-specific A protein. A third mAb 4g3, reacted exclusively with U2 snRNP via a unique epitope on protein B". Two epitopes mapped at the carboxy-terminal region of the B" protein, whereas binding of the third mAb involved both amino- and carboxy-terminal amino acids of the B" protein. Epitope mapping, employing a DNAse I fragment library of the B" cDNA, revealed that the three mAb-reactive sites were discontinuous. Autoantibodies in sera from patients with SLE and other connective tissue diseases competed for binding with the mAb, implying that the mAb define a major autoantibody-reactive region on protein B".

Small nuclear RNA-associated proteins are immunologically related as revealed by mapping of autoimmune reactive B-cell epitopes.

Autoantibodies from a patient with systemic lupus erythematosus, which recognize U1 and U2 small nuclear ribonucleoprotein particles (snRNPs), were used to map B-cell autoepitopes on the U1 snRNP-specific A protein. This protein contains two regions that are highly similar to regions in the U2 snRNP-specific B" protein. A site termed epitope 2 maps in one such region and was found to react with antibodies cross-reactive between A and B". A second site, epitope 1, is situated in a proline-rich region that shows no homology with B". This epitope can bind three different autoantibodies with distinct specificities. Epitope 1-affinity-purified antibodies from different patients react with either (i) the A protein exclusively; (ii) proteins A, B'/B, a synthetic peptide for part of the N polypeptide, and an unidentified protein with a molecular mass of 50 kDa; or (iii) proteins A, B'/B, C, and the N-derived peptide. Comparison of the primary structures of proteins B'/B, N, and C reveals multiple epitope 1-like sequences in all of them. The possibility that these repeating regions act as immunogens in patients with autoimmune disease is discussed.

Marker antibodies in scleroderma and polymyositis: clinical associations.

Sera of 34 patients with progressive systemic sclerosis and of 11 patients with polymyositis/dermatomyositis (PM/DM) were analyzed by the immunoblotting technique for the presence of marker antibodies. The presence of anti-centromere, anti-Topoisomerase-I (anti-Topo-I) and anti-Jo-1 antibodies was found to be highly specific for the CREST syndrome, diffuse scleroderma and PM/DM, respectively, but only of limited sensitivity (78, 44 and 45%, respectively). Anti-Topo-I positive diffuse scleroderma patients had a more severe disease (digital pitting scars and renal insufficiency) than anti-Topo-I negative diffuse scleroderma patients. Anti-Jo-1 was associated with interstitial lung disease. Longitudinal studies showed a constant antibody pattern. Our results confirm the clinical usefulness of these marker antibodies.

Detection of autoantibodies in a quantitative immunoassay using recombinant ribonucleoprotein antigens.

A human cDNA expression library was screened with anti-ribonucleoprotein (RNP) antibodies from patients with connective tissue diseases. Three cDNA clones were isolated encoding 70 kD, A and B" ribonucleoprotein autoantigens which were expressed as beta-galactosidase fusion proteins. Antigens were purified and used to develop sensitive ELISAs suitable for the routine screening of large series of sera from patients with connective tissue diseases. More than 400 sera were tested both by ELISA and by immunoblotting. The ELISA was found to be at least as sensitive as immunoblotting and very specific. Anti-70 kD antibodies were found in 94% of patients with mixed connective tissue disease (MCTD), in 4% of patients with other connective tissue diseases but not in normal controls. Furthermore, the use of recombinant 70 kD antigen enabled us to discriminate between anti-70 kD antibodies present in anti-Sm and in anti-(U1) RNP sera. Recombinant A antigen contained at least two autoantibody-reactive sites; one unique for the A protein and another cross-reactive with anti-B" antibodies. Antibodies reactive with the unique site were found in 83% of MCTD patients, in 4% of patients with other connective tissue diseases and not in normal controls. Antibodies against the cross-reactive B" epitope present on A and B" recombinant antigens, were found in high titres in a small percentage of patients with systemic lupus erythematosus (SLE, 5%) and rheumatoid arthritis (RA, 2%).

Anti-56K: a novel, frequently occurring autoantibody specificity in connective tissue disease.

A novel frequently occurring autoantibody specificity in serum from connective tissue disease patients is described. The autoantibodies as detected by immunoblotting are directed against a 56,000 Dalton (56K) antigen, that after biochemical fractionation predominantly is found in the cytoplasmic fraction of various cell types and tissues. Attempts to localize the antigen more precisely were unsuccessful primarily because these antibodies do not produce a positive immunofluorescence pattern. The antigen in its native form is not associated with DNA or one of the common cytoplasmic or nuclear RNAs in the cell. Immunoprecipitation studies also showed that the protein is not closely associated with other proteins in a multi-component complex. Anti-56K antibodies are found in about 8% of patients with connective tissue disease, most commonly in patients with Sjögren's syndrome (14%). It is not found in patients with mixed connective tissue disease, polymyositis/dermatomyositis or scleroderma and rarely (less than 1%) in healthy control subjects. The fact that 22% of the anti-56K sera also contain La/SS-B antibodies support the idea that this antibody specificity might be characteristic for a subclass of Sjögren's syndrome patients.

Molecular cloning of the cDNA for the human U2 snRNA-specific A' protein.

The A' polypeptide is one of the protein constituents of the U2 snRNP particle. A potentially full-length cDNA clone containing the complete coding sequence for this U2 snRNP-specific protein was isolated by screening of a human lambda gt11 expression vector library with an autoimmune anti-(U1,U2)RNP serum. Monospecific antibodies, eluted from the 140-150 kD fusion protein of this cDNA recombinant, specifically recognized the A' protein on immunoblots and immunoprecipitated U2 snRNP particles from nuclear extracts. The identity of the clone was confirmed by in vitro translation of hybrid-selected mRNA or an RNA transcript synthesized from the cDNA insert. RNA blot analysis showed strong hybridization to a single polyadenylated transcript of 1.3 kb in human cells. The nucleotide sequence of the 1054 bp cDNA contains an open reading frame of 756 bp encoding a polypeptide of 255 amino acids with a predicted molecular weight of 28,444 D. The coding sequence is preceded by a 49 bp 5'-untranslated region and followed by a 226 bp 3'-untranslated region containing a single polyadenylation signal. Most striking feature of the deduced primary structure for the A' protein is a leucine-rich region in the amino-terminal half of the polypeptide. In contrast to the other U2 snRNP-specific protein B", the A' protein does not contain segments homologous to the RNP consensus sequences RNP1 and RNP2, common amino acid motifs found in several RNA-binding proteins. In the A' protein, however, the extremely hydrophilic carboxy terminus may constitute an RNA-binding moiety.

Human U1 snRNP-specific C protein: complete cDNA and protein sequence and identification of a multigene family in mammals.

A complementary DNA clone for the human U1 snRNP-specific C protein has been isolated. The nucleotide sequence of the 733 bp cDNA insert includes a 15 bp 5'-untranslated region, an open reading frame of 477 bp corresponding to 159 amino acids (Mr = 17,373 D), and a 223 bp 3'-untranslated region. The identity of the clone was confirmed by in vitro translation of hybrid-selected mRNA or an RNA transcript synthesized from the cDNA. The in vitro synthesized C protein has a slightly greater mobility on SDS-polyacrylamide gels, indicating that the in vivo product is post-translationally modified. The deduced primary structure contains a segment of high proline and methionine content. A region homologous to the RNP consensus sequence, found in the other two U1 snRNP-specific proteins 70K and A, is absent. Analysis of genomic DNA restriction enzyme digests shows hybridizing fragments in the genome of all vertebrate classes. The results are consistent with multi-copy representation of the C protein gene in mammals, whereas in the other vertebrate classes the related protein seems to be encoded by a single-copy gene.

The use of immunoblotting to detect antibodies to nuclear and cytoplasmic antigens. Clinical and serological associations in rheumatic diseases.

Using the immunoblotting technique, sera from 433 patients with rheumatic diseases were screened for the presence of antibodies against several nuclear and cytoplasmic antigens, such as RNP, Sm, Ro(SSA), La(SSB), CR-19 (centromeric antigen), Topo-1 (Scl-70), Jo-1, histone and 56 kD. At the same time clinical data from these patients were collected without prior knowledge of the immunoblotting results. Syndrome-specific autoantibodies were found for mixed connective tissue disease (antibodies against the RNP related 70 kD antigen), for CREST (anti-CR-19 antibodies), for diffuse scleroderma (anti-Topo-1 antibodies) and for polymyositis (anti-Jo-1 antibodies). Almost all specific autoantibodies were present exclusively in patients with a connective tissue disease. Controls were only in a few cases positive for antihistone and anti-56 kD antibodies. Associations of specific autoantibodies with clinical and laboratory features of the patients were mostly as expected. However, some unexpected associations were found, for example polymyositis and calcinosis with anti-Sm antibodies, sicca symptoms with anti-centromere antibodies and leucopenia with Ro(SSA) and La(SSB).

cDNA cloning of the human U1 snRNA-associated A protein: extensive homology between U1 and U2 snRNP-specific proteins.

Sera from patients with connective tissue diseases often contain antibodies against snRNA-associated proteins. Using one of these sera in an immunological screening of a human lambda gt11 expression vector cDNA library, two cDNA clones for the U1 snRNP-specific A protein, termed lambda HA-1 and lambda HA-2, were isolated. Monospecific antibodies, eluted from the beta-galactosidase fusion protein of either clone reacted with the U1 snRNP-specific A antigen. The identity of the clones was confirmed by in vitro translation of hybrid selected mRNA. RNA blot analysis revealed a single polyadenylated transcript of about 1.4 kb in human cells. A cDNA of 1.2 kb, isolated from the same lambda gt11 expression library by cross-hybridization with a lambda HA-2 restriction fragment, covered the complete coding sequence of the A protein as demonstrated by in vitro translation of an RNA transcript synthesized from this cDNA. The deduced amino acid sequence contains one very hydrophilic region, and internal sequence duplication and a region highly homologous to the RNP consensus sequence that seems to be common to RNA binding proteins. Sequence comparison with the recently cloned U2 snRNP-specific B" protein revealed two extremely homologous regions located in the carboxy-terminal (homology of 86%) and amino-terminal part (homology of 77%) of the proteins. This structural relationship indicates that proteins A and B", although located in different snRNP particles, may have identical functions.

Analysis of a cDNA clone expressing a human autoimmune antigen: full-length sequence of the U2 small nuclear RNA-associated B" antigen.

A U2 small nuclear RNA-associated protein, designated B'', was recently identified as the target antigen for autoimmune sera from certain patients with systemic lupus erythematosus and other rheumatic diseases. Such antibodies enabled us to isolate cDNA clone lambda HB''-1 from a phage lambda gt11 expression library. This clone appeared to code for the B'' protein as established by in vitro translation of hybrid-selected mRNA. The identity of clone lambda HB''-1 was further confirmed by partial peptide mapping and analysis of the reactivity of the recombinant antigen with monospecific and monoclonal antibodies. Analysis of the nucleotide sequence of the 1015-base-pair cDNA insert of clone lambda HB''-1 revealed a large open reading frame of 800 nucleotides containing the coding sequence for a polypeptide of 25,457 daltons. In vitro transcription of the lambda HB''-1 cDNA insert and subsequent translation resulted in a protein product with the molecular size of the B'' protein. These data demonstrate that clone lambda HB''-1 contains the complete coding sequence of this antigen. The deduced polypeptide sequence contains three very hydrophilic regions that might constitute RNA binding sites and/or antigenic determinants. These findings might have implications both for the understanding of the pathogenesis of rheumatic diseases as well as for the elucidation of the biological function of autoimmune antigens.

Monospecific antibodies reveal details of U2 snRNP structure and interaction between U1 and U2 snRNPs.

Monospecific antibodies directed against several U small nuclear ribonucleoprotein (U snRNP) particle proteins were affinity purified from a patient's anti-(U1,U2)RNP serum. These were used to demonstrate that: (i) proteins equivalent to the mammalian U2 snRNP-specific A' and B" proteins are present in Xenopus laevis oocytes; (ii) both proteins A' and B" have the same structural requirements for binding to U2 snRNA; (iii) proteins B, B' and D have the same structural requirement for binding to U2 snRNA; (iv) using very high specific activity RNA probes it is possible to detect a fraction of either U1 or U2 snRNA precipitable by antibodies directed against proteins specific for the other U snRNP, indicating an interaction between U1 and U2 snRNPs. The structural requirements of this interaction were studied for the U2 snRNP. All changes made to U2 snRNA or snRNP structure resulted in loss of the interaction with U1 snRNP.

Further characterization and subcellular localization of Sm and U1 ribonucleoprotein antigens.

Sera from patients with systemic autoimmune diseases often contain antibodies against small nuclear ribonucleoprotein (snRNP) particles. Anti-Sm antibodies react with the entire set of U1, U2, U4, U5 and U6 (U1-U6) RNP particles whereas anti-(U1)RNP sera specifically recognize particles containing U1 RNA. Here we performed semi-quantitative immunoblotting using 16 human anti-Sm, 15 human anti-(U1)RNP sera and two mouse monoclonal antibodies to establish which snRNA-associated proteins carry antigenic determinants. Almost every (15/16) human anti-Sm sera recognized epitopes present on a 28-kDa (B/B') protein doublet and on a 16-kDa (D) polypeptide. Nine anti-(U1)RNP sera also recognized the B/B' doublet, but in all cases a much stronger reaction was observed with one or more of the specifically U1 RNA-associated 70 kDa, A or C antigens. With affinity-purified antibody fractions eluted from individual antigen bands on nitrocellulose blots it is shown that the anti-Sm-reactive polypeptides B/B' and D contain common epitopes. We also report the finding of one human anti-Sm serum with exclusive specificity for the B/B' doublet and a mouse monoclonal anti-Sm antibody recognizing only the D protein, indicating that these antigens also carry unique epitopes. In immunoprecipitation assays, purified anti-B/B' and -D antibodies react with (U1-U6) RNP while purified anti-70 kDa, anti-A and anti-C antibodies precipitate exclusively U1 RNP particles. Finally, we established the subcellular localization of Sm and U1 RNP antigens using a biochemical cell fractionation procedure. Part of the 70 kDa and B/B' antigens were found in a nuclease and high salt-resistant nuclear substructure, usually referred to as nuclear matrix, while the A and D antigens could be extracted completely from HeLa nuclei by ribonuclease treatment and subsequent high salt extraction.