The PPGMRPP repetitive epitope of the Sm autoantigen: Antigenic specificity induced by conformational changes. Application of the Sequential Oligopeptide Carriers (SOCs)

Summary The PPGMRPP sequence, found in several copies in the Sm and U1RNP autoantigens, is the main target of anti-Sm and anti-U1RNP antibodies in systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD) patient's sera. It is also recognized, to a lower extent, by anti-Ro/SSA and anti-La/SSB specificities. The PPGMRPP-NH₂ peptide amide and the PPGMRPP peptide, which is bound to a pentameric sequential oligopeptide carrier (SOC₅), were examined by¹H-NMR spectroscopy and ELISA assays, using sera from patients with autimmune rheumatic diseases. Among the three main conformers found for the free PPGMRPP, the extended one was also identified for PPGMRPP-NH₂ and (PPGMRPP)₅-SOC₅. This can be attributed to the absence of ionic interactions between the Arg-guanidinium and the carboxylate group in the amide and SOC₅-bound forms of the peptide. Immunoassays using sera from various specificities showed an enhanced anti-Sm and anti-U1RNP recognition of PPGMRPP-NH₂ and (PPGMRPP)₅-SOC₅, and lowering of the anti-Ro/SSA and anti-La/SSB reactivity. The presence of multiple conformers of free PPGMRPP may explain the unexpected cross-reactivity to the anti-Ro/La positive sera, while the prevalence of the extended conformation in PPGMRPP-NH₂ and (PPGMRPP)₅-SOC₅ is mainly responsible for the enhanced recognition from the anti-Sm and anti-U1RNP autoantibodies. it is concluded that the antigenic specificity of PPGMRPP-NH₂ and (PPGMRPP)₅-SOC₅ is mainly induced by conformational changes resulting from the conversion of the C-terminal carboxylate group to the amide form.     

The PPGMRPP repetitive epitope of the Sm autoantigen: Antigenic specificity induced by conformational changes. Application of the Sequential Oligopeptide Carriers (SOCs)

The PPGMRPP sequence, found in several copies in the Sm and U1RNPautoantigens, is the main target of anti-Sm and anti-U1RNP antibodies insystemic lupus erythematosus (SLE) and mixed connective tissue disease(MCTD) patient's sera. It is also recognized, to a lower extent, byanti-Ro/SSA and anti-La/SSB specificities. The PPGMRPP-NH₂peptide amide and the PPGMRPP peptide, which is bound to a pentamericsequential oligopeptide carrier (SOC₅), were examined by¹H-NMR spectroscopy and ELISA assays, using sera from patientswith autoimmune rheumatic diseases. Among the three main conformers foundfor the free PPGMRPP, the extended one was also identified for PPGMRPP-NH₂ and (PPGMRPP)₅-SOC₅.This can be attributed to the absence of ionic interactions between theArg-guanidinium and the carboxylate group in the amide andSOC₅-bound forms of the peptide. Immunoassays using sera fromvarious specificities showed an enhanced anti-Sm and anti-U1RNP recognitionof PPGMRPP-NH₂ and(PPGMRPP)₅-SOC₅, and lowering of the anti-Ro/SSAand anti-La/SSB reactivity. The presence of multiple conformers of freePPGMRPP may explain the unexpected cross-reactivity to the anti-Ro/Lapositive sera, while the prevalence of the extended conformation inPPGMRPP-NH₂ and (PPGMRPP)₅-SOC₅is mainly responsible for the enhanced recognition from the anti-Sm andanti-U1RNP autoantibodies. It is concluded that the antigenic specificity ofPPGMRPP-NH₂ and (PPGMRPP)₅-SOC₅ ismainly induced by conformational changes resulting from the conversion ofthe C-terminal carboxylate group to the amide form.     

The position of the LysN epsilon H2-grafted antigens along the sequential oligopeptide carrier, Ac-(Aib-Lys-Aib-Gly)n (SOCn-II), influences the antibody recognition: application to the Sm main autoimmune epitope

A sequential oligopeptide carrier of antigenic peptides is presented, incorporating two Aib residues in each repetitive moiety: Ac-(Aib-Lys-Aib-Gly)(n) (SOC(n) -II; n = 2-4). The conformational study, by (1)H-nmr, CD, and Fourier transform ir spectroscopy, indicated that the SOC(n) -II carrier displays a pronounced 3(10)-helix, compared to the Ac-(Lys-Aib-Gly)(n) (SOC(n) -I) carrier of the same approximately backbone length, previously reported. One of the dominant autoimmune epitopes of the Sm and U1RNP cellular components, the PPGMRPP sequence, was coupled to the Lys-N(epsilon)H(2) groups of the SOC(n) -II carrier and used as antigenic substrate for detecting anti-Sm/U1RNP autoantibodies in ELISA assays. Anti-Sm antibodies are highly specific for systemic lupus erythematosus, while anti-U1RNP are specific for mixed connective tissue disease. The anti-(PPGMRPP)(5)-SOC(n) -II ELISA was compared with the anti-(PPGMRPP)(n) -SOC(n) -I ELISA, provided that both antigenic substrates possess the same amount of the epitope replicates. The significance of the lysine positions along the oligopeptide backbone of the carrier for a favorable antibody recognition of the anchored antigens is also examined.     

Differentiation of RNP- and SM-antibody subsets in SLE and MCTD patients by a new ELISA using recombinant antigens.

Connective tissue diseases often have overlapping clinical features and laboratory abnormalities. The distinctiveness of mixed connective tissue disease (MCTD) as an entity is of scientific interest and practical importance. In order to discriminate between MCTD and SLE patients we used a newly developed, commercially not available ELISA with recombinant antigen expressed in Baculovirus infected cells. This ELISA detects antibodies against RNP and Sm in complex as well as the subsets U1-snRNP 68 kDa, RNP-A, RNP-C (RNP), Sm-BB' and SS-D. We analyzed 66 RNP-positive consecutive patients prediagnosed as SLE or MCTD/overlap-syndrome. 45/66 patients were found to be U1-snRNP-68 kDa positive (27 SLE, 18 MCTD), 51/66 RNP-A [36,15] and 44/66 RNP-C [31,13]. 35/66 had antibodies against Sm-BB' (30 SLE, 5 MCTD), 10/66 against Sm-D (all SLE). 28/66 were found to be U1-snRNP-68 kDa and Sm-BB' positive (23 SLE, 5 MCTD), while 8/66 where U1-snRNP-68 kDa and Sm-D positive (all SLE). The combination of antibodies against 68 kDa, Aand C was exclusively observed in 6 MCTD patients, while the combination against 68 kDa, A, C, Sm-BB' and Sm-D was restricted to 8 patients with SLE. The antibody combination to 68 kDa, A, C and Sm-BB' was also found in 11/20 SLE patients with major organ involvement. In SLE and MCTD, determination of subsets of antibodies against Ul-snRNP-68 kDa and Sm-complex allows a differentiation of patient subgroups with more definite diagnoses and potential prognostic impact.     

Clinical and immunological aspects of autoantibodies to RA33/hnRNP-A/B proteins — A link between RA,SLE and MCTD

Heterogeneous nuclear ribonucleoprotein (hnRNP) complexes are major constituents of the spliceosome. They are composed of approximately 30 different proteins which can bind to nascent pre-mRNA. Among these, the hnRNP-A/B proteins form a subgroup of highly related proteins consisting of two adjacent RNA binding domains (RBD) within the N-terminal parts, whereas the C-terminal halves contain almost 50% glycine residues. These proteins, in particular A2/RA33, are targeted by autoantibodies from patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and mixed connective tissue disease (MCTD). In SLE anti-hnRNP antibodies frequently occur together with antibodies to U1 small nuclear RNP (U1-snRNP) and Sm, other proteins of the spliceosome. Preliminary epitope mapping studies have revealed major antibody binding sites in the RNA binding regions for all three diseases. Nevertheless, there is some indication of disease specific epitope recognition. Studies in animal models have demonstrated anti-RA33/hnRNP-A/B antibodies in lupus-prone mouse strains.Thus, autoantibodies to the spliceosomal hnRNP-A/B proteins are a common feature of RA, SLE, and MCTD. However, these diseases differ in their reactivities to other spliceosomal proteins, especially anti-U1 snRNP and Sm. Therefore, anti-RA33/hnRNP-A/B autoantibodies are not only valuable diagnostic markers but may also allow additional insights into the pathogenesis of rheumatic autoimmune diseases.Abbreviations AS ankylosing spondylitis - hnRNP heterogeneous nuclear ribonucleoprotein - MCTD mixed connective tissue disease - PSA psoriatic arthropathy - RA rheumatoid arthritis - RBD RNA binding domain - SLE systemic lupus erythematosus - snRNP small nuclear ribonucleoprotein     

Purification and characterization of human autoantibodies directed to specific regions on U1RNA; recognition of native U1RNP complexes.

Antibodies against naked U1RNA can be found in sera from patients with overlap syndromes of systemic lupus erythematosus (SLE) in addition to antibodies directed to the proteins of U1 ribonucleoproteins (U1RNP). We investigated the reactivity of these U1RNA specific autoantibodies with the native U1RNP particle both in vitro and inside the cell. For this purpose a method was developed to purify human autoantibodies directed to specific regions of U1RNA. The antibodies are specifically directed to either stemloop II or stemloop IV of U1RNA and do not crossreact with protein components of U1RNP. Both types of antibody are able to precipitate from cell extracts native U1snRNPs containing most, if not all, protein components. Immunofluorescence patterns indicate that the antigenic sites on the RNA, i.e. the stem of stemloop II and the loop of stemloop IV, are also available after fixation of the cells. Immunoelectron microscopy employing anti-stemloop IV antibodies and purified, complete U1snRNP particles showed that stemloop IV is located within the body of the U1RNP complex, which also comprises the Sm site and the common Sm proteins. The anti-U1RNA autoantibodies described in this paper recognize native U1RNP particles within the cell and can therefore be used as tools to study mechanisms involved in splicing of pre-mRNA.     

Structure of the small nuclear RNP particle U1: identification of the two structural protuberances with RNP-antigens A and 70K

We have investigated the structure of the small nuclear RNP (snRNP) U1 by combining EM of complete and partially protein-deficient particles with immunoelectron microscopy employing mAbs against known components of the U1 snRNP. It was found that the two main protuberances of this particle can be identified with the U1-specific proteins A and 70K. The 70K protuberance is the one lying closer to the 5' terminus of the snRNA, as identified by its 5'-terminal m3G cap. The round-shaped main body of U1 snRNP represents its core RNP domain containing the common snRNP proteins. Functional implications of these results are discussed. Our results may also point to the physical basis for the production of autoantibodies directed against specific groups of snRNP proteins. The physical grouping of the common proteins (Sm epitopes) and the specific proteins (RNP epitopes) could result in one or the other being presented to the immune system as is the case in patients suffering from SLE or MCTD, respectively.     

Murine graft vs host disease. A model for study of mechanisms that generate autoantibodies to ribonucleoproteins

We established chronic graft vs host disease in (BALB/c x A/J) F1 mice with the injection of lymphoid cells from the parental A/J strain. These animals developed glomerulonephritis, forefoot edema, alopecia, splenomegaly, and lymphadenopathy to various degrees, and all developed antinuclear antibodies. To determine whether these antibodies were directed against the small nuclear ribonucleoprotein (snRNP) particles that are characteristic targets for autoimmune responses in human rheumatic diseases, sera were studied in the 32P immunoprecipitation and immunoblotting assays. Among 20 mice, antibodies to snRNP developed in 10. These antibodies usually reached maximal levels about 4 wk after induction of graft vs host disease and generally fell thereafter. However, two mice developed antibodies to snRNP between the 10th and 20th wk of follow-up. Sera from six mice strongly recognized the U1 snRNP and an additional serum strongly bound both the U1 and U3 particles. Several sera contained lower levels of antibodies specific for the U3 and possibly pre-U2 snRNP particles. In immunoblots, sera that immunoprecipitated the U1 snRNP bound epitopes located on its 70,000 Da, A, B'/B, and/or C polypeptides. Sera that immunoprecipitated the U3 snRNP recognized a 34,000-Da polypeptide. These polypeptides are known to bear the autoantigenic epitopes that are recognized by human sera containing anti-U1 RNP and anti-U3 RNP autoantibodies. We conclude that chronic graft vs host disease in mice provides a model for the study of the autoimmune responses that characterize human diseases such as mixed connective tissue disease, scleroderma, and SLE.     

High prevalence of autoantibodies to RNA helicase A in Mexican patients with systemic lupus erythematosus

Introduction

Autoantibodies to RNA helicase A (RHA) were reported as a new serological marker of systemic lupus erythematosus (SLE) associated with early stage of the disease. Anti-RHA and other autoantibodies in Mexican SLE patients and their correlation with clinical and immunological features were examined.

Methods

Autoantibodies in sera from 62 Mexican SLE patients were tested by immunoprecipitation of ³⁵S-labeled K562 cell extract and enzyme-linked immunosorbent assay (anti-U1RNP/Sm, ribosomal P, β2GPI, and dsDNA). Anti-RHA was screened based on the immunoprecipitation of the 140-kDa protein, the identity of which was verified by Western blot using rabbit anti-RHA serum. Clinical and immunological characteristics of anti-RHA-positive patients were analyzed.

Results

Anti-RHA was detected in 23% (14/62) of patients, a prevalence higher than that of anti-Sm (13%, 8/62). Prevalence and levels of various autoantibodies were not clearly different between anti-RHA (+) vs. (-) cases, although there was a trend of higher levels of anti-RHA antibodies in patients without anti-U1RNP/Sm (P = 0.07). Both anti-RHA and -Sm were common in cases within one year of diagnosis; however, the prevalence and levels of anti-RHA in patients years after diagnosis did not reduce dramatically, unlike a previous report in American patients. This suggests that the high prevalence of anti-RHA in Mexican patients may be due to relatively stable production of anti-RHA.

Conclusions

Anti-RHA was detected at high prevalence in Mexican SLE patients. Detection of anti-RHA in races in which anti-Sm is not common should be clinically useful. Racial difference in the clinical significance of anti-RHA should be clarified in future studies.     

A murine monoclonal antibody recognizes the 13,000 molecular weight polypeptide of the Sm small nuclear ribonucleoprotein complex

Antibodies to the Sm antigen are closely associated with the rheumatic disease systemic lupus erythematosus (SLE). The Sm antigen exists in the cell as part of a ribonucleoprotein complex containing at least 10 polypeptides and five small nuclear RNA. The major immunoreactive Sm species are three polypeptides of m.w. 27,000, 26,000, and 13,000. By using an MRL/1 mouse, a strain which spontaneously produces a disease with many of the characteristics of human SLE, we have produced an anti-Sm hybridoma specific for the 13,000 m.w. Sm polypeptide. This monoclonal antibody is sufficient to allow for the rapid bulk isolation of the entire class of Sm snRNP, and can be used sequentially with an anti-(U1)RNP monoclonal antibody to subfractionate the Sm snRNP particles.     

snRNP Sm proteins share two evolutionarily conserved sequence motifs which are involved in Sm protein-protein interactions.

The spliceosomal small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/U6 and U5 share eight proteins B', B, D1, D2, D3, E, F and G which form the structural core of the snRNPs. This class of common proteins plays an essential role in the biogenesis of the snRNPs. In addition, these proteins represent the major targets for the so-called anti-Sm auto-antibodies which are diagnostic for systemic lupus erythematosus (SLE). We have characterized the proteins F and G from HeLa cells by cDNA cloning, and, thus, all human Sm protein sequences are now available for comparison. Similar to the D, B/B' and E proteins, the F and G proteins do not possess any of the known RNA binding motifs, suggesting that other types of RNA-protein interactions occur in the snRNP core. Strikingly, the eight human Sm proteins possess mutual homology in two regions, 32 and 14 amino acids long, that we term Sm motifs 1 and 2. The Sm motifs are evolutionarily highly conserved in all of the putative homologues of the human Sm proteins identified in the data base. These results suggest that the Sm proteins may have arisen from a single common ancestor. Several hypothetical proteins, mainly of plant origin, that clearly contain the conserved Sm motifs but exhibit only comparatively low overall homology to one of the human Sm proteins, were identified in the data base. This suggests that the Sm motifs may also be shared by non-spliceosomal proteins. Further, we provide experimental evidence that the Sm motifs are involved, at least in part, in Sm protein-protein interactions. Specifically, we show by co-immunoprecipitation analyses of in vitro translated B' and D3 that the Sm motifs are essential for complex formation between B' and D3. Our finding that the Sm proteins share conserved sequence motifs may help to explain the frequent occurrence in patient sera of anti-Sm antibodies that cross-react with multiple Sm proteins and may ultimately further our understanding of how the snRNPs act as auto-antigens and immunogens in SLE.     

The structure of mammalian small nuclear ribonucleoproteins. Identification of multiple protein components reactive with anti-(U1)ribonucleoprotein and anti-Sm autoantibodies

The Sm small nuclear ribonucleoproteins (snRNPs) from mammalian cells have been characterized as containing U1, U2, U4, U5, and U6 RNA associated with some subset of at least 10 distinct polypeptides (called 68K, A, A', B, B', C, D, E, F, and G) that range in molecular weight from 68,000 to 11,000. Whereas this entire collection of snRNP particles is precipitated by patient anti-Sm autoantibodies, anti-(U1)RNP autoantibodies specifically recognize U1 snRNPs. Here, we have performed immunoblots using the sera from 29 patients and a mouse anti-Sm monoclonal antibody to identify which HeLa cell snRNP proteins carry anti-Sm or anti-(U1)RNP antigenic determinants. Strikingly, every serum surveyed, as well as the monoclonal antibody, recognizes determinants on two or more snRNP protein components. The three proteins, 68K, A, and C, that uniquely fractionate with U1 snRNPs are specifically reactive with anti-(U1)RNP sera in blots. Anti-Sm patient sera and the mouse monoclonal antibody react with proteins B, B', D, and sometimes E, one or more of which must be present on all Sm snRNPs. The blot results combined with data obtained from a refined 32P-labeled RNA immunoprecipitation assay reveal that, in our collection of the sera from 29 patients, anti-Sm rarely exists in the absence of equal or higher titers of anti-(U1)RNP; moreover, (U1)RNP sera often contain detectable levels of anti-Sm. Our findings further define the protein composition of the Sm snRNPs and raise intriguing questions concerning the relatedness of snRNP polypeptides and the mechanism of autoantibody induction.     

Identification of a SmD3 epitope with a single symmetrical dimethylation of an arginine residue as a specific target of a subpopulation of anti-Sm antibodies

Anti-Sm antibodies, identified in 1966 by Tan and Kunkel, are highly specific serological markers for systemic lupus erythrematosus (SLE). Anti-Sm reactivity is found in 5–30% of SLE patients, depending on the autoantibody detection system and the racial background of the SLE population. The Sm autoantigen complex comprises at least nine different polypeptides. All of these core proteins can serve as targets of the anti-Sm B-cell response, but most frequently the B and D polypeptides are involved. Because the BB'Sm proteins share cross-reactive epitopes (PPPGMRPP) with U1 specific ribonucleoproteins, which are more frequently targeted by antibodies that are present in patients with mixed connective tissue disease, the SmD polypeptides are regarded as the Sm autoantigens that are most specific to SLE. It was recently shown that the polypeptides D1, D3 and BB' contain symmetrical dimethylarginine, which is a component of a major autoepitope within the carboxyl-terminus of SmD1. In one of those studies, a synthetic dimethylated peptide of SmD1 (amino acids 95–119) exhibited significantly increased immunoreactivity as compared with unmodified SmD1 peptide. Using immobilized peptides, we confirmed that the dimethylated arginine residues play an essential role in the formation of major SmD1 and SmD3 autoepitopes. Moreover, we demonstrated that one particular peptide of SmD3 represents a more sensitive and more reliable substrate for the detection of a subclass of anti-Sm antibodies. Twenty-eight out of 176 (15.9%) SLE patients but only one out of 449 (0.2%) control individuals tested positive for the anti-SmD3 peptide (SMP) antibodies in a new ELISA system. These data indicate that anti-SMP antibodies are exclusively present in sera from SLE patients. Thus, anti-SMP detection using ELISA represents a new serological marker with which to diagnose and discriminate between systemic autoimmune disorders.     

Identification of a SmD3 epitope with a single symmetrical dimethylation of an arginine residue as a specific target of a subpopulation of anti-Sm antibodies

Anti-Sm antibodies, identified in 1966 by Tan and Kunkel, are highly specific serological markers for systemic lupus erythrematosus (SLE). Anti-Sm reactivity is found in 5-30% of SLE patients, depending on the autoantibody detection system and the racial background of the SLE population. The Sm autoantigen complex comprises at least nine different polypeptides. All of these core proteins can serve as targets of the anti-Sm B-cell response, but most frequently the B and D polypeptides are involved. Because the BB'Sm proteins share cross-reactive epitopes (PPPGMRPP) with U1 specific ribonucleoproteins, which are more frequently targeted by antibodies that are present in patients with mixed connective tissue disease, the SmD polypeptides are regarded as the Sm autoantigens that are most specific to SLE. It was recently shown that the polypeptides D1, D3 and BB' contain symmetrical dimethylarginine, which is a component of a major autoepitope within the carboxyl-terminus of SmD1. In one of those studies, a synthetic dimethylated peptide of SmD1 (amino acids 95-119) exhibited significantly increased immunoreactivity as compared with unmodified SmD1 peptide. Using immobilized peptides, we confirmed that the dimethylated arginine residues play an essential role in the formation of major SmD1 and SmD3 autoepitopes. Moreover, we demonstrated that one particular peptide of SmD3 represents a more sensitive and more reliable substrate for the detection of a subclass of anti-Sm antibodies. Twenty-eight out of 176 (15.9%) SLE patients but only one out of 449 (0.2%) control individuals tested positive for the anti-SmD3 peptide (SMP) antibodies in a new ELISA system. These data indicate that anti-SMP antibodies are exclusively present in sera from SLE patients. Thus, anti-SMP detection using ELISA represents a new serological marker with which to diagnose and discriminate between systemic autoimmune disorders.     

Antiribonucleoprotein antibodies in connective tissue diseases: estimation by counterimmunoelectrophoresis.

One hundred and seventy-two patients with various connective tissue diseases were investigated for the presence of serum antibodies to extractable nuclear antigen (ENA) and its major components, ribonucleo-protein (RNP) and Sm antigen. The counter-immunoelectrophoresis assay allowed independent detection and measurement of antibodies to the different components. All 13 patients with mixed connective tissue disease (MCTD) had anti-RNP antibody in high titres, 16% of patients with systemic lupus erythematosus (SLE) had low titres, and none of the patients with scleroderma had anti-RNP antibody. MCTD seems to be more benign than either SLE or scleroderma. The counterimmunoelectrophoresis assay is a simple and sensitive technique for confirming the diagnosis.     

Autoimmune response to U1 small nuclear ribonucleoprotein (U1 snRNP) associated with cytomegalovirus infection

The induction of autoantibodies to U1 small nuclear ribonucleoprotein (U1 snRNP) complexes is not well understood. We present evidence that healthy individuals with cytomegalovirus (CMV) infection have an increased frequency and quantity of antibodies to ribonucleoprotein, directed primarily against the U1-70k protein. A significant association between the presence of antibodies to CMV and antibodies to the total RNP targeted by the immune response to the spliceosome (to both the Sm and RNP; Sm/RNP) was found for patients with systemic lupus erythematosus (SLE) but not those with mixed connective-tissue disease. CMV thus may play a role in inducing autoimmune responses in a subset of patients with systemic lupus erythematosus.     

Intranuclear localization of a new snRNP-related antigen.

The intranuclear distribution of a new antigen (F78) associated with U snRNPs (small nuclear RNA-protein complexes) was compared with that of the RNP and Sm protein antigens previously identified on individual snRNP particles. Human and rat cells were double stained with human autoantisera and mouse monoclonal antibodies. The binding of the human and mouse antibodies was detected with secondary antibodies conjugated with fluorescein and rhodamine, respectively. The resulting immunofluorescence patterns were compared by digital image analysis. The F78, RNP, and Sm antigens show speckled fluorescence patterns which overlap to a great extent. The F78 pattern, however, also contains two classes of structural elements not present in the RNP pattern. Furthermore, during mitosis expression of the F78 antigen is completely suppressed from early prophase to telophase, while the RNP and Sm antigens are found evenly distributed throughout the cytoplasm of the dividing cells.     

Mapping of multiple B cell epitopes on the 70-kilodalton autoantigen of the U1 ribonucleoprotein complex.

High titer IgG autoantibodies to the 70-kDa polypeptide component (p70) of the U1 ribonucleoprotein (RNP) complex occur in the sera of patients with mixed connective tissue disease, SLE, and related rheumatic diseases. To gain insight into the pathogenesis and diversity of this antibody response we have used recombinant DNA technology to map the linear B cell epitopes on p70. A full length 1.7-kb cDNA clone encoding p70 was isolated from a human placental library and restriction fragments or polymerase chain reaction-generated fragments of the gene subcloned into the bacterial expression vector pGEX. Purified fusion proteins representing specific regions of p70 were immunoblotted with a panel of 70 anti-(U1)RNP+ sera containing anti-p70 antibodies. Six epitopes, four major (A, B, C, and F) and two minor (D and E) were mapped and were located throughout the molecule. The anti-(U1)RNP sera displayed heterogeneity in their pattern of reactivity to the six epitopes although reactivity to epitope C was more frequently associated with SLE rather than mixed connective tissue disease. The identification of multiple B cell epitopes on p70 is consistent with the concept that this self Ag drives the autoantibody response.     

Spliceosomal U snRNP core assembly: Sm proteins assemble onto an Sm site RNA nonanucleotide in a specific and thermodynamically stable manner.

The association of Sm proteins with U small nuclear RNA (snRNA) requires the single-stranded Sm site (PuAU(4-6)GPu) but also is influenced by nonconserved flanking RNA structural elements. Here we demonstrate that a nonameric Sm site RNA oligonucleotide sufficed for sequence-specific assembly of a minimal core ribonucleoprotein (RNP), which contained all seven Sm proteins. The minimal core RNP displayed several conserved biochemical features of native U snRNP core particles, including a similar morphology in electron micrographs. This minimal system allowed us to study in detail the RNA requirements for Sm protein-Sm site interactions as well as the kinetics of core RNP assembly. In addition to the uridine bases, the 2' hydroxyl moieties were important for stable RNP formation, indicating that both the sugar backbone and the bases are intimately involved in RNA-protein interactions. Moreover, our data imply that an initial phase of core RNP assembly is mediated by a high affinity of the Sm proteins for the single-stranded uridine tract but that the presence of the conserved adenosine (PuAU.) is essential to commit the RNP particle to thermodynamic stability. Comparison of intact U4 and U5 snRNAs with the Sm site oligonucleotide in core RNP assembly revealed that the regions flanking the Sm site within the U snRNAs facilitate the kinetics of core RNP assembly by increasing the rate of Sm protein association and by decreasing the activation energy.