Detection of intranuclear clusters of Sm antigens with monoclonal anti-Sm antibodies by immunoelectron microscopy

It has been shown that small nuclear RNA (snRNA) species U1, U2, U4, U5, and U6 are found in the nucleus in the form of small nuclear ribonucleoprotein particles (snRNPs), and that anti-Sm antibodies react with snRNP polypeptides, which are associated with all five snRNAs. We report here a novel intranuclear complex, denoted “Sm cluster,” detected by immunostaining with monoclonal anti-Sm antibodies in HeLa cells.     

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.     

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.     

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.     

Autoantibodies to the U2 small nuclear ribonucleoprotein in a patient with scleroderma-polymyositis overlap syndrome

Autoantibodies directed against the U2 small nuclear ribonucleoprotein (snRNP) have been found in the serum of a patient with scleroderma-polymyositis overlap syndrome. This specificity, called anti-(U2)-RNP, is distinct from all previously described autoantibodies, including those that precipitate related snRNPs: anti-Sm antibodies, which react with the entire set of U1, U2, U4, U5, and U6 snRNPs, and anti-(U1)RNP antibodies, which recognize only U1 snRNPs. From HeLa cell extracts, anti-(U2)RNP immunoprecipitates predominantly one 32P-labeled RNA species, identified as U2 small nuclear RNA, and six [35S]methionine-labeled protein bands, A' (Mr = 32,000), B (Mr = 28,000), D (Mr = 16,000), E (Mr = 13,000), F (Mr = 12,000), and G (Mr = 11,000). Protein blot analysis reveals that the A' protein carries (U2)RNP antigenic determinant(s) and therefore represents a polypeptide unique to the U2 snRNP; the B protein associated with U2 snRNPs may also be unique. Like U1 and the other Sm snRNPs, U2 snRNPs occupy a nuclear, non-nucleolar location and are antigenically conserved from insects to man. An antibody specific for the U2 snRNP will be useful in deciphering the function of this particle.     

Characterization of U small nuclear RNA-associated proteins

Differential immunoaffinity chromatography using a combination of autoimmune antibodies allows for the rapid bulk separation of specific small nuclear ribonucleoproteins (snRNPs). Passage of a HeLa cell extract over a column constructed of human anti-Sm autoantibodies results directly in the elution of complexes containing the small nuclear RNA species, U1, U2, U4, U5, and U6, and nine major polypeptides of molecular weight 69,000, 32,000, 27,000, 26,000, 18,500, 13,000, 11,000 doublet, and less than 10,000. Passage of crude extracts through a column bearing murine monoclonal antibodies directed against the 69,000 molecular weight (U1)RNP peptide gives an enriched population of U1 snRNP particles in the retained material. When the flowthrough material from the (U1)RNP column is passed through an anti-Sm column, the retained material is enriched in U2, U4, U5 plus U6 snRNP complex. The 69,000, 32,000, and 18,500 molecular weight polypeptides are confined to the U1 fraction while the remaining proteins are recovered in both fractions. The procedure is simple and rapid, producing complexes with a high degree of resolution and in sufficient yield to provide a ready source of snRNP complexes for functional studies.     

5′-terminal caps of snRNAs are reactive with antibodies specific for 2,2,7-trimethylguanosine in whole cells and nuclear matrices : Double-label immunofluorescent studies with anti-m3G antibodies and with anti-RNP and anti-Sm autoantibodies

Antibodies specific for 2,2,7-trimethylguanosine (m3G), which do not cross-react with m7G-capped RNA molecules were used to study, by immunofluorescence microscopy, the reactivity of the m3G-containing cap structures of the snRNAs U1 to U5 in situ. In interphase cells, immunofluorescent sites were restricted to the nucleus, whilst nucleoli were free of fluorescence. This indicates that the 5' terminal of most of the nucleoplasmic snRNAs are not protected by an m3G cap-recognizing protein and that the snRNA caps are not necessarily required for the binding of snRNPs to subnuclear structures. The snRNAs in the nucleoplasm appeared as distinct units in the light microscope, and this allowed the comparison of the distribution of snRNP proteins by double label studies with anti-RNP or anti-Sm antibodies within the same cell. The three antibody classes produced superimposable fluorescent patterns. Taking into account that the various IgGs react with antigenic sites on snRNAs or snRNP proteins not shared by all the snRNP species, these data suggest that U1 snRNP particles are distributed in the same way as the other snRNPs in the nucleus. Qualitatively the same results were obtained with DNase-treated nuclear matrices indicating that intact snRNPs are part of the nuclear matrix. Our data are consistent with proposals that the various snRNPs may be involved in processing of hnRNA and that this may take place at the nuclear matrix.     

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.     

Molecular and antigenic nature of isolated Sm

The Sm antigen was isolated and purified from calf thymus nuclear extract by affinity chromatography. The affinity columns were made with serum antibodies from an SLE patient or an anti-Sm monoclonal antibody derived from a hybridoma cell line. Proteins eluted from these two columns had m.w. of 58,000 and 35,000 by SDS polyacrylamide gel electrophoresis. The natural conformation of this antigen appears to be 95,000 in m.w. with the 58,000 particle containing the Sm antigenic determinant. The affinity column-purified antigen detected by the human anti-Sm antibodies is also recognized by anti-Sm antibodies in murine lupus serum, as shown by solid-phase radioimmunoassay. This study 1) demonstrates the molecular and antigenic nature of the Sm antigen and 2) compares the anti-Sm binding capabilities of antibody populations present in sera from SLE patients and from MRL lpr/lpr mice.     

An abundant U6 snRNP found in germ cells and embryos of Xenopus laevis.

The particle state of U snRNPs was analyzed in oocytes, eggs, embryos and testes from Xenopus laevis. In each case both the relative abundance and the composition of some U snRNPs were found to differ from that of somatic cells. U2 and U6 snRNPs were the most prominent U snRNPs in germ cells and early embryos. In particular, the concentration of U6 snRNA was 10-20 times higher than that of U4 snRNA. Most of the U6 snRNA was not associated with U4 snRNA and migrated on sucrose gradients as a U6 snRNP. The structure of this novel U snRNP was analyzed. A single protein of 50 kd was copurified with U6 snRNPs by a combination of gradient fractionation, immunodepletion with anti-Sm antibodies and immunoprecipitation with anti-6-methyl adenosine antibodies. Although the U6 snRNP did not contain Sm proteins it migrated into the nucleus when U6 snRNA was injected into the cytoplasm of oocytes. Two U6 snRNA elements have been identified. The first is essential for nuclear migration in oocytes, but not for the formation of U4/6 snRNPs in vitro and might be the binding site of a U6-specific protein. The second element was required for interaction with U4 snRNPs but not for nuclear targeting.     

Immunoprecipitation distinguishes non-overlapping groups of snRNPs in Schizosaccharomyces pombe.

The large number of snRNAs in the fission yeast Schizosaccharomyces pombe can be divided into four non-overlapping groups by immunoprecipitation with antibodies directed against mammalian snRNP proteins. 1) Of the abundant snRNAs, anti-Sm sera precipitate only the spliceosomal snRNAs U1, U2, U4, U5 and U6. Surprisingly, three Sm-sera tested distinguish between U2, U4 and U5 and U1 from S.pombe; one precipitating only U1 and two precipitating U2, U4 and U5 but not U1. 2) A group of 11 moderately abundant snRNAs are not detectably precipitated by human anti-Sm sera, but are specifically precipitated by monoclonal antibody H57 specific for the human B/B' polypeptides. From Aspergillus nidulans this antibody also precipitates at least 12 snRNAs. 3) Anti-(U3)RNP sera do not precipitate the above snRNAs, but precipitate at least 6 further snRNAs, including the homologues of U3. Both the anti-(U3)RNP sera and H57 also efficiently precipitate a number of discrete non-capped RNAs. 4) A small number of additional snRNAs are not detectably precipitated by any anti-serum tested to date, further analysis may identify antisera specific for these snRNPs. Western blots of purified snRNP proteins were used to identify the S.pombe proteins responsible for these immunoprecipitations. Several Sm-sera decorate a 16.3kD protein which may be a D protein homologue, monoclonal H57 decorates a further protein of 16kD and an anti-(U3)RNP serum decorates the homologue of the 36kD U3-specific protein, fibrillarin.     

Isofocusing of antigenic small nuclear ribonucleoproteins. I. Compositional analysis

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

Characterization of autoantibodies that recognize U4/U6 small ribonucleoprotein particles in serum from a patient with primary Sj?gren's syndrome.

We identified autoantibodies that recognize the U4/U6 snRNPs in a serum from a 63-year-old Japanese patient (TT) with primary Sj?gren's syndrome. This patient's serum immunoprecipitated U4 and U6 sn-RNAs exclusively from 32P-labeled HeLa cell extracts and a newly identified 120-kDa protein along with the Sm core proteins (B'/B, D, E, F, and G) from [35S] methionine-labeled HeLa cell extracts. Immunoblotting demonstrated that only the 120-kDa protein was recognized by this unique serum. In glycerol density gradient centrifugation, the 120-kDa protein reactive with TT serum cosedimented with U4 and U6 snRNAs, suggesting that the 120-kDa protein is a unique component of the U4/U6 snRNP particle. In the same study, the U4/U6 snRNP precipitated by TT serum sedimented only in the lower density, whereas anti-Sm antibodies precipitated U4/U6 snRNAs in a broad range of the gradient. This result suggests the presence of at least two molecular forms of the U4/U6 snRNP particles; larger particles, probably the U4/U5/U6 snRNP complex, and free particles. Thus, the U4/U6 snRNP recognized by TT serum includes the U4 and U6 snRNAs, with Sm core proteins, and the novel 120-kDa protein, and appears to be a free particle not associated with larger complexes.     

Alteration by heat shock and immunological characterization of Drosophila small nuclear ribonucleoproteins

Sera from human patients with systemic lupus erythematosus (SLE) have been shown to react with snRNP particles of both mammals and Drosophila (Mount, S. M. and J. A. Steitz. 1981. Nucleic Acids Res. 9:6351-6368). We have utilized fully characterized monospecific sera and specifically purified antibodies to carry out indirect immunofluorescence experiments with frozen sections of Drosophila embryos. Embryos subjected to severe heat shock before sectioning showed reduced binding of anti-Sm sera. Anti-nRNP sera reacted identically with antigens of heat shocked and non-heat-shocked sections. The reduction in anti-Sm fluorescence was restored by a brief salt wash. These results imply a noncovalent alteration in the conformation of Sm antigens with the administration of heat shock that can revert with exposure to salt. Drosophila antigens have been compared to mammalian standards, showing partial identity with bovine spleen extract (BSE) antigens when reacted with anti-Sm sera. The antigenic relatedness between affinity-purified heat-shocked and non-heat-shocked Drosophila antigens and their mammalian homologues was examined by quantitative ELISA methodology. In all cases, the Drosophila antigens from heat-shocked and non-heat-shocked embryos were identical. We theorize that the heat shock-induced alteration of Sm antigen reverst during extraction. Because the snRNP antigens have been shown to be involved in splicing, and because splicing is inhibited during heat shock (Yost, H. J., and S. Lindquist. 1986. Cell. 45:185-193), our results provide information on the nature and stability of a change in these antigens which may be a central element in control of the heat shock response.     

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.     

Fractionation and characterization of human small nuclear ribonucleoproteins containing U1 and U2 RNAs

Human small nuclear ribonucleoproteins (snRNPs) containing U1 and U2 snRNAs have been isolated from cultured cells by nonimmunological methods. The U1 snRNP population remained immunoprecipitable by systemic lupus erythematosis anti-RNP and anti-Sm antibodies throughout fractionation and contained polypeptides of molecular weights corresponding to those defined as U1 snRNP polypeptides by immunoprecipitation of crude extracts. The purified assemblies contained U1 RNA and nine snRNP polypeptides of molecular weights 67,000 (P67), 30,000 (P30), 23,000 (P23), 21,500 (P22), 17,500 (P18), 12,300 (P12), 10,200 (P10), 9,100 (P9), and 8,500 (P8). P67, P30, and P18 were unique to U1 snRNPs. The U2 snRNP population remained immunoprecipitable by the systemic lupus erythematosis anti-Sm antibody throughout fractionation. The purified U2 assemblies contained six polypeptides of molecular weights corresponding to those defined by immunoprecipitation to be common to U1 and U2 snRNPs including P23, P22, P12, P10, P9, and P8. In addition, U2 snRNPs contained a unique polypeptide of 27,000 Da.     

Newly assembled snRNPs associate with coiled bodies before speckles, suggesting a nuclear snRNP maturation pathway.

BACKGROUND: Small nuclear ribonucleoproteins (snRNPs), which are essential components of the mRNA splicing machinery, comprise small nuclear RNAs, each complexed with a set of proteins. An early event in the maturation of snRNPs is the binding of the core proteins - the Sm proteins - to snRNAs in the cytoplasm followed by nuclear import. Immunolabelling with antibodies against Sm proteins shows that splicing snRNPs have a complex steady-state localisation within the nucleus, the result of the association of snRNPs with several distinct subnuclear structures. These include speckles, coiled bodies and nucleoli, in addition to a diffuse nucleoplasmic compartment. The reasons for snRNP accumulation in these different structures are unclear. RESULTS: When mammalian cells were microinjected with plasmids encoding the Sm proteins B, D1 and E, each tagged with either the green fluorescent protein (GFP) or yellow-shifted GFP (YFP), a pulse of expression of the tagged proteins was observed. In each case, the newly synthesised GFP/YFP-labelled snRNPs accumulated first in coiled bodies and nucleoli, and later in nuclear speckles. Mature snRNPs localised immediately to speckles upon entering the nucleus after cell division. CONCLUSIONS: The complex nuclear localisation of splicing snRNPs results, at least in part, from a specific pathway for newly assembled snRNPs. The data demonstrate that the distribution of snRNPs between coiled bodies and speckles is directed and not random.     

Immunological characterization and intracellular localization of trans-spliceosomal small nuclear ribonucleoproteins in Trypanosoma brucei.

Polyclonal antibodies were raised against purified protein components of the U2 small nuclear ribonucleoprotein (snRNP) from Trypanosoma brucei. Through immunoblot and immunoprecipitation analyses three antisera were characterized that reacted specifically with U2 snRNP proteins of molecular weights 40,000 (anti-40K) and 16,500 (anti-16.5K), and with each of four proteins of molecular weights 14,000, 12,500, 10,000, and 8,500 (anti-CP). Anti-40K antibodies specifically immunoprecipitated the U2 snRNP from trypanosomal extracts, whereas anti-CP antibodies recognized several snRNPs, including the SL RNP and the U2 and U4/U6 snRNPs; in addition, minor RNAs were detected, suggesting that a family of snRNPs with common or related protein components exists in trypanosomes. None of these antibodies cross-reacted significantly with total mammalian snRNP proteins, indicating that the trypanosomal snRNP proteins are immunologically distinct from their mammalian counterparts. Using immunofluorescence microscopy, the snRNP proteins exhibited a differential cellular distribution. Whereas the 40-kDa protein is localized exclusively in the nucleus, with the nucleolus being excluded, a fraction of the common proteins also resides in the cytoplasm.