Ultrastructural distribution of nuclear ribonucleoproteins as visualized by immunocytochemistry on thin sections

The ultrastructural distribution of nuclear ribonucleoproteins (RNP) has been investigated by incubation of thin sections of mouse or rat liver, embedded in Lowicryl K4M or prepared by cryoultramicrotomy, with antibodies specific for RNP. The antibodies were localized by means of a protein A-colloidal gold complex. Anti-small nuclear (sn)RNP antibodies, specific for determinants of the nucleoplasmic snRNP species containing U1, U2, U4, U5, and U6 RNAs, were found associated preferentially with perichromatin fibrils, interchromatin granules, and coiled bodies. This indicates an early association of snRNP with structural constituents containing newly synthesized heterogeneous nuclear RNA. It also suggests a possible structural role of some snRNPs in nuclear architecture. Antibodies against the core proteins of heterogeneous nuclear RNP particles associate preferentially with the border regions of condensed chromatin, and in particular with perichromatin fibrils and some perichromatin granules. These results are discussed in view of recent knowledge about the possible role of nucleoplasmic RNP-containing components in the functions of the cell nucleus.     

Antibodies from patients with mixed connective tissue disease react with heterogeneous nuclear ribonucleoprotein or ribonucleic acid (hnRNP/RNA) of the nuclear matrix

The sera of patients with mixed connective tissue disease (MCTD) have high titers of antibodies directed against nuclear U1-ribonucleoprotein (U1-RNP). This antigen is easily extracted from nuclear preparations with physiologic saline and from tissue sections with 0.1 HCl, leaving the nucleic acids and nuclear matrix behind. When U1-RNP is extracted from HEp-2 cells with 0.1 N HCl, the sera of 32/32 patients with MCTD react with another antigen that is exposed by the extraction procedure. This antigen is not destroyed by trypsin and deoxyribonuclease 1 treatment but is sensitive to both purified ribonuclease A and purified micrococcal nuclease. Absorption studies showed that the antibody reacting with this antigen cannot be absorbed by sheep red blood cells coated with extracts of rabbit thymus that contain U1-RNP. Radioimmunoassay showed that the reaction of the unadsorbed antibody was with heterogeneous nuclear ribonucleoprotein or ribonucleic acid (hnRNP/RNA) and not with transfer RNA or ribosomal RNA. The hnRNP/RNA antigen is demonstrated as discrete particles in the internucleolar chromatin of interphase cells, but in metaphase cells the antigen is diffusely dispersed. The distribution, solubility, and biochemical characteristics suggest that the antigenic moiety is part of the nuclear matrix. Therefore, MCTD sera contain antibodies that react with at least two species of nuclear RNP: small nuclear RNP (snRNP), as described by others, and a high m.w. hnRNP/RNA bound to the nuclear matrix.     

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     

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.     

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.     

Newly identified U4/U6 snRNP-binding proteins by serum autoantibodies from a patient with systemic sclerosis.

We found serum autoantibodies directed against the proteins binding exclusively to U4/U6 of Sm small nuclear ribonucleoprotein particle (snRNP) in serum from a patient (MaS) with systemic sclerosis. Their specificity, called anti-MaS, is distinct from that of known antibodies against U snRNP. The U4 and U6 small nuclear RNA from a 32P-labeled HeLa cell extract and five proteins with Mr 150,000, 120,000, 80,000, 36,000, and 34,000, in addition to Sm core proteins (B, B', D, E, F, and G) from an [35S] methionine-labeled extract, were immunoprecipitated by anti-MaS in isotonic solution. However, the Sm core proteins and U4 and U6 small nuclear RNA were separated from the protein-A-Sepharose facilitated MaS immunoprecipitate by incubation in a solution containing 500 mM NaCl. In immunoblots, anti-MaS antibodies reacted with one protein of Mr 150,000 from a HeLa cell nuclear extract that was fractionated by SDS-PAGE and transferred to a nitrocellulose sheet. The monospecific immunoaffinity purified antibody eluted from the immunoblot band immunoprecipitated U4 and U6 small nuclear RNA and reblotted the protein with Mr 150,000. These data indicate that anti-MaS antibodies recognize at least one antigenic protein that binds exclusively to the U4/U6 snRNP.     

Macromolecular domains containing nuclear protein p107 and U-snRNP protein p28: Further evidence for an in situ nuclear matrix

Summary Polyclonal antibodies have been produced which react with a nuclear protein having a molecular weight of 107kD and a pl of 8.7–8.8 (designated p107). This protein is shown to be a component of the residual ribonucleoprotein (RNP) network of the nuclear matrix. P107 localized exclusively to the nuclear interior but not within nucleolar or chromatin domains. We have taken advantage of this unique probe to examine whether the RNP network of the isolated nuclear matrix has a physical counterpart in situ. We show that RNA, p107, divalent cations and the 28 kD Sm antigen of U-snRNPs are components of in situ macromolecular assemblies. While the morphology and intranuclear distribution of these assemblies are insensitive to the removal of chromatin, they are markedly altered by degradation of RNA. Digestion in situ of RNA in the presence of EDTA followed by extraction with high ionic strength buffers solubilized the components of these assemblies. Electron microscopic and immunobiochemical data are presented which support the concept that the residual RNP network of the nuclear matrix is an isolate of a pre-existing structure, and that perturbations in this internal network can be created by RNA degradation, depletion of essential metal ions and proteolysis.Abbreviations CRLM polyclonal chicken antibody raised against rat liver nuclear matrix - Sm monoclonal antibody specific for the 28 kd protein antigen of U1, U2, U4, U5 and U6 snRNPs - hnRNP ribonucleoprotein particles containing hnRNA - snRNP ribonucleoprotein particles containing snRNA - PBS phosphate buffered saline - PMSF phenylmethylsulfonyl fluoride - PAGE polyacrylamide gel electrophoresis - EDTA ethylenediaminetetraacetic acid - VRC vanadium ribonucleoside complex - BSA bovine serum albumin - DMSO dimethylsulfoxide - HS high salt buffer - LS low salt buffer     

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.     

Intranuclear distribution of SV40 large T-antigen and transformation-related protein p53 in abortively infected cells

The intranuclear localization of SV40 T-antigen (T-Ag) and the cellular protein p53 was studied in SV40 abortively infected baby mouse kidney cells using two complementary methods of ultrastructural immunocytochemistry in combination with preferential staining of nuclear RNP components and electron microscope autoradiography. Both proteins were revealed in association with peri- and interchromatin RNP fibrils containing the newly synthesized hnRNA. In addition, T-Ag and p53 remained bound, at least in part, to the residual internal nuclear matrix following nuclease and salt extractions of infected cells. The localization of T-Ag was different in SV40 lytically infected monkey kidney cells since, in addition to hnRNP fibrils, the viral protein was also associated with cellular chromatin. However, when lytic infection was performed in conditions of blocked viral DNA replication, T-Ag was no longer associated with the cellular chromatin but remained bound to the hnRNP fibrils. We conclude that the transforming and lytic functions of T-Ag can be distinguished by different subnuclear distributions. The significance of the association of T-Ag and p53 with hnRNP fibrils and the internal nuclear matrix is discussed in relation to the role of these structures in the control of cellular mRNA biogenesis.     

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.     

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.     

Ribonucleoprotein organization of eukaryotic RNA. XXX. Evidence that U1 small nuclear RNA is a ribonucleoprotein when base-paired with pre-messenger RNA in vivo

U1 small nuclear RNA is thought to be involved in messenger RNA splicing by binding to complementary sequences in pre-mRNA. We have investigated intermolecular base-pairing between pre-mRNA (hnRNA) and U1 small nuclear RNA by psoralen crosslinking in situ, with emphasis on ribonucleoprotein structure. HeLa cells were pulse-labeled with [3H]uridine under conditions in which hnRNA is preferentially labeled. Isolated nuclei were treated with aminomethyltrioxsalen , which produces interstrand crosslinks at sites of base-pairing between hnRNA and U1 RNA. hnRNA-ribonucleoprotein (hnRNP) particles were isolated in sucrose gradients containing 50% formamide, to dissociate non-crosslinked U1 RNA, and then analyzed by immunoaffinity chromatography using a human autoantibody that is specific for the ribonucleoprotein form of U1 RNA (anti-U1 RNP). After psoralen crosslinking, pulse-labeled hnRNA in hnRNP particles reproducibly bound to anti-U1 RNP. The amount of hnRNA bound to anti-U1 RNP was reduced 80 to 85% when psoralen crosslinking of nuclei was omitted, or if the crosslinks between U1 RNA and hnRNA were photo-reversed prior to immunoaffinity chromatography. Analysis of the proteins bound to anti-U1 RNP after crosslink reversal revealed polypeptides having molecular weights similar to those previously described for U1 RNP. These proteins did not bind to control, non-immune human immunoglobulin G. These results indicate that the subset of nuclear U1 RNA that is base-paired with hnRNA at a given time in the cell is a ribonucleoprotein. This raises the possibility that these proteins, as well as U1 RNA itself, may participate in pre-mRNA splice site recognition by U1 RNP.     

Immunofluorescent localization of the proteins of nuclear ribonucleoprotein complexes

Antibodies were raised in chickens against heterogeneous nuclear RNA (hnRNA)-binding proteins from 30S ribonucleoprotein (RNP) complexes of mouse Taper hepatoma ascites cell nuclei. The antibody preparations were characterized for immunological specificity and purity by double- diffusion gels, binding to specific bands in SDS polyacrylamide gels, and crossed immunoelectrophoresis. Antibodies raised against either whole 30S RNP complexes or purified RNP core proteins had a strong selective affinity for the four 34,000- to 40,000-dalton polypeptides which comprise the major structural proteins of hnRNP. The intracellular distribution of 30S RNP antigens in mouse ascites cells was determined by indirect immunofluorescence microsacopy. In interphase cells immunofluorescent sites were restricted to the nucleus, and nucleoli were free of fluorescence. The chicken anti-mouse- RNP antibodies were also able to react with cells from many different vertebrate species, showing a similar nucleus-restricted localization of the reacting sites. The antibodies also bound chick 30S RNP-proteins and reacted with the nuclei of chick cells. An exception to this was the failure of the antibody to bind to adult chick erythrocytes, suggesting that these major hnRNA binding proteins may be found only in nuclei capable of RNA synthesis.     

U1 SnRNP association with HnRNP involves an initial non-specific splice-site independent interaction of U1 SnRNP protein with HnRNA

Precursor mRNA is complexed with proteins in the cell nucleus to form heterogeneous nuclear ribonucleoprotein (hnRNP), and these hnRNPs are found associated in vivo with small nuclear RNPs (snRNPs) for the processing of pre-mRNA. In order to better characterize the ATP-independent initial association of U1 snRNP with hnRNP, an important early event in assembly of the spliceosome complex, we have determined some of the components essential to an in vitro reassociation of U1 snRNP with hnRNP. U1 snRNP reassociated in vitro with 40S hnRNP particles from HeLa cells and, similar to the in vivo hnRNP/U1 snRNP association, the in vitro interaction was sensitive to high salt concentrations. U1 snRNP also associated with in vitro reconstituted hnRNP in which bacteriophage MS2 RNA, which lacks introns, was used as the RNA component. Purified snRNA alone would not associate with the MS2 RNA-reconstituted hnRNP, however, intact U1 snRNP did interact with protein-free MS2 RNA. This indicates that the U1 snRNP proteins are required for the hnRNP/U1 snRNP association, but hnRNP proteins are not. Thus, the initial, ATP-independent association of U1 snRNP with hnRNP seems to be mediated by U1 snRNP protein(s) associating with hnRNA without requiring a splice-site sequence. This complex may then be further stabilized by intron-specific interactions and hnRNP proteins, as well as by other snRNPs.     

A novel 40S multi-snRNP complex isolated from rat liver nuclei.

Two structurally distinct RNP complexes (MI and MII), each with a sedimentation value of approx. 40S, were isolated from rat liver nuclear extracts by sucrose gradient centrifugation and subsequent native gel electrophoresis of the 40S hnRNP-containing fractions. MII RNP contained the bulk of hnRNA and hnRNP proteins (i.e. the 32-45KD core proteins and polypeptides of 60-80 and 110-130KD). MI RNP was characterized by the exclusive presence of U-snRNAs (U1, U2, U4, U5 and U6), their well known snRNP polypeptides and a number of Sm-associated proteins in the range of 50-210KD. Immunoselection experiments employing a monoclonal antibody with an established specificity for the U2-snRNP-specific B" polypeptide proved that the RNA and protein components characteristic of MI were part of a single multi-snRNP unit. The prominent 200/210KD protein doublet of MI was identified immunochemically as the rat homologue of the yeast PRP8 protein, a known U5-associated splicing component. Based on the major biochemical and immunochemical features of MI and MII RNP complexes, we conclude that MII represents the monomeric 40S hnRNP structure, whereas MI defines a novel multi-snRNP entity.     

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.     

Analysis of small nuclear ribonucleoproteins (RNPs) in Trypanosoma brucei: structural organization and protein components of the spliced leader RNP.

trans splicing in Trypanosoma brucei involves the ligation of the 40-nucleotide spliced leader (SL) to each of the exons of large, polycistronic pre-mRNAs and requires the function of small nuclear ribonucleoproteins (snRNPs). We have identified and characterized snRNP complexes of SL, U2, U4, and U6 RNAs in T. brucei extracts by a combination of glycerol gradient sedimentation, CsCl density centrifugation, and anti-m3G immunoprecipitation. Both the SL RNP and the U4/U6 snRNP contain salt-stable cores; the U2 snRNP, in contrast to other eucaryotic snRNPs, is not stable under stringent ionic conditions. Two distinct complexes of U6 RNA were found, a U6 snRNP and a U4/U6 snRNP. The structure of the SL RNP was analyzed in detail by oligonucleotide-directed RNase H protection and by in vitro reconstitution. Our results indicate that the 3' half of SL RNA constitutes the core protein-binding domain and that protein components of the SL RNP also bind to the U2 and U4 RNAs. Using antisense RNA affinity chromatography, we identified a set of low-molecular-mass proteins (14.8, 14, 12.5, and 10 kDa) as components of the core SL RNP.