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PubMed1993

Role of intermolecular/intrastructural B- and T-cell determinants in the diversification of autoantibodies to ribonucleoprotein particles.

The U1 small nuclear ribonucleoprotein (sn-RNP) particle, which consists of the U1 small RNA and multiple polypeptides, is a central target of the autoimmune response in systemic lupus erythematosus. Autoantibodies to the individual proteins of the U1 snRNP typically co-occur in patients with systemic lupus erythematosus, an observation reconciled by postulating that the intact RNA-protein complex serves as the autoimmunogen and that snRNP-specific autoreactive T cells are necessary for autoantibody production. In this study, we demonstrated that normal mice did not develop antibody responses following immunization with purified self (murine) snRNPs. However, when such mice were coimmunized with self snRNPs in conjunction with the human (foreign) U1 snRNP A protein, they developed autoantibodies directed against individual proteins of the U1 snRNP, in addition to anti-A antibodies; we have previously shown that such mice develop snRNP-specific, autoreactive T cells. Intact snRNPs as a co-immunogen were a prerequisite for antibody expansion, since this response was abrogated by disruption of snRNP particles with pancreatic RNase prior to immunization. These findings indicate that autoreactive helper T cells can drive autoantibody production to the individual proteins of snRNP particles and that such autoantibody responses may require the presence of intact snRNP particles that possess intrastructural B-cell and helper-T-cell determinants. These results also suggest that induction of an immune response to one component of an autoantigenic snRNP complex, possibly through priming with molecular mimics, can induce the diversification of autoantibodies that is characteristic of that found in patients with systemic lupus erythematosus.

Animals↗
PubMed1996

Solution structure of the N-terminal RNP domain of U1A protein: the role of C-terminal residues in structure stability and RNA binding.

The solution structure of a fragment of the human U1A spliceosomal protein containing residues 2 to 117 (U1A117) determined using multi-dimensional heteronuclear NMR is presented. The C-terminal region of the molecule is considerably more ordered in the free protein than thought previously and its conformation is different from that seen in the crystal structure of the complex with U1 RNA hairpin II. The residues between Asp90 and Lys98 form an alpha-helix that lies across the beta-sheet, with residues IIe93, IIe94 and Met97 making contacts with Leu44, Phe56 and IIe58. This interaction prevents solvent exposure of hydrophobic residues on the surface of the beta-sheet, thereby stabilising the protein. Upon RNA binding, helix C moves away from this position, changing its orientation by 135 degrees to allow Tyr13, Phe56 and Gln54 to stack with bases of the RNA, and also allowing Leu44 to contact the RNA. The new position of helix C in the complex with RNA is stabilised by hydrophobic interactions from IIe93 and IIe94 to IIe58, Leu 41, Val62 and His 10, as well as a hydrogen bond between Ser91 and Thr11. The movement of helix C mainly involves changes in the main-chain torsion angles of Thr89, Asp90 and Ser91, the helix thereby acting as a "lid" over the RNA binding surface.

Amino Acid Sequence↗
PubMed1986

Antibodies against small nuclear ribonucleoproteins immunoprecipitate complexes containing ts110 Moloney murine sarcoma virus genomic and messenger RNAs.

Small nuclear ribonucleoproteins (snRNPs) are believed to play a role in processing premessenger RNAs. In this study, snRNPs were immunoprecipitated from extracts of cells infected with ts110 Moloney murine sarcoma virus (ts110 MoMuSV). Both the unspliced 4.0 kb and the spliced 3.5-kb ts110 MoMuSV specific RNA species were found in the immunoprecipitates obtained with monoclonal antibody anti-Sm and polyclonal anti-Sm, anti-(U1) RNP and anti-La sera. Although only a portion of the total ts110 RNAs was present in these immunoprecipitates, immune recognition by the anti-snRNPs was specific and not due to contaminating anti-RNA (at least for the anti-Sm sera) or, to anti-viral protein activities. Genomic 8.3-kb RNA and subgenomic 3.0-kb spliced env mRNA from Moloney murine leukemia virus (MoMuLV) infected cells as well as the cellular actin mRNA were also detected in immunoprecipitates obtained with the same antisera. The fact that pre-mRNAs and mature mRNAs of different origin can be recovered from immunoprecipitates formed with anti-snRNP sera establishes their tight association and confirms the role of snRNPs in mRNA processing.

Actins↗
PubMed1998

Ultrastructural localization of interferon-inducible double-stranded RNA-activated enzymes in human cells.

The protein kinase PKR and the 2',5'-oligoadenylate (2-5A) synthetase are two interferon-induced and double-stranded RNA-activated enzymes which are implicated in the mechanism of action of interferon. Their distribution was undertaken here at the ultrastructural level by the immunogold procedure, following the use of specific monoclonal antibodies directed against PKR and 69- and 100-kDa forms of the 2-5A synthetase. These enzymes were detected as a pool of nonaggregated proteins scattered throughout the cell and as aggregates often associated with electron-dense doughnut-like structures showing a similar aspect whatever their subcellular localization: the cytoplasm, the nuclear envelope, and the nucleus. In general, the 2-5A synthetases were present in much more lower amounts than the PKR, probably due to the difficulty of detecting traces of proteins by electron microscopy. To circumvent this, we used a human lymphoblastoid cell line overexpressing the 69-kDa form of the 2-5A synthetase. In such cells, the synthetase was then clearly observed in both the cytoplasm and the nucleus; isolated or small clusters of gold particles were numerous in the cell mainly over the RNP fibrils of the interchromatin space, nucleolus, and ribosomes. Interestingly, gold particles were also found to be associated with the membranes of nuclear envelope and rough endoplasmic reticulum probably due to the myristilated motif of this form of 2-5A synthetase. Finally, intensely labeled electron-opaque dots sometimes associated with the nuclear pore complexes were present in the nucleus and in the cytoplasm of cells which might suggest their transport from the nucleus to the cytoplasm or reciprocally through the nuclear pore complexes. These observations indicate the wider distribution of the dsRNA-activated enzymes in the cell, thus pointing out their potential implication in as yet undetermined physiological function(s) necessary for various cellular metabolic reactions.

2',5'-Oligoadenylate Synthetase↗
PubMed2025

Preparation of high-purity RNPs of CRISPR-based DNA base editors.

Since their introduction, CRISPR-based DNA base editors (BEs) have become essential in the field of precision genome editing, revolutionizing the correction of pathogenic SNPs for both basic research and therapeutic applications. As this technology advances, more laboratories are implementing these tools into their workflow. The delivery of BEs as BE-guide RNA complexes (RNPs), rather than as mRNA or plasmids, has been shown to exhibit lower off-target effects, establishing it as the preferred method of delivery. However, there are no protocols describing in detail how to obtain high-purity and highly active BE RNPs. Here, we offer a comprehensive guide for the expression, purification, RNP reconstitution, and in vitro activity assessment of TadA-based BEs. The protocol includes guidance on performing activity assays using commercial denaturing gels, which is convenient and uses standard molecular biology equipment. This allows for rapid quality control testing of reconstituted BE RNPs prior to more expensive and time-consuming in vivo genome editing experiments. Overall, this protocol aims to empower more laboratories to generate tailored BE RNPs for diverse in vitro and in vivo applications.

Gene Editing↗
PubMed2006

Xp54 and related (DDX6-like) RNA helicases: roles in messenger RNP assembly, translation regulation and RNA degradation.

The DEAD-box RNA helicase Xp54 is an integral component of the messenger ribonucleoprotein (mRNP) particles of Xenopus oocytes. In oocytes, several abundant proteins bind pre-mRNA transcripts to modulate nuclear export, RNA stability and translational fate. Of these, Xp54, the mRNA-masking protein FRGY2 and its activating protein kinase CK2alpha, bind to nascent transcripts on chromosome loops, whereas an Xp54-associated factor, RapA/B, binds to the mRNP complex in the cytoplasm. Over-expression, mutation and knockdown experiments indicate that Xp54 functions to change the conformation of mRNP complexes, displacing one subset of proteins to accommodate another. The sequence of Xp54 is highly conserved in a wide spectrum of organisms. Like Xp54, Drosophila Me31B and Caenorhabditis CGH-1 are required for proper meiotic development, apparently by regulating the translational activation of stored mRNPs and also for sorting certain mRNPs into germplasm-containing structures. Studies on yeast Dhh1 and mammalian rck/p54 have revealed a key role for these helicases in mRNA degradation and in earlier remodelling of mRNP for entry into translation, storage or decay pathways. The versatility of Xp54 and related helicases in modulating the metabolism of mRNAs at all stages of their lifetimes marks them out as key regulators of post-transcriptional gene expression.

Active Transport, Cell Nucleus↗
PubMed2000

The C-terminal domain of TAP interacts with the nuclear pore complex and promotes export of specific CTE-bearing RNA substrates.

Messenger RNAs are exported from the nucleus as large ribonucleoprotein complexes (mRNPs). To date, proteins implicated in this process include TAP/Mex67p and RAE1/Gle2p and are distinct from the nuclear transport receptors of the beta-related, Ran-binding protein family. Mex67p is essential for mRNA export in yeast. Its vertebrate homolog TAP has been implicated in the export of cellular mRNAs and of simian type D viral RNAs bearing the constitutive transport element (CTE). Here we show that TAP is predominantly localized in the nucleoplasm and at both the nucleoplasmic and cytoplasmic faces of the nuclear pore complex (NPC). TAP interacts with multiple components of the NPC including the nucleoporins CAN, Nup98, Nup153, p62, and with three major NPC subcomplexes. The nucleoporin-binding domain of TAP comprises residues 508-619. In HeLa cells, this domain is necessary and sufficient to target GFP-TAP fusions to the nuclear rim. Moreover, the isolated domain strongly competes multiple export pathways in vivo, probably by blocking binding sites on the NPC that are shared with other transport receptors. Microinjection experiments implicate this domain in the export of specific CTE-containing RNAs. Finally, we show that TAP interacts with transportin and with two proteins implicated in the export of cellular mRNAs: RAE1/hGle2 and E1B-AP5. The interaction of TAP with nucleoporins, its direct binding to the CTE RNA, and its association with two mRNP binding proteins suggest that TAP is an RNA export mediator that may bridge the interaction between specific RNP export substrates and the NPC.

Amino Acid Sequence↗
PubMed1969

The organization of the nucleolus in meristematic plant cells. A cytochemical study.

The architecture of the nucleolus in Allium porum and Triticum vulgare meristematic cells has been investigated by means of digestions with various enzymes. After staining with azure B at pH4, plant nucleoli exhibit lighter regions which, under electron microscopy, correspond to the fibrillar zones characterizing these organelles. Evidence is presented indicating that these latter zones contain coarse convoluted filaments quite similar to the loops first demonstrated by La Cour (24) and which are assumed to originate from the nucleolar-organizing chromosomes. These coarse, 0.2micro wide filaments are remarkably resistant to the action of deoxyribonuclease, ribonuclease, pepsin, trypsin, or of various combinations of these enzymes and, moreover, they show insignificant incorporation of labeled thymidine even after long exposure to this DNA precursor. The clearing action of pepsin on different regions of the nucleolus lends support to the hypothesis that an amorphous material or matrix pervades the mass of this organelle. This effect is particularly striking within the particulate nucleolar zones themselves. Both ribonuclease and trypsin disorganize the RNP (ribonucleoprotein) nucleolar particles. The effect of the latter enzyme on the RNP particles is taken to indicate that they contain proteins particularly susceptible to trypsin which are essential for maintenance of their morphological integrity. Trypsin also interferes with azure B-staining of the nucleolar mass as a whole and, according to radioautographic data, extracts RNA throughout this organelle. Accordingly, the hypothesis is considered that RNA is complexed with proteins not only within the particulate nucleolar portions, as is already well known, but also in the fibrillar zones.

Autoradiography↗
PubMed1982

Antibodies from patients with connective tissue diseases bind specific subsets of cellular RNA-protein particles.

We characterized the RNA-containing antigens precipitated by sera from 260 patients with positive antinuclear antibodies. 49 individuals, most of whom had systemic lupus erythematosus or Sjögren's syndrome, possessed antibodies that precipitated the previously identified RNP, Sm, Ro, and La antigens either singly or in combinations. These antigens, which are located on discrete sets of small nuclear or cytoplasmic RNA-protein particles, exhibited a number of antigenic interrelationships. One patient's serum recognized a new particle containing a small RNA which we have called Th; it also precipitated the Ro complexes. Other patients with systemic lupus erythematosus, hepatitis B virus infection, juvenile rheumatoid arthritis, myositis, and rheumatoid arthritis had antibodies that precipitated specific subsets of ribosomal RNA and transfer RNA. One patient's serum contained a monoclonal immunoglobulin G that precipitated ribosomes. Most of these antibodies identified antigenic determinants constituted at least in part of protein. The specificity of the proteins bound to particular cellular RNA, probably explains the exquisite precision with which antibodies from rheumatic disease patients discriminate among RNA subsets. Such sera should be useful probes for investigating specific roles that different RNA and RNA-protein complexes play in cellular metabolism.

Antibodies, Antinuclear↗
PubMed2004

Beta-catenin associates with human parainfluenza virus type 3 ribonucleoprotein complex and activates transcription of viral genome RNA in vitro.

Several studies have indicated that human parainfluenza virus type 3 (HPIV-3) requires polymeric actin for transcription of its genome RNA in vitro and in vivo. In the current study, we have identified beta-catenin, an actin-bound protein, as one of the transcriptional activators for HPIV-3 genome RNA. Beta-catenin was packaged within the purified HPIV-3 virions and was associated with the HPIV-3 ribonucleoproteins (RNP) from infected cells. Moreover, purified beta-catenin interacted with bacterially expressed HPIV-3 nucleocapsid protein (N) and phosphoprotein (P) fused to glutathione S-transferase (GST). Double-labeled immunofluorescent confocal microscopic analysis revealed colocalization of beta-catenin with HPIV-3 RNP at cell periphery in infected cells. The HPIV-3 RNP-associated beta-catenin functioned as a transactivator of HPIV-3 genome, because purified beta-catenin stimulated transcription of viral RNP in an in vitro transcription assay. These results demonstrate that beta-catenin, a multifunctional protein that is involved in cell-cell adhesion and embryogenesis, acts as one of the transcriptional activators of HPIV-3 genome RNA.

Cytoskeletal Proteins↗
PubMed1991

Pathways for the nuclear transport of proteins and RNAs.

The nuclear pore complex catalyses the import and export of both proteins and RNAs. The molecular mechanisms of RNA and protein translocation through the nuclear pore are likely to be similar; however, their signals and targeting apparatus may differ. Recent insights into RNA transport have come from studies of kinetic control mechanisms and the preconditions for translocation that include processing, RNP assembly, and a targeting function for 5' caps.

Journal Article↗
PubMed1975

Effect of RNA synthesis on the binding of 3H-cortisol to nuclear ribonucleoprotein particles from rat liver carrying DNA-like RNA in vivo.

3H-cortisol was found to associate with rat liver nuclear 30S ribonucleoprotein particles carrying D-RNA in vivo. No interaction was detectable when RNA synthesis was inhibited by alpha-amanitine. The association appears to be specifically for RNP carrying RNA synthesized after the administration of cortisol to adrenalectomized rats. The DNA/protein/RNA ratio of rat liver nuclei was not effected by alpha-amanitine under our conditions. However, the drug caused a 5-10 fold decrease in nuclear uptake of cortisol. The results are discussed in relation to a supposed transfer of cortisol-receptor complexes from the chromatin template to the nascent RNA chains.

Adrenalectomy↗
PubMed2002

Large-scale induced fit recognition of an m(7)GpppG cap analogue by the human nuclear cap-binding complex.

The heterodimeric nuclear cap-binding complex (CBC) binds to the 5' cap structure of RNAs in the nucleus and plays a central role in their diverse maturation steps. We describe the crystal structure at 2.1 A resolution of human CBC bound to an m(7)GpppG cap analogue. Comparison with the structure of uncomplexed CBC shows that cap binding induces co-operative folding around the dinucleotide of some 50 residues from the N- and C-terminal extensions to the central RNP domain of the small subunit CBP20. The cap-bound conformation of CBP20 is stabilized by an intricate network of interactions both to the ligand and within the subunit, as well as new interactions of the CBP20 N-terminal tail with the large subunit CBP80. Although the structure is very different from that of other known cap-binding proteins, such as the cytoplasmic cap-binding protein eIF4E, specificity for the methylated guanosine again is achieved by sandwiching the base between two aromatic residues, in this case two conserved tyrosines. Implications for the transfer of capped mRNAs to eIF4E, required for translation initiation, are discussed.

Amino Acid Sequence↗
PubMed2006

Essential domains for ribonucleoprotein complex formation required for retrotransposition of telomere-specific non-long terminal repeat retrotransposon SART1.

Non-long terminal repeat (LTR) retrotransposons are major components of the higher eukaryotic genome. Most of them have two open reading frames (ORFs): ORF2 encodes mainly the endonuclease and reverse transcriptase domains, but the functional features of ORF1 remain largely unknown. We used telomere-specific non-LTR retrotransposon SART1 in Bombyx mori and clarified essential roles of the ORF1 protein (ORF1p) in ribonucleoprotein (RNP) formation by novel approaches: in vitro reconstitution and in vivo/in vitro retrotransposition assays using the baculovirus expression system. Detailed mutation analyses showed that each of the three CCHC motifs at the ORF1 C terminus are essential for SART1 retrotransposition and are involved in packaging the SART1 mRNA specifically into RNP. We also demonstrated that amino acid residues 555 to 567 and 285 to 567 in the SART1 ORF1p are crucial for the ORF1p-ORF1p and ORF1p-ORF2p interactions, respectively. The loss of these domains abolishes protein-protein interaction, leading to SART1 retrotransposition deficiency. These data suggest that systematic formation of RNP composed of ORF1p, ORF2p, and mRNA is mainly mediated by ORF1p domains and is a common, essential step for many non-LTR retrotransposons encoding the two ORFs.

Amino Acid Motifs↗
PubMed2000

[A novel method for screening anti-inner ear autoantibody in patients with autoimmune diseases].

OBJECTIVE: To appraise the clinical value of a newly established method, rapid electric field immobilizing liquid phase molecule dot blot analysis (REILMD), for screening anti-inner ear autoantibody in patients with autoimmune diseases. METHODS: Seventy-one patients with 11 kinds of autoimmune diseases were chosen for the study. Both the general immunity and autoantibodies were tested. In the processes of detection of anti-inner ear autoantibody, REILMD was used for screening, and then the Western blot was used to define the molecular weight of inner ear antigen recognized by the positive autoantibody. RESULTS: Acceleration of erythrocyte sedimentation rate (ES), positive rheumatoid factor (RF), increases in C reactive protein (CRP), IgG and circulating immune complex (CIC) were found in most cases with rheumatoid arthritis (RA) and systemic lupus erythematodes (SLE). Some of these patients had increased IgA, IgM and C4. Two of 16 RA had anti-double-stranded DNA (dsDNA) and anti-mitochondria and 4/16 had anti-nucleus antibodies. Eleven of 16 SLE had antinucleus, 7/13 had anti-ribonucleoprotein (RNP), anti-Sjögren syndrome A (SSA) and anti-dsDNA, 3/13 had anti-smooth muscle (Sm) and 1/13 had anti-DNA topoisomerase I (Scl)-70, striated muscle and stomach acid cell antibodies. No autoantibody was detected in AS. Anti-inner ear autoantibody existed in 9 out of 71 patients (13%) with autoimmune diseases, in 2 of 21 patients (10%) with sudden deafness and only in 1 of 48 control subjects (2%, coronary heart disease). The anti-inner ear autoantibody was positive in 5 of 16 (31%) patients with SLE and 1 each in RA, AS, Behset's disease and streptococcus infection syndrome. In patients with positive anti-inner ear antibody, 67% had anti-nucleus antibody, 50% had anti-RNP and dsDNA antibody. The molecules recognized by the positive anti-inner ear antibody were defined as 52,000, 36,000, 31,000 and 15,000 molecules of inner ear antigen. CONCLUSION: REILMD is a feasible and easy method for screening anti-inner ear autoantibody. Several autoimmune diseases, particularly SLE, may be implicated in damage to the inner ear.

Adolescent↗
PubMed1995

Autoimmune response to the spliceosome. An immunologic link between rheumatoid arthritis, mixed connective tissue disease, and systemic lupus erythematosus.

OBJECTIVE: To assess the significance of autoantibodies to RA33, the A2 protein of the heterogeneous nuclear ribonucleoproteins (hnRNP), and to the related hnRNP proteins A1, B1, and B2 in rheumatic diseases. METHODS: Using a partially purified preparation of hnRNP-A and hnRNP-B proteins, sera from 303 patients with various rheumatic diseases were investigated by immunoblotting. For the analysis of cross-reactivities, autoantibodies were affinity purified by blot elution. RESULTS: Anti-A2/RA33 was found in 35% of rheumatoid arthritis (RA) patients, 38% of mixed connective tissue disease (MCTD) patients, 23% of systemic lupus erythematosus (SLE) patients, and, apart from single exceptions, not in patients with other rheumatic diseases. All anti-A2/RA33-positive sera were also reactive with B1 and B2, and anti-A2/RA33 antibodies cross-reacted with both proteins. Antibodies to hnRNP-A1 were found less frequently; moreover, the majority of anti-A1-positive sera also contained anti-A2/RA33 antibodies. In anti-A1, anti-A2/RA33 double-positive sera, cross-reactivity between the 2 antibodies was generally observed. In SLE patients, the presence of anti-A2/RA33 was correlated with the presence of anti-(U1) small nuclear RNP (snRNP) and anti-Sm (P < 0.0001 and P < 0.005, respectively), but there was no evidence for cross-reactivity between antibodies to hnRNP and antibodies to snRNP antigens. CONCLUSION: Since both hnRNPs and snRNPs are essential components of the spliceosome, the data show that the immune systems of patients with RA, SLE, and MCTD react to this functional complex. However, compared with MCTD and SLE patients, RA patients have a more restricted immune response to the spliceosome: they react to hnRNP proteins, particularly to hnRNP-A2/RA33, but not to snRNPs.

Arthritis, Rheumatoid↗
PubMed1992

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.

Antibody Specificity↗