Identification of the Ro and La antigens in the endoribonuclease VII--ribonucleoprotein complex.
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ABSTRACT:
SUBMITTER: Bachmann M
PROVIDER: S-EPMC1147831 | biostudies-other | 1987 Apr
REPOSITORIES: biostudies-other
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Project description:Ro, or Sjogren syndrome type A (SS-A), antigen is the most prevalent of the human systemic autoimmune specificities and exists as an inabundant ribonucleoprotein complex (RNP) composed of a 60,649-Da protein, as defined here by cDNA cloning, and the human Y RNAs. The recombinant 60-kDa Ro protein and human Y1 RNA were reconstituted in vitro, and the binding was enhanced by divalent cations. A region of the Ro amino acid sequence revealed a resemblance to the RNP consensus motif found in most RNA-binding proteins. In addition, Ro contained a potential "zinc-binding finger" motif that was distinct from the RNP consensus region and that may participate in the interaction with human Y RNAs or with other proteins. The recombinant Ro fusion protein also proved useful for the detection of autoantibodies in the sera of patients with autoimmune disorders. Possible functions of the Ro RNPs and their relationship to RNA polymerase III transcription are discussed.
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Project description:In total 3 mice brain tissues were used for this experiment. Tissue from each mouse brain was divided for immunoprecipitation with 5ug of either rabbit anti-TDP-43 antibody (Abcam) or normal rabbit IgG (Sigma) .The antibodies were first incubated with the lysate overnight at 4 degrees C, after which 50 uL of protein G Dynabeads(tm) (Invitrogen(tm)) added and the solution incubated for 1 hour at 4 degrees C with rotation. Following several washing with washing buffer (Invitrogen(tm)), the protein-RNA complex was eluted from 20 uL of bead-protein complex using 10 uL of elution buffer (Invitrogen(tm)) and seperated on a 10% NuPage(tm) Bis-Tris gel (Invitrogen(tm)). RNA was isolated from the remaining 30 uL of protein-bead complex using TRIzol reagent (Invitrogen) followed by DNaseI treatment (Ambion). The TDP-43 immunoprecipitated RNA was converted to cDNA, fragmented and biotin labelled using WT cDNA synthesis & amplification kit and Terminal Labeling Kit (Affymetrix(tm)) for Affymetrix(tm) GeneChip(tm) Mouse Gene 1.0 ST and 3'-IVT expression analysis kit (Affymetrix(tm)) for GeneChip(tm) Mouse 430_2 arrays according to the standard protocol. Labelled RNA was hybridised to arrays in a hybridization oven (Affymetrix(tm)) at 45 degrees C rotating at 60 rpm for 17 hours and scanned using the Affymetrix(tm) GeneChip Scanner. Successful hybridisation to the microarray was checked using Expression Console software (Affymetrix(tm)) and the data (.CEL files) transferred to Partek Genomics Suite for statistical analysis. TDP-43 and IgG immunoprecipitated RNA samples were each hybridised to 3 GeneChip Mouse Gene 1.0 ST and 3 GeneChip Mouse 430 (n = 6 for each group).
2012-02-06 | E-MEXP-3500 | biostudies-arrayexpress
Project description:Sam68, Src associated in mitosis of 68 kDa, is a known RNA-binding protein and a signaling adaptor protein for tyrosine kinases. However, the proteins associated with Sam68 and the existence of a Sam68 complex, its mass, and regulation are, however, unknown. Herein we identify a large Sam68 complex with a mass >1 MDa in HeLa cells that is composed of approximately 40 proteins using an immunoprecipitation followed by a mass spectrometry approach. Many of the proteins identified are RNA-binding proteins and are known components of a previously identified structure termed the spreading initiation center. The large Sam68 complex is a ribonucleoprotein complex, as treatment with RNases caused a shift in the molecular mass of the complex to 200-450 kDa. Moreover, treatment of HeLa cells with phorbol 12-myristate 13-acetate or epidermal growth factor induced the disassociation of Sam68 from the large complex and the appearance of Sam68 within the smaller complex. Actually, in certain cell lines such as breast cancer cell lines MCF-7 and BT-20, Sam68 exists in equilibrium between a large and a small complex. The appearance of the small Sam68 complex in cells correlates with the ability of Sam68 to promote the alternative splicing of CD44 and cell migration. Our findings show that Sam68 exists in equilibrium in transformed cells between two complexes and that extracellular signals, such as epidermal growth factor stimulation, promote alternative splicing by modulating the composition of the Sam68 complex.
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Project description:Here we report the rapid identification of the proteins of the spliceosomal U1 small nuclear ribonucleoprotein (snRNP) from the yeast Saccharomyces cerevisiae by searching mass spectrometric data in genomic sequence databases. The U1 snRNP, containing a histidine-tagged 70K protein, was isolated from cell extracts by anti m3G-cap immunoaffinity and subsequent nickel nitrilotriacetic acid chromatography. A U1 snRNP fraction containing 20 proteins was obtained. Further purification by glycerol gradient centrifugation identified nine U1 snRNP specific and six common proteins. The U1 snRNP proteins were partially sequenced by nanoelectrospray mass spectrometry, and their genes were identified in the data base via multiple peptide sequence tags. Apart from the already known common proteins D1, D3, F, and G, the D2 and E homologs were also identified. The same six common proteins were detected in core U2 snRNP, which was purified and analyzed separately. The biochemical association of these six proteins with yeast snRNPs is shown here for the first time. Intriguingly, the Sm B/B' homolog was not detected. In addition to the well characterized yeast U1 specific proteins [U1-70K (Snp1p), U1-A (Mud1p), Prp39p, and Prp40p] the homolog of the U1-C protein was identified together with four additional novel U1 specific proteins, which are not found in mammalian U1. This is the first time that the components of a multiprotein complex from an organism with a sequenced genome have been characterized by mass spectrometry. The technique should be applicable to any protein complex that can be biochemically purified from an organism whose genome is known.
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Project description:Maturation of chloroplast psaA pre-mRNA from the green alga Chlamydomonas reinhardtii requires the trans-splicing of two split group II introns. Several nuclear-encoded trans-splicing factors are required for the correct processing of psaA mRNA. Among these is the recently identified Raa4 protein, which is involved in splicing of the tripartite intron 1 of the psaA precursor mRNA. Part of this tripartite group II intron is the chloroplast encoded tscA RNA, which is specifically bound by Raa4. Using Raa4 as bait in a combined tandem affinity purification and mass spectrometry approach, we identified core components of a multisubunit ribonucleoprotein complex, including three previously identified trans-splicing factors (Raa1, Raa3, and Rat2). We further detected tscA RNA in the purified protein complex, which seems to be specific for splicing of the tripartite group II intron. A yeast-two hybrid screen and co-immunoprecipitation identified chloroplast-localized Raa4-binding protein 1 (Rab1), which specifically binds tscA RNA from the tripartite psaA group II intron. The yeast-two hybrid system provides evidence in support of direct interactions between Rab1 and four trans-splicing factors. Our findings contribute to our knowledge of chloroplast multisubunit ribonucleoprotein complexes and are discussed in support of the generally accepted view that group II introns are the ancestors of the eukaryotic spliceosomal introns.
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Project description:Antibody-mediated rejection (AMR) causes more than 50% of late kidney graft losses. In addition to anti-human leukocyte antigen (HLA) donor-specific antibodies, antibodies against non-HLA antigens are also linked to AMR. Identifying key non-HLA antibodies will improve our understanding of AMR.MethodsWe analyzed non-HLA antibodies in sera from 80 kidney transplant patients with AMR, mixed rejection, acute cellular rejection (ACR), or acute tubular necrosis. IgM and IgG antibodies against 134 non-HLA antigens were measured in serum samples collected pretransplant or at the time of diagnosis.ResultsFifteen non-HLA antibodies were significantly increased (P < 0.05) in AMR and mixed rejection compared with ACR or acute tubular necrosis pretransplant, and 7 at diagnosis. AMR and mixed cases showed significantly increased pretransplant levels of IgG anti-Ro/Sjögren syndrome-antigen A (SS-A) and anti-major centromere autoantigen (CENP)-B, compared with ACR. Together with IgM anti-CENP-B and anti-La/SS-B, these antibodies were significantly increased in AMR/mixed rejection at diagnosis. Increased IgG anti-Ro/SS-A, IgG anti-CENP-B, and IgM anti-La/SS-B were associated with the presence of microvascular lesions and class-II donor-specific antibodies (P < 0.05). Significant increases in IgG anti-Ro/SS-A and IgM anti-CENP-B antibodies in AMR/mixed rejection compared with ACR were reproduced in an external cohort of 60 kidney transplant patients.ConclusionsThis is the first study implicating autoantibodies anti-Ro/SS-A and anti-CENP-B in AMR. These antibodies may participate in the crosstalk between autoimmunity and alloimmunity in kidney AMR.
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