Cloning of a complementary DNA coding for the 100-kD antigenic protein of the PM-Scl autoantigen.

Gě, Qún; Frank, Mark Barton; O’Brien, Charles A.; Targoff, Ira N.

doi:10.1172/jci115895

Cited by 73 publications

(41 citation statements)

References 25 publications

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“…The estimated molecular masses of the proteins found in these studies were 110, 90, 80, 39,37,33,30,27,26,22, and 20 kDa. Comparison of these molecular masses with those of the proteins characterized in the present study suggested that hRrp40, hRrp41 and hRrp46 correspond to the 30-, 27-, and 26-kDa proteins, respectively.…”

Section: Cloning Of Human Homologues Of Yeast Rrp40pmentioning

confidence: 66%

Three Novel Components of the Human Exosome

Brouwer

Allmang

Raijmakers

et al. 2001

Journal of Biological Chemistry

101

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The yeast exosome is a complex of 3 3 5 exoribonucleases. Sequence analysis identified putative human homologues for exosome components, although several were found only as expressed sequence tags. Here we report the cloning of full-length cDNAs, which encode putative human homologues of the Rrp40p, Rrp41p, and Rrp46p components of the exosome. Recombinant proteins were expressed and used to raise rabbit antisera. In Western blotting experiments, these decorated HeLa cell proteins of the predicted sizes. All three human proteins were enriched in the HeLa cells nucleus and nucleolus, but were also clearly detected in the cytoplasm. Size exclusion chromatography revealed that hRrp40p, hRrp41p, and hRrp46p were present in a large complex. This cofractionated with the human homologues of other exosome components, hRrp4p and PM/ Scl-100. Anti-PM/Scl-positive patient sera coimmunoprecipitated hRrp40p, hRrp41p, and hRrp46p demonstrating their physical association. The immunoprecipitated complex exhibited 3 3 5 exoribonuclease activity in vitro. hRrp41p was expressed in yeast and shown to suppress the lethality of genetic depletion of yeast Rrp41p. We conclude that hRrp40p, hRrp41p, and hRrp46p represent novel components of the human exosome complex.In both bacteria and eukaryotes, the processing and degradation of many RNA species involves multiprotein complexes (reviewed in Refs. 1-4). The Escherichia coli degradosome includes the endoribonuclease E (RNase E), the 3Ј 3 5Ј exonuclease polynucleotide phosphorylase, the DEAD box RNA helicase RhlB, and several additional proteins whose role is unclear (5-7). Related complexes are implicated in RNA processing in chloroplasts and mitochondria (8 -10). The yeast exosome contains at least 11 components, which are known or predicted to be 3Ј 3 5Ј exoribonucleases (11,12). Ten of these (Rrp4p, Rrp40 -46p, Mtr3p, and Csl4p) have been demonstrated to be encoded by essential genes. These 10 components were found in both cytoplasmic and nuclear complexes, whereas the nonessential RRP6 gene product was detected only in the nucleus (11, 13).The 3Ј processing of many RNAs is affected by the absence or mutation of exosome components. The nuclear exosome is implicated in the processing of ribosomal RNA (rRNA), spliceosomal small nuclear RNAs, and small nucleolar RNAs, as well as the degradation of pre-rRNA spacers and unspliced pre-mRNAs (12-22). The cytoplasmic exosome complex is involved in the 3Ј 3 5Ј pathway of mRNA degradation (22). The activity of the exosome complex may be regulated by cofactors including, for example, the putative ATP-dependent DEVH box RNA helicases Dob1p and Ski2p (23,24).Human cells also contain a multiprotein complex that is related to the yeast exosome (11). This complex, initially designated as the polymyositis/scleroderma (PM/Scl) 1 overlap syndrome particle and herein referred to as the human exosome, was reported to contain 11 (25) to 16 (26) subunits ranging from 20 to 110 kDa. Two proteins of this complex were identified as autoantigens, which are tar...

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Section: Cloning Of Human Homologues Of Yeast Rrp40pmentioning

confidence: 66%

Three Novel Components of the Human Exosome

Brouwer

Allmang

Raijmakers

et al. 2001

Journal of Biological Chemistry

101

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“…The subunits PM/Scl-75, PM/Scl-100, and hRrp4p have been shown to carry the main autoantigenic determinants for polymyositis/ scleroderma overlap syndrome autoantibodies (4,5). Following the identification of two of its subunits, PM/Scl-75 and PM/Scl-100 (6,7), and their characterization as putative exoribonucleases (8), the PM/Scl complex was shown to be related to the yeast exosome, a complex containing ϳ10 exoribonucleases (9).…”

mentioning

confidence: 99%

The Association of the Human PM/Scl-75 Autoantigen with the Exosome Is Dependent on a Newly Identified N Terminus

Raijmakers¹,

Egberts²,

Venrooij³

et al. 2003

Journal of Biological Chemistry

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The exosome is a complex of 3 3 5 exoribonucleases that functions in a variety of cellular processes, all concerning the processing or degradation of RNA. Paradoxically, the previously described cDNA for the human autoantigenic exosome subunit PM/Scl-75 (Alderuccio, F., Chan, E. K., and Tan, E. M. (1991) J. Exp. Med. 173, 941-952) encodes a polypeptide that failed to interact with the exosome complex. Here, we describe the cloning of a more complete cDNA for PM/Scl-75 encoding 84 additional amino acids at its N terminus. We show that only the longer polypeptide is able to associate with the exosome complex. This interaction is most likely mediated by protein-protein interactions with two other exosome subunits, hRrp46p and hRrp41p, one of which was confirmed in a mammalian two-hybrid system. In addition we show that the putative nuclear localization signal present in the C-terminal region of PM/Scl-75 is sufficient, although not essential for nuclear localization of the protein. Moreover, the deletion of this element abrogated the nucleolar accumulation of PM/Scl-75, although its association with the exosome was not disturbed. This suggests that this basic element of PM/ Scl-75 plays a role in targeting the exosome to the nucleolus.The PM/Scl-75 protein (encoded by gene PMSCL1) was the first protein of the polymyositis/scleroderma (PM/Scl) 1 complex to be characterized (1). The PM/Scl complex was originally described as a complex consisting of 11-16 proteins, some of which are autoantigenic in autoimmune patients. Autoantibodies to this complex are most frequently found in patients suffering from PM/Scl overlap syndrome but are also present in some patients with myositis or scleroderma alone (2, 3). The subunits PM/Scl-75, PM/Scl-100, and hRrp4p have been shown to carry the main autoantigenic determinants for polymyositis/ scleroderma overlap syndrome autoantibodies (4, 5). Following the identification of two of its subunits, PM/Scl-75 and PM/Scl-100 (6, 7), and their characterization as putative exoribonucleases (8), the PM/Scl complex was shown to be related to the yeast exosome, a complex containing ϳ10 exoribonucleases (9).The exosome functions in a variety of processes involving the 3Ј 3 5Ј processing or degradation of RNA. Among these processes are the maturation of 5.8 S rRNA (10 -12), the processing of many small nuclear and nucleolar RNAs (13-15), and the turnover of different types of mRNAs (16,17), especially the AU-rich element containing mRNAs (18,19). PM/Scl-75 has been suggested to be an AU-rich element binding protein, involved in the recruitment of the exosome to this class of mRNAs (19).Like five other exosome proteins, PM/Scl-75 contains an RNase PH domain, which is homologous to the prokaryotic 3Ј-5Ј exoribonuclease RNase PH. Based on mutual interactions between exosome components and structural similarity with the bacterial protein polynucleotide phosphorylase (PNPase), a component of the bacterial degradosome, recently a model for the structure of the human exosome was generated. In this mo...

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“…Binding of AUF1 seems to be essential to form the exosome, and AUF1 is part of mRNA complexes that are degraded or exported. Interestingly, 2 human exosome components, PM-Scl-100 and PM-Scl-75, are recognized by sera from patients with the polymyositis-scleroderma overlap syndrome (30,31). However, the complete absence of anti-AUF1 antibodies in patients with scleroderma or polymyositis/ dermatomyositis along with the absence of antiexosomal antibodies in patients with SLE suggest that patients with SLE do not target AUF1 contained in exosomal complexes, while mRNA decay complexes do not appear to form major target structures in scleroderma and related disorders.…”

Section: Discussionmentioning

confidence: 99%

AUF1, the regulator of tumor necrosis factor α messenger RNA decay, is targeted by autoantibodies of patients with systemic rheumatic diseases

Skriner¹,

Hueber²,

Süleymanoglu³

et al. 2008

Arthritis & Rheumatism

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Objective. To investigate which members of the heterogeneous nuclear RNP (hnRNP) family are targeted by autoantibodies from patients with systemic rheumatic diseases.Methods. Using a semipurified preparation of natural hnRNP proteins, 365 sera from patients with rheumatic diseases and control subjects were screened by immunoblotting for the presence of autoantibodies. Bacterially expressed recombinant hnRNP D (AUF1) proteins were used for confirming the data obtained. Binding of RNA and autoantibody to AUF1 was investigated by gel retardation assays. Expression of AUF1 in cultivated cells and synovial tissue was analyzed by indirect immunofluorescence and immunohistochemistry.Results. Autoantibodies to AUF1 proteins were detected in 33% of patients with systemic lupus erythematosus, 20% of patients with rheumatoid arthritis, 17% of patients with mixed connective tissue disease, and <10% of patients with other rheumatic disorders. Epitope mapping studies showed the autoantibodies to be directed to conformational epitopes in the N-terminal RNA-binding part of AUF1. However, autoantibody binding did not interfere with RNA binding as assessed by gel-shift assays. Immunohistochemical studies revealed AUF1 to be expressed in the cytoplasm of RA synovial tissue as compared with nuclear staining in osteoarthritis and normal synovium, particularly in macrophages of the lining layer and in fibroblasts of the sublining areas.Conclusion. These data identify AUF1 proteins as novel autoantigens in SLE and related autoimmune disorders. Because AUF1 proteins are major components of messenger RNA stability complexes, our findings suggest that these complexes form a novel macromolecular target structure for autoantibodies in rheumatic autoimmune diseases.

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Cloning of a complementary DNA coding for the 100-kD antigenic protein of the PM-Scl autoantigen.

Cited by 73 publications

References 25 publications

Three Novel Components of the Human Exosome

Three Novel Components of the Human Exosome

The Association of the Human PM/Scl-75 Autoantigen with the Exosome Is Dependent on a Newly Identified N Terminus

AUF1, the regulator of tumor necrosis factor α messenger RNA decay, is targeted by autoantibodies of patients with systemic rheumatic diseases

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