Autoantibodies directed to novel components of the PM/Scl complex, the human exosome

The autoantigenic polymyositis/scleroderma (PM/Scl) complex was recently shown to be the human homologue of the yeast exosome, which is an RNA-processing complex. Our aim was to assess whether, in addition to targeting the known autoantigens PM/Scl-100 and PM/Scl-75, autoantibodies also target recently identified components of the PM/Scl complex. The prevalence of autoantibodies directed to six novel human exosome components (hRrp4p, hRrp40p, hRrp41p, hRrp42p, hRrp46p, hCsl4p) was determined in sera from patients with idiopathic inflammatory myopathy (n = 48), scleroderma (n = 11), or the PM/Scl overlap syndrome (n = 10). The sera were analyzed by enzyme-linked immunosorbent assays and western blotting using the affinity-purified recombinant proteins. Our results show that each human exosome component is recognized by autoantibodies. The hRrp4p and hRrp42p components were most frequently targeted. The presence of autoantibodies directed to the novel components of the human exosome was correlated with the presence of the anti-PM/Scl-100 autoantibody in the sera of patients with idiopathic inflammatory myopathy (IIM), as was previously found for the anti-PM/Scl-75 autoantibody. Other clear associations between autoantibody activities were not found. These results further support the conception that the autoimmune response may initially be directed to PM/Scl-100, whereas intermolecular epitope spreading may have caused the autoantibody response directed to the associated components.     

Three novel components of the human exosome

The yeast exosome is a complex of 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' --> 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.     

Clinical evaluation of autoantibodies to a novel PM/Scl peptide antigen

Synovial fluid from patients with various arthritides contains procoagulant, cell-derived microparticles. Here we studied whether synovial microparticles modulate the release of chemokines and cytokines by fibroblast-like synoviocytes (FLS). Microparticles, isolated from the synovial fluid of rheumatoid arthritis (RA) and arthritis control (AC) patients (n = 8 and n = 3, respectively), were identified and quantified by flow cytometry. Simultaneously, arthroscopically guided synovial biopsies were taken from the same knee joint as the synovial fluid. FLS were isolated, cultured, and incubated for 24 hours in the absence or presence of autologous microparticles. Subsequently, cell-free culture supernatants were collected and concentrations of monocyte chemoattractant protein-1 (MCP-1), IL-6, IL-8, granulocyte/macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF) and intracellular adhesion molecule-1 (ICAM-1) were determined. Results were consistent with previous observations: synovial fluid from all RA as well as AC patients contained microparticles of monocytic and granulocytic origin. Incubation with autologous microparticles increased the levels of MCP-1, IL-8 and RANTES in 6 of 11 cultures of FLS, and IL-6, ICAM-1 and VEGF in 10 cultures. Total numbers of microparticles were correlated with the IL-8 (r = 0.91, P < 0.0001) and MCP-1 concentrations (r = 0.81, P < 0.0001), as did the numbers of granulocyte-derived microparticles (r = 0.89, P < 0.0001 and r = 0.93, P < 0.0001, respectively). In contrast, GM-CSF levels were decreased. These results demonstrate that microparticles might modulate the release of chemokines and cytokines by FLS and might therefore have a function in synovial inflammation and angiogenesis.     

Clinical evaluation of autoantibodies to a novel PM/Scl peptide antigen

Anti-PM/Scl antibodies represent a specific serological marker for a subset of patients with scleroderma (Scl) and polymyositis (PM), and especially with the PM/Scl overlap syndrome (PM/Scl). Anti-PM/Scl reactivity is found in 24% of PM/Scl patients and is found in 3–10% of Scl and PM patients. The PM/Scl autoantigen complex comprises 11–16 different polypeptides. Many of those proteins can serve as targets of the anti-PM/Scl B-cell response, but most frequently the PM/Scl-100 and PM/Scl-75 polypeptides are targeted. In the present study we investigated the clinical relevance of a major alpha helical PM/Scl-100 epitope (PM1-α) using a newly developed peptide-based immunoassay and compared the immunological properties of this peptide with native and recombinant PM/Scl antigens. In a technical comparison, we showed that an ELISA based on the PM1-α peptide is more sensitive than common techniques to detect anti-PM/Scl antibodies such as immunoblot, indirect immunofluorescence on HEp-2 cells and ELISA with recombinant PM/Scl polypeptides. We found no statistical evidence of a positive association between anti-PM1-α and other antibodies, with the exception of known PM/Scl components. In our cohort a negative correlation could be found with anti-Scl-70 (topoisomerase I), anti-Jo-1 (histidyl tRNA synthetase) and anti-centromere proteins. In a multicenter evaluation we demonstrated that the PM1-α peptide represents a sensitive and reliable substrate for the detection of a subclass of anti-PM/Scl antibodies. In total, 22/40 (55%) PM/Scl patients, 27/205 (13.2%) Scl patients and 3/40 (7.5%) PM patients, but only 5/288 (1.7%) unrelated controls, tested positive for the anti-PM1-α peptide antibodies. These data indicate that anti-PM1-α antibodies appear to be exclusively present in sera from PM/Scl patients, from Scl patients and, to a lesser extent, from PM patients. The anti-PM1-α ELISA thus offers a new serological marker to diagnose and discriminate different systemic autoimmune disorders.     

The changing landscape of the clinical value of the PM/Scl autoantibody system

Autoantibodies to the polymyositis/scleroderma (PM/Scl) complex have been associated with systemic sclerosis and PM/Scl overlap syndrome. The report of Hanke and colleagues in a recent issue of Arthritis Research and Therapy is the first to describe the separate evaluation of anti-PM/Scl-75c and PM/Scl-100 autoantibodies and their relationship to clinical manifestations of systemic sclerosis. Several observations are of paramount interest, but are not in general agreement with earlier studies. These include the prevalence of anti-PM/Scl antibodies in systemic sclerosis, the association with certain clinical manifestations and prognosis of patients. This report will hopefully trigger systematic multi-centre studies to confirm and/or elucidate the novel line immunoassay and clinical associations.     

Autoantibodies to components of the mitotic apparatus

Autoantibodies directed to a variety of cellular antigens and organelles are a feature of autoimmune diseases. They have proven useful in a clinical setting to establish diagnosis, estimate prognosis, follow disease progression, alter therapy, and initiate new investigations. Cellular and molecular biologists have used autoantibodies as probes to identify molecules involved in key cellular processes. One of the most interesting sets of autoantibodies are those that target antigens within the mitotic apparatus (MA). The MA includes chromosomes, spindle microtubules and centrosomes. The identification, localization, function, and clinical relevance of MA autoantigens is the focus of this review. Abbreviations: ATP – adenosine triphosphate; CENP – centromere protein; CREST – calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia; HMG – high mobility group; IB – intercellular bridge; IIF – indirect immunofluorescence; MAPs – microtubule associated proteins; NuMA – nuclear mitotic apparatus; NOR – nucleolar organizer; PBC – primary biliary cirrhosis; PM – polymyositis; Pol I, II, III – RNA polymerases; RA-rheumatoid arthritis; SLE – systemic lupus erythematosus; SS – Sjögren's syndrome; SSc – systemic sclerosis; topo – topoisomerase.     

Caspase-mediated cleavage of the exosome subunit PM/Scl-75 during apoptosis

Recent studies have implicated the dying cell as a potential reservoir of modified autoantigens that might initiate and drive systemic autoimmunity in susceptible hosts. A number of subunits of the exosome, a complex of 3'→5' exoribonucleases that functions in a variety of cellular processes, are recognized by the so-called anti-PM/Scl autoantibodies, found predominantly in patients suffering from an overlap syndrome of myositis and scleroderma. Here we show that one of these subunits, PM/Scl-75, is cleaved during apoptosis. PM/Scl-75 cleavage is inhibited by several different caspase inhibitors. The analysis of PM/Scl-75 cleavage by recombinant caspase proteins shows that PM/Scl-75 is efficiently cleaved by caspase-1, to a smaller extent by caspase-8, and relatively inefficiently by caspase-3 and caspase-7. Cleavage of the PM/Scl-75 protein occurs in the C-terminal part of the protein at Asp369 (IILD³⁶⁹↓G), and at least a fraction of the resulting N-terminal fragments of PM/Scl-75 remains associated with the exosome. Finally, the implications of PM/Scl-75 cleavage for exosome function and the generation of anti-PM/Scl-75 autoantibodies are discussed.     

Structure and regulation of the nuclear exosome targeting complex guides RNA substrates to the exosome

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Identification of two novel components of the human NDC80 kinetochore complex

Proper kinetochore function is essential for the accurate segregation of chromosomes during mitosis. Kinetochores provide the attachment sites for spindle microtubules and are required for the alignment of chromosomes at the metaphase plate (chromosome congression). Components of the conserved NDC80 complex are required for chromosome congression, and their disruption results in mitotic arrest accompanied by multiple spindle aberrations. To better understand the function of the NDC80 complex, we have identified two novel subunits of the human NDC80 complex, termed human SPC25 (hSPC25) and human SPC24 (hSPC24), using an immunoaffinity approach. hSPC25 interacted with HEC1 (human homolog of yeast Ndc80) throughout the cell cycle and localized to kinetochores during mitosis. RNA interference-mediated depletion of hSPC25 in HeLa cells caused aberrant mitosis, followed by cell death, a phenotype similar to that of cells depleted of HEC1. Loss of hSPC25 also caused multiple spindle aberrations, including elongated, multipolar, and fractured spindles. In the absence of hSPC25, MAD1 and HEC1 failed to localize to kinetochores during mitosis, whereas the kinetochore localization of BUB1 and BUBR1 was largely unaffected. Interestingly, the kinetochore localization of MAD1 in cells with a compromised NDC80 function was restored upon microtubule depolymerization. Thus, hSPC25 is an essential kinetochore component that plays a significant role in proper execution of mitotic events.     

The association of the human PM/Scl-75 autoantigen with the exosome is dependent on a newly identified N terminus

The exosome is a complex of 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 human SKI complex regulates channeling of ribosome-bound RNA to the exosome via an intrinsic gatekeeping mechanism

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Musing on the structural organization of the exosome complex

The exosome complex of 3'-->5' exoribonucleases functions in both the precise processing of 3' extended precursor molecules to mature stable RNAs and the complete degradation of other RNAs. Both processing and degradative activities of the exosome depend on additional cofactors, notably the putative RNA helicases Mtr4p and Ski2p. It is not known how these factors regulate exosome function or how the exosome distinguishes RNAs destined for processing events from substrates that are to be completely degraded. Here we review the available data concerning the modes of action of the exosome and relate these to possible structural arrangements for the complex. As no detailed structural data are yet available for the exosome complex, or any of its constituent enzymes, this discussion will rely heavily on rather speculative models.     

C1D and hMtr4p associate with the human exosome subunit PM/Scl-100 and are involved in pre-rRNA processing

The exosome is a complex of 3'-5' exoribonucleases and RNA-binding proteins, which is involved in processing or degradation of different classes of RNA. Previously, the characterization of purified exosome complexes from yeast and human cells suggested that C1D and KIAA0052/hMtr4p are associated with the exosome and thus might regulate its functional activities. Subcellular localization experiments demonstrated that C1D and KIAA0052/hMtr4p co-localize with exosome subunit PM/Scl-100 in the nucleoli of HEp-2 cells. Additionally, the nucleolar accumulation of C1D appeared to be dependent on PM/Scl-100. Protein-protein interaction studies showed that C1D binds to PM/Scl-100, whereas KIAA0052/hMtr4p was found to interact with MPP6, a previously identified exosome-associated protein. Moreover, we demonstrate that C1D, MPP6 and PM/Scl-100 form a stable trimeric complex in vitro. Knock-down of C1D, MPP6 and KIAA0052/hMtr4p by RNAi resulted in the accumulation of 3'-extended 5.8S rRNA precursors, showing that these proteins are required for rRNA processing. Interestingly, C1D appeared to contain RNA-binding activity with a potential preference for structured RNAs. Taken together, our results are consistent with a role for the exosome-associated proteins C1D, MPP6 and KIAA052/hMtr4p in the recruitment of the exosome to pre-rRNA to mediate the 3' end processing of the 5.8S rRNA.     

The human exosome: an autoantigenic complex of exoribonucleases in myositis and scleroderma

The anti-PM/Scl autoantibodies are known to characterize a subset of autoimmune patients with myositis, scleroderma (Scl), and the PM/Scl overlap syndrome. The major autoantigens that are recognized by anti-PM/Scl autoantibodies are designated PM/Scl-100 and PM/Scl-75. These autoantigens have been reported to associate into a large complex consisting of 11 to 16 proteins and to play a role in ribosome synthesis. Recently, it was discovered that the PM/Scl complex is the human counterpart of the yeast (Saccharomyces cerevisiae) exosome, which is an RNA-processing complex consisting of 11 3' → 5' exoribonucleases. To date, 10 human exosome components have been identified, although only some of these were studied in more detail. In this review, we discuss some recent advances in the characterization of the PM/Scl complex.     

MYCN recruits the nuclear exosome complex to RNA polymerase II to prevent transcription-replication conflicts

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Structural basis for RNA surveillance by the human nuclear exosome targeting (NEXT) complex

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