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Clin Exp Immunol. Dec 2002; 130(3): 532–540.
PMCID: PMC1906554

Autoantibodies against small nucleolar ribonucleoprotein complexes and their clinical associations

Abstract

Sera from patients suffering from systemic autoimmune diseases such as systemic lupus erythematosus (SLE) and systemic sclerosis (SSc) have been shown to contain reactivities to nuclear components. Autoantibodies specifically targeting nucleolar antigens are found most frequently in patients suffering from SSc or SSc overlap syndromes. We determined the prevalence and clinical significance of autoantibodies directed to nucleolar RNA-protein complexes, the so-called small nucleolar ribonucleoprotein complexes (snoRNPs). A total of 172 patient sera with antinucleolar antibodies were analysed by immunoprecipitation. From 100 of these patients clinical information was obtained by chart review. Autoantibodies directed to snoRNPs were detected not only in patients suffering from SSc and primary Raynaud's phenomenon (RP), but also in patients suffering from SLE, rheumatoid arthritis (RA) and myositis (PM/DM). Antibodies against box C/D small snoRNPs can be subdivided in antifibrillarin positive and antifibrillarin negative reactivity. Antifibrillarin-positive patient sera were associated with a poor prognosis in comparison with antifibrillarin negative (reactivity with U3 or U8 snoRNP only) patient sera. Anti-Th/To autoantibodies were associated with SSc, primary RP and SLE and were found predominantly in patients suffering from decreased co-diffusion and oesophagus motility and xerophthalmia. For the first time autoantibodies that recognize box H/ACA snoRNPs are described, identifying this class of snoRNPs as a novel autoantigenic activity. Taken together, our data show that antinucleolar patient sera directed to small nucleolar ribonucleoprotein complexes are found frequently in other diseases than SSc and that categorization of diagnoses and clinical manifestations based on autoantibody profiles seems particularly informative in patient sera recognizing box C/D snoRNPs.

Keywords: nucleolus, snoRNP, systemic autoimmune disease, Th/ To autoantigen

Introduction

Antinuclear antibodies (ANA) have been demonstrated to occur frequently in systemic autoimmune diseases such as systemic lupus erythematosus (SLE), systemic sclerosis (SSc), Sjögren's syndrome (SjS), polymyositis (PM) and dermatomyositis (DM). Antibodies targeted to nucleolar autoantigens such as fibrillarin, Th/To, PM-Scl, NOR-90/UBF, RNA polymerase I, Ku and DNA topoisomerase I (Scl70) are found most frequently in patients suffering from SSc or SSc overlap syndromes (reviewed in [14]). The occurrence of these antibodies in patient sera is helpful in establishing the diagnosis and prognosis of the disease. For example, autoantibodies directed against DNA topoisomerase I identify a subgroup of SSc with early diffuse disease and pulmonary involvement (reviewed in [1]) and autoantibodies against fibrillarin identify a subgroup of SSc with a poor prognosis [5].

In the past decade, our understanding of nucleolar processes, the most prominent of which is the biogenesis of ribosomes, and the macromolecular complexes involved increased dramatically. It has been shown that the precursor ribosomal RNA, encoding mature 18S, 5·8S and 25S-28S rRNA is processed and modified extensively (reviewed in [68]). The most important trans-acting factors in these processes are the small nucleolar ribonucleoprotein particles (snoRNPs). These snoRNPs can be divided into three groups based on conserved sequence elements in their RNA components: box C/D snoRNPs, box H/ACA snoRNPs and RNase MRP/RNase P ([9], reviewed in [6,7]), see Fig. 1.

Fig. 1
Schematic representation of the structure and function of autoantigenic small nucleolar ribonucleoprotein complexes. In the nucleolus (shaded area) the biogenesis of ribosomes takes place. Three of the four ribosomal RNAs (18S, 5·8S and 25/28S) ...

Most of the box C/D snoRNPs have been demonstrated to function in the 2′-O-ribose methylation of pre-rRNA, whereas U3, U8, U13 and U22 box C/D snoRNPs have been demonstrated or suggested to function in the cleavage of pre-rRNA at the 5′- and 3′-end of mature 18S rRNA (reviewed in [6,7]). Until now three proteins have been identified that are shared by this class of snoRNPs: fibrillarin, Nop56 and Nop5/58 [1012]. The box H/ACA snoRNPs have been implicated in the conversion of uridine residues in pre-rRNA to pseudouridines [9,13] (see Fig. 1). At present, four proteins have been shown to specifically associate with this class of snoRNPs: hGar1, Nap57/dyskerin, hNHP2 and hNOP10 [1416]. The RNase MRP and RNase P complexes contain the Th/To autoantigen [1719]) and are the only representatives of the third class of snoRNPs. Both ribonucleoprotein particles function as endoribonucleases and have been shown to function in the cleavage of pre-rRNA to mature rRNA and the processing of pre-tRNA, respectively (reviewed in [20]) (see Fig. 1).

The aim of this study was to assess whether these three classes of small nucleolar ribonucleoprotein complexes are targeted by autoantibodies present in sera of patients suffering from systemic autoimmune diseases. The clinical relevance of the occurrence of these autoantibodies was investigated using chart reviews of these patients.

Patients and methods

Patients

Most patient sera were obtained from the department of Rheumatology of the University Medical Centre St Radboud, Nijmegen and the St Maartens Hospital, Nijmegen and tested routinely for the presence of autoantibodies by indirect immunofluorescence on HEp-2 cells and by counter-immunoelectrophoresis. From this collection (consisting of more than 4500 patients’ sera) a total of 172 sera were selected that displayed moderate and strong antinucleolar reactivities, when analysed by indirect immunofluorescence on HEp-2 cells using a 40-fold serum dilution and a FITC-antihuman IgG conjugate [21]. In addition to the antinucleolar reactivities, approximately 5% and 50% of these patients’ sera contained also anticytoplasmic or antinuclear reactivities, respectively. When the total collection of 4500 patients’ sera was taken into account, the prevalence of antinucleolar reactivity for individual disease groups was below 7%, except for SSc and SjS, for which 25% and 11% of the sera, respectively, were positive.

From 100 patients clinical information could be obtained by chart review (26 from the St Maartens Hospital and 74 from the University Medical Centre St Radboud). Patient's diagnosis was set by trained rheumatologists and chart review was independently confirmed by a trained rheumatologist (FvdH, DJdR).

The patients’ age and sex were recorded along with clinical findings, which include all signs, symptoms and serology contributing to the classification criteria of rheumatoid arthritis (RA) [22], SLE [23], SSc [24], SjS [25] and diagnostic criteria of DM and PM by Peter and Bohan [26,27]. Often-occurring non-specific signs such as Raynaud's phenomenon (RP), sicca complaints, pulmonary function test abnormalities, pulmonary hypertension, motility abnormalities of oesophagus and gut, myocarditis and other (autoimmune) diseases such as diabetes, vasculitic disorders, thyreoiditis, spondyalarthropathies and gout were also recorded.

SnoRNP immunoprecipitation analysis

Total HeLa cell extracts were prepared as described previously [28]. For each immunoprecipitation with patient serum, 20 µl serum was incubated with 20 µl of a 50% suspension of protein A-agarose beads in IPP500 (500 mm NaCl, 10 mm Tris-HCl pH 8·0, 0·05% NP-40) for 1 h at room temperature. Beads were washed twice with IPP500 and once with IPP150 (150 mm NaCl, 10 mm Tris-HCl pH 8·0, 0·05% NP-40). HeLa extract was added, the mixture was incubated for 2 h at 4°C and the beads were washed three times with IPP150. To analyse co-precipitating RNAs, the RNA was isolated by phenol–chloroform extraction and ethanol precipitation. RNAs were resolved on a denaturing polyacrylamide gel and blotted onto Hybond-N membranes (Amersham Pharmacia Biotech, Roosendaal, The Netherlands). Northern blot hybridizations with 32P-labelled oligonucleotides were performed as described by Sambrook et al. [29]. The following oligonucleotides were used to analyse the following RNAs: RNase P: 5′-GGG-AGA-GCC-CTG-TTA-GGG-CCG-CCC-3′; RNase MRP: 5′-CCC-CGT-GTG-GTT-GGT-GCG-CGG-ACA-C-3′; U3: 5′-CGA-AGC-TTG-TA C-CAC-TCA-GAC-CGC-GTT-CTC-3′; U8: 5′-CAT-GTT-CTA-ATC-TGC-CCT-CCG-GAG-GAG-G-3′; U22: 5′-ACA-GGC-TCT-GGG-ACT-AGG-ACA-GAG-AG-3′; U17: 5′-AGA-GCC-CTA-TGC-TCC-CCT-ACG-CCA-C-3′; E3: 5′-GCA-GGG-GGA-ACG-ACA-ACA-CAG-CAC-3′; U1: 5′-GCG-CGA-ACG-CAG-TCC-CCC-ACT-ACC-AC-3′; Y1: 5′-CTA-AGC-TTA-AAA-GAC-TAG-TCA-AGT-GCA-GT-3′

Western blot analysis with recombinant human brillarin

Recombinant (His)6-tagged human fibrillarin was expressed in and purified from Sf-9 cells using the Bac-to-Bac baculovirus expression system according to the manufacturer's protocol (Gibco-BRL, Breda, The Netherlands). For Western blot analysis approximately 1 ng of recombinant protein was loaded onto a 13% SDS-PAGE and transferred to nitrocellulose. Patient sera and sera of healthy individuals as controls were used in a 5000-fold dilution. Detection was performed using horseradish peroxidase-conjugated rabbit-antihuman IgG (Dako Immunoglobulins, Glostrup, Denmark) as secondary antibody and visualization by chemiluminescence.

Anti PM/Scl100 autoantibodies

Reactivity with PM/Scl100 was determined by ELISA as described previously [30].

Results

Anti-nucleolar patient sera recognize various small nucleolar RNPs

To determine the presence of autoantibodies directed against small nucleolar ribonucleoprotein complexes a random serum collection was tested for the presence of antinucleolar antibodies by immunofluorescence using HEp-2 cells, as described previously [21]. The 172 antinucleolar sera obtained were analysed further by immunoprecipitation using a total HeLa cell extract. Co-precipitating RNAs were isolated and analysed by Northern blot hybridization for the presence of RNase MRP/RNase P RNA, U3, U8 and U22 (box C/D) snoRNAs and U17 and E3 (box H/ACA) snoRNAs. We also analysed for reactivities directed to the U1 snRNP complex (probing for U1 snRNA) and the Ro RNPs (probing for Y1 scRNA) as these activities are common in mixed connective tissue disease (MCTD) and Sjögren's syndrome/SLE, respectively.

A typical example of a Northern blot analysis is depicted in Fig. 2. The upper two panels show that in most cases sera that immunoprecipitate RNase MRP RNA also precipitate RNase P RNA (lanes 2, 4, 13 and 14), although one serum appeared to recognize RNase MRP specifically (lane 17). The recognition of both the RNase MRP and RNase P complexes is generally referred to as Th/To specificity. Lane 11 shows an example of a serum recognizing U3, U8 and U22 box C/D snoRNPs, suggesting that a poly-peptide that is common to this class of snoRNPs is autoantigenic. Lane 17 suggests that a protein common to the box H/ACA sno-RNPs is autoantigenic as well, because both box H/ACA sno-RNPs E3 and U17 are immunoprecipitated by this patient serum.

Fig. 2
Northern blot analysis of antinucleolar sera. Immunoprecipitations using total HeLa cell extract with 21 patient sera (lanes 1–21) are shown. Co-precipitated RNAs were isolated and analysed by Northern blot hybridization with probes specific for ...

Table 1 summarizes the results of the Northern blot analyses and the occurrence of autoantibodies against PM/Scl100, as detected by enzyme-linked immunosorbent assay (ELISA) analyses of this group of 172 antinucleolar patient sera. These data will be described below in more detail for each of the snoRNPs.

Table 1
Summary of Northern blot hybridization data

Anti-nucleolar activity is found in patients suffering from SLE, SSc, RA and primary RP

Clinical information of 100 patients with antinucleolar activity was obtained by chart review. The recognition of nucleolar complexes by autoantibodies in this patient group is specified in Table 1. A comparison of the occurrence of these autoantibodies in this group (n = 100) with that in the total group (n = 172) shows that the patient data group is a good representation of the total group.

Table 2 shows the diagnoses of the patients in this group. As expected, based on literature data, patients with antinucleolar antibodies suffer from SSc (n = 14), PM (n = 2), DM (n = 2), primary RP (n = 10) and SSc-overlap syndromes (n = 2). Surprisingly, antinucleolar antibodies were also found in patients diagnosed with SLE (n = 11), SjS (n = 4), RA (n = 20), MCTD (mixed connective tissue disease; n = 4) and a group of different other diseases (n = 27), including gout, M. Buerger, M. Kahler, M. Reiter, Crohn's disease, ankylosing spondylitis.

Table 2
Diagnoses of patients with antinucleolar autoantibodies

Fibrillarin is autoantigenic in patient sera recognizing box C/D snoRNPs

In 9% of the antinucleolar patient sera (see Table 1), autoantibodies co-precipitating box C/D snoRNAs were found (a serum is designated anti-box C/D snoRNP when U3, U8 and U22 snoRNAs are all immunoprecipitated). Three proteins have been reported to be associated with all box C/D snoRNPs (fibrillarin, Nop56 and Nop5/58), suggesting that one or more of these proteins is targeted by these sera. Previous studies indicate that sera with antinucleolar activity frequently contain autoantibodies against fibrillarin [5,31]. To investigate the recognition of fibrillarin by the patient sera precipitating the box C/D snoRNPs, Western blot analyses were performed using recombinant fibrillarin. As shown in Fig. 3, lanes 11–18, all patient sera that are able to immunoprecipitate box C/D snoRNPs efficiently recognize fibrillarin, whereas with sera that recognize merely U8 or U3 snoRNP, only background levels of antifibrillarin reactivity were observed (lanes 1–10). The former group will now be referred to as antifibrillarin-positive patient sera.

Fig. 3
Recognition of fibrillarin by antinucleolar sera. Recombinant fibrillarin was separated by SDS-PAGE and transferred to nitrocellulose membranes. Strips of these membranes were incubated with patient sera that coimmunoprecipitate either U3 snoRNA (lanes ...

Antifibrillarin antibodies have been reported to occur in patients suffering from primary RP, SSc and SSc-overlap syndromes [3,5]. Chart review confirmed that antifibrillarin-positive sera (n = 8) can be found in SSc (n = 1) and primary RP (n = 2), see Table 2. In addition, antifibrillarin-positive sera were found in patients suffering from SLE (n = 3), RA (n = 1) and undefined connective tissue disease (UCTD) (n = 1). Clinical manifestations of antifibrillarin positive patients were studied in more detail; see Table 3. Antifibrillarin-positive patient sera appeared to be associated particularly with manifestations suggesting a more poor prognosis, such as pleuritis, pericarditis, renal failure and myocarditis.

Table 3
Clinical manifistations per group of antinucleolar patient sera

Identification of reactivity to U3 and U8 snoRNPs only

The analyses of this cohort of antinucleolar sera showed for the first time that 5–10% of these sera contained reactivity to either U3 snoRNP only or to U8 snoRNP only; see Table 1. These sera do not show detectable reactivity to fibrillarin, as illustrated in Fig. 3, lanes 1–10. The ‘anti-U3 snoRNP only’ antibodies (n = 5) were found to be present in patients suffering from DM (n = 1), RA (n = 2), RA with sicca complaints (n = 1) and fibromyalgia (n = 1); see Table 2. ‘Anti-U8 snoRNP only’ antibodies (n = 6) are found in patients suffering from similar diseases, i.e. PM (n = 1), RA (n = 2), deforming osteoarthritis (n = 1), arthralgies (n = 1) and juvenile chronic arthritis (JCA) (n = 1). In more detail, the patient sera that recognize U3 or U8 snoRNP only (n = 11) are associated with arthritis (n = 7), polymyositis (n = 2), RP (n = 2) and sicca complaints (n = 4, e.g. xerostomia and xerophthalmia), see Table 3. In addition, ‘anti-U8 snoRNP only’ patient sera associate with pleuritis (n = 1), anaemia (n = 3) and lymphopenia (n = 3), diabetes (n = 1) and vasculitis (n = 1).

In summary, antifibrillarin-positive and -negative anti-box C/D snoRNP patient sera appear to be associated with two patient groups with different manifestations. The group of antifibrillarin-positive sera seems to be associated with a poorer prognosis than the antifibrillarin-negative patient sera, suggesting that such analyses may contribute to a more reliable prognosis for these patients.

Identification of box H/ACA snoRNPs as a new nucleolar autoantigen

One antinucleolar patient serum was found to co-immunoprecipitate both U17 and E3 box H/ACA snoRNAs, indicating that components of this class of snoRNPs (albeit with low frequency) can also be autoantigenic in patients suffering from connective tissue diseases (see Fig. 2, lane 17 and Table 1). At present, four proteins have been identified that are known to associate with all box H/ACA snoRNPs: hGar1, NAP57/dyskerin, hNHP2 and hNOP10 [1416]. Immunoprecipitations using these four proteins translated in vitro did not generate conclusive data on the direct target of the autoantibodies (our unpublished observations). It is possible that the autoantibodies in this serum react with another, as yet unidentified, common subunit of box H/ACA snoRNPs. A chart review revealed that this patient suffered from gout and polyarticular non-erosive arthritis.

Anti-Th/To autoantibodies are associated with different connective tissue diseases

The Th/To autoantigen (RNase MRP/RNase P) has been reported to be recognized by SSc patients with a frequency of 10–14%[1,3,3234]. In this population of random antinucleolar sera, we detected autoantibodies with anti-Th/To specificity in 8% of the cases (n = 14). Co-precipitation of RNase MRP and RNase P RNAs was also observed in 15 other antinucleolar patient sera, but these sera also precipitated Ro RNP complexes. This suggests strongly that anti-La antibodies are responsible for the precipitation of the RNase MRP and RNase P RNAs by these sera, because the La autoantigen is known to associate (transiently) with all RNA polymerase III products, including the RNAs associated with RNase MRP, RNase P and Ro RNPs.

The well-known association of the anti-Th/To specificity with SSc and primary RP is supported by our data (Table 2). In addition, anti-Th/To reactivity was found in one patient with SLE, one patient with PM and one patient with severe weight loss. Chart review of these seven patients (Table 3) showed that the Th/To autoantigen is associated primarily with manifestations of RP (n = 5), decreased codiffusion (n = 4), oesophagus hypomotility (n = 4) and with sicca complaints (n = 6, of whom five suffered from xerophthalmia).

Forty per cent of antinucleolar reactivity remains uncharacterized

In addition to antibodies targeted to small nucleolar ribonucleoprotein complexes, antibodies to the PM/Scl100 autoantigen, which are also known to result in nucleolar staining in immunofluorescence, were also detected (n = 44; see Table 1). PM/Scl100 reactivity was found in patients suffering from DM, PM, SSc and DM/SSc overlap syndromes, as has been reported previously ([30] and references therein). In addition, reactivity against PM/Scl100 was detected in sera from patients suffering from RA, SLE, SjS and MCTD; see Table 1.

All antinucleolar patient sera were tested further for the presence of reactivity against the non-nucleolar U1 snRNP and Ro RNPs. Antibodies directed against U1 snRNP were detected in 23 sera, whereas 31 contained anti-Ro RNP reactivity. The presence of these antibodies associated with SLE, MCTD and SLE, SSc and SjS, respectively. However, in 61 of the antinucleolar sera none of the tested activities could be detected, suggesting that these sera recognize other (possibly yet uncharacterized) autoantigens.

Discussion

This study describes the analysis of a group of 172 antinucleolar sera from autoimmune patients for the presence of autoantibodies directed against small nucleolar ribonucleoprotein particles. Other investigators have reported previously that the existence of antinucleolar reactivities is particularly associated with SSc (reviewed in [13,5]). In contrast to these studies, which addressed the presence of antinucleolar reactivities in a particular disease, we selected patients’ sera based upon the presence of antinucleolar reactivities irrespective of the patients’ diagnoses. Chart review of 100 of these patients indicated that the occurrence of antinucleolar reactivity is not restricted to SSc, but is also found in patients suffering from other diseases. The association of antinucleolar reactivities with other systemic autoimmune diseases (i.e. SLE, PM/DM and RA) has been mentioned previously in two other studies, which reported the presence of these reactivities in control patient sera [35,36]. In addition, Fujii and coworkers showed that of 91 patient's sera containing antinucleolar reactivity, 21 were diagnosed with SLE, 21 with SSc, 14 with RA and 13 with SjS [37]. Taken together, our observations and those of other groups indicate that antinucleolar reactivities are not associated specifically with SSc and thus lowers the relevance of antinucleolar autoantibody detection for diagnostic purposes.

Autoantibodies precipitating the U3 snoRNA and possibly other box C/D snoRNPs have been reported in 5–10% of sera from patients suffering from primary RP, SSc and SSc-overlap syndromes. Antifibrillarin autoantibodies have been reported not only in SSc, but also in MCTD, SLE, RA and SjS [5,31]. The high frequencies of antifibrillarin reactivities in these diseases reported by Kasturi and coworkers [31], however, was neither confirmed by our data nor by those of Arnett and coworkers [5]. Only 9% of the antinucleolar sera precipitated box C/D snoRNPs, which appeared to correlate with antifibrillarin reactivity.

Our data indicate that two types of anti-box C/D snoRNP autoantibodies exist, one that is reactive with fibrillarin (i.e. precipitating the whole group of box C/D snoRNPs) and another that recognizes individual box C/D snoRNPs such as U3 and U8 snoRNP (this group is also referred to as ‘U3 only’ or ‘U8 only’). The fact that anti-U3-positive sera devoid of antifibrillarin reactivity exist implies that antifibrillarin-positive sera should not be referred to as anti-U3 positive. This distinction may be important for the diagnosis and prognosis of these patients, because our data and the work of Arnett and coworkers [5] indicate that antibodies directed to fibrillarin are associated with severe SSc, whereas antibodies directed to individual box C/D snoRNPs appear to be associated with arthritis and polymyositis. To investigate the potential clinical relevance of autoantibodies targeted to individual box C/D snoRNPs in arthritis and polymyositis, studies with larger groups of patients’ sera with these specificities have to be performed.

The presence of autoantibodies against fibrillarin in the group of sera targeting box C/D snoRNPs does not imply that autoantibodies directed to other protein subunits shared by all box C/D snoRNPs do not exist. These putative autoantigens include Nop5/58 and Nop56 [11,12]. Possible targets of patients’ sera directed against U3 snoRNP include only U3–55K, Mpp10 and human homologues of IMP3 and IMP4 [3840]. Other patients’ sera could be identified as ‘anti-U8 snoRNP only’. At present, only one protein is known that specifically binds U8 snoRNP: X29 [41]. The X29 protein has been identified in Xenopus laevis, but a homologous human protein is also likely to exist. Identification of the primary targets of box C/D snoRNP specific autoantibodies awaits a better and more complete characterization of the composition of these ribonucleoprotein complexes.

Autoantibodies against box H/ACA snoRNPs were detected in one patient suffering from gout and arthritis. The existence of autoantibodies against this class of snoRNPs has not been reported previously. Four proteins are known to associate specifically with this class of box H/ACA snoRNPs: hGar1, NAP57/dyskerin, hNHP2 and hNOP10 [1416]. We were unable to obtain conclusive data on which of these proteins is autoantigenic. We also cannot exclude the possibility that post-translational modifications are important for the recognition of (one of) these proteins by autoantibodies. Post-translational modifications (e.g. methylation or deimination of arginines) have been shown to be of critical importance for the efficient recognition of Sm proteins and RA-specific autoantigens [42,43]. Alternatively, an as yet unidentified box H/ACA snoRNP associated protein may be targeted by these autoantibodies. The generation of autoantibodies directed to box H/ACA snoRNPs appears to be a rare phenomenon. Northern blot analysis, however, may not be sensitive enough to detect low-titred autoanti-bodies in antinucleolar sera, because box H/ACA snoRNPs are much less abundant than, for example, U3 and U8 snoRNPs. Both the further characterization of the composition of box H/ACA snoRNPs and the identification of more patient sera with anti-box H/ACA snoRNP reactivities will be required to shed more light on the specificity of this new class of autoantibodies.

The observed prevalence of autoantibodies directed against the Th/To-autoantigen in SSc patients is in accordance with reported frequencies (8–14%) ([5,30,32,34], reviewed in [1,2]). Because we analysed antinucleolar patient sera rather than SSc sera, the present results demonstrate that anti-Th/To antibodies are also present in other autoimmune diseases, e.g. SLE and PM. Both Yamane and coworkers and Okano and coworkers showed that anti-Th/To antibodies are associated with localized SSc [32,34]. In our study, anti-Th/To antibodies are associated with RP, decreased codiffusion, oesophagus hypomotility and xerophthalmia, supporting the previous findings. The major autoantigen associated with the Th/To antigen is referred to as Th-40. This 40 kDa protein has been identified using immunoprecipitation of proteins from radioactively labelled cell extracts and UV-cross-linking analysis [4446]. Although three proteins (hPop1, Rpp30 and Rpp38) of the RNase MRP and RNase P complexes have been reported to be recognized by Th/To-sera, none of them seems to represent the Th-40 autoantigen ([47,48], reviewed in [20]), leaving the question of the identity of this autoantigen unanswered.

Surprisingly, a large group of patient sera (35–47%) did not recognize any of the tested small (nucleolar) ribonucleoprotein complexes. It seems likely to assume that in these sera autoantibodies are directed at present to other nucleolar autoantigens such as Ku, B23, NOR-90/UBF, RNA polymerase I or DNA topoisomerase I. Because none of these proteins/complexes is known to be stably associated with (sno)RNAs, autoantibodies targeting these antigens will not be detected with the RNA immunoprecipitation technique. These proteins/complexes have been reported to be autoantigenic in 10–25% of SSc sera (reviewed in [1,2]). Future research will have to evaluate whether these or other as yet unidentified autoantigens are targeted by patient sera that do not recognize small nucleolar ribonucleoprotein complexes.

Acknowledgments

We would like to thank Ben de Jong and Eugenie Terwindt for technical assistance and Femke van Karnebeek and Iris Ketel for assisting with the chart review. We also thank Vanda Pogacic and Dr Witold Filipowicz (Friedrich Miescher Institute, Basel, Switzerland) for the kind gift of the cDNAs of hGar1, dyskerin, hNHP2 and hNOP10. This research has been supported financially by the Council for Chemical Sciences of the Netherlands Organization for Scientific Research (CW-NWO).

REFERENCES

1. Fritzler MJ. Autoantibodies in scleroderma. J Dermatol. 1993;20:257–68. [PubMed]
2. Pollard KM, Reimer G, Tan EM. Autoantibodies in scleroderma. Clin Exp Rheumatol. 1989;7:S57–S62. [PubMed]
3. Kipnis RJ, Craft J, Hardin JA. The analysis of antinuclear and antinucleolar autoantibodies of scleroderma by radioimmunoprecipitation assays. Arthritis Rheum. 1990;33:1431–7. [PubMed]
4. Fimiani C. Systemic sclerosis: a woman disease. Mod Asp Immunobiol. 2001;1:233–7.
5. Arnett FC, Reveille JD, Goldstein R, et al. Autoantibodies to fibrillarin in systemic sclerosis (scleroderma). An immunogenetic, serologic, and clinical analysis. Arthritis Rheum. 1996;39:1151–60. [PubMed]
6. Tollervey D. Trans-acting factors in ribosome synthesis. Exp Cell Res. 1996;229:226–32. [PubMed]
7. Tollervey D, Kiss T. Function and synthesis of small nucleolar RNAs. Current Opinion Cell Biol. 1997;9:337–42. [PubMed]
8. Venema J, Tollervey D. Ribosome synthesis in Saccharomyces cerevisiae. Annu Rev Genet. 1999;33:261–311. [PubMed]
9. Balakin AG, Smith L, Fournier MJ. The RNA world of the nucleolus: two major families of small RNAs defined by different box elements with related functions. Cell. 1996;86:823–34. [PubMed]
10. Aris JP, Blobel G. cDNA cloning and sequencing of human fibrillarin, a conserved nucleolar protein recognized by autoimmune antisera. Proc Natl Acad Sci USA. 1991;88:931–5. [PMC free article] [PubMed]
11. Newman DR, Kuhn JF, Shanab GM, Maxwell ES. Box C/D snoRNA-associated proteins: two pairs of evolutionarily ancient proteins and possible links to replication and transcription. RNA. 2000;6:861–79. [PMC free article] [PubMed]
12. Lyman SK, Gerace L, Baserga SJ. Human Nop5/Nop58 is a component common to the box C/D small nucleolar ribonucleoproteins. RNA. 1999;5:1597–604. [PMC free article] [PubMed]
13. Ganot P, Caizergues-Ferrer M, Kiss T. The family of Box ACA small nucleolar RNAs is defined by an evolutionarily conserved secondary structure and ubiquitous sequence elements essential for RNA accumulation. Genes Dev. 1997;11:941–56. [PubMed]
14. Meier UT, Blobel G. NAP57, a mammalian nucleolar protein with a putative homolog in yeast and bacteria. J Cell Biol. 1994;127:1505–14. [PMC free article] [PubMed]
15. Dragon F, Pogacic V, Filipowicz W. In vitro assembly of human H/ACA small nucleolar RNPs reveals unique features of U17 and telomerase RNAs. Mol Cell Biol. 2000;20:3037–48. [PMC free article] [PubMed]
16. Pogacic V, Dragon F, Filipowicz W. Human H/ACA small nucleolar RNPs and telomerase share evolutionarily conserved proteins NHP2 and NOP10. Mol Cell Biol. 2000;20:9028–40. [PMC free article] [PubMed]
17. Gold HA, Craft J, Hardin JA, Bartkiewicz M, Altman S. Antibodies in human serum that precipitate ribonuclease P. Proc Natl Acad Sci USA. 1988;85:5483–7. [PMC free article] [PubMed]
18. Gold HA, Topper JN, Clayton DA, Craft J. The RNA processing enzyme RNase MRP is identical to the Th RNP and related to RNase P. Science. 1989;245:1377–80. [PubMed]
19. Bartkiewicz M, Gold H, Altman S. Identification and characterization of an RNA molecule that copurifies with RNase P activity from HeLa cells. Genes Dev. 1989;3:488–99. [PubMed]
20. van Eenennaam H, Jarrous N, van Venrooij WJ, Pruijn GJM. Architecture and function of the human endonucleases RNase P and RNase MRP. IUBMB Life. 2000;49:265–72. [PubMed]
21. Humbel RL. Detection of antinuclear antibodies by immunofluorescence. In: van Venrooij WJ, Maini RN, editors. Manual of biological markers of disease A2. Dordrecht: Kluwer Academic Publishers; 1993. pp. 1–16.
22. Arnett FC, Edworthy SM, Bloch DA, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum. 1988;31:315–24. [PubMed]
23. Tan EM, Cohen AS, Fries JF, et al. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 1982;25:1271–7. [PubMed]
24. Masi AT. Preliminary criteria for the classification of systemic sclerosis (scleroderma) Arthritis Rheum. 1980;23:581–90. [PubMed]
25. Vitali C, Bombardieri S, Moutsopoulos HM, et al. Preliminary criteria for the classification of Sjögren's syndrome. Results of a prospective concerted action supported by the European Community. Arthritis Rheum. 1993;36:340–7. [PubMed]
26. Bohan A, Peter JB. Polymyositis and dermatomyositis (first of two parts) N Engl J Med. 1975;292:344–7. [PubMed]
27. Bohan A, Peter JB. Polymyositis and dermatomyositis (second of two parts) N Engl J Med. 1975;292:403–7. [PubMed]
28. Verheijen R, Wiik A, De Jong BA, et al. Screening for auto-antibodies to the nucleolar U3- and Th (7–2) ribonucleoproteins in patients’ sera using antisense riboprobes. J Immunol Meth. 1994;169:173–82. [PubMed]
29. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. New York: Cold Spring Harbor Laboratory Press; 1989.
30. Brouwer R, Hengstman GJ, Vree Egberts W, et al. Autoantibody profiles in the sera of European patients with myositis. Ann Rheum Dis. 2001;60:116–23. [PMC free article] [PubMed]
31. Kasturi KN, Hatakeyama A, Spiera H, Bona CA. Antifibrillarin autoantibodies present in systemic sclerosis and other connective tissue diseases interact with similar epitopes. J Exp Med. 1995;181:1027–36. [PMC free article] [PubMed]
32. Okano Y, Medsger TAJ. Autoantibody to Th ribonucleoprotein (nucleolar 7–2 RNA protein particle) in patients with systemic sclerosis. Arthritis Rheum. 1990;33:1822–8. [PubMed]
33. Reddy R, Tan EM, Henning D, Nohga K, Busch H. Detection of a nucleolar 7–2 ribonucleoprotein and a cytoplasmic 8–2 ribonucleoprotein with autoantibodies from patients with scleroderma. J Biol Chem. 1983;258:1383–6. [PubMed]
34. Yamane K, Ihn H, Kubo M, et al. Antibodies to Th/To ribonucleoprotein in patients with localized scleroderma. Rheumatology. 2001;40:683–6. [PubMed]
35. Reimer G, Steen VD, Penning CA, Medsger TAJ, Tan EM. Correlates between autoantibodies to nucleolar antigens and clinical features in patients with systemic sclerosis (scleroderma) Arthritis Rheum. 1988;31:525–32. [PubMed]
36. Okano Y, Steen VD, Medsger TAJ. Autoantibody to U3 nucleolar ribonucleoprotein (fibrillarin) in patients with systemic sclerosis. Arthritis Rheum. 1992;35:95–100. [PubMed]
37. Fujii T, Mimori T, Akizuki M. Detection of autoantibodies to nucleolar transcription factor NOR 90/hUBF in sera of patients with rheumatic diseases, by recombinant autoantigen-based assays. Arthritis Rheum. 1996;39:1313–8. [PubMed]
38. Pluk H, Soffner J, Lührmann R, van Venrooij WJ. cDNA cloning and characterization of the human U3 small nucleolar ribonucleoprotein complex-associated 55-kilodalton protein. Mol Cell Biol. 1998;18:488–98. [PMC free article] [PubMed]
39. Lee SJ, Baserga SJ. Imp3p and Imp4p, two specific components of the U3 small nucleolar ribonucleoprotein that are essential for pre-18S rRNA processing. Mol Cell Biol. 1999;19:5441–52. [PMC free article] [PubMed]
40. Westendorf JM, Konstantinov KN, Wormsley S, et al. M phase phosphoprotein 10 is a human U3 small nucleolar ribonucleoprotein component. Mol Biol Cell. 1998;9:437–49. [PMC free article] [PubMed]
41. Tomasevic N, Peculis B. Identification of a U8 snoRNA-specific binding protein. J Biol Chem. 1999;274:35914–20. [PubMed]
42. Brahms H, Raymackers J, Union A, de Keyser F, Meheus L, Luhrmann R. The C-terminal RG dipeptide repeats of the spliceosomal Sm proteins D1 and D3 contain symmetrical dimethylarginines, which form a major B-cell epitope for anti-Sm autoantibodies. J Biol Chem. 2000;275:17122–9. [PubMed]
43. Schellekens GA, De Jong BA, van den Hoogen FH, van de Putte LB, van Venrooij WJ. Citrulline is an essential constituent of antigenic determinants recognized by rheumatoid arthritis-specific autoantibodies. J Clin Invest. 1998;101:273–81. [PMC free article] [PubMed]
44. Reimer G, Raska I, Scheer U, Tan EM. Immunolocalization of 7-2-ribonucleoprotein in the granular component of the nucleolus. Exp Cell Res. 1988;176:117–28. [PubMed]
45. Yuan Y, Tan E, Reddy R. The 40-kilodalton to autoantigen associates with nucleotides 21–64 of human mitochondrial RNA processing/7-2 RNA in vitro. Mol Cell Biol. 1991;11:5266–74. [PMC free article] [PubMed]
46. Li K, Smagula CS, Parsons WJ, et al. Subcellular partitioning of MRP RNA assessed by ultrastructural and biochemical analysis. J Cell Biol. 1994;124:871–82. [PMC free article] [PubMed]
47. Lygerou Z, Pluk H, van Venrooij WJ, Seraphin B. hPop1: an autoantigenic protein subunit shared by the human RNase P and RNase MRP ribonucleoproteins. EMBO J. 1996;15:5936–48. [PMC free article] [PubMed]
48. Eder PS, Kekuda R, Stolc V, Altman S. Characterization of two scleroderma autoimmune antigens that copurify with human ribonuclease P. Proc Natl Acad Sci USA. 1997;94:1101–6. [PMC free article] [PubMed]
49. Maxwell ES, Fournier MJ. The small nucleolar RNAs. Annu Rev Biochem. 1995;64:897–934. [PubMed]
50. Kiss T. Small nucleolar RNA-guided post-transcriptional modification of cellular RNAs. EMBO J. 2001;20:3617–22. [PMC free article] [PubMed]

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