Autoimmunity Reviews 2 (2003) 313–321
Autoantigenicity of nucleolar complexes Tim J.M. Welting, Reinout Raijmakers, Ger J.M. Pruijn* Department of Biochemistry 161, Nijmegen Centre for Molecular Life Sciences, University of Nijmegen, P.O. Box 9101, NL-6500 HB Nijmegen, The Netherlands Accepted 4 February 2003
Abstract Autoantibodies targeting nucleolar autoantigens (ANoA) are most frequently found in sera from patients with systemic sclerosis (SSc, also designated scleroderma) or with SSc overlap syndromes. During the last decade an extensive number of nucleolar components have been identified and this allowed a more detailed analysis of the identity of nucleolar autoantigens. This review intends to give an overview of the molecular composition of the major (families of) autoantigenic nucleolar complexes, to provide some insight into their functions and to summarise the data concerning their autoantigenicity. 䊚 2003 Elsevier Science B.V. All rights reserved. Keywords: Antinucleolar antibodies; Nucleolar autoantigens; Nucleolus; snoRNP; Systemic sclerosis
1. Introduction The nucleolus of eukaryotic cells was first described in the early 19th century. It typically has the highest concentration of mass of any part of the cell. The nucleolus is the site of rRNA synthesis and ribosome assembly, processes in which a large variety of enzymatic activities are involved. Autoantibodies targeting nucleolar antigens such as fibrillarin, ThyTo, PMyScl, NOR-90yUBF and RNA polymerase I are most frequently found in patients suffering from SSc or SSc overlap syndromes. The occurrence of these antibodies in patient sera may be helpful in establishing the diagnosis and prognosis of the disease. For example, autoantibodies directed against fibrillarin iden*Corresponding author. Tel.: q31-243-616-847; fax: q31243-540-525. E-mail address:
[email protected] (G.J. Pruijn).
tify a subgroup of SSc with a poor prognosis. The increased knowledge on the composition of the nucleolus and the enzymatic macromolecular complexes residing in the nucleolus, allows a more detailed analysis of the molecules targeted by autoantibodies. The molecular dissection of these complexes and the characterisation of their components often leads to the identification of new autoantigenic molecules, as for example the hPop1 protein, which is associated with the RNase P and RNase MRP complexes and the U3 snoRNP specific U3-55k protein. The analysis of such molecules may lead to the discovery of new correlations between the production of autoantibodies and clinical symptoms with diagnosticyprognostic value. 2. The nucleolus In an eukaryotic nucleus, a number of subnuclear structures can be discerned, such as nucle-
1568-9972/03/$ - see front matter 䊚 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S1568-9972Ž03.00029-6
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oli, nuclear speckles and Cajal (coiled) bodies. The most pronounced sub-nuclear compartment, which is even visible by using an ordinary lightmicroscope, is the nucleolus. Like other nuclear bodies, the nucleoli lack membrane boundaries. The nucleolus functions as an assembly and transcription centre for the biosynthesis of ribosomes w1x. The majority of the ribosomal RNA genes are transcribed in this sub-nuclear compartment and the primary transcripts are subsequently processed, modified and together with at least eighty imported ribosomal protein subunits, assembled into the two ribosomal subunits. In an actively growing HeLa cell approximately 400 000 ribosomal proteins enter the nucleolus each minute, which means that approximately 10 000 ribosomal subunits are synthesised per minute and a similar number of newly assembled ribosomal subunits have to be exported to the cytoplasm in the same period of time. Because the nucleolus represents the highest concentration of mass in the cell, one might think that nucleoli are quite rigid structures, but the opposite is true. Recent data show that nucleoli and their associated constituents are highly dynamic and that their formation requires ongoing transcription of the ribosomal DNA w1x. Electron microscopy reveals that in a nucleolus several substructures can be discerned. Transcription of rRNA genes is believed to take place at the transition zone between the fibrillar centre and the dense fibrillar component, whereas the cascade of pre-rRNA processing events and the assembly of ribosomal subunits progresses from the dense fibrillar centre to the surrounding granular region w1x. Nucleoli are only present in nuclei of interphase cells. When a cell enters mitosis, rDNA transcription is silenced and as a result ribosome biosynthesis stops. Because nucleolus formation is dependent on rDNA transcription, the nucleolus disassembles during mitosis. At the end of mitosis, in the newly formed nuclei, nucleoli reassemble by the recruitment of nucleolar components to so-called nucleolar organising regions (NORs), concomitant with the initiation of transcription by RNA polymerase I. Besides its major function in the biosynthesis of ribosomes, it has been shown that nucleoli also participate in other RNA processing and complex assembly events. In the nucleolus a number of
small RNAs, transcribed by RNA polymerase III, are processed and assembled into ribonucleoprotein complexes. A subset of pre-tRNAs is processed in this sub-nuclear compartment and the maturation of the spliceosomal U6 snRNA occurs in the nucleolus w2,3x. Furthermore, the nucleolus plays a role in mRNA production w4x, the maturation of the signal recognition particle (SRP) RNA and the partial assembly of the SRP with a subset of its protein constituents. Interestingly, three other small RNAs, which are transcribed by RNA polymerase III outside the nucleolus in the nucleoplasm, are transported to the nucleolus: 5S rRNA, which is incorporated into large ribosomal subunits, RNase MRP RNA, the RNA component of the ribonucleoprotein enzyme RNase MRP, which participates in pre-rRNA processing, and RNase P RNA which is structurally related to RNase MRP RNA and is involved in the processing of pre-tRNA w5x. In addition to its involvement in the biosynthetic processes described above, the nucleolus represents a sequestering compartment for regulatory proteins, such as mitogenic growth factors and cell-cycle and growth-factor related proteins. 3. Nucleolar macromolecular complexes As described above, ribosome biosynthesis is the major function of the nucleolus. To perform this task properly, the nucleolus has an impressive machinery at its disposal. The onset of ribosome biosynthesis is the transcription of pre-rRNA in the nucleolus by RNA polymerase I. This prerRNA transcript is polycistronic and by means of a complex series of endo- and exonuclease activities, the pre-rRNA is processed to 18S, 5.8S and 28S rRNA (Fig. 1). To obtain fully functional RNAs, they undergo extensive covalent nucleotide modification. The cleavage reactions and nucleotide modifications are mediated by a large group of ribonucleoprotein complexes, termed small nucleolar RNPs (snoRNPs), and a protein complex possessing 39™59 exoribonuclease activity, called the exosome w6x. Based on structural and functional differences, snoRNPs can be divided into three classes. Box CyD snoRNPs are involved in 29-Omethylation and endonucleolytic cleavage, box Hy
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Fig. 1. Nucleolar macromolecular complexes involved in ribosomal RNA maturation. Schematic representation of the different processing steps that occur in the nucleolus to generate mature ribosomal RNAs (18S, 5.8S and 28S). Methylation (Me), pseudouridinylation (C), endoribonucleolytic cleavages and exoribonucleolytic processing are catalysed by nucleolar complexes of the box CyD snoRNP family, the box HyACA snoRNP family, RNase MRPyRNase P, and the exosome, respectively. The lower part of the figure shows schematic structures of these complexes. The black elements represent RNA molecules, in which the conserved boxes are indicated. The dotted line in the RNase MRPyRNase P RNA depicts a long range basepairing interaction. The coloured elements represent the (common) protein components of these complexes. In the case of the exosome the hCs14 protein is attached to the backside of the complex and thus not visible in this model. The way in which the PMyScl-100 and the hDis3 (which is not included in the figure) proteins associate with the exosome is unknown. Proteins specifically associated with the U3 snoRNP and the telomerase complex are listed below the schematic models for these complexes.
ACA snoRNPs mediate pseudouridinylation and RNase MRP and P are involved in endonucleolytic processing events w5,7x. 3.1. Box CyD snoRNPs This class of snoRNPs is characterised by the presence of two short conserved sequence elements in the snoRNAs, referred to as box C and box D. The box CyD snoRNPs function in posttranscriptional 29-O-methylation of pre-rRNA w8x. All box
CyD snoRNPs share protein subunits that are typical for this class of snoRNPs and for some box CyD snoRNPs specific proteins have been identified (Fig. 1). The major box CyD specific core protein is fibrillarin, which is likely to be the methyltransferase responsible for the 29-O-methylation of pre-rRNAs. Other conserved core proteins of the box CyD snoRNP complexes are Nop58, Nop56 and 15.5K w7x. Besides their function in 29-O-methylation, some box CyD snoRNPs have been demonstrated or suggested to function
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in the cleavage of pre-rRNA, e.g. the U3 and U8 snoRNPs w9x. Recently, it has been shown that the U3 snoRNP-specific hU3-55k protein requires the 15.5K. protein for its association with the U3 complex. In the past few years, other human U3 snoRNP specific proteins have been identified, like Mpp10, Imp3 and Imp4 w10x. 3.2. Box HyACA snoRNPs The box HyACA snoRNAs are characterised by the presence of a common ‘hairpin–hinge–hairpintail’ structure and contain the conserved boxes H and ACA. The box HyACA snoRNAs direct the pseudouridinylation of certain uridine nucleotides in pre-rRNA by means of complementary sequences present in the hairpin structures w11x. Until now, four box HyACA snoRNP associated proteins have been described, GAR1, dyskerin, NOP10 and NHP2 (Fig. 1) w7,12x. These proteins, as well as a box HyACA motif, are also found in the telomerase complex, a ribonucleoprotein reverse transcriptase responsible for adding one strand of simple sequence DNA repeats to the ends of linear chromosomes w13x. Besides the common box Hy ACA proteins, two telomerase specific proteins have been identified, TEP1 and the reverse transcriptase hTERT. Other box HyACA snoRNPspecific protein components have not been identified yet. 3.3. RNase MRP and RNase P RNase MRP and RNase P represent a third class of small nucleolar RNPs. RNase P is a precursortRNA processing enzyme which is ubiquitously present in every investigated organism. RNase MRP however, is restricted to eukaryotic cells. Most probably, RNase MRP and RNase P are evolutionarily related. In spite of the low sequence homology between the RNA subunits of RNase MRP and RNase P, their secondary structures indicate that they adopt similar conformations w5x. Until now, ten proteins have been described that associate with both complexes (Fig. 1) and up to now no human RNase MRP or RNase P specific proteins have been described. Recently, it has been shown that certain mutations in conserved struc-
tural regions and upstream sequence elements of the human RNase MRP RNA gene can lead to the pleiotropic disorder Cartilage-Hair Hypoplasia w14x. 3.4. Exosome The PMyScl particle or exosome is a complex consisting of at least 11 protein subunits, all of which have been cloned, sequenced and partially characterised. The 39™59 exoribonuclease activity of the exosome plays a major role in the degradation and processing of many RNAs, not only in nucleolus, but also in other cellular compartments. Its subunits contain 39™59 exoribonuclease activity andyor RNA-binding properties. The exosome is both structurally and functionally homologous to PNPase, the major component of the bacterial degradosome. Both complexes assemble into a ring-like structure (Fig. 1). For two exosome components, PMyScl-100 and hDis3p, no significant sequence homology with bacterial degradosome components has been found and it is still unknown how these proteins associate with the exosome. PMyScl-100 and hDis3p may be associated with the human exosome only in specific subcellular compartments w15,16x. 3.5. Other nucleolar macromolecular complexes Many other complexes of macromolecules reside in the nucleoli, some of which are also part of the ribosome synthesis machinery (e.g. RNA polymerase I), whereas the function and composition of others, like the p105-p42 complex, is still not or poorly documented. 4. Nucleolar autoantigens Because of the dynamic nature of the nucleolus it is difficult to define true nucleolar proteins or complexes. In general, a patient serum is considered reactive with nucleolar components when in immunofluorescence experiments (usually on HEp-2 cells) the staining of the nucleolar compartment is at least as high as that of the surrounding nucleoplasm. These types of reactivities are most common in patients with SSc or SSc overlap
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syndromes. Since the nucleolus contains different compartments with specific functions and components, the staining of specific subnucleolar regions can give a clue on the identity of the antigen that is targeted. A list of autoantigenic nucleolar proteins and complexes that have been reported is shown in Table 1 and the most extensively characterised antigens are discussed in more detail below. SSc is a chronic, multisystem connective tissue disorder, characterised by a variety of circulating autoantibodies. The specificity of these autoantibodies can be associated with clinical subgroups of SSc. For example, anti-centromere antibodies are often present in patients with limited skin involvement whereas anti-topoisomerase I antibodies identify a subgroup with early diffuse and pulmonary involvement. The presence of more of these reactivities in the same serum is very uncommon and the prevalence of most reactivities is generally quite low. 4.1. Techniques used to detect anti-nucleolar autoantibodies As described above the main technique to determine whether a patient serum contains anti-nucleolar reactivity is indirect immunofluorescence (IIF). With this method it is, however, difficult to discriminate between reactivities with different nucleolar autoantigens. To identify the target of the autoantibodies, other methods can be used like ELISA, Western blotting or immunoprecipitation using radioactively labelled autoantigens or metabolically labelled cell extracts. In the case of autoantibodies targeting antigens associated with ribonucleoprotein complexes (snoRNPs), frequently immunoprecipitations are performed and the coprecipitation of specific RNAs is monitored by Northern blot hybridisation. 4.2. Box CyD snoRNPs Autoantibodies reactive with box CyD snoRNPs, present in patients with SSc, systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), can be divided in two groups, those capable of co-precipitating all box CyD snoRNAs and
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those co-precipitating a specific snoRNA (e.g. only U3 or only U8). Of all anti-nucleolar sera, 9–52% have been reported to co-precipitate all box CyD snoRNAs, suggesting that these sera contain antibodies against proteins associated with all box Cy D snoRNAs (fibrillarin, Nop56, Nop58 and 15.5K). Several studies have shown that in these sera often antibodies against fibrillarin are present, although also other common box CyD snoRNP proteins might be targets of autoantibodies w17,18x. When autoantibodies specifically co-precipitate only one of the box CyD snoRNAs, the target must be one of the snoRNP-specific protein components. For anti-U3 snoRNP-specific sera, it has been shown that autoantibodies against the U3 snoRNP specific protein hU3-55K are present. In addition, both anti-hU3-55K and anti-hMpp10 autoantibodies have been found in anti-fibrillarin positive sera w18x. Besides U3 snoRNP specific sera, also sera have been found that specifically co-precipitate the U8 snoRNA, but the fact that no U8 specific human proteins have been characterised yet, makes it difficult to identify the target of these autoantibodies. In patients with SSc, the presence of anti-box CyD snoRNP autoantibodies is often correlated with diffuse cutaneous involvement and a poor prognosis w17x. 4.3. Box HyACA snoRNPs Autoantibodies against box HyACA snoRNPs are only rarely present in patient sera. In one patient suffering from gout and arthritis, antibodies were detected capable of co-precipitating at least two of the box HyACA snoRNAs, U22 and E7, suggesting that these antibodies are reactive with a common box HyACA snoRNP component. These antibodies, however, were not reactive with in vitro translated GAR1, dyskerin, NHP2 and NOP10, suggesting that either another, yet unidentified, common box HyACA protein is the target of these antibodies or that post-translational modification of these proteins is required for their reactivity w17x. Another box HyACA snoRNP, the human telomerase complex, might be autoantigenic in patients with hepatocellular carcinoma or other types of cancer w19x. In addition, the human telomerase complex can associate with the La and
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Table 1 Nucleolar autoantigens Autoantigen
Autoantigenic subunits
Diseaseydisorder*
References
Box CyD snoRNPs U3 snoRNP U8 snoRNP
Fibrillarin hU3-55K hMpp10 Unknown Unknown hTERT hPop1 Rpp25 Rpp38 Rpp30 hPop5 Rpp14 PMyScl-100 PMyScl-75 hRrp4p hRrp42p hRrp46p – Subunits shared with RNA pol II and III – – – – – – – Ku DNA-PKcs – – Unknown
SSc (4–13%), SLE, RA SSc, DM, RA
w17,18x w18x w18x w17x w17x w19x w17,24x
Box HyACA snoRNPs Telomerase RNase PyMRP
Exosome (PMyScl)
Nucleolin (C23) RNA polymerase I NOR90yhUBF Nop52 Nucleophosmin (B23) ASE-1 Ngp-1 NOA36 No55 DNA-PK La Topo IyScl-70 (p105-p42)
RA, PM Gout, arthritis HCC SSc (5–14%), RP, SLE, PM
PMySSc (24–31%), PM (5–8%), SSc (3%) PMySSc, PM, SSc PMySSc, PM, SSc Cancer SLE, GVHySSc SSc (22%)
w26x w26x w27x w28x w29x
SSc, SS, RA N.D. Breast cancer SLE Breast cancer RA Interstitial cystitis, prostate cancer (15%) PM, SSc SLE, PM, SSc SLE (20%), SS (;40–60%), SSc (5%) SSc (;25%) RA, SSc
w30x w31x w32x w33x w34x w35x w36x w37x w38x w29,39x w29x w40x
w15,25x
* Numbers between brackets indicate the percentages of patients that have been reported to be positive for reactivity with the autoantigen. The abbreviations used are: SSc, systemic sclerosis; SLE, systemic lupus erythematosus; RA, rheumatoid arthritis; PM, polymyositis; RP, primary Raynaud’s phenomenon; HCC, hepatocellular carcinoma; GVH, graft vs. host disease; SS, Sjogren’s syndrome.
Ku autoantigens in vivo w20,21x and the complex contains a protein immunologically related to the human autoantigen Ro60 w22x. 4.4. RNase MRP and RNase P Autoantibodies recognising RNase MRP and RNase P (referred to as anti-Thyanti-To) are predominantly found in patients suffering from SSc (5–14%), often with limited cutaneous involvement, but a poor prognosis. In addition, these antibodies are also present in other autoimmune
diseases like primary Raynaud’s phenomenon (RP), systemic lupus erythematosus (SLE) and polymyositis (PM) w17x. Since these autoantibodies are capable of immunoprecipitating the RNAs associated with RNase P and RNase MRP from HeLa cell extracts, but not the corresponding in vitro transcribed naked RNAs, the reactivity most likely is directed to one of the shared proteins subunits of these complexes. A reactivity commonly observed in these sera is directed to a protein of 40 kDa, originally designated Th40 w23x. This antigen was recently shown to be
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identical to the Rpp38 protein subunit of RNase PyMRP. Antibodies against the RNase PyMRP subunits Rpp38, Rpp30, hPop5 and Rpp14 proteins are found in approximately half of the anti-ThyTo positive sera and the majority of the sera are reactive with the Rpp25 and hPop1 protein subunits w23x. Especially antibodies to hPop1 have been shown to be highly specific for patients with SSc andyor RP w24x. Although no human proteins specific for either RNase P or RNase MRP have been described, one serum capable of co-precipitating only the RNase MRP RNA and not the RNase P RNA has been reported w23x. 4.5. Exosome In approximately 24–31% of the patients with the overlap syndrome of polymyositis and scleroderma (PMySSc or PMyScl) antibodies are found directed to the human exosome, also called the PMyScl complex. The prognosis for disease development in patients with these autoantibodies is generally good w15x. In addition, these antibodies are found in ;6% of all myositis patients w25x and ;3% of all SSc patients w15x. These autoantibodies are mostly reactive with the protein subunits PMyScl-100 and PMyScl-75 and to a lesser extent with hRrp4p and hRrp42p w26x. Until recently, it was believed that anti-PMyScl-75 reactivity was found only in a subset of the PMyScl100 positive sera, but recent data show that the recombinant PMyScl-75 protein used for these tests was incomplete and that the complete PMy Scl-75 protein is even more autoantigenic than PMyScl-100 (Raijmakers et al., manuscript in preparation). Finally, autoantibodies recognising the exosome subunit hRrp46p have recently been found in 10–33% of patients suffering from melanoma, lung cancer, or prostate cancer w27x. 5. Concluding remarks Many different nucleolar proteins have been identified that are the target of autoantibodies in SSc and other (autoimmune) diseases. Most of these proteins are associated with enzymatic complexes that play a role in the biogenesis of ribosomes, in particular of the ribosomal RNAs. The
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prevalence and clinical relevance of autoantibodies directed to the majority of these nucleolar proteins still has to be investigated. Approximately 60% of patient sera containing anti-nucleolar reactivity target the major (families of) autoantigenic nucleolar complexes, box CyD snoRNPs, box HyACA snoRNPs, RNase MRPyRNase P or the exosome. The remainder of the antinucleolar sera are directed either to other nucleolar autoantigens listed in Table 1 or to yet unidentified antigens w17x. Many of the identified nucleolar autoantigens still need to be characterised at the molecular level and with regard to their clinical relevance. As a consequence, the techniques currently used to identify the targets (immunoprecipitation and especially ELISA) fail to detect a portion of the autoantibodies. Because the role of post-translational modification of autoantigenic molecules in the recognition by autoantibodies becomes more and more important, future research will also be focussed on this aspect of nucleolar autoantigens. Acknowledgments The authors wish to thank Walther van Venrooij (Department of Biochemistry, University of Nijmegen, The Netherlands) for helpful comments on the manuscript. This work was supported by the Council for Chemical Sciences of the Netherlands Organisation for Scientific Research (CW-NWO). Take-home messages ● ANoA predominate in but are not restricted to systemic sclerosis (SSc) and SSc overlap syndromes. ● ANoA target many different autoantigens which are present in all compartments of the nucleoli, but may also reside in other subcellular compartments. ● The major nucleolar autoantigenic molecules are associated with complexes involved in the biosynthesis of ribosomes. ● More studies using larger patient groups are required to reveal the value of ANoA in the clinical differentiation between diseases and inflammatory conditions.
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The World of Autoimmunity; Literature Synopsis Cytotoxic T lymphocyte-associated antigen 4 blockade in patients with metastatic melanoma Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is an immunoregulatory molecule capable of downregulating T cell activation. Phan et al. (Proc Natl Acad Sci USA 2003;100:8372) treated 14 patients with metastatic melanoma by using serial intravenous administration of a fully human anti-CTLA-4 antibody (MDX-010) in conjunction with subcutaneous vaccination with two modified HLA-A*0201-restricted peptides from the gp100 melanoma-associated antigen, gp100:209–217(210M) and gp100:280–288 (288V). Following this treatment protocol, autoimmune manifestations occurred in six patients (43%), which included dermatitis, enterocolitis, hepatitis and hypophysitis. Objective cancer regression was noted in 3 of 14 patients (21%; two complete and one partial responses). This study provides hope for use of anti-CTLA-4 in cancer immunotherapy, and further emphasizes the association between cancer and autoimmunity.
Vitiligo in a restricted isolated community Birlea et al. (Pigment Cell Res 2003;16:603) studied 39 subjects having vitiligo in a rural Romanian community of 1710 subjects (around 2.3% prevalence). Most patients had generalized and progressive form of vitiligo, and familial aggregation could be detected in 36% of them. Associated autoimmune diseases in a significant portion of these patients were thyroid diseases, rheumatoid arthritis, and diabetes mellitus. Using structured questionnaires, the authors concluded that the disease appeared to worsen during summer and work load, and thus the presence of vitiligo in this community might be related to genetics, and also to environmental factors such as sun exposure and stress.