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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Objective

To characterize the molecular identity of the Th/To autoantigen, which is targeted by autoantibodies in scleroderma and which is associated with the human RNase MRP and RNase P ribonucleoprotein complexes.

Methods

Proteins immunoprecipitated by anti-Th/To+ patient antisera from biotinylated total HeLa cell extracts were analyzed by immunoblotting. The association of autoantigenic proteins with the RNase MRP complex was analyzed by reconstitution experiments and ultraviolet crosslinking. The reactivity of patient sera with all known RNase MRP/RNase P proteins was analyzed by immunoprecipitation of the individual recombinant proteins.

Results

The previously defined Th40 autoantigen appeared to be identical to the Rpp38 protein. Paradoxically, Rpp38 did not bind to the P3 domain of the RNase MRP RNA, as suggested by previously published data for Th40, and only half of the anti-Th/To+ sera contained anti-Rpp38 reactivity. Two other RNase MRP/RNase P subunits, Rpp20 and Rpp25, were found to interact with the P3 domain. The previously reported 40-kd species associated with this domain appeared to consist of Rpp20 and/or Rpp25 associated with a nuclease-resistant RNA fragment. Finally, we demonstrated that almost all tested anti-Th/To+ patient sera contained autoantibodies to Rpp25 and hPop1, indicating that these proteins harbor the most frequently targeted Th/To determinants.

Conclusion

Our data unequivocally define the identity of the Th/To autoantigen and demonstrate that Th/To autoepitopes are found on several protein subunits of RNase MRP/RNase P.

Intracellular macromolecular complexes are important targets of autoantibodies found in the sera of patients with systemic autoimmune diseases. Autoantibodies directed against components of the nucleolus are often found in patients with scleroderma or scleroderma overlap syndromes (1). Examples of such nucleolar autoantigens are RNA polymerase I, fibrillarin, PM–Scl-100, and Th/To (for review, see ref. 1).

Anti-Th/To antibodies have been reported to occur in 4–13% of scleroderma patients (2, 3) and are defined as autoantibodies that are able to immunoprecipitate the human RNase MRP and RNase P ribonucleoprotein particles (3–5). The RNase MRP ribonucleoprotein particle was originally identified by its capacity to cleave a mitochondrial RNA in vitro to generate RNA primers for mitochondrial DNA replication (6). In addition, RNase MRP has been shown to cleave at site A3 within the first internal transcribed spacer of the precursor of ribosomal RNA in Saccharomyces cerevisiae (7–9).

In many respects, the RNase MRP complex is related to the RNase P complex. The RNase P complex is required for the removal of the 5′-end of precursor transfer RNA and is suggested to be involved in the processing of the precursor ribosomal RNA in internal transcribed spacer 2 (for review, see ref. 10). The RNase MRP and RNase P complexes both function as site-specific endonucleases, contain an RNA component that has been proposed to adopt a similar secondary structure, and share several protein subunits (for review, see ref. 10). Currently, 10 proteins have been reported to be associated with the human RNase MRP and/or RNase P complexes: hPop1, hPop5, Rpp14, Rpp20, Rpp21, Rpp25, Rpp29/hPop4, Rpp30, Rpp38, and Rpp40 (11–18).

The first autoantigenic polypeptide associated with RNase MRP/RNase P was identified via immunoprecipitations with patient sera using 35S-methionine– or 3H-leucine–labeled HeLa cell extracts (3, 19–22) and has been designated Th40, referring to its apparent molecular weight of 40 kd. Yuan and colleagues subsequently showed that a protein with a molecular weight of 40 kd could be ultraviolet-crosslinked (UV-crosslinked) to the P3 domain of RNase P and MRP RNA (23), suggesting that Th40 is bound to this domain. In a previous study, we showed that 2 proteins with apparent molecular weights of 20 kd and 25 kd, respectively, can be UV-crosslinked to the P3 domain of RNase MRP RNA (nucleotides 22–67; see Figure 1) and that a protein with a molecular weight of ∼40 kd, Rpp38, is bound to a centrally located region of RNase MRP RNA (nucleotides 86–176; see Figure 1) (24).

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Figure 1. Secondary structure of the human RNase MRP RNA. This structure is based on phylogenetic comparison and chemical modification data (28). The P3 domain (nucleotides 22–67) and the central domain (nucleotides 86–176) are shaded.

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In this study, we determined the identity of the Th40 autoantigen and identified the proteins carrying Th/To determinants as well as their binding sites on RNase MRP RNA.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Immunoprecipitation using biotinylated HeLa cell extracts.

Total HeLa cell extract was prepared as described previously (25). The extract was dialyzed against 50 mM NaHCO3, pH 8.3, 0.5 mM dithioerythritol (DTE), and 0.5 mM phenylmethylsulfonyl fluoride (PMSF) prior to the addition of 1 mg NHS-LC-biotin (Pierce, Rockford, IL) per 20 mg of total protein. After incubation on ice for 2 hours, the reaction was stopped with 50 mM Tris HCl, pH 8.0. Finally, the buffer was changed to 10 mM Tris HCl, pH 8.0, 0.5 mM DTE, 0.5 mM PMSF, 0.1M KCl, 0.01% Nonidet P40 (NP40), and 20% glycerol by dialysis.

For each immunoprecipitation, 20 μl patient 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% NP40) for 1 hour 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% NP40). Biotinylated HeLa extract was added and incubated for 2 hours at 4°C. After 4 washes with IPP150, coprecipitating proteins were eluted with sodium dodecyl sulfate (SDS)–sample buffer, resolved by 13% SDS–polyacrylamide gel electrophoresis (SDS-PAGE), and blotted onto nitrocellulose membranes.

Detection of biotinylated proteins was performed using horseradish peroxidase (HRP)–conjugated streptavidin (Dako, Glostrup, Denmark) and visualization by chemiluminescence.

In vitro reconstitution.

In vitro–transcribed 32P-labeled RNase MRP RNA (full-length and deletion mutants MRP22–67, MRP1–82, and MRP 86–176), and U3 small nucleolar RNA (U3 snoRNA) were allowed to associate with their protein components in an in vitro reconstitution assay and analyzed as described previously (24).

UV-crosslinking.

UV-crosslinking experiments to study RNA–protein interactions were performed as described previously (24). After reconstitution and UV irradiation, the reconstituted complexes were treated with either 10 μg of RNase A for 1 hour at 37°C or 100 units of RNase T1 for 30 minutes at 30°C per immunoprecipitation. The RNA–protein complexes were subjected to immunoprecipitation, analyzed by SDS-PAGE, and visualized by autoradiography.

Immunoblotting analysis.

Patient sera were used in a 5,000-fold dilution, polyclonal rabbit sera were used in a 100-fold dilution, and mouse monoclonal antibodies SW5 (anti-La) and 9A9 (anti-U1A and anti-U2B′′; Euro-Diagnostica, Arnhem, The Netherlands) were used in 50-fold and 100-fold dilutions, respectively. Detection was performed using HRP-conjugated rabbit anti-human IgG, swine anti-rabbit IgG, or rabbit anti-mouse IgG (Dako) as secondary antibodies and visualization by chemiluminescence.

Recombinant (His)6–tagged Rpp38 was obtained by expression of Rpp38 complementary DNA (cDNA) in pQE-30 (Qiagen, Leusden, The Netherlands) in M15[pREP4] cells according to the manufacturer's instructions.

In vitro translation.

For in vitro transcription-translation of hPop1, hPop4, and hPop5, the hPop1–pT7–7TT, VSV–hPop4, and VSV–hPop5 DNA constructs (11, 15, 16) were linearized using Sal I and Fsp I. The cDNA of Rpp30 (17) was subcloned in pGEM-3Zf(+) and linearized using Xba I. The open reading frames of Rpp14, Rpp20, Rpp21, Rpp25, and Rpp40 were subcloned using a polymerase chain reaction–based strategy using cDNA constructs as templates (12–14) in pCR4-TOPO (Invitrogen, Leek, The Netherlands) and linearized using Sma I digestion. In vitro transcription was performed as described previously, using either T7 or T3 RNA polymerase (24). In vitro translation of these transcripts was performed in the presence of 35S-methionine using rabbit reticulocyte lysates (Promega, Leiden, The Netherlands) or wheat germ extracts.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Th40 autoantigen is identical to Rpp38.

Sera displaying antinucleolar reactivities from 172 patients were screened for their capacity to coimmunoprecipitate RNase MRP RNA, RNase P RNA, U3 snoRNA, U8 snoRNA, U22 snoRNA, U1 small nuclear RNA (U1 snRNA), and Y1 small cytoplasmic RNA (van Eenennaam H, et al: unpublished observations). Sera that immunoprecipitated RNase MRP and RNase P RNA were designated anti-Th/To+ sera. To study the polypeptides precipitated by these anti-Th/To+ sera, immunoprecipitations with biotinylated total HeLa cell extracts were performed. Coprecipitating proteins were analyzed by Western blot analysis and visualized using HRP-conjugated streptavidin. As shown in Figure 2A, lanes 1 and 2, anti-Th/To+ sera coprecipitated several polypeptides, the most prominent being a 40-kd protein. This 40-kd protein was not precipitated by control patient sera (lanes 3–12), which displayed other patterns of coprecipitating proteins. Identical immunoprecipitation patterns to those shown in Figure 2A, lanes 1 and 2 were observed using 3 reference anti-Th/To+ sera, indicating that the 40-kd band coprecipitated by anti-Th/To+ sera by definition represents the Th40 autoantigen (results not shown).

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Figure 2. Immunoprecipitation and immunoblotting of the Th40 autoantigen. A, Autoantigens were immunoprecipitated from a biotinylated HeLa cell extract using patient sera containing the following reactivities: anti-Th/To (lanes 1 and 2, sera designated Th4 and Th8, respectively), anti–U3 small nucleolar RNP (U3 snoRNP; lanes 3 and 4), anti–Ro RNP (lanes 5 and 6), anti–U22 snoRNP (lanes 7 and 8), anti–U8 snoRNP (lanes 9 and 10), and anti–U1 small nuclear RNP (lanes 11 and 12). Coprecipitating proteins were resolved by 13% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, blotted to nitrocellulose, and visualized with horseradish peroxidase (HRP)–conjugated streptavidin. On the left, the positions of molecular weight markers are indicated and the arrow points to the band designated Th40. B, The blot shown in A was probed with antibodies specific for Rpp40, Rpp38, hPop4, La, and U1A. Bound antibodies were visualized by incubation with HRP-conjugated secondary antibodies followed by chemiluminescence. The arrows point to the position of the band designated Th40 (see A).

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To shed more light on the identity of the coprecipitating proteins, the Western blot shown in Figure 2A was reprobed with antibodies directed against Rpp40, Rpp38, and hPop4 (protein subunits of the RNase MRP and RNase P complexes), against the La protein (a component of the Ro RNPs), and against the U1A protein (a component of the U1 snRNPs). As shown in Figure 2B, lanes 1 and 2, Rpp40, Rpp38, and hPop4 were specifically coprecipitated by the anti-Th/To+ sera. The specific immunoprecipitation of the La protein by the anti–Ro RNP RNA sera (lanes 5 and 6) and of U1A by the anti–U1 snRNP sera (lanes 11 and 12) further substantiated the specificity of the assay. Overlays of the total protein signals (Figure 2A, lanes 1 and 2) and the immunoblotting signals (Figure 2B, lanes 1 and 2) indicated that the Th40 autoantigen is identical to the Rpp38 protein.

Th40/Rpp38 binds the central domain of RNase MRP RNA.

Previously, we showed that Rpp38 binds to the central domain of the RNase MRP RNA (24). Paradoxically, Yuan and colleagues have demonstrated that the major Th/To autoantigen, with an apparent molecular weight of 40 kd, could be UV-crosslinked to the P3 domain of RNase MRP and P RNA (23). To clarify this issue, in vitro–transcribed 32P-labeled MRP22–67 (containing the P3 domain), MRP86–176 (containing the central domain), and U3 snoRNA as a control were incubated with a HeLa cell extract, and reconstituted complexes were analyzed by immunoprecipitation using the sera from 12 anti-Th/To+ patients (Th1–Th12). As shown in Figure 3A, almost all anti-Th/To+ sera coimmunoprecipitated the P3 domain of RNase MRP RNA (MRP22–67), whereas none of them immunoprecipitated the U3 control RNA. Only 5 of the sera efficiently immunoprecipitated the central domain of RNase MRP RNA (lanes 4, 5, 7, 10, and 11), which would be consistent with the presence of anti-Rpp38 reactivity in the sera of these 5 patients. The presence of anti-Rpp38 autoantibodies in these sera was confirmed by Western blot analysis using recombinant Rpp38 protein (Figure 3B). Taken together, these results show that the Th40/Rpp38 protein binds to the central domain of RNase MRP RNA and that another, more frequently targeted, autoantigenic component interacts with the P3 domain.

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Figure 3. In vitro reconstitution of RNase MRP complexes. A,32P-labeled U3 small nucleolar RNA, MRP86–176, and MRP22–67 RNA were incubated in HeLa cell extracts, and reconstituted complexes were immunoprecipitated using 12 anti-Th/To+ patient sera (Th1–Th12, lanes 1–12). Coprecipitating RNAs were isolated from the immunoprecipitates and analyzed by gel electrophoresis and autoradiography. In lane 13, RNA isolated from 10% of the input fraction from the Th7 precipitation was loaded. B, Anti-Rpp38 reactivity present in the anti-Th/To+ patient sera (Th1–Th12, lanes 1–12) was determined by immunoblotting using recombinant Rpp38 protein.

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Rpp20 and Rpp25 interact with the P3 domain of RNase MRP RNA.

Our previous UV-crosslinking experiments showed that 2 polypeptides with molecular weights of 20 kd and 25 kd associate with the P3 domain of the RNase MRP RNA (24). Reconstitution experiments using rabbit antisera raised against Rpp20 (13) and Rpp25 (18) demonstrated that both Rpp20 and Rpp25 can bind to the P3 domain (results not shown), suggesting that these proteins represent the previously observed UV-crosslinked species (24). To investigate this issue, UV-crosslinking experiments using 32P-labeled MRP22–67 RNA, followed by immunoprecipitations using Th7 and control patient antibodies and analysis on a high-resolution SDS-PAGE gel were performed. The results in Figure 4A, lane 4 show that the band previously referred to as MRP25 (24) is actually composed of 3 bands, whereas MRP20 is a single band. Both anti-Rpp20 and anti-Rpp25 antibodies immunoprecipitated these 4 bands, confirming that Rpp20 and Rpp25 represent the previously identified crosslinked proteins (lanes 2 and 3). The multiple bands observed in this experiment probably represent the Rpp20 and Rpp25 proteins covalently attached to different nuclease-resistant RNA fragments. Preimmune rabbit sera and control patient sera did not immunoprecipitate any UV-crosslinked proteins (lanes 1 and 5).

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Figure 4. Ultraviolet-crosslinking (UV-crosslinking) of Rpp20 and Rpp25 to the P3 domain of RNase MRP RNA. A, Reconstituted complexes containing 32P-labeled MRP22–67 RNA were subjected to UV-crosslinking and RNase A treatment, followed by immunoprecipitation using preimmune rabbit serum (lane 1), anti-Rpp20 serum (lane 2), anti-Rpp25 serum (lane 3), patient serum Th7 (lane 4), and control patient serum (lane 5). Coprecipitating proteins were resolved on a high-resolution 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel, and crosslinked proteins were visualized by autoradiography. The positions of MRP20 and MRP25 are indicated on the right. B, UV-crosslinked complexes containing 32P-labeled MRP22–67 RNA were treated with either RNase A (lanes 1–3) or RNase T1 (lanes 4–6) and subsequently immunoprecipitated with patient sera Th7 (lanes 2 and 5) and Th8 (lanes 3 and 6) or control sera (lanes 1 and 4). Coprecipitating proteins were analyzed by 13% SDS-PAGE and visualized by autoradiography. Note that the resolution of the gel shown here is lower than that of the gel in A. The positions of molecular weight markers are shown on the left. The positions of MRP20 and MRP25 are indicated on the right, and the open arrows mark the position of the 40-kd doublet obtained after UV-crosslinking and RNase T1 treatment.

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The fact that Th40/Rpp38 does not and Rpp20 and Rpp25 do bind to the P3 domain of RNase MRP RNA raised the question as to why other investigators previously observed a 40-kd species after UV-crosslinking using the P3 domain (23). To clarify this issue, we performed a UV-crosslinking experiment under the conditions applied by Yuan and colleagues. The main difference in the experimental conditions of our analyses was the use of RNase T1 rather than RNase A to degrade the RNA after UV irradiation. RNase T1 cleaved single-stranded regions of RNA after a G residue, while RNase A cleaved single-stranded RNAs preferentially after a C or U residue. As shown in Figure 4B, lanes 5 and 6, the RNase T1 digestion resulted in a doublet migrating at ∼40 kd, consistent with previously reported data (23). UV-crosslinking followed by RNase A treatment, which was performed in parallel, resulted in the immunoprecipitation of the 20- and 25-kd species (lanes 2 and 3), confirming our previous results (24). Similar findings were obtained when rabbit anti-Rpp20 and anti-Rpp25 antibodies were used instead of patient antibodies (results not shown).

From these data, we conclude that the difference in apparent molecular weights of the crosslinked species is due to the use of different ribonucleases. In the case of RNase T1, the protein(s) remained most likely covalently bound to a relatively large part of the MRP22–67 RNA, whereas in the case of RNase A, only a few nucleotides remained bound to the protein(s). The resistance of the P3 domain to RNase T1 digestion was confirmed by incubating in vitro–transcribed MRP22–67 in a HeLa cell extract, followed by treatment with either RNase A or RNase T1, immunoprecipitation with anti-Th/To+ sera, and RNA analysis by gel electrophoresis. While the size of MRP22–67 was barely affected by RNase T1 treatment, RNase A degraded MRP22–67 to small (oligo)nucleotides (results not shown).

Rpp25 and hPop1 are the most targeted autoantigenic RNase MRP components.

The results described above demonstrate that anti-Th/To+ sera are frequently reactive with proteins associated with the P3 domain and that Rpp20 and Rpp25 bind to this fragment of RNase MRP RNA. To investigate the antigenicity of these proteins, in vitro–translated, 35S-labeled Rpp20 and Rpp25 were subjected to immunoprecipitations with anti-Th/To+ patient sera and 2 human control sera. As shown in Figure 5, panel G, Rpp20 was detectably immunoprecipitated only by Th5, and not by the other 11 anti-Th/To+ sera. In contrast, the majority of the anti-Th/To+ patient sera immunoprecipitated in vitro–translated Rpp25 (panel E), indicating that Rpp25 represents an important Th/To autoantigen bound to the P3 domain of RNase MRP RNA.

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Figure 5. Immunoprecipitation of RNase MRP proteins by anti-Th/To+ patient sera. 35S-labeled in vitro–translated hPop1 (panel A), Rpp40 (panel B), Rpp30 (panel C), hPop4 (panel D), Rpp25 (panel E), Rpp21 (panel F), Rpp20 (panel G), hPop5 (panel H), and Rpp14 (panel I) were subjected to immunoprecipitation with patient sera Th1–Th12 (lanes 2–13), 2 control patient sera (lanes 14 and 15), and rabbit antisera that were raised against each of these proteins (lane 1). Coprecipitating proteins were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and autoradiography.

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In addition, all other protein subunits of the RNase MRP and/or RNase P complexes, except Rpp38, the recognition of which is documented in Figure 3, were tested in similar immunoprecipitation assays using Th1–Th12. As shown in Figure 5, panels A–D, F, H, and I, these proteins were recognized with variable frequencies. Almost all anti-Th/To+ sera recognized hPop1 (panel A), about half of the sera recognized Rpp30 (panel C), hPop5 (panel H), and Rpp14 (panel I), and reactivity with Rpp40, hPop4, and Rpp21 was observed in only a minority of these sera (panels B, D, and F).

In conclusion, these data indicate that patient sera precipitating RNase MRP and RNase P complexes (anti-Th/To+) most frequently recognize the hPop1 and Rpp25 proteins contained in these complexes.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

The results of previous studies demonstrated that patient sera containing reactivity directed to both the RNase MRP and RNase P complexes were able to immunoprecipitate a protein of ∼40 kd, named Th40 (3, 19–21). Here, we demonstrate that Th40 is identical to the Rpp38 protein. Although almost all anti-Th/To+ patient sera were able to (co)immunoprecipitate the Th40 autoantigen, only approximately half of the anti-Th/To+ sera actually contained reactivity directed to the Rpp38 protein (see Figure 3). The immunoprecipitation, even under relatively stringent conditions, of Th40/Rpp38 by patient sera that do not recognize the Rpp38 protein is apparently caused by the recognition of other autoantigenic components in the RNase MRP and RNase P complexes. The RNase P holoenzyme has been shown to be stable up to concentrations of 500 mM KCl (17), suggesting that under these conditions the intact complexes, rather than individual protein subunits, are coprecipitated.

Rpp38 binds to the region between nucleotides 86 and 176 of the RNase MRP RNA (results not shown; see ref. 24). The interaction of Rpp38 with the corresponding domain of the RNase P RNA is suggested by the observation that Rpp38 was able to interact with the intact RNase P RNA but not with a fragment comprising nucleotides 1–74 (26). These observations seem to contradict what has been reported by Yuan et al (23) and Liu et al (27). However, we demonstrated that the reported 40-kd doublet observed after UV-crosslinking actually represents Rpp25 and/or Rpp20 covalently linked to the (almost) complete P3 domain, because this domain is rather resistant to RNase T1 cleavage. This is consistent with the results of RNase protection experiments described by Liu and coworkers (27) and is not surprising in view of the almost complete absence of single-stranded guanosines in this part of RNase MRP RNA (see Figure 1).

The association of Rpp25 with the P3 domain and of Rpp38 with the central domain of the RNase MRP RNA has been suggested to occur in the RNase P complex as well. Data reported by Guerrier-Takada et al (18) and by Jiang et al (26) indeed support these locations for the Rpp25 and Rpp38 binding sites on the RNase P RNA. No direct binding of recombinant Rpp20 and the P3 domain of RNase P RNA could be demonstrated, suggesting that other factors may be involved in stabilizing this interaction (18). The observation that almost no reactivity against Rpp20 was found in anti-Th/To+ antisera, while the majority of these sera were shown to contain reactivity against Rpp25, suggests that Rpp20 is most probably coprecipitated with anti-Rpp25 when anti-Th/To+ patient sera are used in reconstitution and UV-crosslinking experiments. This strongly suggests that Rpp25 may function as a stabilizing factor for the interaction of Rpp20 with the P3 domain.

The ability of patient sera to immunoprecipitate the RNase MRP and RNase P complexes has been designated anti-Th/To reactivity (3–5). The availability of cDNA clones encoding most, if not all, protein subunits of these complexes now allows a more detailed analysis of the anti-Th/To autoantibody specificities present in patient sera. The results of our study demonstrate that all protein components may be targeted by anti-Th/To autoantibodies, although the frequencies of recognition display large differences. The anti-Th/To reactivity can now be subclassified according to the presence of autoantibodies against each of the identified RNase MRP/RNase P protein subunits or according to the recognition of a specific combination of these subunits. The analysis of a larger collection of anti-Th/To+ sera will be required to investigate the potential clinical relevance of this subclassification and to determine whether such a classification may aid in establishing the diagnosis and/or prognosis of patients showing anti-Th/To reactivity.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

The authors thank T. Welting for technical assistance and Drs. H. Pluk and R. Hoet for the development of procedures to biotinylate cell extracts. We also thank Drs. C. Guerrier-Takada, S. Altman (Yale University, New Haven, Connecticut), and N. Jarrous (Hebrew University–Hadassah Medical School, Jerusalem, Israel) for their kind gifts of rabbit antisera raised against the RNase P proteins and cDNA encoding these protein subunits. We are grateful to Drs. R. Karwan (Vienna University Institute of Tumor Biology, Vienna, Austria), J. Craft (Yale University), and T. Mimori (Kyoto University, Kyoto, Japan) for providing us with anti-Th/To+ reference patient sera.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES