Identification of autoantibodies to RNA polymerase II. Occurrence in systemic sclerosis and association with autoantibodies to RNA polymerases I and III

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Identification of autoantibodies to RNA polymerase II. Occurrence in systemic sclerosis and association with autoantibodies to RNA polymerases I and III
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  Identification of Autoantibodies to RNA Polymerase   Occurrence in Systemic Sclerosis and Association with Autoantibodies to RNA Polymerases   and III Michito Hirakata, * Yutaka Okano,t Uttam Pati, Akira Suwa, * Thomas A. Medsger, Jr.,t John A. Hardin, andJoe Craft**Section of Rheumatology, Department of Internal Medicine,Yale University School of Medicine, New Haven, Connecticut 06510; tDivision of Rheumatology and Clinical Immunology, Departmentof Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261; and ODepartment of Medicine, Medical College of Georgia, Augusta,Georgia30912-3100 Abstract In this study, autoantibodies to RNA polymerase II from sera of patients with systemic sclerosis havebeen identified and characterized. These antibodies immunoprecipitated polypep- tides of 220 kD  IIA)and145 kD  IIC),the two largest sub- units of RNA polymerase II, and bound both subunits in immu- noblots. These polypeptides wereimmunoprecipitated by the anti-RNA polymerase II monoclonal antibody 8WG16, which recognizes the carboxyl-terminal domain of the 220-kD sub- unit, and their identity to theproteins bound by human sera was confirmed in immunodepletion studies. Sera with anti-RNA polymerase II antibodies also immunoprecipitated proteins that were consistent with components of RN polymerases I and III. In vitro transcription experiments showed that the hu- man antibodies werean effectiveinhibitor of RN polymerase II activity.  n indirect immunofluorescence studies, anti-RNA polymerase II autoantibodies stainedthe nucleoplasm, as ex- pected from the known location of RNA polymerase II, and colocalized withthe anti-RNA polymerase II monoclonal anti- body. The human sera alsostainedthenucleolus,thelocation of RNA polymerase I. From a clinical perspective,theseantibod- ies werefound in 13 of 278 patients with systemic sclerosis, including 10 with diffuse and three with limited cutaneous dis- ease, but were not detected in sera from patients with otherconnective tissue diseases and from normal controls. We con- clude that anti-RNA polymerase II antibodies are specific to patients with systemic sclerosis, and that they are apparently associated with antibodies to RNA polymerases I and III. These autoantibodies may be useful diagnostically and as a probe for further studies of thebiologicalfunction of RNA poly- merases.  J. Clin. Invest. 1993. 91:2665-2672.) Key words: RNA polymerases   autoantibodies   antinuclear antibodies. systemic sclerosis   autoimmunity IntroductionThe three RN polymerases  RNAP) catalyze the transcrip- tion of different sets of genes into RNA. RNAP I synthesizes Addresscorrespondence to Joe Craft, M.D., Section of Rheumatology,610 LCI, Yale University School of Medicine, Box 3333, 333 Cedar St., New Haven, CT 06510. Receivedfor publication 30 July 1993 and in revisedform 28 De- cember 1992. ribosomal R precursors in nucleoli. RNAP II synthesizes theprecursors of mRNAs and most of the smallnuclear RNAs which are found in ribonucleoprotein  RNP particlesthat me- diate pre-mRNA splicing. RNAP III synthesizessmall RNAs, including 5S ribosomal RNAs and transfer RNAs. Thesemul- timeric enzymes are comprised of 8-14 polypeptide subunits, dependingupon the species from which they are isolated  1, 2); however, the details of their complex structures are still being defined. In mammalian cells, RNAP II contains two large polypep- tides  220 kD and 145 kD) thatare highly conserved across species, and atleast six smaller subunits   1, 2). Several of the latter may beshared among RNAP I,II, and III, whereas the larger two subunits are unique to RNAP 11   1-5). The largest 220-kD polypeptide of RNAP II migrates as aphosphorylated subunit of 240 kD, a nonphosphorylated subunit of 220kD, and a proteolyticderivative of 180 kD, referredto as IIO, IIA, and IIB, respectively, when analyzed in SDS-polyacrylamide gels  4, 6-8). Recent studies have revealed that the 220-kD subunitcontains a unique carboxyl-terminal domain  CTD consisting of a Tyr-Ser-Pro-Thr-Ser-Pro-Ser heptapeptide that is repeated 52times in mammalian cells  2, 4, 7, 9). The CTD is released by proteolysis generating the 1 80-kD subunit  10, 11). Thissequence is highly phosphorylated, required for cell viability, and thought to mediate promoter specific transcrip- tion 12, 13). Thompson and colleagues havedeveloped a monoclonal antibody referred toas 8WG16  anti-CTD mAb that reacts with this highly conserved carboxyl-terminal repeat of the 220-kD subunit   14, 15). Some patientswith the connective tissue disease systemic sclerosis  scleroderma) have been shown to produce autoanti- bodies that bind RN polymerase I 16, 17). Corresponding autoantibodies to RNAP II and III have not yet been identified.  n thepresent study, we have identified and characterized auto antibodies from sera of patients with systemic sclerosisthat recognize RNAP II subunits. Methods Human autoimmune sera and RNAP II monoclonal antibody. Sera were obtained from 84 patients with systemic sclerosis  diffuse cutane- ous systemic sclerosis, limited cutaneous systemic sclerosis, and sys- temic sclerosis overlap syndromes) who were identified from a review of patient records of the Section of Rheumatology, Yale University 1. Abbreviations used in this paper: CMV, cytomegalovirus; CTD, car- boxyl-terminal domain; MCTD, mixed connective tissue disease; PM/ DM, polymyositis or dermatomyositis; RNAP, RN polymerase; RNP, ribonucleoprotein; snRNP, smallnuclear RNP; TBS, Tris-buff- ered saline. Autoantibodies to RNA Polymerase II 2665 J. Clin. Invest. © TheAmerican Society forClinical Investigation, Inc. 0021-9738/93/06/2665/08  2.00 Volume 91, June 1993, 2665-2672  School of Medicine, andfrom 194 patients withsystemic sclerosis who were seen at theUniversity of Pittsburgh Schoolof Medicineduring1986-1988. All patients seen at Yale University and 167 of the 194 patients seen at University of Pittsburgh fulfilled the preliminary crite-ria of the American College of Rheumatology for classification asdefi- nite systemic sclerosis   18 . Sera from 217 patients with other systemic autoimmune diseases, including SLE  n = 126 19 , polymyositis/ dermatomyositis  PM/DM n = 64)  20 , andmixed connective tissue disease  MCTD n =27)  2 1  , and sera from 30 normal individ-uals wereused as controls. A murine monoclonal antibody  8WG16; anti-CTD mAb 14 , raised against wheat germ RNAP II, which reacts with the carboxyl-terminal repeat domain of the largest subunitof RNAP II, was a kind gift from Dr. Nancy E. Thompson  McArdle Laboratory for Cancer Research, University of Wisconsin).This mAb binds the 220-kD large subunit of RNAP II in immunoblotsand a synthetic heptapeptide repeat corresponding to the CTD region of this polypeptide. It also inhibits promoter-specific transcription by RNAP II  14, 15 . Indirect immunofluorescence. Human sera were diluted 1:40 in Tris-buffered saline  TBS)   150 mM NaCl, 10 mM Tris HCl, pH 7.4 , and were screened by indirect immunofluorescence  22 using HEp-2 cells  Immunoconcepts,Sacramento, CA) with goat anti-human IgG fluorescein isothiocyanate conjugate  Sigma, St. Louis, MO and goat anti-mouse IgG Texas r conjugate Tago, Inc., Burlingame, CA). Each well wasviewed at a magnification of 1,000 on an immunofluores- cence microscope  Carl Zeiss, Oberkochen, Germany) to detect cyto- plasmic, nuclear, and nucleolar staining. Preparation of radiolabeled cell extracts. HeLa cells  2-2.5 X 105 cells/ml were radiolabeled for 14 h in deficient media, as previously described, with[35S ]methionine   10 qCi/ml cells ICN Biomedical Inc., Irvine, CA for analysis of proteins and with [32P]orthophosphate   10 pCi/ml cells ICN Biomedical Inc. for RNA analysis 22,23 . Cells were collected by centrifugation, washed in TBS   10 mM Tris Cl, pH 7.5, 150 mM NaCI),and sonicated three times each for 40 s in NET-2 buffer  50 mM Tris Cl, pH 7.5, 150 mM NaCl, 0.05 Nonidet P-40) with a sonifier  Branson Sonic, Danbury, CT) at setting 3. The sonicatedlysate was centrifuged at 15,000 g for 10 min to remove cellular debris. Immunoprecipitation of radiolabeled cell extracts. Immunoprecipi- tation of radiolabeled cell extracts was performed as previously de- scribed  22-24 . Briefly, 10 ,ul of patient sera was mixed with 2 mg of protein A-sepharose CL-4B  Pharmacia Inc., Piscataway, NJ) in 500 /II of immunoprecipitation buffer  IPP)   10 mM Tris Cl, pH 8.0, 500 mM NaCI, 0.1 Nonidet P-40) and incubatedwith end-over-end rotation  Labquake shaker; Lab Industries,Berkeley, CA) for 2 h at 4°C. The sepharose particles with adsorbed IgG were washed four times in 500 1AI of IPP buffer using 10-s spins in a microfuge tube, and resuspended in 400  ul of NET-2 buffer. For protein studies, antibody-coated sepharose beads were mixed with 400 ,l of [35S]methionine-labeled extracts  8 X 106 cells and rotated at 4°C for 2 h. Afterfour washes with NET-2, the sepharose beadswere resuspended in SDS sample buffer  2 SDS, 10 glycerol, 62.5 mM Tris Cl, pH 6.8, 0.005 bromophenol blue . After heating  90°C for5 min), the proteins were fractionated onSDS- 10 polyacrylamide gels, enhanced with 0.5 M sodium salicylate, and dried; labeled proteins wereanalyzed by autoradiography. Immuno- precipitated RNAs wereanalyzed as previouslydescribed  22, 23 . Antigen depletion. [ 35S ] methionine-labeled HeLa cell extracts were incubatedwith 20-160 tg of anti-CTD mAb bound to protein A Sepha- rose CL-4B for 2 h at 4°C to depletethe extracts of RNAP II. The depleted supernatant was preincubated with protein A-sepharose to absorb any antibody excess, and thenused in immunoprecipitation asthe antigen source for patient anti-RNAP II sera. Inthe reciprocal experiment, [35S ] methionine-labeled HeLa cell extracts were absorbed with 40 gig of IgG isolated froman anti-RNAP II human serum as described and then probed with the anti-CTD mAb. The same amount of IgG from a normal human serumandfrom an SLE serum with the anti-Sm specificity  25)were included ascontrols. In vitro transcription from the adenovirus major late promoter andfrom the cytomegalovirus  CMV) immediate early promoter. Dignam extracts from HeLa cells wereused for studying transcription from both these promoters  26 . For transcription from the adenovirus ma- jor late promoter, the plasmid vector pBR 322 containing the 877-bp fragment with the major late promoter region  from position -680 to position + 197 was prepared as previouslydescribed 27 . The gel- purified fragmentwas used as a template in a transcription assay to generate a single 197-nucleotide transcript. Transcription from the CMV immediate early promoter Promega, Madison, WI) was carried out according to the protocol of the manufacturer; the CMV run-off product was363 nucleotides. Purification of RNAP II subunits.Purification of RNA polymerase II from calf thymus was carried out by a modification of the method of Hodo and Blatti  28 , except that all buffers contained 0.1 Nonidet P-40. Fractions from the phosphocellulose column containing RNAP II activity were pooled and applied to a heparin-agarose column equili- brated with buffer containing 60 mM ammonium sulfate. Active frac- tions were eluted with buffer containing 500 mM ammonium sulfate. The activefractions werepooled and passed through a SephadexA-25 column. RNAP II was further purified by glycerol gradient centrifuga- tion. RNAP II polypeptides were analyzed on gradient  7-15 ) poly- acrylamide gels and the 145-kD polypeptide of RNAP II was separated from the gel byan electroeluter  Bio Rad, Richmond, CA). Purifica- tion of the 220-kD polypeptide of RNAP II from HeLa cells was carried out by immunoaffinity chromatography with the anti-CTD mAb 8WG 16 14, 15 . The purified 145-kDand 220-kD subunits wereused as substratesfor immunoblots. Immunoblots. Immunoblots were performed by a modificationof the Towbin et al. procedure  29 . Results Immunoprecipitation. Serum samples from 278 patients with systemic sclerosis and 217 patients with other connective tissue diseases, and samples from 30normalblooddonors were testedfor the presence of antibodiesagainst RNAP II in radioimmu- noprecipitation assays. An initial prototype serum Wa was identified, whichimmunoprecipitated polypeptides of 240,220, and 145 kD, which correspond to known subunits of RNAP 11  1, 2 Figs. 1 and 2, lane 2 . The anti-CTD mAb 8WG 16 also immunoprecipitated the same polypeptides  Figs. 1 and 2, lane 1 . It should be noted that this antibodybinds to the CTD of the 220- and 240-kD subunits, and that the latter polypeptide runs as a smearon gels since it is highly phosphor- ylated. The 145-kD subunit is included in the immunoprecipi- tates formed with this mAb, since it is part of the multimeric RNAP II complex   1, 2 . It should also be noted that the anti- CTD mAb immunoprecipitated the 23-kD subunit  Fig. 1, lane 1 , one of the smallersubunits shared among all three polymerases  1, 2 . In contrastto thefindings with the  WG 16 mAb andserum Wa, control sera and a control mAb  the Y 12 anti-Sm mouse mAb did not immunoprecipitate any of these polypeptides  Fig. 2, lanes 3-7). The band of   200 kD bound by anti-Sm antibodies that migrates slightly faster than the 220- kD subunitof RNAP II is a component of the 20S U5 snRNP  30)  Fig. 2, compare lanes 3 and 4with lanes 1 and 2 . Among the 278 sera from patients with systemic sclerosis, 13 producedimmunoprecipitates containing these same poly- peptides, as demonstrated by nine representative sera  Fig. 1, lanes 2-10). 12 of the13 sera  including the prototype serum Wa also immunoprecipitated six additionalpolypeptides with 2666 Hirakata, Okano, Pati, Suwa, Medsger, Hardin, and Craft  -Q C) Mr  ac E j F f)I Human sera kD 2 ; ~~~~~~0 mv .W * ONtE~ *W.~_ 97.4 69- ii  6 30   --   2 3 4 5 6 7 8 9 10 11 121314 Figure 1 [ S ]methionine-labeled HeLa cell proteins immunoprecipitated with hu- man sera and the anti-CTD mAb, followed by resolution in an SDS-7 polyacrylamide gel  24). The anti-CTD mAb immunopre- cipitated RNAP II polypeptides of 240, 220, and 145 kD  lane 1). The anti-RNAP II prototype serum Wa  lane2) and other representative sera from patients with sys- temic sclerosis immunoprecipitated the identical polypeptides  lanes 3-10).These sera also immunoprecipitated polypeptides of 190,155,138,126, 44, and 23 kD  lanes 2-8 and 10), except for oneserum  lane 9), which did not react with the 190-,126-, and44-kD polypeptides. Sera presumably containing anti-RNAP I/RNAP III anti- bodies immunoprecipitated the same six polypeptides, but not those of 240, 220, and 145 kD  lanes 11-14). M, shows mo- lecular weight markers; asterisks and arrows denote the240-, 220-, and 145-kD subunits of RNAP II -o I- m Mr icz -o E c\1J -j C/) cn YIcu l IO   S I -M 200-  M __ 97.4 69- _ 46- _   1 2 3 4 567 Figure 2. [35S]methionine-labeled HeLa cell proteins immunoprecipi- tated with the anti-CTD mAb, human sera, and the Y12 anti-Sm mAb, followed by resolution in an SDS-7 polyacrylamide gel  24). The anti-CTD mAb immunoprecipitated RNAP II polypeptides of 240,220, and 145 kD  lane 1); the bands at   100 and70 kD rep- resent degradationproducts that were not reproducibly seen in other immunoprecipitatesformed with the anti-CTD mAb. The anti- RNAP II prototype serum Wa immunoprecipitated the identical polypeptides as the CTD mAb  lane 2). The Y12 anti-Sm mAb and an anti-Sm patient serum immunoprecipitated the U5-specific dou- blet of 200 kD (30) and other snRNP proteins lanes 3 and 4). An anti-La patient serum immunoprecipitated the 48-kDLa protein  lane 5), an anti-Ku patient serum immunoprecipitated the70- and 80-kD Ku polypeptides lane 6), and a normal human serum (NHS) did not immunoprecipitate any specific polypeptides. M, shows mo lecular weight markers. molecular masses of 190, 155, 138,126,44, and 23kD, which correspond to known subunits of RNAP I and RNAP III  1)  Fig. 1 examples in lanes 2-8and 10). In contrast, the serum shown in lane 9 immunoprecipitated only the 155-,138-, and23-kD polypeptides, alongwith the 240-,220-, and145-kD subunits of RNAP II Sera from another 54 patients with systemic sclerosis im- munoprecipitated the six smaller proteins (190, 155, 138, 126,44, and 23 kD), without immunoprecipitationof the240-, 220-, and145-kD subunits of RNAP II  Fig. 1 examples inlanes 11-14). None of thecontrol specimens, consisting of 30normal human sera and217 autoimmune sera from patients with SLE, MCTD, and PM/DM were able to immunoprecipi- tate the RNAP II profile, or t 190-,155-,138-,126-,44-, and23-kD proteins which, by their size, areconsistent with compo- nents of RNAP I and III When studies were carried out with HeLa cells labeled with [32P]orthophosphate  22, 23), no small RNAs were specifically immunoprecipitated by 13 anti- RNAP II sera, in contrastto their ready immunoprecipitation by positivecontrol sera  anti-Sm, anti-U   RNP, anti-Ro, and anti-La; data not shown). Immunodepletion studies. To confirm that the anti-CTD mAb and the putative anti-RNAP II sera identified above rec- ognized the same cell components, extracts depleted of RNAP II by absorptionwith the anti-CTD mAb next were used in immunoprecipitation experiments. This mAb depleted ex- tracts of the 240-,220-, and145-kD polypeptidesrecognized by the prototype serum Wa in a dose-dependent manner  Fig. 3, lanes 3-11);however, polypeptides of 190,155,138,126, 44, and 23 kD, presumedcomponents of RNAP-I and III were still immunoprecipitated. Identical results wereobtained with two other sera that immunoprecipitated the RNAP II subunits, Autoantibodies to RNA Polymerase II 2667 kD  D C c E  9_ c O0   Z .9 CL a a) E -0 U co Mr Im a £2 E H C X 2X4X8X   ::: D 200- w 97.4- bI 69- 46- 30- 4IM   234 5   78 9 10 11 along with the putative polypeptides of RNAP I and III. Ina reciprocal fashion, when HeLa cell extracts were absorbed with serum Wa and then probed with the anti-CTD mAb, the RNAP II subunits of 240, 220, and 145 kD were not immuno- precipitated  Fig. 3, lane 2). In contrast, depletion of radiola- beled HeLa cellextracts using autoantibodies of different im- munologic specificities  e.g., anti-Sm antibodies) did not affect specific antigen recognition by the anti-CTD mAb  Fig. 3, lane 1). These findings indicate that the epitope boundby the mono- clonal antibody to RNAP II resides on the same macromolecu- lar structure recognized by the autoantibodies from sera of pa- tients with systemic sclerosis. Similar immunodepletion studies using human sera con- firmed thatthe six smaller polypeptides  190, 155, 138,126, 44, and 23 kD immunoprecipitatedby the anti-RNAP II sera were identicalto those bound by the additional 54 sera from systemic sclerosis patients that did not bind RNAP II  data not shown . Immunofluorescence studies. Indirect immunofluores- cence was next used to identify the cellular location of the antigen s) bound by the sera studied here. As shown by a repre- sentative example, all 13 systemic sclerosis serathat immuno- precipitated RNAP II subunits produced nucleoplasmic and nucleolar staining that was indistinguishable from that ob- served with a control antibody to RNAP I  Fig. 4, A and B, respectively). In contrast, staining produced with the anti- CTD mAb was limited to the nucleoplasm  Fig. 4, C). There- fore, to furtherdefine the site of binding of the human anti- RNAP II antibodies, double immunostainingwasperformed using the patient sera and the anti-CTD mAb. The location of the both antibodies was then determined with goat anti-hu- man IgG conjugated to fluorescein isothiocyanate and goat £2   C-CZ C E° 0 E~~~~   Co  E C/ 0 A m o in: 0 X2X4X8X 0 -a CTD rAD  x = 20 Lig ~~~~~~~~~~~~~~~~~3S Figure 3. Depletion of RNAP II from [355J- methionine-labeled HeLa cell extracts by the anti- CTD mAb and human anti-RNAP II antibodies. The anti-CTD mAb immunoprecipitated the RNAP II large subunits  240-,220-, and 145-kD polypeptides; denoted by asterisks and arrows) in a dose-dependent manner  lanes 3-6 , and effectivelydepletedthese proteins from solution before immunoprecipitation with the prototype human serum Wa  lanes 7-11). Immunoprecipitation studies shown in lanes 8-11 wereperformed with the serum Wa and the superna- tants were derived from the immunoprecipitates shown in lanes3-6. A control immunoprecipitation performed with an anti-Sm serum from a patient with SLE is shown in lane 12. The supernatant of the latter immunoprecipitation still contained RNAP II large subunits  240-, 220-, and 145-kD polypeptides; de- noted by asterisks), which couldbe immunoprecipi- tated with the anti-CTD mAb  lane 1), whereas thesupernatant absorbedwith serum Wa from lane7 did 12 not contain these polypeptides lane 2). anti-mouse IgG conjugated to Texas red. As shownby the orange fluorescence, the nucleoplasm was stained by both the anti-CTD mAb and anti-RNAP II sera  Fig. 4, D . In contrast, cells stained with normal human sera did not produce immuno- fluorescence, while those stained with anti-Sm antibodies  from human sera and the Y12 mAb produced only finely speckled nuclear fluorescence  data not shown . Inhibition of transcription from the adenovirus major late promoter and from the CMV immediate early promoter with IgGfrom systemic sclerosis sera. These studies were designed to test the ability of anti-RNAP II antibodies from patients to inhibit promoter-directed transcription in vitro. In these tran- scription assays, 10 Ail ofa Dignam extract was incubated for 30 min with IgG  from 50 ,l of patient serum bound to protein A-sepharose beads, before the addition of NTPs and the DN template. The beadsthen were removed with centrifugation and the supernatant was used for the transcription from the DN segments containing the adenovirus major late promoter and the CMV immediate early promoter. RN was purified andanalyzedon a 5 polyacrylamide/7 M urea gel  26). IgGfrom serum Wa and from asecond serum that immunoprecipi- tated RNAP II subunits significantly inhibited RNAP II activ- ity  Fig. 5 A,lane2; Fig.5 B, lanes 3 and 4). In contrast, normal human IgG  Fig. 5 A and B, lane1) andIgGfrom an anti-Sm patient serum did not produce any inhibition Fig. 5B, lane 2). In this assaysystem, no transcription was detected when a-amanitin was used at aconcentration of   gg/ml, which inhibits RNAP II activity, but not RNAP I activity  datanot shown 27). Immunoreactivity ofpatient anti-RNAP II antibodieswith the 220- and 145-kD polypeptides of RNA polymerase I. In immunoblots, 9 of 13 anti-RNAP II sera bound the 220-kD 2668 Hirakata, Okano, Pati, Suwa, Medsger, Hardin, and Craft  Figure 4. Immunofluorescence pattern of HEp-2 cells stained with serum Wa and the anti-CTD mAb. Serum Wa demonstrated nucleoplasmic and nucleolar staining  A . A serum whichimmunoprecipitated the apparent subunits of RNAP I and RNAP III but did not recognize the large subunits of RNAP II demonstrated the same pattern  B . Staining with the anti-CTD mAb was limited to the nucleoplasm C . Double staining with serum Wa and the anti-CTD mAb showed nucleoplasmic staining  note the orange color produced by thecolocalization of serum Wa  fluorescein isothiocyanate and the anti-CTD mAb  Texas red D . polypeptide and 11 of 13 recognized the 145-kD polypeptide of RNAP II  including three with weak reactivity , as demon- strated by representative examples  Fig. 6, lanes 2-5 . Only   of the13 sera with antibodies to R P II as determinedby immunoprecipitation did not bind either of the 220- or 145-kD subunits in immunoblots. Control anti-Sm, anti-La, anti-Ku, and normal human sera did not bind either polypeptide  Fig. 6, lanes6-9, respectively . Discussion These studies demonstrate the presence of antibodies to R P II through several different strategies. First, we found 13 sera, all from patients with systemic sclerosis, which were able to immunoprecipitate the 240-,220-, and 145-kD polypeptide componentsof R P II. As demonstrated by an immunode- pletion assay, these were the same polypeptides immunoprecip- itated by the anti-RNAP II mAb 8WG 16. In addition,the prototype serum Wa and a second human serum were able to inhibit RNAP II function in in vitro transcription assays. Fi- nally, 12 of these13 sera recognized eitherthe 220- or 145-kD subunits in immunoblots, providing further evidence thatthese sera were not merely immunoprecipitating RNAP II via binding to subunits common to this polymerase and RNAP I and/or RNAP III. It is noteworthy that the majority of the serathat immuno precipitated RNAP II also bound the 220-kD polypeptide in immunoblots. Binding to a specific site of this protein canbe inferred from thepresent studies. First, it is important to note that the R P II specific mAb 8WG16  14,15 , as well as another R P II mAb  31   recognize the heptapeptide repeat of the CTD of the large 220-kD subunit, indicating that this region acts as an immunogenic epitope in animals. The 8WG16 mAb immunoprecipitates the 240-kD  110 , 220-kD  IIA , and 145-kD  IIC RNAP II subunits, butnot the 180- kD  IIB polypeptidederived from IIA by proteolysis, which deletes the CTD. This same pattern ofimmunoprecipitation was exhibited by the systemic sclerosis sera in our study sug- gesting thatthese sera recognized the CTD but not the amino- terminal region corresponding to the 1 80-kD subunit. In addi- tion, in preliminary studies we have recently expressedthe CTD as a glutathione-S-transferase fusion protein, and have shown that representative anti-RNAP II patient sera recog- nized this fusionprotein in immunoblots  32 . Thus, a portion of the autoantigenicepitopes may reside on the heptapeptide repeats within the CTD. It has become clear thatthis domain Autoantibodies to RNA Polymerase II 2669
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