Autoantibodies to RNA polymerases recognize multiple subunits and demonstrate cross-reactivity with RNA polymerase complexes

Objective. To determine the subunit specificity of autoantibody directed to RNA polymerases (RNAP) I, II, and III, which is one of the major autoantib ody responses in patients with systemic sclerosis (SSc), Methods. Thirty-two SSc sera with anti-RNAP antibodies (23 with anti-RNAP I /III, 5 with anti-RNAP I/III and II, and 4 with anti-RNAP II alone) were an alyzed by immunoblotting using affinity-purified RNAP and by immunoprecipit ation using S-35-labeled cell extracts in which RNAP complexes were dissoci ated. Antibodies bound to individual RNAP subunits were eluted from prepara tive immunoblots and were further analyzed by immunoblotting and immunoprec ipitation, Results. At least 15 different proteins were bound by antibodies in anti-RN AP-positive SSc sera in various combinations. All 9 sera immunoprecipitatin g RNAP II and all 28 sera immunoprecipitating RNAP I/III recognized the lar ge subunit proteins of RNAP II and III, respectively. Reactivity to RNAP I large subunits was strongly associated with bright nucleolar staining by in direct immunofluorescence. Affinity-purified antibodies that recognized a 6 2-kd subunit protein crossreacted with a 43-kd subunit protein and immunopr ecipitated both RNAP I and RNAP III. Antibodies that recognized a 21-kd sub unit protein obtained from sera that were positive for anti-RNAP I/III and II antibodies immunoprecipitated both RNAP II and RNAP III. Conclusion. Anti-RNAP antibodies recognize multiple subunits of RNAP I, II, and III. Moreover, the results of this study provide the first direct evid ence that antibodies that recognize shared subunits of human RNAPs or epito pes present on different human RNAP subunits are responsible for the recogn ition of multiple RNAPs by SSc sera.