Recent study of autoantibody responses seen inpatients with autoimmune connective tissue diseases has centered on the molecular analysis of the relevant autoantigens. Despite advances in the understanding of the molecular biology of many of these targets, the ribonucleoprotein containing the Ro determinants (the Ro-RNP) remains poorly characterized. The present study proposes a detailed examination of the protein-RNA and protein-protein interactions within the R0-RNP through a series of reconstitution experiments using recombinant Ro proteins. Initially, engineered cDNA clones will be constructed, with subsequent transcription and translation (both in vitro and in vivo) generating the recombinant substrates for binding. Interactions detected by this approach will be further defined on the molecular level by in vitro mutagenesis of the full-length cDNAs. The function of the Ro-RNP, as well as the stoichiometry of binding within the particle, will be examined by using transfection experiments to alter the levels of expression of individual Ro proteins in human cells. The use of deletion mutants to direct the intra-cellular assembly of mosaic particles will test theories about the coordinate control of particle assembly. The origin and pathogenetic significance of antibodies to the Ro-RNP are unknown, although clinical and immunogenetic associations with this antibody response have been reproducibly identified. Patient-specific reactivity profiles, generated by analysis of the ability of sera to recognize epitopes expressed on the recombinant polypeptides, will be correlated with previously obtained clinical and immunogenetic data from over 200 patients with anti-Ro antibodies. The resulting statistical associations will help to define the context within which specific patterns of reactivity occur against the Ro antigen.
