RNA-LABELED RO AND LA RIBONUCLEOPROTEIN COMPLEXES REASSEMBLED IN-VITRO - CHARACTERIZATION BY GEL SHIFT ANALYSIS

Ro and La RNP complexes were reassembled from in vitro labelled hY5 RN A and HeLa cell extracts. These complexes were then visualized through retardation of migration of labelled hY5 RNA in non-denaturing polyac rylamide gels. Three major complexes (named A, B, and C) were formed w hen crude cellular extracts (S100 fraction) were used. Using monospeci fic anti-60-kD Ro (Ro60) and anti-La antibodies to retard RNPs contain ing these antigens during migration in the gels, the three major compl exes were shown to contain Ro60 (C), La (B), or both proteins (A). The specificity of RNA-protein interactions in the reassembled complexes was further demonstrated using two 3'-shortened hY5 RNA transcripts la cking the La-binding site (hY5-Alu I RNA) and both the Ro60 and La-bin ding sites (hY5-Hha I RNA). hY5-Hha I RNA still formed a single, minor complex when incubated with S100 extract, suggesting interaction with a yet undefined protein. In addition, we used the capacity of specifi c antibodies to retard the migration of the ressembled complexes to de sign a detection assay for anti-Ro and anti-La autoantibodies. Using 8 4 human sera, our assay was shown to approximate the specificity and s ensitivity of an immunoprecipitation assay where P-32-labelled cell ex tracts are used as source of antigens. Our assay may be used to detect low levels of antibodies to conformational determinants on Ro60 and L a proteins in human sera and antibody preparations.