Further characterization and subcellular localization of Sm and Ul ribonucleoprotein antigens

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Abstract
Sera from patients with systemic autoimmune diseases often contain antibodies against small nuclear ribonucleoprotein (snRNP) particles. Anti‐Sm antibodies react with the entire set of U1, U2, U4, U5 and U6 (Ul‐U6) RNP particles whereas anti‐(U1)RNP sera specifically recognize particles containing U1 RNA. Here we performed semi‐quantitative immunoblotting using 16 human anti‐Sm, 15 human anti‐(U1)RNP sera and two mouse monoclonal antibodies to establish which snRNA‐associated proteins carry antigenic determinants. Almost every (15/16) human anti‐Sm sera recognized epitopes present on a 28‐kDa (B/B') protein doublet and on a 16‐kDa (D) polypeptide. Nine anti‐(U1)RNP sera also recognized the B/B' doublet, but in all cases a much stronger reaction was observed with one or more of the specifically U1 RNA‐associated 70 kDa, A or C antigens. With affinity‐purified antibody fractions eluted from individual antigen bands on nitrocellulose blots it is shown that the anti‐Sm‐reactive polypeptides B/B' and D contain common epitopes. We also report the finding of one human anti‐Sm serum with exclusive specificity for the B/B' doublet and a mouse monoclonal anti‐Sm antibody recognizing only the D protein, indicating that these antigens also carry unique epitopes. In immunoprecipitation assays, purified anti‐B/B' and ‐D antibodies react with (Ul‐U6) RNP while purified anti‐70 kDa, anti‐A and anti‐C antibodies precipitate exclusively U1 RNP particles. Finally, we established the subcellular localization of Sm and U1 RNP antigens using a biochemical cell fractionation procedure. Part of the 70 kDa and BIB' antigens were found in a nuclease and high salt‐resistant nuclear substructure, usually referred to as nuclear matrix, while the A and D antigens could be extracted completely from HeLa nuclei by ribonuclease treatment and subsequent high salt extraction.