Autoantigenic epitopes on hY5 Ro RNA are distinct from regions bound by the 60-kDa Ro and La proteins

We recently reported the identification in human anti-Ro serum of Abs specifically immunoprecipitating deproteinized hY5 RNA. In the present report, we characterized the epitopes recognized by anti-hY5 RNA Abs. Using deletion and site-directed mutagenesis of hY5 cDNA and in vitro transcribed RNAs with intact and 3'-shortened ends, we have defined two conformational antigenic determinants distinct from the regions known to bind Ro and La proteins. One of these epitopes (epitope A) is present in the middle portion of hY5 RNA and is dependent on the presence of a four-nucleotide sequence (AACC at position 58-61) that may form a single-stranded loop. Deleting these four nucleotides or modifying the stem structures proximal or distal to this loop abolishes recognition of the mutated RNAs by Abs. The second epitope (epitope B) requires the presence of another four-nucleotide sequence (CUUG at position 74-77) in between the Ro and La binding sites. Deleting this CUUG sequence or modifying nucleotides on the 5' side of the stem structure below the Ro60 binding site severely compromises the interaction with Abs. Since Abs to deproteinized hY RNAs are restricted to anti-hY5 RNA and target determinants not involved in interactions with known hY5 RNA-binding proteins, human RohY5 particles may play a direct role in the immunization process, leading to the production of anti-hY5 RNA autoantibodies.