In vitro RNA selection of an autoimmune epitope on stem-loop II of U1 RNA

Autoantibodies to U1 RNA occur frequently in sera from patients with SLE and SLE-overlap syndromes. These autoantibodies have been previously shown to recognize major epitopes on stem-loops II and IV of U1 RNA. To further define these recognition sites, in vitro RNA selection was performed to identify the individual nucleotides which form the antibody binding site. Serum autoantibodies were used in this procedure to select synthetic variants from a library of RNA oligomers with a central region of 25 degenerate nucleotides in a linear context. A consensus sequence was derived from the autoantibody-selected RNA ligands that corresponded to a region of stem-loop II. It contained six continuous nucleotides (U63-C64-C65,-A66-X-U68) and one presumed discontinuous nucleotide (U79). In vitro selection of RNA ligands from simpler sublibraries consisting of oligomers with random loops and fixed U1 stem II sequences yielded no consensus sequence, suggesting that autoimmune recognition occurs independent of loop nucleotides. Competition assays confirmed the specificity of these binding reactions. The structural nature of this autoimmune U1 RNA epitope is compatible with a model of autoantibody production based on stimulation by native small nuclear ribonucleoprotein particles.