BIOCHEMICAL ANALYSIS OF A NOVEL U1 RNA-ANTIBODY COMPLEX

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This proposal outlines a research effort to further our understanding of the structural rules that dictate protein-RNA interaction. The role of specific amino acids and nucleotides in the formation of an antibody-RNA complex will be delineated using antibodies that bind to stem-loop II of U1 RNA. The study of RNA-protein interactions in the context of anti-U1 RNA antibodies will allow for relatively simple assessment of the RNA binding site due to the small size of antigen binding regions and the known structures of several antibodies. Experiments in this proposal are designed to: 1) characterize naturally occurring U1 RNA-stem loop II antibodies and produce genetically engineered U1 RNA-binding antibody fragments (Fabs); 2) analyze the RNA binding sites contained in the naturally occurring and recombinant Fabs by evaluation of amino acid sequence, antibody specificity and binding characteristics; 3) determine the role of specific amino acids in binding U1 RNA by the use of molecular modeling and site-directed mutagenesis; 4) elucidate the ribonucleotides critical to antibody binding using a rapid selection technique and; 5) initiate NMR and crystallographic studies with the long-term goal of elucidating the structure of an RNA antibody and/or antibody-RNA complex. U1 RNA, as well as many other cellular RNAs, exist in the cell as ribonucleoproteins functioning in RNA processing and gene regulation. These ribonucleoproteins are often targeted by the immune system in patients with autoimmune diseases such as systemic lupus erythematosus (SLE), Sjogren's syndrome, and rheumatoid arthritis. This research will improve our understanding of RNA-protein recognition and gene regulation. A better understanding of these processes will help discern the cellular disregulation associated with autoimmune disease. In addition, information gained will impact on our knowledge of the structure of RNA-reactive antibodies and will have future application in the design of novel therapeutic antibodies and nucleic acid autoantibody inhibitors.