Ro ribonucleoprotein (Ro RNP) is the target of an aggressive autoimmune response in many patients with systemic lupus erythematosus (SLE) and Sjogren's syndrome (SS). Limited specificity of autoimmune responses in these disorders is consistent with the Ro RNP being important in the pathology and/or etiology of the autoimmune dysregulation found in these diseases. The function of Ro RNP is not known, though some structural data are available such as the sequences for the human (h) Y RNAs and the associated 60 kD Ro peptide. However, neither the secondary structures of the Y RNAs nor the conserved primary regions of Y RNA sequence between species have been established. Such data would have potential to reveal Ro RNP function, as was the case for U particle function in hnRNA splicing. The investigators propose to clone, sequence, and analyze the known Y RNAs from mice, rats, rabbits, cattle, ducks, and trout (16 Y RNAs). Also, toads, drosophila, lobster, a nematode, and yeast will be explored for the presence of Y-like RNAs and Ro-like mRNA. Finally, in order to define sufficient diversity of h Y5-like sequences it may be necessary to recover homologs of h Y5 from primates in addition to man. Technology to be used has been recently developed in the investigators' laboratory for these low abundance RNAs and with which hY4 RNA has been sequenced. Immunoprecipitated Y RNA will be polyadenylated followed by cDNA synthesis. After poly-g tailing, intervening cDNA or Y RNA will be expanded by polymerase chain reaction, cloned, and sequenced. With sequences categorized according to each type of Y RNA obtained, the preserved base pairing pattern will be used to construct the evolutionarily conserved secondary structure. Conserved primary sequence of homologous Y RNAs will provide short sequences with which to evaluate the existing DNA sequence data bases in an effort to find a pattern or recurring complementary sequences with potential functional significance. Such a result would lead toward elucidating a role for Ro RNPs in the molecular metabolism of cells. In the final phase of this project the investigators will develop a model system in which to generate and test hypotheses of Ro RNP function. Anti-sense DNA oligonucleotides prepared according to the 60 kD Ro and hY sequences will be evaluated for their capacity to inhibit Ro RNP formation. If Ro RNP formation is inhibited then the function of Ro RNP will be characterized in this model. If not, then the suitability of yeast as a model will be evaluated. If neither the anti-sense DNA oligos nor yeast provide suitable models, the investigators will transfect tissue culture cells with plasmid constructs that produce intracellular anti-sense RNAs designed to inhibit the products of the 60 kD Ro and hY genes. These experiments they believe will provide important structural and functional information that should carry them toward a comprehensive understanding of the Ro RNP antigen.
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