Abstract Peterson MCB-9808637 The mouse immunoglobulin mu gene is an established model system for studying regulated RNA processing during B lymphocyte development. This gene contains a site subject to either cleavage-polyadenylation or splicing; the efficiencies of these two competing reactions must be balanced. The relative use of these two processing options is modulated as B cells mature to plasma cells; plasma cells produce relatively more RNA cleaved and polyadenylated at the mu-s polyA site. Evidence to date has suggested that the gene structure, rather than Ig gene-specific sequences, is required for regulation. The regulatory mechanism must, therefore, involve changes in components of the general RNA processing machinery. Cleavage-polyadenylation activity increases as B cells mature to plasma cells. In addition, preliminary evidence suggests that splicing activity differs between B cells and plasma cells. Thus, the mechanism regulating mu processing likely involves changes in both the cleavage-polyadenylation and splicing activities. This project will extend current studies to further the understanding of the mechanism regulating mu alternative RNA processing, of splice site choice, and of cleavage-polyadenylation site usage in general. Toward these ends, the changes in splicing activity will be explored by two different approaches. Sequences that contribute to differential splicing activity will be identified using a splice enhancer-requiring gene. These sequences will be identified in the mu gene and in a chimeric gene whose splicing is regulated in B cells and plasma cells. Any sequences that activate splicing will be used to identify RNA binding proteins by a combination of RNA gel shift, UV cross-linking, and antibody identification procedures. A second approach will use the suppression subtraction hybridization technique to find genes encoding products relevant to RNA processing whose expression differs between B cells and plasma cells. The cleavage-polyadenylation reaction will be further studied by examining sequences from the mu-s polyA site that potentially affect cleavage-polyadenylation activity in novel ways. Sequences far downstream and upstream from the polyA site both contribute to its activity. To understand better the function of these sequences, additional site-directed mutational analyses and in vitro cleavage-polyadenylation and binding assays will be performed. To determine if the upstream sequences are involved in interactions between splice and cleavage-polyadenylation reactions, in vitro assays using conditions that couple these reactions will be performed. As regulation is predicted to involve changes in general splicing and cleavage-polyadenylation factors, these proposed approaches will provide information on splice and cleavage-polyadenylation enhancing sequences and factors in general, as well as contribute to the understanding of the regulatory mechanisms operating during lymphocyte maturation. Alternative RNA processing is a mechanism by which eukaryotic cells expand the coding capacity of their genomes: more than one product can be made from a single gene. Alternative RNA processing is often regulated such that different mRNA's are made in different cell types or during different stages of cellular development. How this alternative RNA processing is regulated is only beginning to be understood. This project examines the alternative processing of the immunoglobulin gene - the alternate products are either a secreted protein or a membrane-associated protein whose relative abundance changes during B lymphocyte development. Information gained from this research will contribute to our knowledge of RNA processing reactions in general and of the regulation of these reactions.
