The general goal of the proposed research is to understand the role of nucleolar nonribosomal, nonhistone proteins in the assembly of ribosomes. Two of these proteins, nucleolin (Mr 110,000, also called C23) and B23 (Mr 38,000) have been extensively studied in this laboratory. Recent work showed that two forms of B23 arise from alternative splicing at the MRNA level. The major focus of the proposal is to investigate the expression, properties, interaction with nucleic acids and possible functions of the two B23 isoforms.
The specific aims are: (a) To examine the expression of the two B23 isoforms at the protein and messenger RNA levels in rat tissues, in murine erythroleukemia cells during differentiation and in mouse fibroblasts after refeeding following serum starvation. (b) To express the two forms of protein B23 in bacteria for producing large quantities for studies described below. (c) Using the bacterially expressed proteins, structure-function relationships in B23 isoforms will be studied with regard to interaction with nucleic acids, cooperativity and protein-- protein interactions. Specific amino acid residues in B23 and nucleolin interacting with nucleic acids will be determined. The regions of the protein required for interaction with nucleic acids will be examined by deletion studies. (d) The association of B23 with RNA in preribosomal RNP particles will be examined after cross-linking the protein to RNA in cells. The cross-linked RNA sequences will be identified by hybridization to cloned fragments of a gene for preribosomal RNA. The specificity of RNA binding by protein B23 will be studied in vitro using synthetically produced segments of preribosomal RNA. The interactions with other nucleolar proteins will be investigated. (e) The effects of over- and underexpression of protein B23 on nucleolar and cellular physiology will be examined. Inducible vectors containing the cDNA's for protein B23 isoforms will be introduced into mammalian cells so that the protein may be overproduced. Similar vectors which produce antisense RNA to B23 mRNA will be used to reduce expression. The rates of ribosomal RNA synthesis, the effects on preribosomal RNA processing, the cellular distribution of protein B23 and the nucleolar morphology at the ultrastructural level will be examined before and after these treatments. As time permits attempts will be made to overexpress protein B23 with mutations in functionally critical segments. These latter studies are hoped to elucidate the role of protein B23 isoforms in regulation of ribosome assembly and in nucleolar structure. This proposed work should aid in understanding the molecular basis of alteration in the nucleolus in neoplasia, chemotherapy and in autoimmune diseases.
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