STRUCTURE AND FUNCTION OF NUCLEOLAR NONHISTONE PROTEINS

拟议研究的总体目标是了解 核仁非核糖体、非组蛋白蛋白在核糖体组装中的作用。 其中两种蛋白质，核仁素（Mr 110，000，也称为C23）和B23（Mr 38,000）在这个实验室进行了广泛的研究。 最近的工作 表明B23的两种形式是由mRNA的选择性剪接产生的 水平 该提案的主要重点是调查表达， 性质、与核酸的相互作用以及它们的可能功能。 两种B23亚型。 具体目标是：（a）审查 大鼠B23两种亚型在蛋白质和信使RNA水平的表达 在分化过程中的小鼠红白血病细胞中， 血清饥饿后再喂食小鼠成纤维细胞。(b)到 在细菌中表达两种形式的B23蛋白， 以下所述研究的数量。(c)使用细菌表达的 蛋白质，B23亚型的结构-功能关系将被研究 关于与核酸的相互作用，协同性和蛋白质-- 蛋白质相互作用 B23和核仁素中的特定氨基酸残基 将确定与核酸的相互作用。 的区域 与核酸相互作用所需的蛋白质将通过 删除研究。(d)前核糖体RNP中B23与RNA的结合 将在细胞中将蛋白质交联到RNA后检查颗粒。 将通过杂交鉴定交联的RNA序列， 前核糖体RNA基因的克隆片段。 RNA的特异性 蛋白B23的结合将在体外研究， 产生前核糖体RNA片段。 与他人的互动 将研究核仁蛋白。(e)过度的影响 蛋白B23的低表达对核仁和细胞生理学的影响将是 考察 含有蛋白B23同种型cDNA的诱导型载体 将被引入哺乳动物细胞中， 生产过剩。 产生B23 mRNA的反义RNA的类似载体将 用来减少表达。 核糖体RNA合成速率， 对前核糖体RNA加工的影响， 蛋白B23和核仁形态在超微结构水平上将 在这些治疗前后进行检查。 如果时间允许， 将使B23蛋白过表达，B23蛋白在功能上发生突变， 关键部分。 这些后面的研究希望阐明的作用 蛋白B23亚型在核糖体组装和核仁中的调节 结构 这项拟议的工作应有助于了解分子 肿瘤、化疗和化疗中核仁改变的基础 自身免疫性疾病

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.