STRUCTURE AND FUNCTION OF NUCLEOLAR NONHISTONE PROTEINS

这项拟议研究的总体目标是了解 核仁非核糖体、核糖体组装中的非组蛋白。 其中两种蛋白质是核仁蛋白(Mr 110,000，也称为C23)和B23(MR 38,000)已在本实验室进行了广泛的研究。近期工作 显示了两种形式的B23来自于mRNA的选择性剪接 水平。该提案的主要重点是调查表达， 酶的性质、与核酸的相互作用及其可能的功能 两个B23亚型。具体目标是：(A)审查 大鼠蛋白质和信使RNA水平上的两种B23亚型 组织，在小鼠红白血病细胞分化过程中和在 血清饥饿后再喂养的小鼠成纤维细胞。(B)至 两种形式的B23蛋白在细菌中的高效表达 下文所述的研究数量。(C)使用细菌表达的 将研究B23亚型中的蛋白质、结构-功能关系 关于与核酸的相互作用，协作性和蛋白质-- 蛋白质的相互作用。B23和核仁中的特定氨基酸残基 与核酸的相互作用将被确定。世界各地的 与核酸相互作用所需的蛋白质将通过 删除研究。(D)胸前RNP中B23与RNA的关系 在将蛋白质与细胞中的RNA交联后，将对颗粒进行检查。 交叉连接的RNA序列将通过杂交来鉴定 乳房前核糖核酸基因的克隆片段。核糖核酸的专一性 蛋白质B23的结合将在体外研究，使用合成的 产生了胸前核糖核酸的片段。与其他人的互动 将对核仁蛋白进行研究。(E)过度和 蛋白质B23在核仁和细胞生理上的低表达将是 检查过了。含B23蛋白亚型基因的诱导型载体 将被引入哺乳动物细胞中，因此这种蛋白质可能 生产过剩。产生B23反义RNA的类似载体将 用来减少表达。核糖体RNA合成的速率， 对胸前RNA加工的影响，细胞分布 蛋白B23与核仁超微结构形态 在这些治疗前后进行检查。在时间允许的情况下 将使蛋白质B23在功能上发生突变而过表达 关键细分市场。这些后一项研究希望能阐明 蛋白质B23亚型在核糖体组装和核仁中的调节作用 结构。这项拟议的工作应该有助于理解分子 核仁改变在肿瘤、化疗和非小细胞肺癌中的基础 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.