Nucleolin Function in Amphibian Oocyte Nuclei

该项目的目标是确定核仁特异性蛋白质--核仁蛋白的功能。虽然核素被认为与核糖体前RNA结合，但它在将新生核糖体RNA加工成18S、5.8S和28S核糖体RNA方面的实际功能尚不清楚。非洲爪哇核仁由具有不同功能特征的独特模块组成；氨基末端第三个包含几个交替的酸性和碱性结构域，而羧基末端三分之二包含四个RNA结合域。非洲爪哇卵母细胞将被注入体外合成的转录本，这些转录本编码表位标记的全长非洲爪哇核仁或表位标记的核仁截短肽；随后将表位标记的核仁截断免疫定位到灯刷染色体环的核糖核蛋白矩阵或多个核仁的亚区，将强烈表明功能关联。在已建立的非洲爪哇前rRNA加工提取物中，核素的大分子结合将通过免疫沉淀法从提取物中分离出含有核仁素的核糖核蛋白复合体来确定。与其他核仁蛋白或核仁特异的小核RNA的关联将扩大我们对核仁功能的理解。从炮制提取物中去除核仁也将测试核仁素在最初的Pre-rRNA加工反应中的功能意义。这些处于细胞和分子生物化学交界处的研究，将促进我们对真核细胞核功能的基础知识。在真核细胞中，大多数遗传物质被隔离在细胞核中，DNA在细胞核中被复制和转录成RNA，而RNA又被输送到细胞核外，参与蛋白质的生物合成。三种不同类型的RNA直接在细胞质中的蛋白质生物合成中发挥作用：信使RNAs，它包含新生蛋白质特定氨基酸序列的“密码”；Transfer RNAs，它是提供正确序列中正确氨基酸的“解码器”；以及核糖体RNAs，它是核糖体的结构组件，它是将氨基酸“缝合”在一起的机器。虽然信使RNA序列散布在细胞核的DNA中，但编码核糖体RNA的基因聚集在细胞核的一个可在显微镜下定义的结构区域中，称为核仁。除了核糖体基因外，核仁还含有一些核仁特异性蛋白，这些蛋白被认为参与了核糖体RNA的转录或组装，但其具体功能尚未确定。在这个项目中，目标是确定一种名为核仁蛋白的特定核仁蛋白的功能。这项工作将促进对真核细胞的基本过程的理解。

The objective of this project is to characterize the functions of nucleolin, a nucleolar specific protein. Although nucleolin is thought to bind pre-ribosomal RNA, its actual function in processing nascent ribosomal RNA into 18S, 5.8S, and 28S ribosomal RNA remains unknown. Xenopus nucleolin consists of unique modules with distinct functional characteristics; the amino terminal third contains several alternating acidic and basic domains, while the carboxyl terminal two-thirds contains four RNA-binding domains. Xenopus oocytes will be injected with in vitro synthesized transcripts that encode either epitope-tagged full length Xenopus nucleolin or epitope-tagged nucleolin truncation peptides; subsequent immunolocalization of epitope-tagged nucleolin truncations to either the ribonucleoprotein matrices of lampbrush chromosome loops or subregions of multiple nucleoli will strongly indicate functional associations. Nucleolin's macromolecular associations within an established Xenopus pre-rRNA processing extract will be determined by immunoprecipitating nucleolin- containing ribonucleoprotein complexes from the extract. Associations with other nucleolar proteins or with nucleolar- specific small nuclear RNAs will expand our understanding of nucleolin function. Immunodepletion of nucleolin from the processing extract will also test the functional significance of nucleolin in the initial pre-RRNA processing reaction. These studies, which are at the interface of cellular and molecular biochemistry, will advance our fundamental knowledge of the function of the nucleus of the eukaryotic cell. %%% In eukaryotic cells, most of the genetic material is sequestered in the nucleus of the cell, and it is in the nucleus that the DNA is both replicated and transcribed into RNA, which in turn is transported out of the nucleus to function in the biosynthesis of proteins. Three different kinds of RNA function directly in protein biosynthesis in the cytoplasm: messenger RNAs, which contain the "codes" for the specific amino acid sequences of the nascent proteins; transfer RNAs, which are the "decoders" that supply the correct amino acids in the correct sequence; and ribosomal RNAs, which are structural components of the ribosomes, the machines which "sew" the amino acids together. While messenger RNA sequences are scattered throughout the DNA of the nucleus, the genes coding for ribosomal RNAs are grouped together in a microscopically-definable structural region of the nucleus called the nucleolus. The nucleolus contains, in addition to the ribosomal genes, a number of nucleolar-specific proteins which are presumed to be involved in either transcription or assembly of ribosomal RNAs, but for which specific functions have not yet been determined. In this project, the goal is to determine the function of one specific nucleolar protein, called nucleolin. This work will advance the understanding of a fundamental process of eukaryotic cells.