STRUCTURE AND FUNCTION OF NUCLEAR RNP PARTICLES

核RNP粒子的结构和功能

• 批准号: 3292172
• 负责人: GIDEON DREYFUSS
• 金额: $ 21.16万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 1991-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3292172
• 关键词: Escherichia coli; chemical structure function; crosslink; density gradient ultracentrifugation; eukaryote; gene expression; genetic library; genetic mapping; genetic transcription; heterogeneous nuclear RNA; heterogeneous nuclear ribonucleoprotein; laboratory mouse; laboratory rabbit; messenger RNA; molecular cloning; molecular sieving; monoclonal antibody; nucleic acid sequence; polynucleotides; protein sequence; radiotracer; tissue /cell culture

In eukaryotic cells, messenger RNAs (mRNAs), the functional translatable intermediates of gene expression, are formed by extensive processing of primary gene transcripts (hnRNAs). We and others have recently demonstrated that hnRNAs exist in the nucleus in association with a specific set of proteins to form unique structures termed hnRNP particles. The hnRNP particles, one of the most abundant structures in the nucleus, are the sites of RNA processing. The RNP proteins are essential for the packaging, processing and function of these polynucleotides. The RNA-associated proteins can be photochemically crosslinked to it in intact cells, and the crosslinked complexes can be readily isolated. We have immunized mice with crosslinked hnRNP complexes and have generated monoclonal antibodies against several of the major hnRNP proteins in divergent eukaryotes. With these antibodies we have begun to characterize the hnRNP proteins and have isolated the hnRNP complex. One of the antibodies to the prominent hnRNP C proteins (41K and 43K) specifically inhibits the splicing of mRNA precursors in vitro. We shall investigate the structure and function of the hnRNP proteins and the hnRNP particles in order to understand their role in mRNA formation and in the packaging and organization of hnRNA in the nucleus. The antibodies will be used to isolate the cDNA clones for the hnRNP proteins by immunological screening of expression vector libraries. The amino acid sequence of the proteins will be determined form the cDNA nucleotide sequence and their mRNAs and their genes will be characterized. Additional antibodies to hnRNP proteins will be obtained and characterized. The interaction of hnRNP proteins with specific RNAs synthesized in vitro will be studied in order to understand the assembly and disassembly of the hnRNP complex -- knowledge which is necessary for understanding the structure and formation of hnRNP particles and the process of nucleo-cytoplasmic transport of mRNA. The function of the proteins and the hnRNP complexes in transcription and in RNA splicing will be studied in in vitro systems. The ability to isolate pure and intact hnRNP particles and the availability of antibodies to individual hnRNP proteins will enable the further characterization of their structure and their role in mRNA biogenesis.

在真核细胞中，信使RNA(MRNAs)是功能可翻译的 基因表达的中间产物，是由大量的 初级基因转录本(HnRNA)。我们和其他人最近 证明了hnRNAs存在于细胞核中，与 形成独特结构的一组特定蛋白质，称为hnRNP颗粒。 HnRNP颗粒是原子核中最丰富的结构之一， 是RNA加工的场所。 RNP蛋白是包装、加工和功能所必需的 这些多核苷酸。RNA相关蛋白可以是 在完整的细胞中以光化学的方式与之交联，而交联物 络合物很容易被分离出来。我们用交联剂免疫小鼠 HnRNP复合体，并产生了针对几种 发散真核生物中的主要hnRNP蛋白。有了这些抗体，我们 已经开始鉴定hnRNP蛋白并分离出hnRNP 很复杂。针对主要hnRNP C蛋白的抗体之一(41K和 43K)在体外特异性地抑制mRNA前体的剪接。 我们将研究hnRNP蛋白的结构和功能 HnRNP颗粒，以了解它们在mRNA形成中的作用 以及细胞核内hnRNA的包装和组织。这个 将使用抗体来分离hnRNP蛋白的cdna克隆。 通过对表达载体文库的免疫学筛选。氨基酸 蛋白质的序列将从cDNA核苷酸中确定 序列及其mRNAs和它们的基因将被描述。其他内容 将获得针对hnRNP蛋白的抗体并对其进行鉴定。这个 HnRNP蛋白与体外合成的特定RNA的相互作用 为了了解hnRNP的组装和拆卸 复杂性--理解结构和结构所必需的知识 HnRNP颗粒的形成与核质过程 信使核糖核酸转运。蛋白质和hnRNP复合体在体内的功能 转录和RNA剪接将在体外系统中进行研究。这个 分离纯净和完整的hnRNP颗粒的能力以及 针对单个hnRNP蛋白的抗体将使进一步的 它们的结构特征及其在信使核糖核酸生物发生中的作用。