RIBONUCLEOPROTEINS, MRNA, AND CYTOSKELETAL STRUCTURES

核糖核蛋白、mRNA 和细胞骨架结构

• 批准号: 3280299
• 负责人: GIDEON DREYFUSS
• 金额: $ 12.09万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-05-01 至 1991-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3280299
• 关键词: HeLa cells; RNA binding protein; affinity chromatography; complementary DNA; crosslink; cytoskeleton; eukaryote; gene expression; genetic manipulation; genetic transcription; genetic translation; heterogeneous nuclear ribonucleoprotein; laboratory mouse; laboratory rabbit; messenger RNA; molecular genetics; monoclonal antibody; muscle proteins; nucleic acid probes; polynucleotides; protein engineering; protein sequence; radioimmunoassay; ribonucleoproteins

Messenger RNAs (mRNAs), the intermediates of gene expression which direct protein synthesis, are found in eukaryotic cells associated with a specific subset of cytoplasmic proteins. The mRNA-protein complexes, termed mRNP particles (mRNPs), are distinct structural entitles different in protein composition, structure, function and subcellular compartmentalization from nuclear pre-mRNA-protein (hnRNP) particles. A plethora of recent observations indicate that mRNP proteins play a cardinal role in mRNA transport, translation, stability and localization. The mRNP proteins can be photochemically crosslinked to the mRNA in intact cells and the crosslinked complexes can be readily isolated. We have studied these complexes in normal and virus-infected cells across eukaryotes. In all of these, the most abundant protein is a 72,000 dalton protein which is crosslinked to the poly(A) tail of the mRNA. The poly(A) binding protein is a highly conserved, poorly immunogenic protein that has been the focus of much interest because it appears to play a key role in mRNA metabolism. We have immunized mice with crosslinked purified mRNP complexes to produce antibodies to the mRNP proteins. Antibodies were obtained to the poly(A) binding protein from yeast, and we have begun to characterize the protein and have isolated the gene encoding it. The antibodies and the gene are the first probes for this protein from any organism and they open the way of its molecular and genetic characterization. We shall investigate the structure, function, properties and localization of the mRNP proteins and the mRNP complexes with particular emphasis on the poly(A) binding protein. Additional antibodies to mRNP proteins will be produced. The cDNAs encoding these proteins will be isolated by immunological screening of expression vector libraries. The complete amino acid sequence of the proteins will be determined by nucleotide sequencing of the cDNAs, and their mRNAs and genes will be characterized. The interaction of the proteins with RNA will be studied in detail. The subcellular localization of the proteins will be examined by immunocytochemical techniques. The functions of the proteins will be investigated in vivo by gene disruption and mutagenesis in yeast and by introduction of antibodies into living animal cells, and in vitro using cell free systems for protein synthesis and mRNA transcription, splicing and polyadenylation. From these studies a better picture of ho mRNA is formed, maintained and functions in animal cells is likely to emerge.

信使RNA（mRNA），基因表达的中间体 在真核细胞中， 与细胞质蛋白质的特定子集相关。 的 mRNA-蛋白质复合物，称为mRNP颗粒（mRNP）， 蛋白质组成不同的不同结构蛋白， 结构、功能和亚细胞区室化 核前mRNA蛋白（hnRNP）颗粒。 过多的 最近的观察表明，mRNP蛋白质发挥着重要的作用， 在mRNA转运、翻译、稳定性和定位中的作用。 mRNP蛋白可以光化学交联至 完整细胞中的mRNA和交联的复合物可以是 容易隔离。 我们研究了这些复杂的正常和 真核生物中的病毒感染细胞。 在所有这些中， 丰富的蛋白质是一种72，000道尔顿的蛋白质，其与 mRNA的poly（A）尾。 poly（A）结合蛋白是一种 高度保守，免疫原性差的蛋白质， 这是一个非常感兴趣的焦点，因为它似乎在以下方面发挥了关键作用： mRNA代谢 我们用交联的 纯化的mRNP复合物以产生针对mRNP的抗体 proteins. 获得针对poly（A）结合蛋白的抗体 我们已经开始研究这种蛋白质的特性， 已经分离出编码它的基因。抗体和基因 是第一个从任何生物体中检测这种蛋白质的探针， 开辟了其分子和遗传表征的道路。 我们将研究它的结构、功能、性质和 mRNP蛋白和mRNP与 特别强调poly（A）结合蛋白。 额外 将产生针对mRNP蛋白的抗体。 的cdna 编码这些蛋白质将通过免疫学方法分离。 筛选表达载体文库。 完整氨基酸 蛋白质的序列将通过核苷酸序列来确定。 将对cDNA及其mRNA和基因进行测序， 表征了蛋白质与RNA的相互作用将是 详细研究 蛋白质的亚细胞定位将 用免疫细胞化学技术检测。 的功能 将通过基因破坏在体内研究蛋白质， 在酵母中诱变和通过将抗体引入活的 动物细胞，并在体外使用无细胞系统的蛋白质 合成和mRNA转录、剪接和聚腺苷酸化。 从这些研究中形成了ho mRNA的更好的图像， 在动物细胞中维持和发挥功能的可能性。

项目成果

Characterization of heterogeneous nuclear RNA-protein complexes in vivo with monoclonal antibodies.

使用单克隆抗体表征体内异质核 RNA-蛋白质复合物。

• DOI: 10.1128/mcb.4.6.1104-1114.1984
• 发表时间: 1984
• 期刊: Molecular and cellular biology
• 影响因子: 5.3
• 作者: Dreyfuss,G;Choi,YD;Adam,SA
• 通讯作者: Adam,SA

The ribonucleoprotein structures along the pathway of mRNA formation.

核糖核蛋白沿着 mRNA 形成途径构建。

• DOI: 10.3109/07435808909036348
• 发表时间: 1989
• 期刊: Endocrine research
• 影响因子: 2.1
• 作者: Dreyfuss,G;Choi,YD;Adam,SA
• 通讯作者: Adam,SA

Molecular cloning of cDNA for the nuclear ribonucleoprotein particle C proteins: a conserved gene family.

核核糖核蛋白颗粒 C 蛋白 cDNA 的分子克隆：保守基因家族。

• DOI: 10.1073/pnas.83.7.2007
• 发表时间: 1986
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Nakagawa,TY;Swanson,MS;Wold,BJ;Dreyfuss,G
• 通讯作者: Dreyfuss,G