MRNA SYNTHESIS IN ANIMAL CELLS--3' END FORMATION

动物细胞中的 mRNA 合成--3 端形成

• 批准号: 2175347
• 负责人: James L. Manley
• 金额: $ 29.66万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-04-01 至 1998-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2175347
• 关键词: RNA binding protein; RNA biosynthesis; animal genetic material tag; chemical cleavage; clone cells; genetic mapping; genetic regulation; high performance liquid chromatography; intermolecular interaction; messenger RNA; molecular cloning; polyadenylate; posttranscriptional RNA processing; precursor mRNA; protein purification; recombinant proteins; transcription factor

The principal goal of this research project is to understand how pre-mRNA polyadenylation occurs. Work during the previous granting period has shown that this is an unexpectedly complex reaction, requiring approximately 10 distinct polypeptides. The factors involved include two multisubunit RNA binding complexes, CPSF and CstF, two cleavage factors, CFI and ll, and poly(A) polymerase (PAP). Both in vitro and in vivo assays will be employed to study the mechanism and regulation of polyadenylation. The following specific aims are proposed: 1. Purification and cloning of remaining polyadenylation factors. While a number of these have already been purified and/or cloned, several remain. Most notable are two partially purified proteins, cleavage factors I and ll, one or both of which almost certainly constitute the actual endonuclease that cleaves the pre-mRNA. These proteins will be purified to homogeneity, most likely from calf-thymus, and cDNAS cloned using protein microsequence information. 2. Biochemistry of polyadenylation. The multiple protein-RNA and protein- protein interactions required for polyadenylation will be investigated in detail. These include binding of CPSF, CstF and PAP to RNA; the protein- protein interactions responsible for the cooperative RNA binding of CPSF and CstF; interactions of PAP with CPSF; and the interaction of the cleavage factors with other components. 3. In vivo analysis of polyadenylation. Efforts will be made to develop in vivo systems to study polyadenylation. Several approaches will be employed to study interactions between polyadenylation factors in transiently transfected cells. Drosophila homologues of selected genes will be isolated and their chromosomal locations determined. Any genes that map to positions of previously described mutants that might conceivably play a role in RNA processing will be investigated further. A chicken B cell line that undergoes high levels of homologous recombination will be used to determine whether selected genes are required for viability. 4. Regulation of polyadenylation. This will be studied from two perspectives: First, changes in the expression of the 64 kD subunit of CstF during B cell development will be studied as a specific example. The possibility that such changes influence the alternative processing of IgM (membrane vs. secreted) pre-mRNA will be investigated. Second, possible regulation of poly(A) polymerase, and appropriate other factors, including 64kD, will be examined more generally. Distinctive features of their expression, such as the existence of alternatively spliced mRNAs, and structure (e.g., each are phosphoproteins) suggest that their activities may be regulated. Expression patterns and activities in various cell types will be investigated, and the significance of any differences examined.

该研究项目的主要目标是了解前MRNA 聚腺苷酸化发生。在上一个授予期间的工作已显示 这是一个意外复杂的反应，大约需要10个 不同的多肽。所涉及的因素包括两个多亚基RNA 结合复合物，CPSF和CSTF，两个切割因子CFI和LL，以及 聚（A）聚合酶（PAP）。体外和体内测定都将是 用于研究聚腺苷酸化的机理和调节。这 提出了以下特定目标： 1。剩余的聚腺苷酸化因子的纯化和克隆。而a 这些数量已经被纯化和/或克隆，保留了几个。 最值得注意的是两种部分纯化的蛋白质，切割因子I和 LL，几乎可以肯定构成实际的 核酸内切酶切割前MRNA。这些蛋白质将被纯化为 同质性，最有可能来自小牛 - 胸骨和使用蛋白质克隆的cDNA Microsequence信息。 2。聚腺苷酸化的生物化学。多蛋白-RNA和蛋白质 聚腺苷酸化所需的蛋白质相互作用将在 细节。这些包括CPSF，CSTF和PAP与RNA的结合；蛋白质 - 负责CPSF合作RNA结合的蛋白质相互作用 和CSTF； PAP与CPSF的相互作用；以及 与其他组件的切割因子。 3。聚腺苷酸化的体内分析。将努力发展 研究聚腺苷酸化的体内系统。将采用几种方法 研究瞬时聚腺苷酸因子之间的相互作用 转染的细胞。选定基因的果蝇同源物将是 分离及其染色体位置。任何映射到的基因 可能可以想象的先前描述的突变体的位置 将进一步研究RNA处理中的作用。鸡肉B细胞系 进行高水平的同源重组将用于 确定是否需要选定的基因才能生存。 4。多腺苷酸化的调节。这将从两个 观点：首先，64 kd亚基的表达变化 B细胞开发过程中的CSTF将作为一个特定示例进行研究。这 这种变化会影响IgM的替代处理 （膜与分泌的）将研究前mRNA。第二，可能 调节聚（a）聚合酶，以及适当的其他因素，包括 64KD，将进行更广泛的检查。他们的独特特征 表达，例如存在剪接的mRNA和 结构（例如，每种都是磷蛋白）表明它们的活性 可能受到监管。各种细胞类型的表达模式和活动 将研究，以及所检查的任何差异的重要性。