MRNA SYTHESIS IN ANIMAL CELLS--3END FORMATION

动物细胞中的 mRNA 合成--3END 形成

• 批准号: 2612915
• 负责人: James L. Manley
• 金额: $ 33.77万
• 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 2002-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2612915
• 关键词: HeLa cells; RNA binding protein; RNA biosynthesis; animal genetic material tag; cell line; chemical cleavage; clone cells; complementary DNA; genetic mapping; genetic regulation; high performance liquid chromatography; intermolecular interaction; messenger RNA; molecular cloning; molecular site; polyadenylate; posttranscriptional RNA processing; precursor mRNA; protein purification; protein sequence; recombinant proteins; tissue /cell culture; transcription factor

The experiments described in this proposal are designed to provide insights into the mechanism and regulation of pre-mRNA polyadenylation. The following five specific aims are proposed. 1. Purification and characterization of polyadenylation factors. Efforts to purified CF1 and especially CFII from HeLa nuclear extract will be continued. cDNA clones will be isolated and recombinant proteins and antibodies produced. Studies to understand the role of hPTS will be continued, and the possibility that additional polypeptides are required for 3' cleavage will be explored. 2. Analysis of protein-protein and protein- RNA interactions. Studies to understand the RNA binding specificities of CstF-64 and its yeast homologue RNA15 will be continued. The region of CPSF-160 responsible for AAUAAA binding, which appears to represent a novel RN binding motif, will be identified. Protein-protein interactions will be investigated, concentrating on the subunits of CstF, which all contain interesting domains. The possible existence and significance of overlapping for identical binding sites in CstF-64 for CstF-77 and hPTA will be investigated. In vitro protein binding assays will be employed when the interacting proteins 3. In vivo analysis of polyadenylation. Studies employing the chicken B cell line DT40 will be continued. A cell line with both alleles of CstF-64 disrupted, containing a tetracycline-repressible cDNA as the only source of CstF-64, will be further analyzed. The basis and specificity of a dramatic decrease in IgM H-chain mRNA accumulation will be determined and the effects of varies CstF-64 concentrations on alternative ploy (A) site selection studied further. Efforts to construct a similar line containing tet-repressible PAP will continue. This line will be used to investigate the functional significance of PAP isoforms produced by alternative splicing, and the requirement of PAP hyperphosphorylation during the cell cycle. 4. Regulation of polyadenylation. Studies examining the possible ability of CPEB to enhance polyadenylation by stabilizing CPSF binding through a direct interaction with CPSF-160 will be continued. Experiments to understand the basis for regulation of PAP activity by p34 cdc2/cyclin B phosphorylation will be continued, including analysis of the role of direct cyclin binding to PAP and the mechanism by which hyperphosphorylation inhibits PAP activity. The effects of PAP phosphorylation on protein binding will be explored, using the well-define U1A-PAP interaction as a model. 5. Polyadenylation and transcription. Recent studies suggesting a possible link between transcription and polyadenylation will be continued, first by establishing a coupled transcription-polyadenylation system. The importance of CPSF recruitment by TFIID for efficient 3' end formation will be investigated, as will the possibility that dissociation of CPSF from RNA polymerase II as the poly (A) signal contributes to subsequent transcription termination. Finally, the CstF-64 tet-repressible cell line will be used to determine if depletion of CstF-64 affects splicing of a poly(A) site proximity intron, transcription termination downstream of the poly (A) site, and/or transcription initiation.

本提案中描述的实验旨在提供 对前体mRNA多聚腺苷酸化的机制和调节的见解。 提出了以下五个具体目标。1.纯化及 多聚腺苷酸化因子的表征。努力纯化CF 1和 特别是HeLa细胞核提取物的CFII将继续进行。 cDNA 克隆将被分离，重组蛋白和抗体将被分离， 制作。 将继续研究hPTS的作用， 3 ′端需要额外多肽的可能性 将探索裂缝。 2.分析蛋白质-蛋白质和蛋白质- RNA相互作用 了解RNA结合特异性的研究 CstF-64及其酵母同源物RNA 15的研究将继续进行。 该地区 CPSF-160负责AAUAAA结合，这似乎代表了 一个新的RN结合基序，将被确定。 蛋白质 相互作用将被调查，集中在亚基的 CstF，它们都包含有趣的域。 可能的存在和 CstF-64中相同结合位点重叠的意义 将研究CstF-77和hPTA。 体外蛋白结合试验 当相互作用蛋白3.的体内分析 聚腺苷酸化 使用鸡B细胞系DT 40的研究将 请继续。 CstF-64的两个等位基因都被破坏的细胞系， 含有四环素阻遏的cDNA作为唯一的 CstF-64，将进一步分析。 A的基础和特殊性 将测定IgM H链mRNA积累的显著减少 以及不同浓度的CstF-64对替代策略的影响（A） 进一步研究选址问题。 建设类似线路的努力 含有tet抑制性PAP的药物将继续存在。 这条线将用于 研究PAP亚型的功能意义， 选择性剪接和PAP过度磷酸化的需要 在细胞周期中。 4.多聚腺苷酸化的调节。 研究 检查CPEB增强聚腺苷酸化的可能能力， 通过与CPSF-160的直接相互作用稳定CPSF结合， 请继续。 了解PAP调节基础的实验 p34 cdc 2/细胞周期蛋白B磷酸化的活性将继续， 包括分析细胞周期蛋白直接结合PAP的作用， 过度磷酸化抑制PAP活性的机制。的 将探索PAP磷酸化对蛋白结合的影响， 使用定义明确的U1 A-PAP相互作用作为模型。5.聚腺苷 和转录。 最近的研究表明， 转录和多聚腺苷酸化将继续进行，首先通过 建立偶联的转录-多聚腺苷酸化系统。 的 TFIID招募CPSF对于有效3'端形成的重要性 将被调查，因为将有可能解离CPSF 从RNA聚合酶II作为聚（A）信号有助于随后的 转录终止 最后，CstF-64 tet抑制细胞 线将用于确定CstF-64的耗尽是否影响剪接 poly（A）位点邻近内含子，转录终止下游 poly（A）位点和/或转录起始。