Histone pre-mRNA processing in Drosophila Melanogaster

果蝇中的组蛋白前体 mRNA 加工

• 批准号: 7276469
• 负责人: Mindy M Steiniger
• 金额: $ 5.29万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2008-12-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7276469
• 关键词: Affinity; Binding; Biochemical; Biological Models; Biological Process; Cell Cycle; Cell Death; Cell Line; Cell Survival; Collaborations; Complex; DNA Damage; DNA biosynthesis; DNA chemical synthesis; Defect; Drosophila genus; Drosophila melanogaster; Elements; Ensure; Escherichia coli; Goals; Heterogeneous Nuclear RNA; Histones; Homologous Gene; Human; Immunoprecipitation; In Vitro; Laboratories; Lactamase; Lead; Link; Location; Malignant Neoplasms; Mammals; Messenger RNA; Modeling; Mutation; North Carolina; Nuclear Extract; Phase; Polyadenylation; Process; Production; Proteins; Proteolysis; Proteome; RNA; RNA Processing; Radiolabeled; Reaction; Recombinant Proteins; Recombinants; Recruitment Activity; Regulation; Role; Site; Specificity; Structure; System; Techniques; Universities; X-Ray Crystallography; Yeasts; crosslink; endonuclease; mRNA Cleavage and Polyadenylation Factors; mRNA Precursor; protein E; radiotracer; research study; stem; tumorigenesis

DESCRIPTION (provided by applicant): RNA processing is essential for cell viability and many of proteins invoved in this phenomenon have only recently been elucidated. Production of mature histone mRNA from its precursor mRNA provides an excellent model system in which to study RNA processing because of a well-established in vitro system. The endonuclease responsible for cleavage of the histone precursor mRNA in mammals has recently been defined by the Marzluff Laboratory as cleavage and polyadenylation specificity factor (CPSF)-73. Since this discovery, biochemical and biophysical characterization of mammalian CPSF-73 has proven difficult because recombinant protein is unavailable. In an attempt to circumvent this problem, we have cloned the Drosophila melanogaster CPSF-73 homolog and successfully purified recombinant protein from E. coli. Here, I propose to functionally characterize D. melanogaster CPSF-73 by developing an in vitro system using nuclear extracts from the Dml-2 cell line and recombinant CPSF-73. By supplementing these nuclear extracts with recombinant CPSF-73, we hope to establish that CPSF-73 functions as the endonuclease for histone pre-mRNA processing in D. Melanogaster. Once this has been established, I will attempt to physically characterize the interaction between CPSF-73 and its substrate RNA. To determine the location of CPSF-73 binding, CPSF-73 will be UV-cross-linked to RNA substrates radiolabeled at different locations. The CPSF-73 residues involved in this interaction will be determined by LC/MS/MS analysis. Also, we have established a collaboration with Dr. Matt Redinbo (University of North Carolina at Chapel Hill) to determine the crystal structure of CPSF-73. Finally, CPSF-73 must be recruited to the histone pre-mRNA via interaction with other proteins in the cleavage complex. We hope to determine the identity of these other proteins using RNA affinity and immunoprecipitation techniques and to functionally characterize them using the previously developed in vitro system. Histone pre-mRNA processing is coordinated with the cell cycle to ensure that histones are abundant during DNA synthesis. Any defect in histone pre-mRNA processing can disrupt histone protein production can lead to oncogenesis, DNA damage, cell cycle defects and cell death. Therefore, a better understanding of histone pre-mRNA processing will lead to a better understanding of cancer.

描述（由申请人提供）：RNA加工对于细胞活力至关重要，并且直到最近才阐明了这种现象中的许多蛋白质。从其前体mRNA产生成熟的组蛋白mRNA，提供了一个出色的模型系统，可以在其中研究RNA处理，因为体外系统已建立良好。最近，胶凝实验室定义了哺乳动物中组蛋白前体mRNA裂解的核酸内切酶为裂解和聚腺苷酸化特异性因子（CPSF）-73。自从这一发现以来，事实证明，哺乳动物CPSF-73的生化和生物物理表征被证明很困难，因为重组蛋白不可用。为了解决这个问题，我们已经克隆了果蝇Melanogaster CPSF-73同源物，并从大肠杆菌中成功纯化了重组蛋白。在这里，我建议通过使用DML-2细胞系和重组CPSF-73的核提取物开发体外系统来在功能上表征D. Melanogaster CPSF-73。通过用重组CPSF-73补充这些核提取物，我们希望确定CPSF-73在D. melanogaster中的组蛋白前MRNA加工起作用。一旦建立了这一点，我将尝试实际表征CPSF-73及其底物RNA之间的相互作用。为了确定CPSF-73结合的位置，CPSF-73将与在不同位置在不同位置的RNA底物进行UV-CROSS连接。与此相互作用有关的CPSF-73残基将通过LC/MS/MS分析确定。此外，我们已经与Matt Redinbo博士（北卡罗来纳大学教堂山分校）建立了合作，以确定CPSF-73的晶体结构。最后，必须通过与裂解复合物中其他蛋白质相互作用将CPSF-73募集到组蛋白Pre-MRNA中。我们希望使用RNA亲和力和免疫沉淀技术确定这些其他蛋白质的身份，并使用先前开发的体外系统来表征它们。 组蛋白前MRNA加工与细胞周期协调，以确保在DNA合成过程中组蛋白丰富。组蛋白前MRNA加工中的任何缺陷都可以破坏组蛋白蛋白的产生，都会导致肿瘤发生，DNA损伤，细胞周期缺陷和细胞死亡。因此，对组蛋白前MRNA处理的更好理解将导致对癌症的更好理解。