Histone pre-mRNA processing in Drosophila Melanogaster

果蝇中的组蛋白前体 mRNA 加工

基本信息

批准号：
7489898
负责人：
Mindy M Steiniger
金额：
$ 2.53万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
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/7489898
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

项目摘要

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，并且这种现象中涉及的许多蛋白质最近才被阐明。由于成熟的体外系统，从其前体 mRNA 生产成熟组蛋白 mRNA 提供了一个极好的模型系统，可用于研究 RNA 加工。 Marzluff 实验室最近将哺乳动物中负责切割组蛋白前体 mRNA 的核酸内切酶定义为切割和聚腺苷酸化特异性因子 (CPSF)-73。自这一发现以来，哺乳动物 CPSF-73 的生化和生物物理表征已被证明很困难，因为重组蛋白无法获得。为了解决这个问题，我们克隆了黑腹果蝇CPSF-73同源物，并成功从大肠杆菌中纯化了重组蛋白。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对黑腹果蝇 CPSF-73 进行功能表征。通过用重组 CPSF-73 补充这些核提取物，我们希望确定 CPSF-73 作为黑腹果蝇组蛋白前体 mRNA 加工的核酸内切酶。一旦确定了这一点，我将尝试从物理角度表征 CPSF-73 与其底物 RNA 之间的相互作用。为了确定 CPSF-73 结合的位置，CPSF-73 将与在不同位置进行放射性标记的 RNA 底物进行 UV 交联。参与这种相互作用的 CPSF-73 残基将通过 LC/MS/MS 分析确定。此外，我们还与 Matt Redinbo 博士（北卡罗来纳大学教堂山分校）建立了合作关系，以确定 CPSF-73 的晶体结构。最后，CPSF-73 必须通过与裂解复合物中的其他蛋白质相互作用而被招募到组蛋白前体 mRNA 中。我们希望使用 RNA 亲和力和免疫沉淀技术来确定这些其他蛋白质的身份，并使用先前开发的体外系统对它们进行功能表征。组蛋白前体 mRNA 加工与细胞周期协调，以确保 DNA 合成过程中组蛋白丰富。组蛋白前体 mRNA 加工中的任何缺陷都会破坏组蛋白的产生，从而导致肿瘤发生、DNA 损伤、细胞周期缺陷和细胞死亡。因此，更好地了解组蛋白前体 mRNA 加工将有助于更好地了解癌症。