A Novel Factor Involved in Translation of Histone mRNA and Subsets of PolyA mRNA

参与组蛋白 mRNA 和 PolyA mRNA 子集翻译的新因子

• 批准号: 7408758
• 负责人: RACHEL S. LERNER
• 金额: $ 4.96万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-01 至 2009-06-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7408758
• 关键词: Affect; Binding; Binding Proteins; Biochemical; Cell Cycle; Cell Death; Cell physiology; Collaborations; Complex; DNA Damage; DNA biosynthesis; DNA chemical synthesis; Data; Defect; Elements; Elongation Factor; Ensure; Essential Genes; Eukaryotic Initiation Factors; Fibrinogen; Genetic Translation; Goals; Heating; Histones; Immunoprecipitation; Laboratories; Lead; Malignant Neoplasms; Mammalian Cell; Mass Spectrum Analysis; Mediating; Messenger RNA; Methods; Microarray Analysis; Molecular; Mutation; Names; Phase; Play; Poly A; Poly(A) Tail; Poly(A)-Binding Proteins; Process; Production; Protein Binding; Proteins; RNA Interference; RNA-Binding Proteins; Reporter; Repression; Ribosomes; Role; Structure; Techniques; Testing; Translation Process; Translational Activation; Translations; Universities; Xenopus oocyte; Yeasts; in vivo; novel; stem; tissue/cell culture; translation assay; translation factor; tumorigenesis; yeast two hybrid system

DESCRIPTION (provided by applicant): Histone mRNA levels are regulated coordinately with the cell cycle, accumulating at the beginning of Sphase and being degraded at the end of S-phase. These metazoan replication-dependent histone mRNAs are unique, in that unlike other mRNAs, they are not polyadenylated, instead ending in a conserved stemloop structure. This unique 3' end is bound by the stemloop binding protein (SLBP), which plays a role in histone mRNA processing, translation, and degradation. My studies focus on understanding the role of a novel protein discovered in the Marzluff lab which binds SLBP, named SLIP1. Preliminary data has shown that SLIP1 is involved in histone mRNA translation and possibly in the translation of a subset of poly (A) mRNAs, and is an essential gene. The broad long-term objectives of this proposal are to define the molecular details of the role of SLIP1 as a translation factor and to determine what cellular mRNAs in addition to histone mRNAs require SLIP1 for translation. To characterize SLIP1 as a translation factor, I will employ molecular and biochemical techniques. In vivo translation assays using both Xenopus oocytes and mammalian cells will determine mRNA cis-elements important for SLIP1-mediated translation. Additional biochemical approaches will be used to examine SLIP1 binding to general translation factors and ribosomes. To identify SLIP1 binding proteins, I will perform both a yeast-two-hybrid screen using SLIP1 as bait, and immunoprecipitations of SLIP1 followed by mass spectrometry. Proteins identified will be validated by a translation assay to confirm a functional interaction between SLIP1 and proteins identified. Celluar mRNAs translationally regulated by SLIP1 will be identified through immunoprecipitation of SLIP1-RNP complexes, followed by microarray analysis of precipitated mRNAs. Microarray analysis will be accomplished through a collaboration with Dr. Michael Whitfield's lab (Dartmouth University), who have expertise with microarray analysis and have previously collaborated with the Marzluff lab. mRNAs identified through this approach will be tested by in vivo translation assays to determine the affect of SLIP1 on their translation. Histone mRNA levels are coordinated with the cell cycle to ensure that histone proteins are abundant during DNA synthesis. Mutations causing defects in histone protein production can lead to oncogenesis, DNA damage, cell cycle defects, and cell death. Thus an understanding of factors regulating histone mRNA translation will contribute to our understanding of cancer.

描述（申请人提供）：组蛋白mRNA水平与细胞周期协调调节，在期开始时积累，在s期结束时降解。这些后生动物复制依赖的组蛋白mrna是独特的，因为与其他mrna不同，它们不是聚腺苷化的，而是以保守的茎环结构结束。这个独特的3'端与茎环结合蛋白（SLBP）结合，在组蛋白mRNA的加工、翻译和降解中起作用。我的研究重点是了解在Marzluff实验室发现的一种结合SLBP的新蛋白的作用，名为SLIP1。初步资料表明，SLIP1参与组蛋白mRNA的翻译，并可能参与poly (a) mRNA亚群的翻译，是一个必需基因。本提案的长期目标是定义SLIP1作为翻译因子作用的分子细节，并确定除了组蛋白mrna之外，哪些细胞mrna需要SLIP1进行翻译。为了将SLIP1描述为翻译因子，我将采用分子和生化技术。使用爪蟾卵母细胞和哺乳动物细胞进行体内翻译试验将确定对slip1介导的翻译重要的mRNA顺式元件。其他的生化方法将用于检测SLIP1与一般翻译因子和核糖体的结合。为了鉴定SLIP1结合蛋白，我将使用SLIP1作为诱饵进行酵母-双杂交筛选，并对SLIP1进行免疫沉淀，然后进行质谱分析。鉴定的蛋白质将通过翻译实验验证，以确认SLIP1与鉴定的蛋白质之间的功能相互作用。通过对SLIP1- rnp复合物的免疫沉淀，鉴定由SLIP1翻译调节的细胞mrna，然后对沉淀的mrna进行微阵列分析。微阵列分析将通过与Michael Whitfield博士的实验室（达特茅斯大学）合作完成，该实验室具有微阵列分析方面的专业知识，并曾与Marzluff实验室合作。通过这种方法鉴定的mrna将通过体内翻译试验进行测试，以确定SLIP1对其翻译的影响。组蛋白mRNA水平与细胞周期协调，以确保组蛋白在DNA合成过程中丰富。引起组蛋白产生缺陷的突变可导致肿瘤发生、DNA损伤、细胞周期缺陷和细胞死亡。因此，了解组蛋白mRNA翻译的调节因子将有助于我们对癌症的理解。