TRANSLATIONAL INITIATION FACTOR EIF4E FAMILY MEMBERS IN C ELEGANS

线虫中的翻译起始因子 EIF4E 家族成员

• 批准号: 8363824
• 负责人: ROBERT E. RHOADS
• 金额: $ 0.54万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363824
• 关键词: Affinity Chromatography; Animal Model; Binding; Binding Proteins; Biochemical; Biological; Caenorhabditis elegans; Cytoplasmic Granules; Data; Eukaryotic Initiation Factor-4E; Family member; Funding; Goals; Grant; Individual; Knock-out; Mass Spectrum Analysis; Messenger RNA; National Center for Research Resources; Organism; Peptide Initiation Factors; Phenotype; Phosphorylation; Phosphorylation Site; Physiological; Principal Investigator; Process; Protein Binding; Protein Biosynthesis; Proteins; RNA Cap-Binding Proteins; Regulation; Research; Research Infrastructure; Resources; Ribosomes; Role; Sepharose; Site; Source; System; Translating; United States National Institutes of Health; cost; prevent

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The long-term goals are to understand the biochemical mechanisms, physiological regulation, and biological roles of the mRNA cap-binding protein eIF4E, which is involved in recruitment of mRNA to the ribosome. This process determines the rate of protein synthesis, the spectrum of mRNAs translated, and the rate of mRNA turnover. The project centers on two aspects of eIF4E: 1) the role of its phosphorylation and 2) proteins that bind to it. For the first, although the rate of protein synthesis and phosphorylation of eIF4E are highly correlated, the biochemical consequences of eIF4E phosphorylation remain elusive and controversial. The most definitive data will come from genetically tractable animal models like C. elegans. We have discovered and extensively characterized five family members of eIF4E in C. elegans, termed IFE-1, IFE-2, etc. We propose to determine the phosphorylation sites in each of the five IFEs by mass spectrometry. Then we will alter the site in one particular IFE to prevent phosphorylation and determine the result of expressing the modified form in knockout worms with regard to protein synthesis, spectrum of mRNAs translated, and overall phenotype of the organism. For the second, at least one of the IFEs is specifically bound to a protein, PGL-1, resident in P-granules, which are markers of the germline. In other systems, eIF4E-binding proteins are responsible for biological actions of eIF4E. We propose to identify proteins that co-purify with individual IFE family members by performing m7GTP-Sepharose affinity chromatography, comparing wild-type C. elegans with strains lacking each of the five IFEs.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 长期目标是了解mRNA帽结合蛋白eIF 4 E的生化机制、生理调节和生物学作用，该蛋白参与mRNA向核糖体的募集。 这一过程决定了蛋白质合成的速率、mRNA翻译的范围和mRNA周转的速率。该项目围绕eIF 4 E的两个方面：1）其磷酸化的作用和2）与其结合的蛋白质。首先，尽管eIF 4 E的蛋白质合成速率和磷酸化高度相关，但eIF 4 E磷酸化的生化后果仍然难以捉摸且有争议。 最确定的数据将来自遗传学上易于处理的动物模型，如C。优雅的 我们已经在C. elegans，被称为IFE-1，IFE-2等。我们建议通过质谱法确定五个IFE中的每一个的磷酸化位点。 然后，我们将改变一个特定IFE中的位点以防止磷酸化，并确定在敲除蠕虫中表达修饰形式的结果，包括蛋白质合成、翻译的mRNA谱和生物体的总体表型。 对于第二种情况，至少一种IFE特异性结合于P颗粒中的蛋白质PGL-1，其是生殖系的标记。 在其他系统中，eIF 4 E结合蛋白负责eIF 4 E的生物学作用。 我们建议通过进行m7 GTP-Sepharose亲和层析来鉴定与IFE家族成员共纯化的蛋白质，比较野生型C。具有缺乏五种IFE中的每一种的菌株的秀丽线虫。