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.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 长期目标是了解mRNA帽结合蛋白EIF4E的生化机制，生理调节和生物学作用，该蛋白与将mRNA募集到核糖体中有关。 该过程决定了蛋白质合成的速率，被翻译的mRNA谱和mRNA更新速率。该项目集中在EIF4E的两个方面：1）其磷酸化的作用和2）与之结合的蛋白质。 首先，尽管EIF4E的蛋白质合成速率和磷酸化速率高度相关，但EIF4E磷酸化的生化后果仍然难以捉摸和有争议。 最明确的数据将来自诸如秀丽隐杆线虫（C. ofemans）等遗传障碍动物模型。 我们已经发现并广泛地表征了秀丽隐杆线虫中五个家族成员，称为IFE-1，IFE-2等。我们建议通过质谱法确定五个IFE的磷酸化位点。 然后，我们将在一个特定的IFE中改变位点以防止磷酸化，并确定在蛋白质合成，翻译的mRNA谱和生物体的整体表型方面表达敲除蠕虫中修饰形式的结果。 第二，至少一个IFE与蛋白质PGL-1特别结合，居民是p颗粒，这是种系的标志物。 在其他系统中，EIF4E结合蛋白负责EIF4E的生物作用。 我们建议通过执行M7GTP-Sepharose亲和力色谱法鉴定与单个IFE家族成员共纯化的蛋白质，将野生型C.秀丽隐杆线虫与缺乏五个IFE的菌株进行比较。