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: 8169820
• 负责人: ROBERT E. RHOADS
• 金额: $ 0.18万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-12 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8169820
• 关键词: Affinity Chromatography; Animal Model; Binding; Binding Proteins; Biochemical; Biological; Caenorhabditis elegans; Computer Retrieval of Information on Scientific Projects Database; Cytoplasmic Granules; Data; Family member; Funding; Goals; Grant; Individual; Institution; Knock-out; Mass Spectrum Analysis; Messenger RNA; Organism; Peptide Initiation Factors; Phenotype; Phosphorylation; Phosphorylation Site; Physiological; Process; Protein Binding; Protein Biosynthesis; Proteins; RNA Cap-Binding Proteins; Regulation; Research; Research Personnel; Resources; Ribosomes; Role; Sepharose; Site; Source; System; Translating; United States National Institutes of Health; prevent

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 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，该蛋白参与 mRNA 向核糖体的募集。 这个过程决定了蛋白质合成的速率、翻译的 mRNA 的范围以及 mRNA 周转的速率。该项目集中于 eIF4E 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，尽管蛋白质合成速率和 eIF4E 磷酸化高度相关，但 eIF4E 磷酸化的生化后果仍然难以捉摸且存在争议。 最明确的数据将来自像秀丽隐杆线虫这样的遗传易驯化动物模型。 我们发现并广泛表征了秀丽隐杆线虫中 eIF4E 的五个家族成员，称为 IFE-1、IFE-2 等。我们建议通过质谱法确定五个 IFE 中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 谱和生物体的整体表型。 对于第二种情况，至少有一个 IFE 与存在于 P 颗粒中的蛋白质 PGL-1 特异性结合，P 颗粒是种系的标记。 在其他系统中，eIF4E 结合蛋白负责 eIF4E 的生物学作用。 我们建议通过进行 m7GTP-Sepharose 亲和层析，将野生型线虫与缺乏五个 IFE 的菌株进行比较，来鉴定与各个 IFE 家族成员共纯化的蛋白质。