TRANSLATIONAL INITIATION FACTOR EIF4E FAMILY MEMBERS IN C ELEGANS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 长期的目标是了解mRNA帽结合蛋白eIF4E的生化机制、生理调节和生物学作用，eIF4E涉及到核糖体的mRNA募集。这一过程决定了蛋白质合成的速度、翻译的mRNAs的谱以及mRNAs的周转率。该项目围绕eIF4E的两个方面展开：1)其磷酸化作用；2)与其结合的蛋白质。首先，尽管eIF4E的蛋白质合成速度和磷酸化程度高度相关，但eIF4E磷酸化的生化后果仍然难以捉摸，也存在争议。最确定的数据将来自易遗传的动物模型，如线虫。我们已经在线虫中发现并广泛鉴定了5个eIF4E家族成员，分别命名为IFE-1、IFE-2等。我们建议用质谱仪来确定这5个IFE的磷酸化位点。然后，我们将改变一个特定的IFE中的位置，以防止磷酸化，并确定在敲除蠕虫中表达修饰形式的结果，涉及蛋白质合成、翻译的mRNAs谱以及生物体的整体表型。对于第二种，至少有一种IFE与一种驻留在P颗粒中的蛋白质PGL-1特异性结合，P颗粒是生殖系的标志。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-Sephose亲和层析来鉴定与单个IFE家族成员共同纯化的蛋白质，比较野生型线虫和缺乏这五种IFE的菌株。

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.