Regulation of Eukaryotic Protein Synthesis Initiation

描述（由申请人提供）：长期目标是了解将mRNA募集到核糖体的起始因子的生化机制、生理调节和生物学作用，这是一个决定蛋白质合成速率、mRNA翻译谱和mRNA周转速率的过程。研究集中在mRNA帽结合蛋白eIF 4 E，eIF 4 E的一种调节机制，eIF 4 E在mRNA周转中的作用，以及eIF 3，将eIF 4 E（通过eIF 4G）连接到40 S核糖体亚基的因子。目的1阐明eIF 4 E五种异构体中的三种（IFE）在C.优雅我们将使用生物信息学和生物化学方法来发现IFE-1，-2和-4的功能，包括对翻译依赖于每个IFE的mRNA进行全基因组分析。我们将IFE特异性翻译与mRNA结构特征和IFE特异性结合蛋白相关联。目的二是研究eIF 4 E磷酸化及其激酶MNK-1在C.优雅我们将试图通过确定IFE-2磷酸化对IFE-2依赖性mRNA翻译和表型性状的影响来解决矛盾的报告，我们将描述假定的C。elegans MNK-1在酶学和整个动物水平上的表达。在目标3中，我们将通过合成对体内去帽有抗性的新型帽类似物和通过改变eIF 4 E与Dcp 1/Dcp 2之间帽结合的竞争来研究eIF 4 E在培养的哺乳动物细胞中的mRNA周转中的作用。在目的4中，我们将继续我们的观察，即哺乳动物eIF 3的亚基之一eIF 3e形成eIF 4G的至少部分结合位点。我们将生物化学表征eIF 4G-eIF 3e相互作用和eIFSe在翻译中的作用。我们还将尝试了解MMTV插入eIFSe基因导致小鼠乳腺肿瘤的机制。这些研究与癌症相关，因为eIF 4 E的过表达导致细胞的恶性转化，人类肿瘤含有大大升高的eIF 4 E水平，并且eIF 4 E是新型抗癌疗法的靶标。此外，MMTV整合到eIFSe基因中是开发最好的实验癌症模型之一。此外，eIF 4 E在生殖细胞成熟和早期胚胎发育中起关键作用，其影响导致出生缺陷的异常。最后，一种eIF 4 E同种型是正常精子发生所需的，这可能揭示不育症。

DESCRIPTION (provided by applicant): The long-term goals are to understand the biochemical mechanisms, physiological regulation, and biological roles of initiation factors that recruit mRNA to the ribosome, a process that determines rate of protein synthesis, spectrum of mRNAs translated, and rate of mRNA turnover. The research is centered on the mRNA cap-binding protein elF4E, one mechanism by which elF4E is regulated, the role of elF4Ein mRNA turnover, and elF3, the factor that links elF4E (via elF4G) to the 40S ribosomal subunit. Aim 1 is to elucidate the physiological roles of three of the five elF4E isoforms (termed IFEs) in C. elegans. We will use bioinformatics and biochemical approaches to discover the functions of IFE-1, -2, and -4 including genome wide analysis of mRNAs whose translation is dependent on each IFE. We will correlate IFE-specific translation with mRNA structural features and with IFE-specific binding proteins. Aim 2 is to examine the function of elF4E phosphorylation and its putative kinase, MNK-1, in C. elegans. We will attempt to resolve contradictory reports by determining the effect of IFE-2 phosphorylation on translation of IFE-2-dependent mRNAs and phenotypic traits, and we will characterize the putative C. elegans MNK-1 at the enzymological and whole animal levels. In Aim 3, we will investigate the role of elF4E in mRNA turnover in cultured mammalian cells by synthesizing novel cap analogs that are resistant to in vivo decapping and by altering the competition for cap binding between elF4E and Dcp1/Dcp2. In Aim 4, we will follow up on our observation that elF3e, one of the subunits of mammalian elF3, forms at least part of the binding site for elF4G. We will biochemically characterize the elF4G-elF3e interaction and role of elFSe in translation. We will also attempt to learn the mechanism by which MMTV insertion into the elFSe gene causes mammary tumors in mice. These studies have relevance to cancer because overexpression of elF4E causes malignant transformation of cells, human tumors contain greatly elevated levels of elF4E, and elF4E is the target of novel anti-cancer therapies. Also, MMTV integration into the elFSe gene is one of the best developed experimental cancer models. Furthermore, elF4E plays a critical role in both germ cell maturation and early embryonic development, which impacts on abnormalities resulting in birth defects. Finally, one elF4E isoform is required for normal spermatogenesis, which may shed light on infertility conditions.