Regulation of Eukaryotic Protein Synthesis Initiation

描述(由申请人提供)：长期目标是了解将mRNA招募到核糖体的起始因子的生化机制、生理调节和生物学作用，这一过程决定了蛋白质合成速度、翻译的mRNAs谱和mRNAs周转率。研究集中在mRNA帽结合蛋白elF4E和elF3，elF4E是调节elF4E的一种机制，elF4E在mRNA周转中的作用，以及elF3是将elF4E(通过elF4G)连接到40S核糖体亚基的因子。目的1阐明5种elF4E亚型中的3种(称为IFE)在线虫中的生理作用。我们将使用生物信息学和生化方法来发现IFE-1、-2和-4的功能，包括对其翻译依赖于每个IFE的mRNAs的全基因组分析。我们将把IFE特异性翻译与mRNA结构特征和IFE特异性结合蛋白联系起来。目的2研究线虫中elF4E磷酸化及其可能的蛋白激酶MNK-1的功能。我们将试图通过确定IFE-2磷酸化对依赖于IFE-2的mRNAs的翻译和表型特征的影响来解决相互矛盾的报道，并从酶学和整个动物水平对推测的线虫MNK-1进行鉴定。在目标3中，我们将通过合成新的抵抗体内去顶的CAP类似物，并通过改变elF4E与Dcp1/Dcp2之间的CAP结合竞争，来研究elF4E在培养的哺乳动物细胞mRNA转换中的作用。在目标4中，我们将继续我们的观察，即哺乳动物elF3的一个亚基elF3e至少形成了elF4G的部分结合位点。我们将对elF4G-elF3e的相互作用和elFSe在翻译中的作用进行生化表征。我们还将尝试了解MMTV插入elFSe基因导致小鼠乳腺肿瘤的机制。这些研究与癌症相关，因为elF4E的过度表达会导致细胞恶性转化，人类肿瘤中含有大量升高的elF4E，而elF4E是新的抗癌治疗的靶点。此外，将MMTV整合到elFSe基因中是发展最好的实验癌症模型之一。此外，elF4E在生殖细胞成熟和早期胚胎发育中都发挥着关键作用，后者影响导致出生缺陷的异常。最后，正常的精子发生需要一个elF4E亚型，这可能有助于揭示不育条件。

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.