Functional Characterization of a Novel Mammalian Homologue of eIF4E

Jagus MCB 9808401本研究的目的是表征一种与翻译起始因子eIF4E具有同源性的新蛋白的功能。目的是确定该蛋白在mRNA募集调节中的作用。实验策略旨在确定这种eif4样蛋白（4E-LP）是否刺激或抑制mrna的募集进行翻译，并鉴定与4E-LP相互作用以执行其功能的蛋白质。这将在这一重要步骤上扩展我们对基因表达控制的理解。已建立了人、小鼠和斑马鱼4E-LP的cDNA序列。序列比较表明，4E-LP存在于广泛的动物和植物物种中，并且高度保守。Northern的分析表明，4E-LP的mRNA存在于迄今为止测试的所有组织中，并且在早期发育过程中受到调节。同样，该蛋白似乎存在于所有被测试的组织中，尽管其含量低于eIF4E。与eIF4E一样，4E-LP可以绑定帽结构并与eIF4G相互作用。与eIF4E不同，4E-LP在羧基端没有调节磷酸化位点。目前尚不清楚4E-LP的作用是作为促进特定mrna（或特定生理条件下的所有mrna）翻译的替代起始因子，还是该蛋白以调节能力调节eIF4E的活性（通过与eIF4E相互作用蛋白竞争）。在斑马鱼发育模型中，我们研究了4E-LP在早期发育过程中对基因表达的调控作用，并从中获得了eIF4E和eIF4G。eIF4E通过控制mRNA的募集在基因表达调控中起关键作用。eIF4E通过结合5' m7Gppp帽结构来招募rna。eIF4E的活性受到高度调控，该因子已被证明是生长因子受体和原癌基因转导信号的主要靶标。eIF4E与eIF4G结合起作用。这些因子一起将mrna招募到核糖体中进行翻译。eIF4E和eIF4G对mrna募集的调节受MAP激酶和雷帕霉素敏感信号通路的调控，这些信号通路控制eIF4E的磷酸化和调节性eIF4E结合蛋白1,4e - bp1。eIF4E/4G功能的调控也可以通过与eIF4G具有同源性的蛋白的干预来实现。这些蛋白要么侵入eIF4E与eIF4G的相互作用（eIF4E结合蛋白或4e - bp），要么隔离募集mRNA所需的其他起始因子（NAT1、ap -5或p97）。4E-LP的发现为mRNA募集的调控增加了另一个方面，并对多种细胞功能的调控具有重要意义。Jagus 2。非技术细胞蛋白质的生产始于模板遗传密码，以mRNA的形式复制。mRNA编码的信息在核糖体上被翻译成蛋白质。控制核糖体对mRNA的募集是调控动物细胞基因表达的一种重要形式。失去对这一步骤的控制可能导致发育异常和生长控制的丧失。核糖体募集mRNA需要几种蛋白质因子，其中最重要的是翻译因子eIF4E。eIF4E识别并结合到mRNA的5'端帽结构上。eIF4E受到可逆修饰（磷酸化）和与其他蛋白相互作用的严格调控。本研究的目的是表征一种与eIF4E具有同源性的新蛋白4E-LP。编码4E-LP的mRNA存在于所有测试组织中，并在早期发育过程中受到调节。与eIF4E一样，4E-LP可以结合帽结构并与翻译的其他成分相互作用。最大的问题是它是作为eIF4E的类似物起作用还是作为eIF4E功能的竞争性抑制剂起作用。4E-LP在斑马鱼发育系统中的发育意义将被研究。的翻译。最大的问题是它是作为eIF4E的类似物起作用，还是作为eIF4E功能的竞争性抑制剂起作用。我们将研究4E-LP在斑马鱼发育系统中的发育意义。4 Jagus， R. C.项目描述NSF FORM 1360 (1/94

Jagus MCB 9808401 1. Technical The aim of this study is to characterize the function(s) of a novel protein that shares homology with the translational initiation factor eIF4E. The goal is to establish the role(s) of the protein in the regulation of mRNA recruitment. The experimental strategy is designed to determine whether this eIF4E-like protein (4E-LP) stimulates or inhibits the recruitment of mRNAs for translation and identify the proteins with which 4E-LP interacts to perform its function(s). This will extend our understanding of the control of gene expression at this important step. cDNA sequences have been established for human, mouse and zebrafish 4E-LP. Sequence comparisons have shown the 4E-LP is present in a wide range of animal and plant species and is highly conserved. Northern analysis has shown that the mRNA for 4E-LP is present in all tissues tested to date and is under regulation during early development. Similarly, the protein appears to be in all tissues tested, although at a lower level than eIF4E. Like eIF4E, 4E-LP can bind cap structures and interact with eIF4G. Unlike eIF4E, 4E-LP does not have the regulatory phosphorylation site at the carboxy-terminus. It remains to be determined whether the role of 4E-LP is that of an alternate initiation factor which facilitates the translation of specific mRNAs (or all mRNAs under specific physiological conditions), or whether the protein functions in a regulatory capacity to modulate the activity of eIF4E (by competing with eIF4E interacting proteins). The developmental regulation which suggests a role for 4E-LP in the regulation of gene expression during early development, is investigated in the zebrafish developmental model, from which eIF4E and eIF4G have been obtained. eIF4E plays a pivotal role in the regulation of gene expression by controlling mRNA recruitment. eIF4E recruits RNAs by binding to the 5' m7Gppp cap structure. The activity of eIF4E is highly regulated and the factor has been shown to be a major t arget of signals transduced from growth factor receptors and proto-oncogenes. eIF4E functions in conjunction with eIF4G, to which it binds. Together these factors recruit mRNAs to the ribosomes for translation. The regulation of the recruitment of mRNAs by eIF4E and eIF4G is regulated by the MAP kinase and rapamycin-sensitive signalling pathways which control phosphorylation of eIF4E and the regulatory eIF4E binding protein 1, 4E-BP1. Regulation of eIF4E/4G function can also occur through the intervention of proteins that share homology with eIF4G. These proteins either encroach upon the interaction of eIF4E with eIF4G (eIF4E binding proteins or 4E-BPs) or sequester other initiation factors required for the recruitment of mRNA (NAT1, DAP-5, or p97). The discovery of 4E-LP adds yet another facet to the regulation of mRNA recruitment and has implications for the regulation of diverse cellular functions. Jagus 2. Non-technical The production of cellular proteins begins with the template genetic code that is copied in the form of mRNA. The information encoded by mRNA is translated on the ribosomes into proteins. Control of the recruitment of mRNA by ribosomes is an important form of regulation of gene expression in animal cells. Loss of control of this step can lead to developmental abnormalities and the loss of growth control. Several protein factors are required for the recruitment of mRNA by the ribosome, the most important of which is the translation factor eIF4E. eIF4E recognizes and binds to the cap structure at the 5' end of mRNA. eIF4E is under heavy regulation by reversible modification (phosphorylation) and by interaction with other proteins. The aim of this study is to characterize a novel protein, 4E-LP, that shares homology with eIF4E. The mRNA encoding 4E-LP is present in all tissues tested and is under regulation during early development. Like eIF4E, 4E-LP can bind to cap structures and interact with other components of translation. The big question is whether it function as an eIF4E look-alike or as a competitive inhibitor of eIF4E function. The developmental significance of 4E-LP will be investigated in zebrafishdevelopmepmental systems.of translation. The big questio n is whether it functions as an eIF4E look-alike or as a competive inhibitor of eIF4E function. The developmental significance of 4E-LP will be investigated in the zebrafish developmental system. Jagus, R., Ph.D. A. PROJECT SUMMARY 4 Jagus, R. C. PROJECT DESCRIPTION NSF FORM 1360 (1/94) 5