Investigating the role of 4E-T, an eIF4E-binding protein resident in P-bodies, in gene expression control

DNA在细胞核中被复制成信使RNA，然后转移到细胞质中，mRNA被翻译成蛋白质。mrna以不同的速率被翻译成蛋白质，从而导致蛋白质水平的巨大差异，这在一定程度上解释了为什么肌肉细胞和红细胞虽然共享相同的基因，但却如此不同。mRNA翻译的调控主要由eIF4E控制，eIF4E结合mRNA 5'端不寻常的帽状结构。eIF4E很重要，因为它与eIF4G相互作用，后者将核糖体（翻译机制）招募到mrna上。存在替代的eIF4E结合蛋白，如4e - bp，它们阻止eIF4G与eIF4E结合并减少翻译。我建议研究最近发现的一种名为4E-T的eif4e结合蛋白，它也与eIF4G竞争eif4e结合。有趣的是，4E-T和eIF4E的一小部分集中在显微镜下可见的点状细胞质“体”中，称为p体，缺乏核糖体，被认为是mrna储存和返回翻译的地方，或者mrna降解的地方。尽管对p体的研究兴趣浓厚，但它们的作用仍然是个谜，尽管它们影响病毒复制等。我们和其他研究人员发表的和未发表的工作表明，4E-T调节mrna的一个子集，但不知道它们编码的是哪种蛋白质，也不知道4E-T是否必须在p体中发挥这种控制作用，也不知道它在那里结合了什么蛋白质，也不知道它通过磷酸化修饰来实现这种控制意味着什么。这些都是我们想要回答的问题，在培养中使用人类细胞。我们认为这项工作是非常及时的，因为异常水平的eIF4E（及其磷酸化）与细胞生长、增殖和癌症发展有关，因此研究与eIF4E相互作用的因子是很重要的。此外，p体的作用仍然相对未被探索，我们的工作将对我们理解mrna在这些病灶中的调节方式做出重大贡献。最后，我们有幸与来自莱斯特MRC毒理学部门的安妮·威利斯教授合作，承担4E-T mRNA靶点的部分分析，并从那里的翻译资源数据库中扩展和受益，该数据库包含我们和其他研究人员的类似实验数据。本研究新颖、集中、及时，其结果将有助于我们理解和利用eIF4E对翻译起始的调控及其相互作用因素。

DNA is copied into messenger RNA in the nucleus, and following transfer to the cytoplasm, mRNA is translated into protein. mRNAs are translated into protein at different rates, leading to vastly different protein levels, which in part explains why muscle and red blood cells, for example, are so distinct though sharing the same genes. Regulation of mRNA translation is principally controlled by eIF4E, which binds the unusual cap structure at the 5' end of mRNA. eIF4E is important because it interacts with eIF4G which recruits ribosomes, the translational machinery, to mRNAs. Alternative eIF4E-binding proteins exist, such as 4E-BPs and these prevent eIF4G binding to eIF4E and reduce translation. I propose to investigate a more recently discovered eIF4E-binding protein called 4E-T, which also competes with eIF4G for eIF4E-binding. Interestingly, 4E-T and a fraction of eIF4E is concentrated in punctate cytoplasmic "bodies" visible in the microscope, called P-bodies, which lack ribosomes, and are thought to be sites where mRNAs are stored and returned to translation, or where mRNAs are degraded. Despite considerable interest, the role of these P-bodies remains enigmatic, though they influence for example virus replication. Published and unpublished work from us and other investigators show that 4E-T regulates a sub-set of mRNAs, but it is not known what sort of proteins they encode, nor is it known whether 4E-T has to be in P-bodies to exert this control, what proteins it binds there, or what its modification by phosphorylation means for this control. These are the questions we want to answer, using human cells in culture. We believe this work is highly timely since aberrant levels of eIF4E (and its phosphorylation) has been implicated in cell growth, proliferaion and in cancer development, and therefore investigation of a factor that interacts with eIF4E is important. Moreover, the role of P-bodies is still relatively unexplored, and our work will make a significant contribution to our understanding of how mRNAs are regulated in these foci. Last, we're fortunate to collaborate with Professor Anne Willis from the MRC Toxicology Unit, Leicester, to undertake a part of the analysis of 4E-T mRNA targets, and to extend and benefit from a Translational Resource database there, containing data from our and similar experiments from other investigators. The proposed work is novel, focused and timely, and the outcomes will contribute to our understanding and possible exploitation of the regulation of translation initiation by eIF4E and its interacting factors.