Functional Dissection of the Roles of eIF4A in Translational Control

蛋白质合成是将储存在我们DNA中的遗传信息通过信使RNA （mRNA）中间体翻译成决定细胞性质的功能性蛋白质的过程。许多mrna含有抑制结构，这些抑制结构需要在mrna有效地翻译成蛋白质之前解绕。此外，许多这些mrna编码的蛋白质在调节细胞生长和发育中很重要，这些蛋白质水平的扰动可能导致癌症或细胞死亡。真核起始因子4A （Eukaryotic initiation factor 4A, eIF4A）是一种能够通过解旋酶活性解开这些抑制mRNA结构的蛋白质，并允许翻译装置获得mRNA编码的信息。eIF4A是真核生物中最丰富的翻译起始因子，已被证明在调控一些参与细胞生长和分化的基因中起关键作用。然而，eIF4A对全局基因表达的影响尚未得到详细的研究。拟议的研究将使用几种策略来识别其调控因eIF4A抑制而改变的基因，并描述实现这种调控的机制。有趣的是，eIF4A的丰度远远大于已知与之相互作用的因子的丰度，这表明eIF4A可能在细胞中具有其他额外的作用。我们将使用多种方法抑制培养细胞中的eIF4A功能，其中包括特异性和快速作用的eIF4A小分子抑制剂；利用与eIF4A结合的RNA分子阻止其发生重要构象变化的新方法；以及抑制eIF4A不同功能的野生型和突变型蛋白的表达。然后，我们可以区分由于eIF4A功能受损而导致行为改变的mrna和不受影响的mrna。表达水平受影响最大的mrna可以使用cDNA微阵列技术进行鉴定，然后克隆以进一步分析。重要的是，我们可以比较使用不同实验方法鉴定的靶标的差异，并确定eIF4A是否具有独立于解旋酶活性的功能。在少数已经被确定为eIF4A介导调控靶点的信息中，其中许多具有通过一种不常见的机制启动蛋白质合成的能力，该机制涉及将翻译机制招募到mRNA内部的一个点，这一过程被称为帽独立翻译起始。新发现的eIF4A介导的翻译控制靶点支持帽独立翻译的能力将被测试。

Protein synthesis is the process by which inherited information stored in our DNA is translated, via a messenger RNA (mRNA) intermediate, into functional proteins which determine the nature of a cell. Numerous mRNAs contain inhibitory structures which need to be unwound before the mRNAs can be efficiently translated into protein. Moreover, many of these mRNAs encode proteins that are important in regulating cell growth and development, and perturbations in the levels of these proteins can lead to cancer or cell death. Eukaryotic initiation factor 4A (eIF4A) is a protein which can unwind these otherwise inhibitory mRNA structures by virtue of its helicase activity, and allow the translation apparatus access to the information encoded by the mRNA. eIF4A is the most abundant eukaryotic translation initiation factor, and has been shown to be critical in the regulation of a number of genes involved in control of cell growth and differentiation. However, the influence of eIF4A on global gene expression has not been examined in detail. The proposed research will use several strategies to identify genes whose regulation is altered in response to eIF4A inhibition, and to characterise the mechanisms by which such regulation is achieved. It is interesting that the abundance of eIF4A is much greater than that of the factors it is known to interact with, suggesting that eIF4A may have other additional roles in the cell. We will suppress eIF4A function in cultured cells using a number of methods, among them specific and fast acting small molecule inhibitors of eIF4A; a novel approach employing an RNA molecule that binds to eIF4A to prevent it making important conformational changes; and expression of wild type and mutant proteins that inhibit different aspects of eIF4A function. We can then differentiate between mRNAs whose behaviour changes as a result of the compromising of eIF4A function and those mRNAs that are unaffected. mRNAs whose level of expression is most affected can be identified using cDNA microarray technology, and then cloned for further analysis. Importantly, we can compare the differences in targets identified using different experimental approaches and determine whether eIF4A has functions independent of its helicase activity. Of the small number of messages that have already been identified as targets for eIF4A mediated regulation, many of them have the ability to initiate protein synthesis by an uncommon mechanism involving recruitment of the translation machinery to a point internal to the mRNA in a process known as cap-independent tranlation initiation. The ability of newly identified targets of eIF4A mediated tranlational control to support cap-independent translation will be tested.