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蛋白质的过程。许多mRNA含有抑制性结构，在mRNA可以有效地翻译成蛋白质之前需要解旋。此外，这些mRNA中的许多编码在调节细胞生长和发育中重要的蛋白质，并且这些蛋白质水平的扰动可导致癌症或细胞死亡。真核起始因子4A（eIF 4A）是一种蛋白质，其可以凭借其解旋酶活性解开这些原本抑制性的mRNA结构，并允许翻译装置访问由mRNA编码的信息。eIF 4A是最丰富的真核生物翻译起始因子，并且已经显示在参与控制细胞生长和分化的许多基因的调节中是关键的。然而，eIF 4A对整体基因表达的影响尚未详细研究。拟议的研究将使用几种策略来识别其调节在响应eIF 4A抑制时发生改变的基因，并阐明实现这种调节的机制。有趣的是，eIF 4A的丰度比已知与其相互作用的因子的丰度大得多，这表明eIF 4A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eIF 4A功能，其中包括eIF 4A的特异性和快速作用的小分子抑制剂;采用与eIF 4A结合的RNA分子以防止其发生重要构象变化的新方法;以及抑制eIF 4A功能不同方面的野生型和突变蛋白的表达。然后，我们可以区分由于eIF 4A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微阵列技术鉴定，然后克隆用于进一步分析。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eIF 4A是否具有独立于其解旋酶活性的功能。在已经被鉴定为eIF 4A介导调节的靶点的少量信息中，它们中的许多具有通过不常见的机制启动蛋白质合成的能力，所述机制涉及在称为帽非依赖性翻译起始的过程中将翻译机器募集到mRNA内部的点。将测试新鉴定的eIF 4A介导的翻译控制的靶标支持帽非依赖性翻译的能力。

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.