Functional Dissection of the Roles of eIF4A in Translational Control

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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.