Functional Dissection of the Roles of eIF4A in Translational Control

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.

Protein synthesis represents a direct and rapidly responsive target for regulation of gene expression, although our understanding of the processes involved is only beginning to reveal their full complexity. Many mRNAs contain long structured 5' UTRs constituting a significant barrier to ribosome scanning necessary for translation initiation. mRNAs for proteins involved in regulation of growth, differentiation and metabolic pathways, and in protection from external damage, are more likely to contain long structured 5' UTRs, and these transcripts are thought to be poorly translated under normal conditions. eIF4A is the most abundant eukaryotic translation initiation factor, present in the cell at many times the concentration of its known binding partners. It is the prototypical DEAD-box RNA helicase and is proposed to unwind secondary structure in mRNAs, allowing the scanning 40S ribosomal subunit to reach the initiation codon. eIF4A overexpression, and also repression of Pdcd4 (which inhibits eIF4A), is associated with dysregulation of cell growth and differentiation. The aims of this project are to characterise the roles of eIF4A in the translational regulation of cap-dependent and IRES-mediated translation of cellular mRNAs. We will identify mRNAs that are targets for translational regulation by eIF4A. To do this we will use a number of parallel strategies to inhibit eIF4A functions, followed by sucrose density sedimentation and cDNA micro-array analysis to compare the translational status of large numbers of mRNAs in treated and control cells. By using multiple methods to inhibit different aspects of eIF4A function, we can establish whether eIF4A has functions independent of its translation initiation associated helicase activity, as may be suggested by its abundance. mRNAs whose translational status is most affected will be selected for further characterisation.