The role of eIF4G in translation initiation and cell cycle progression

Critical information stored in the gene sequences of the genetic material (DNA) has to be decoded by the cell to produce a wide variety of essential proteins, as and when they are required. The sequence of each gene specifies the sequence of a given protein, with many proteins themselves in turn responsible for the synthesis of other types of structures in the cell. The general transfer of information from DNA to protein is carried out by the messenger RNA (mRNA), which is a copy of the DNA sequence and has to be decoded by a complex, highly regulated machine termed a ribosome, in a process known as translation. To work efficiently, accurately, and to allow the ribosome to function in the best interests of the cell, this machinery requires other proteins (translation initiation factors) that interact with each other, the mRNA and the ribosome in a highly regulated manner. One of these, eIF4G, acts as a scaffold protein, onto which the ribosome and several other initiation factors assemble. In mammalian cells, two slightly different genes contain the sequence for eIF4G, and the two genes also express different variants of the final protein. We wish to determine how these distinct proteins cause differences in how the ribosome and other initiation factors assemble onto and regulate the translation of different mRNAs during cell growth and division. This study will improve our understanding of the different stages of gene expression, and may also lead to the specific control of certain mRNAs that are deregulated during diseases, including cancer.

The regulation of translation initiation is a key point in gene expression, and can rapidly alter the temporal and spatial expression of either the whole transcriptome or specific mRNAs, without requiring new transcription. Central to translation initiation are the multi-domain proteins eIF4GI/II, which act as a scaffold for the assembly of the initiation complex via contacts with the mRNA cap-binding protein eIF4E, the RNA helicase, eIF4A, the ribosome-binding eIF3; the eIF4E kinase, Mnk1; and the poly(A)-binding protein (PABP). The two mammalian eIF4G proteins exist as different variants, and we propose to identify the particular roles in translation during the cell cycle of the different variants of eIF4GI versus eIF4GII. Exciting preliminary work to reduce eIF4GI expression in HeLa cells has revealed that this results in a reduction in overall translation rates whilst leading to detectable changes in the proteome as analysed by 2D gel electrophoresis. Silencing of eIF4GI expression also leads to aberrations in the progress of the cell cycle, causing defects in mitosis. In the proposed work, we will investigate the expression and/or modification of specific cell cycle proteins affected as a consequence of reduced expression of eIF4GI. In addition, we propose to examine the expression of eIF4GI/II isoforms during the cell cycle using a combination of Quantitative RT-PCR, polysome analysis and immunoblotting. This will be complemented by the use of siRNA-mediated knockout of eIF4GI and eIF4GII, either separately or in combination to determine the contribution to global and specific mRNA translation that these proteins have in different cell types. In further work, we propose to use cDNA microarrays to identify polysome-associated mRNAs that remain translated under conditions of reduced expression of eIF4G, with the new University of Sussex core proteomics facility being used to identify the effects that down-regulation of eIF4G have on the proteome. Reintroducing siRNA-resistant cDNAs expressing individual isoforms, fragments and binding partner mutants of eIF4GI and eIF4GII will enable the translational capacities of eIF4G isoforms and variants to be determined and will also contribute to work to identify novel binding partners of the eIF4G proteins.