Initiation of protein synthesis in eukaryotes is a complex process requiring more than 12 different initiation factors, comprising over 30 polypeptide chains. The functions of many of these factors have been established in great detail; however, the precise role of some of them and their mechanism of action still remain not well understood. eIF2A is a single chain 65 kDa protein that was initially believed to serve as the functional homologue of prokaryotic IF2, since eIF2A and IF2 catalyze biochemically similar reactions, i.e. they stimulate initiator methionyl-tRNA (Met-tRNAMeti) binding to the small ribosomal subunit. However, subsequent identification of a heterotrimeric 126 kDa factor, eIF2(????????) showed that this factor and not eIF2A is primarily responsible for the binding of Met-tRNAMeti to 40S ribosomal subunits in eukaryotes. In mammals, four stress- activated kinases reduce the level of active eIF2 by phosphorylating the eIF2??subunit and, consequently, reducing the global level of translation. However, translation of many cellular and viral proteins appeared to be resistant to eIF2? phosphorylation despite requiring Met-tRNAMeti. It was found that a subset of factors, including eIF2A, can promote efficient recruitment of Met-tRNAMeti to 40S/mRNA complexes under conditions of inhibition of eIF2 activity, or its absence. Recently, eIF2A was also reported to be involved in non-AUG dependent initiation in higher eukaryotes and the control of antigen presentation by major histocompatibility complex (MHC) class I molecules, the integrated stress response and tumor initiation and progression. All of these events were affected by eIF2A silencing in cellular models. Yet, the precise role of eIF2A in vivo, as well as the precise mechanism of its action still remain largely enigmatic. There is a fundamental gap in our understanding of how eIF2A functions in mammalian systems in vivo and ex vivo. To fill in this gap above and to continue the physical and functional characterization of a eukaryotic/mammalian eIF2A, we have created a viable homozygous eIF2A-total knockout mouse strain and obtained recombinant eIF2A expressed in E. coli cells. The ultimate goal of this proposal is to understand the function of eIF2A in vivo and in vitro. This goal will be achieved by a combination of in vitro, ex vivo and in vivo (mouse model) approaches. The outcome of this proposal will be important for understanding the basic mechanisms of the translational control of gene expression in higher eukaryotes, especially as part of the stress response.
Translational control is a principal mechanism for the dynamic regulation of gene expression in higher eukaryotes and it plays a pivotal role in development, differentiation, cell cycle progression, cell growth, apoptosis, and stress. As such, deregulation of translation has been shown to be associated with a wide range of human diseases, including but not limited to diabetes and cancer. This proposal employs a combination of in vitro, ex vivo and in vivo (knockout mouse model) approaches and is aimed at understanding of the molecular mechanism(s) by which a non-canonical eukaryotic initiation factor 2A (eIF2A), which stimulates initiator methionyl-tRNA binding to the small ribosomal subunit under stress conditions, functions and affects the physiological and pathophysiological processes in mammals.