Genetic and biochemical studies to understand the role of eIF2A in non canonical translation initiation under stress conditions using yeast model

Abstract

newlinexi newlineABSTRACT newlineMostly under stress conditions, highly conserved eukaryotic initiation factor (eIF) 2A stimulates initiator methionyl-tRNA (Met-tRNAi) binding to P-site of 40S ribosomal subunit in a codon-dependent manner and helps in protein synthesis via a non-canonical pathway. eIF2A, a 71.3 kDa protein in yeast, has mRNA, 40S as well as Met-tRNAi binding activity which mostly occur in response to stresses like nutrient depletion, viral infection, heme deprivation and during unfolded protein response following the initiation of endoplasmic reticulum stress when the and#945;-subunit of eIF2 gets phosphorylated which leads to the decrease in overall rate of translation. eIF2A provides an alternative initiation pathway to stimulate the internal translation initiation of specific viral and cellular mRNAs refractory to eIF2and#945; phosphorylation during stress conditions. Seven different eukaryotic species have homologs of eIF2A, indicating that eIF2A has some conserved roles. Human and yeast (S. cerevisiae) eIF2A share a 28 % identity and a 58 % similarity. In this study we wanted to know how eIF2A initiates non-canonical translation initiation under stress and what are the domains of eIF2A responsible for its interactions with mRNA, tRNAi and ribosome? So, we used yeast as a model system where different conserved residues of eIF2A were mutated to understand their role in non-canonical translation initiation. We found that 586-642 residues are involved in the formation of and#945;-helices structure of eIF2A. Lys503 is critical for eIF2A function because it might be involved in the interaction of eIF2A with tRNAi. The Short Linear Motif (SLiM) (507-14) is partially required for the interaction of eIF2A with tRNAi, whereas WN240-41 residues have also been linked to be involved in combating stress. We also found that eIF2A overexpression is toxic to cells under normal and stress conditions. As a result, eIF2A has been proposed as a possible target for novel anti-cancer or anti-viral therapies. newlineKeywords: eIF2A, stress, yeast, non-canonical translation initiation. newlinexi