Yeast eIF2A plays a minimal role in translation initiation in vivo
Earlier investigations have associated mammalian eIF2A with Met-tRNAi binding to the 40S subunit and its recruitment to specialized mRNAs in a GTP-independent manner. Additionally, eIF2A has been implicated in non-AUG start codon initiation, particularly under conditions where eIF2 function is attenuated by phosphorylation of its a-subunit during stress or starvation. However, the precise role of eIF2A in vivo translation remains unclear. Moreover, it's uncertain if the conserved ortholog in budding yeast can functionally substitute for eIF2 during stress. To address these questions, we conducted ribosome profiling on a yeast deletion mutant lacking eIF2A, alongside isogenic wild-type (WT) cells, both in the presence or absence of eIF2a phosphorylation induced by amino acid starvation. Overall design: This study includes a total of 16 libraries comprised of 8 RNA-Seq libraries (mRNA) and 8 ribosome footprint profiling libraries (ribo). The samples used for library preparation were sourced from two biological replicates of both the BY4742 (WT) and yeast eIF2A? deletion mutant F2247 (eIF2A?). The strains were cultured at 30ºC in synthetic complete (SC) medium to log-phase (A600 = 0.5–0.6). Additionally, the WT and eIF2A? strains were grown in SC media lacking isoleucine and valine (SC-Ile/Val) to log-phase (A600 = 0.5–0.6). Subsequently, they were treated with the antimetabolite Sulfometuron methyl (SM) at 1 µg/mL for 25 min to induce eIF2a phosphorylation.