Altered proteome in translation initiation fidelity defective eIF5G31R mutant causes oxidative stress and DNA damage.

The recognition of the AUG start codon and selection of an open reading frame (ORF) is fundamental to protein biosynthesis. Defect in the fidelity of start codon selection adversely affect proteome and have a pleiotropic effect on cellular function. Using proteomic techniques, we identified differential protein abundance in the translation initiation fidelity defective eIF5G31R mutant that initiates translation using UUG codon in addition to the AUG start codon. Consistently, the eIF5G31R mutant altered proteome involved in protein catabolism, nucleotide biosynthesis, lipid biosynthesis, carbohydrate metabolism, oxidation-reduction pathway, autophagy and re-programs the cellular pathways. The utilization of the upstream UUG codons by the eIF5G31R mutation caused downregulation of uridylate kinase expression, sensitivity to hydroxyurea, and DNA damage. The eIF5G31R mutant cells showed lower glutathione levels, high ROS activity, and sensitivity to H2O2.

Ribosomal mutation in helix 32 of 18S rRNA alters fidelity of eukaryotic translation start site selection.

The 40S ribosome plays a critical role in start codon selection. To gain insights into the role of its 18S rRNA in start codon selection, a suppressor screen was performed that suppressed the preferential UUG start codon recognition (Suppressor of initiation codon: Sui- phenotype) associated with the eIF5G31R mutant. The C1209U mutation in helix h32 of 18S rRNA was found to suppress the Sui- and Gcn- (failure to derepress GCN4 expression) phenotype of the eIF5G31R mutant. The C1209U mutation suppressed Sui- and Gcd- (constitutive derepression of GCN4 expression) phenotype of eIF2ßS264Y , eIF1K60E , and eIF1A-ΔC mutation. We propose that the C1209U mutation in 40S ribosomal may perturb the premature head rotation in 'Closed/PIN ' state and enhance the stringency of translation start site selection.