Translation initiation factor eIF3 promotes programmed stop codon readthrough.

Programmed stop codon readthrough is a post-transcription regulatory mechanism specifically increasing proteome diversity by creating a pool of C-terminally extended proteins. During this process, the stop codon is decoded as a sense codon by a near-cognate tRNA, which programs the ribosome to continue elongation. The efficiency of competition for the stop codon between release factors (eRFs) and near-cognate tRNAs is largely dependent on its nucleotide context; however, the molecular mechanism underlying this process is unknown. Here, we show that it is the translation initiation (not termination) factor, namely eIF3, which critically promotes programmed readthrough on all three stop codons. In order to do so, eIF3 must associate with pre-termination complexes where it interferes with the eRF1 decoding of the third/wobble position of the stop codon set in the unfavorable termination context, thus allowing incorporation of near-cognate tRNAs with a mismatch at the same position. We clearly demonstrate that efficient readthrough is enabled by near-cognate tRNAs with a mismatch only at the third/wobble position. Importantly, the eIF3 role in programmed readthrough is conserved between yeast and humans

The candidate antimalarial drug MMV665909 causes oxygen-dependent mRNA mistranslation and synergises with quinoline-derived antimalarials

To cope with growing resistance to current antimalarials, new drugs with novel modes of action are urgently needed. Molecules targeting protein synthesis appear to be promising candidates. We identified a compound (MMV665909) from the MMV Malaria Box of candidate antimalarials that could produce synergistic growth inhibition with the aminoglycoside antibiotic paromomycin, suggesting a possible action of the compound in mRNA mistranslation. This mechanism of action was substantiated with the yeast cell model using available reporters of mistranslation and other genetic tools. Mistranslation induced by MMV665909 was oxygen-dependent, suggesting a role for reactive oxygen species (ROS). Overexpression of Rli1 (a ROS-sensitive, conserved FeS protein essential in mRNA translation) rescued inhibition by MMV665909, consistent with the drug’s action on translation fidelity being mediated through Rli1. The MMV drug also synergised with major quinoline-derived antimalarials which can perturb amino acid availability or promote ROS stress: chloroquine, amodiaquine and primaquine. The data collectively suggest translation-fidelity as a novel target of antimalarial action and support MMV665909 as a promising drug candidate

Structural integrity of the PCI domain of eIF3a TIF32 is required for mRNA recruitment to the 43S pre initiation complexes

Transfer of genetic information from genes into proteins is mediated by messenger RNA (mRNA) that must be first recruited to ribosomal pre-initiation complexes (PICs) by a mechanism that is still poorly understood. Recent studies showed that besides eIF4F and poly(A)-binding protein, eIF3 also plays a critical role in this process, yet the molecular mechanism of its action is unknown. We showed previously that the PCI domain of the eIF3c/NIP1 subunit of yeast eIF3 is involved in RNA binding. To assess the role of the second PCI domain of eIF3 present in eIF3a/TIF32, we performed its mutational analysis and identified a 10-Ala-substitution (Box37) that severely reduces amounts of model mRNA in the 43–48S PICs in vivo as the major, if not the only, detectable defect. Crystal structure analysis of the a/TIF32-PCI domain at 2.65-Å resolution showed that it is required for integrity of the eIF3 core and, similarly to the c/NIP1-PCI, is capable of RNA binding. The putative RNA-binding surface defined by positively charged areas contains two Box37 residues, R363 and K364. Their substitutions with alanines severely impair the mRNA recruitment step in vivo suggesting that a/TIF32-PCI represents one of the key domains ensuring stable and efficient mRNA delivery to the PICs