Abstract 2472 Investigating the role of eIF3 in translation initiation through long-read sequencing

Initiation is the rate-limiting and most highly-regulated step of translation by the ribosome.Misregulation of this translation can lead to the development of certain cancers and is observed in viral infections and other human diseases.Various eukaryotic initiation factors (eIFs) contribute to the formation of a ribosomal pre-initiation complex (PIC), its attachment to the end of an mRNA to be translated, and its subsequent scanning to locate the start site of translation.mRNA attachment, scanning, and start-codon recognition together comprise mRNA recruitment, the defining feature of translation initiation in eukaryotes.eIF3 is the largest and most complex eIF and is required for mRNA recruitment.In budding yeast (S. cerevisiae), eIF3 comprises five essential subunits conserved across eukaryotes but whose individual mechanistic contributions to mRNA recruitment remain unknown.We have previously demonstrated that the eIF3a subunit, which extends domains near the channels where mRNA enters and exits the PIC, is primarily responsible for RNA binding by eIF3.And yet, how these domains of eIF3a contribute to RNA binding and mRNA recruitment remains a mystery.To investigate these roles directly, we have designed expression plasmids to enable purification of eIF3a variants containing mutations in either its entry-or exit-channel arms.In parallel, we are performing fluorescence anisotropy experiments to monitor RNA binding and how it is affected by these mutations within the context of the full eIF3 complex.Going forward, we will investigate the effects of these mutations on RNA binding by eIF3a outside the context of the full complex and explore additional eIF3a mutations.These experiments will shed light on how the two arms of eIF3a contribute to key events during translation initiation.