The mechanism and regulation of mRNA recruitment during eukaryotic translation initiation

PROJECT SUMMARY The efficiency with which messenger RNAs (mRNAs) are translated into proteins by the ribosome is a fundamental determinant of gene expression. This efficiency is often determined during the mRNA recruitment step of translation by the ribosome. Consequently, this step is a crucial point of control for gene expression. In eukaryotes, mRNA recruitment is an elaborate, multi-step, and highly regulated process that depends upon the activities of ~13 eukaryotic initiation factors (eIFs). Dysregulation of eIF activity and mRNA recruitment has been causally linked to tumorigenesis, tumor growth, drug resistance, and metastasis in an increasing list of human cancers. Consequently, several eIFs and their roles in mRNA recruitment are emerging as very attractive anticancer drug targets, with an existing, FDA-approved, eIF-targeting compound already having been successfully repurposed as an anticancer therapy. In order to expand and fully exploit this therapeutic potential, however, it is necessary to understand the molecular events that underlie eIF function and mRNA recruitment. Here, we will use a highly purified, fluorophore-labeled, Saccharomyces cerevisiae in vitro translation system that we developed and that includes a full-length, site-specifically labeled eIF4G and a fully reconstituted, site-specifically labeled eIF3, reagents that have been difficult to generate. With these reagents in hand, we will use state-of-the-art, single-molecule fluorescence microscopy and cryogenic electron microscopy (cryo-EM), including a pioneering, time-resolved cryo-EM approach developed by our collaborator, Dr. Joachim Frank, to directly observe and characterize the dynamics of mRNA recruitment during eukaryotic translation initiation. In Aim 1, we will investigate the mechanism through which the multi-component eIF4F complex activates different classes of mRNAs for loading onto ribosomal 43S pre-initiation complexes (PICs), and how changes to the composition of the eIF4F complex can alter which classes of mRNAs are activated. We hypothesize that the structural dynamics of the activated mRNA complex are critical for mRNA loading and will quantify how these dynamics contribute to mRNA selection. In Aim 2, we will investigate how the multi-component eIF3 complex interacts with different classes of mRNAs and/or the 43S PIC in order to facilitate mRNA activation and/or loading onto a 43S PIC, as well as how biologically active subcomplexes of eIF3 can modulate these activities. A large- scale structural rearrangement of 43S PIC-bound eIF3 is thought to control its mRNA loading activity and we will therefore characterize how this rearrangement facilitates formation of the 48S PIC on mRNAs of different classes. In Aim 3, we will investigate the mechanism through which eIF1A and eIF5B mediate mRNA start-codon recognition within a 48S PIC. Start-codon recognition by eIF1A has recently been associated with a large-scale rearrangement of the 48S PIC in which eIF5B and initiator transfer RNA (Met-tRNAi) are repositioned in preparation for joining of the large subunit to the 48S PIC to form the elongation-competent 80S IC. We will characterize these 48S PIC dynamics and determine their role in start-codon recognition and subunit joining.