Regulation of human eIF4A activity in health and disease: Mechanisms of canonical and aberrant translation initiation

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Translation of eukaryotic mRNAs starts with the recognition of the 5’-cap by the translation initiation factor complex eIF4F, comprised of the cap-binding protein eIF4E, the scaffold protein eIF4G, and the DEAD-box helicase eIF4A. eIF4A is believed to unwind secondary structures in the 5’-untranslated region (5’-UTR) during ribosomal scanning towards the start codon. RNA unwinding by eIF4A is tightly linked to its conformational cycle, driven by ATP binding and hydrolysis. Yeast eIF4A acts as a conformational sensor and a regulatory hub in translation initiation: other translation initiation factors, as well as the 5’-UTR itself, modulate the conformational landscape of eIF4A, and thereby regulate its activities. Translation initiation in humans employs a similar set of translation initiation factors. However, the mechanisms of regulation of eIF4A activities and conformational dynamics by the functional interaction of these factors and the consequences for translation initiation and gene expression are not understood. Transcriptome-wide studies have identified a set of eIF4A-dependent mRNAs, among them many oncogene mRNAs. eIF4A also plays a key role in repeat-associated translation on mRNAs lacking an AUG start codon (RAN translation), a process linked to a number of neurological disorders. To understand the mechanism of eIF4A-dependent translation initiation and the coupling of its conformational cycle to ATPase and RNA unwinding activities and translation efficiencies, we propose to investigate the regulation of human eIF4A by other translation initiation factors and by 5’-UTRs, both in canonical and aberrant translation initiation. We will investigate the effects of eIF4B, 4E, 4G, and 4H, of model 5’-UTRs and of selected 5’-UTRs of eIF4A-dependent genes on the kinetics of eIF4A conformational changes in single-molecule FRET experiments. We will also determine the effect of these factors and the 5’-UTRs on the ATPase and RNA unwinding activities of eIF4A. Translation efficiencies of reporter genes under the control of these 5’-UTRs will be determined in in vitro translation assays to correlate eIF4A conformational dynamics with gene expression. To understand the role of eIF4A in aberrant translation initiation, we will investigate the mechanisms of RAN translation and the translation of oncogenes. Altogether, these studies will reveal the effect of other translation initiation factors and 5’-UTRs on eIF4A conformational dynamics, the link between conformational dynamics of eIF4A and translation efficiencies, and the mechanisms of regulation of eIF4A activities in health and disease.