A multi-level approach to understanding the regulation of translation initiation

Abstract

mRNA translation is an extremely complex process required for life. Translation consumes vast amounts of cellular resources, and organisms have evolved tight regulatory mechanisms to control this process, which are often deregulated in cancer and other disease states. Initiation, as the rate-limiting step in translation, is particularly well regulated. Two kinase pathways that respond to cellular stresses, the GCN2 and mTORC1 pathways, sense amino acid insufficiency to inhibit translation initiation at distinct points. GCN2 is activated in response to amino acid deprivation and inhibits formation of the ternary complex, comprising elF2, GTP, and the initiator methionyl-tRNA, which is required for recognition of the start codon. Although translation of most mRNAs is greatly suppressed when GCN2 is activated, mRNAs with certain cis elements escape inhibition. In contrast, the mTORC1 pathway is inhibited by the lack of amino acids, which ultimately results in the disruption of eIF4F, a multiprotein initiation factor complex that coordinates the recruitment of the small ribosomal subunit to the 5' end of mRNA. Like a decrease in the amount of ternary complex, disruption of eIF4F also suppresses translation of most mRNAs; however, the translation of a subset of mRNAs harboring a 5'TOP motif is even more dramatically reduced when mTORC1 is inhibited. Here we describe the translational program downstream of amino acid insufficiency, and present evidence of a novel uORF in murine ATF4 whose ribosome occupancy is regulated by the presence of amino acids. We identify the 4EBPs as the mTORC1 substrates that mediate the major effects of mTORC1 inhibition on translation of mRNAs both globally and on 5'TOP mRNAs specifically. Although we cannot mechanistically explain the dependence of 5'TOP mRNA translation on mTORC1 activity, we uncover a surprising role of the cap-proximal sequence in eIF4E recruitment. We systematically assess how the juxtacap sequence modulates eIF4E binding and translation, and present a model whereby the juxtacap sequence dictates the cap-proximal RNA secondary structure in an mRNA-context-dependent manner.