Critical cis-parameters influence STructure Assisted RNA Translation (START) initiation on non-AUG codons in eukaryotes

In eukaryotes, translation initiation is a highly regulated process, which combines cis- regulatory sequences located on the messenger RNA along with trans-acting factors like eukaryotic initiation factors (eIF). One critical step of translation initiation is the start codon recognition by the scanning 43S particle, which leads to ribosome assembly and protein synthesis. In this study, we investigated the involvement of secondary structures downstream the initiation codon in the so-called START (Structure-Assisted RNA translation) mechanism on AUG and non-AUG translation initiation. The results demonstrate that downstream secondary structures can efficiently promote non-AUG translation initiation provided that they are stable enough to stall a scanning 43S particle and that they are located at an optimal distance from this non-AUG codon to trigger and stabilize the codon-anticodon base-pairing in the P site. The required stability of the downstream structure for efficient translation initiation varies in distinct cell types. We extended this study to genome-wide analysis of the Homo sapiens alternative translation initiation sites and discovered 556 of them starting with an AUG and 506 starting with a non-AUG that contained a downstream RNA structure at an optimal distance and with a predicted stability of at least -15 kcal/mol. We validated the impact of these structures on translation initiation for several selected uORFs.