Suppression of upstream ORF translation is not a widespread mechanism of translational stimulation by yeast helicase Ded1.

Ded1 is an essential DEAD-box helicase in yeast that broadly stimulates translation initiation and is critical for messenger RNAs (mRNAs) with structured 5' untranslated regions (UTRs). We have evaluated the proposal that Ded1 stimulates translation primarily by preventing initiation at upstream open-reading-frames (uORFs) associated with stable secondary structures. By Ribo-seq analysis under experimental conditions designed to suppress artifactual 5'UTR translation, we found that reduced translation of the main open-reading-frames (mORFs) in native mRNAs is generally not accompanied by increased 5'UTR translation in ded1 mutant cells, and that the presence of translated uORFs in yeast mRNAs generally does not confer heightened dependence on Ded1 for efficient translation of mORFs. Results from a high-throughput reporter assay examining native 5'UTRs reinforce the importance of Ded1 in initiation from structured 5' UTRs and show that impairing Ded1 has minimal effects on translational repression by uORFs. Our results demonstrate that, in cells growing vegetatively in rich medium, translational stimulation by suppression of inhibitory uORFs is restricted to a minority of Ded1 targets, and that unwinding of 5' UTR secondary structures per se is the principal mechanism for Ded1 stimulation of translation initiation.