Pervasive downstream RNA hairpins dynamically dictate start-codon selection

Translational reprogramming allows organisms to adapt to changing conditions. Upstream start codons (uAUGs), which are prevalently present in mRNAs, have crucial roles in regulating translation by providing alternative translation start sites(1-4). However, what determines this selective initiation of translation between conditions remains unclear. Here, by integrating transcriptome-wide translational and structural analyses during pattern-triggered immunity in Arabidopsis, we found that transcripts with immune-induced translation are enriched with upstream open reading frames (uORFs). Without infection, these uORFs are selectively translated owing to hairpins immediately downstream of uAUGs, presumably by slowing and engaging the scanning preinitiation complex. Modelling using deep learning provides unbiased support for these recognizable double-stranded RNA structures downstream of uAUGs (which we term uAUG-ds) being responsible for the selective translation of uAUGs, and allows the prediction and rational design of translating uAUG-ds. We found that uAUG-ds-mediated regulation can be generalized to human cells. Moreover, uAUG-ds-mediated start-codon selection is dynamically regulated. After immune challenge in plants, induced RNA helicases that are homologous to Ded1p in yeast and DDX3X in humans resolve these structures, allowing ribosomes to bypass uAUGs to translate downstream defence proteins. This study shows that mRNA structures dynamically regulate start-codon selection. The prevalence of this RNA structural feature and the conservation of RNA helicases across kingdoms suggest that mRNA structural remodelling is a general feature of translational reprogramming.

Automated summary

This study investigated the pattern-triggered immunity response in Arabidopsis and found that upstream open reading frames (uORFs) in mRNA play a crucial role in immune-induced translation. In the absence of infection, hairpin structures downstream of uAUGs lead to the selective translation of these uORFs. After immune challenge, induced RNA helicases break down these structures, allowing ribosomes to bypass uAUGs and translate downstream defense proteins. This research reveals the role of mRNA structures in dynamically regulating start-codon selection and suggests that mRNA structural remodelling is a general feature of translational reprogramming.