Upstream ORFs are prevalent translational repressors in vertebrates

Regulation of gene expression is fundamental in establishing cellular diversity and a target of natural selection. Untranslated mRNA regions (UTRs) are key mediators of post‐transcriptional regulation. Previous studies have predicted thousands of ORFs in 5′ UTRs, the vast majority of which have unknown function. Here, we present a systematic analysis of the translation and function of upstream open reading frames (uORFs) across vertebrates. Using high‐resolution ribosome footprinting, we find that (i) uORFs are prevalent within vertebrate transcriptomes, (ii) the majority show signatures of active translation, and (iii) uORFs act as potent regulators of translation and RNA levels, with a similar magnitude to miRNAs. Reporter experiments reveal clear repression of downstream translation by uORFs/oORFs. uORF number, intercistronic distance, overlap with the CDS, and initiation context most strongly influence translation. Evolution has targeted these features to favor uORFs amenable to regulation over constitutively repressive uORFs/oORFs. Finally, we observe that the regulatory potential of uORFs on individual genes is conserved across species. These results provide insight into the regulatory code within mRNA leader sequences and their capacity to modulate translation across vertebrates. Synopsis Upstream open reading frames (uORFs) are found in many genes, but their functional impact is unclear. This study explores their abundant translation, conservation, and potent role in shaping the vertebrate translational landscape. uORFs are pervasive and actively translated throughout vertebrate transcriptomes. uORFs and oORFs act as potent regulators of downstream translation and mRNA stability. Sequence features such as AUG context and number modulate the activity of uORFs in vivo . uORF regulation is conserved across vertebrate species. Graphical Abstract Upstream open reading frames (uORFs) are found in many genes, but their functional impact is unclear. This study explores their abundant translation, conservation, and potent role in shaping the vertebrate translational landscape.