Abstract

Abstract Regulation of gene expression is fundamental in establishing cellular diversity and a target of natural selection. Untranslated mRNA regions ( UTR s) are key mediators of post‐transcriptional regulation. Previous studies have predicted thousands of ORF s in 5′ UTR s, the vast majority of which have unknown function. Here, we present a systematic analysis of the translation and function of upstream open reading frames ( uORF s) across vertebrates. Using high‐resolution ribosome footprinting, we find that (i) uORF s are prevalent within vertebrate transcriptomes, (ii) the majority show signatures of active translation, and (iii) uORF s act as potent regulators of translation and RNA levels, with a similar magnitude to mi RNA s. Reporter experiments reveal clear repression of downstream translation by uORF s/ oORF s. uORF number, intercistronic distance, overlap with the CDS , and initiation context most strongly influence translation. Evolution has targeted these features to favor uORF s amenable to regulation over constitutively repressive uORF s/ oORF s. Finally, we observe that the regulatory potential of uORF s 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.