The exon-intron gene structure upstream of the initiation codon predicts translation efficiency.

Introns in mRNA leaders are common in complex eukaryotes, but often overlooked. These introns are spliced out before translation, leaving exon-exon junctions in the mRNA leaders (leader EEJs). Our multi-omic approach shows that the number of leader EEJs inversely correlates with the main protein translation, as does the number of upstream open reading frames (uORFs). Across the five species studied, the lowest levels of translation were observed for mRNAs with both leader EEJs and uORFs (29%). This class of mRNAs also have ribosome footprints on uORFs, with strong triplet periodicity indicating uORF translation. Furthermore, the positions of both leader EEJ and uORF are conserved between human and mouse. Thus, the uORF, in combination with leader EEJ predicts lower expression for nearly one-third of eukaryotic proteins.