Abstract

Upstream open reading frames (uORFs) are critical regulators of messenger RNA translation, yet their evolutionary dynamics remain poorly understood. Here, we analyze uORF evolution across Drosophila species and uncover pervasive birth–death turnover. This process is characterized by a persistent excess of upstream start codon (uATG) gains over losses, shaped by the interplay of mutational input and natural selection. We find that the evolutionary conservation of uATGs is strongly associated with translational evidence, indicating a tight coupling between uORF retention and translational output. Lineage-specific uATGs are linked to reduced translation of downstream coding sequences, revealing lineage-dependent regulatory effects. We further identify evolutionary compensation between uATG gain and loss events within genes, supported by functional assays demonstrating frequent and condition-dependent effects on translation. At the population level, canonical uORF variants show signatures of population-specific selection, suggesting a role for uORF turnover in local adaptation. Together, our results reveal how natural selection, translational regulation, and evolutionary turnover jointly shape the uORF landscape in Drosophila.