loading . . . Evolutionary emergence and preservation of microproteins encoded by upstream ORFs The analysis of ribosome profiling (Ribo-Seq) data has provided evidence that many eukaryotic mRNAs contain translated upstream or downstream ORFs (uORFs/dORFs), but the biological significance of this translation activity remains, for the most part, unknown. One of the principal limitations has been the lack of Ribo-Seq data from several closely related species, precluding the identification of cases in which translation is phylogenetically conserved. Here, by combining Ribo-Seq data from 100 different experiments, we identify 2,332 translated uORFs and 1,008 translated dORFs in S. cerevisiae, which result in microproteins that tend to be highly hydrophobic or positively charged. To study their phylogenetic conservation, we have generated Nanopore direct RNA sequencing data, together with Ribo-Seq data, from six additional Saccharomyces species, spanning an evolutionary period of around 16 million years. We have identified 195 translated S. cerevisiae uORFs that are also translated in other Saccharomyces species; these uORFs are translated at levels comparable to the main coding sequence and display signatures of purifying selection at the level of the encoded microproteins. In contrast, dORFs are translated at very low levels and they are rarely conserved, suggesting much more limited microprotein functionalization. We have also discovered that uORF translation is associated with the formation of alternative transcript isoforms encompassing the region containing the uORFs but not the main protein coding sequence, implying that some microproteins can be produced independently of the main protein product. This work significantly advances our understanding of how initially pervasive uORF translation can result in new microproteins, providing many new candidates for further functional studies. ### Competing Interest Statement The authors have declared no competing interest. European Research Council, https://ror.org/0472cxd90, 101052538 Ministerio de Ciencia, Innovación y Universidades, PID2021- 122726NBI00, PGC2018-094091- B-I00, PID2022-136939OBI00, CEX2024-001431-M, MICIU/AEI/10.13039/501100011033 Generalitat de Catalunya, https://ror.org/01bg62x04, 2021SGR00042, 2021SGR00176 https://www.biorxiv.org/content/10.64898/2026.03.05.709866v1.supplementary-material
