Protein evidence of unannotated ORFs inDrosophilareveals unappreciated diversity in the evolution of young proteins

De novogene origination, where a previously non-genic genomic sequence becomes genic through evolution, has been increasingly recognized as an important source of evolutionary novelty across diverse taxa. Manyde novogenes have been proposed to be protein-coding, and in several cases have been experimentally shown to yield protein products. However, the systematic study ofde novoproteins has been hampered by doubts regarding the translation of their transcripts without the experimental observation of protein products. Using a systematic, ORF-focused mass-spectrometry-first computational approach, we identify almost 1000 unannotated open reading frames with evidence of translation (utORFs) in the model organismDrosophila melanogaster, 371 of which have canonical start codons. To quantify the comparative genomic similarity of these utORFs acrossDrosophilaand to infer phylostratigraphic age, we further develop a synteny-based protein similarity approach. Combining these results with reference datasets on tissue- and life-stage-specific transcription and conservation, we identify different properties amongst these utORFs. Contrary to expectations, the fastest-evolving utORFs are not the youngest evolutionarily. We observed more utORFs in the brain than in the testis. Most of the identified utORFs may be ofde novoorigin, even accounting for the possibility of false-negative similarity detection. Finally, sequence divergence after an inferredde novoorigin event remains substantial, raising the possibility thatde novoproteins turn over frequently. Our results suggest that there is substantial unappreciated diversity inde novoprotein evolution: many more may exist than have been previously appreciated; there may be divergent evolutionary trajectories; andde novoproteins may be gained and lost frequently. All in all, there may not exist a single characteristic model ofde novoprotein evolution, but rather complex origins and evolutionary trajectories forde novoproteins.