Molecular and functional characterization of the Drosophila melanogaster conserved smORFome

Short polypeptides encoded by small open reading frames (smORFs) are ubiquitously found in eukaryotic genomes and are important regulators of physiology, development, and mitochondrial processes. Here, we focus on a subset of 298 smORFs that are evolutionarily conserved between Drosophila melanogaster and humans. Many of these smORFs are conserved broadly in the bilaterian lineage, and ∼182 are conserved in plants. We observe remarkably heterogeneous spatial and temporal expression patterns of smORF transcripts—indicating wide-spread tissue-specific and stage-specific mitochondrial architectures. In addition, an analysis of annotated functional domains reveals a predicted enrichment of smORF polypeptides localizing to mitochondria. We conduct an embryonic ribosome profiling experiment and find support for translation of 137 of these smORFs during embryogenesis. We further embark on functional characterization using CRISPR knockout/activation, RNAi knockdown, and cDNA overexpression, revealing diverse phenotypes. This study underscores the importance of identifying smORF function in disease and phenotypic diversity. [Display omitted] •Bioinformatic searches reveal fly-human conserved smORF genes•Conserved fly smORFs express in different embryonic stages and tissues•Conserved fly smORFs are enriched for mitochondrial domains•A collection of genetic tools to manipulate fly smORFs reveals diverse phenotypes Bosch et al. provide a community resource to characterize short protein-coding genes conserved between flies and humans. Their study reveals unique developmental expression patterns and connections to mitochondrial function. Surprisingly, given the degree of conservation, most tested conserved smORFs are non-lethal upon deletion.