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.

Two neuronal peptides encoded from a single transcript regulate mitochondrial complex III in Drosophila.

Naturally produced peptides (<100 amino acids) are important regulators of physiology, development, and metabolism. Recent studies have predicted that thousands of peptides may be translated from transcripts containing small open-reading frames (smORFs). Here, we describe two peptides in Drosophila encoded by conserved smORFs, Sloth1 and Sloth2. These peptides are translated from the same bicistronic transcript and share sequence similarities, suggesting that they encode paralogs. Yet, Sloth1 and Sloth2 are not functionally redundant, and loss of either peptide causes animal lethality, reduced neuronal function, impaired mitochondrial function, and neurodegeneration. We provide evidence that Sloth1/2 are highly expressed in neurons, imported to mitochondria, and regulate mitochondrial complex III assembly. These results suggest that phenotypic analysis of smORF genes in Drosophila can provide a wealth of information on the biological functions of this poorly characterized class of genes.