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The emergence of small open reading frame (sORF)-encoded peptides (SEPs) is rapidly expanding the known proteome at the lower end of the size distribution1,2. Here, we show that the mitochondria proteome is enriched for proteins smaller than 100 a.a. (defined as SEPs). Using a mitochondrial prediction and validation pipeline for small open-reading-frame (sORF)-encoded peptides (SEPs), we report the discovery of 16 endogenous mitochondrial SEPs (mito-SEPs) associated with oxidative phosphorylation (OXPHOS). Through functional prediction, proteomics, metabolomics and metabolic flux modeling, we demonstrate that BRAWNIN (BR), a 71 amino acid peptide encoded by theC12orf73gene, is essential for respiratory chain complex III (CIII) assembly. In human cells, BR is induced by the energy-sensing AMPK pathway, and its depletion impairs mitochondrial ATP production.In vivo, BR is enriched in muscle tissues and its maternal zygotic deletion in zebrafish causes complete CIII loss, resulting in severe growth retardation, lactic acidosis and early death. Our findings demonstrate that BR is essential for oxidative phosphorylation across vertebrate species. We propose that mito-SEPs are an untapped resource for essential regulators of oxidative metabolism.

SummaryEmerging and neglected diseases pose challenges as their biology is frequently poorly understood, and genetic tools often do not exist to manipulate the responsible pathogen. Organism agnostic sequencing technologies offer a promising approach to understand the molecular processes underlying these diseases. Here we apply dual RNA-seq toOrientia tsutsugamushi(Ot), an obligate intracellular bacterium and the causative agent of the vector-borne human disease scrub typhus. Half the Ot genome is composed of repetitive DNA, and there is minimal collinearity in gene order between strains. Integrating RNA-seq, comparative genomics, proteomics, and machine learning, we investigated the transcriptional architecture of Ot, including operon structure and non-coding RNAs, and found evidence for wide-spread post-transcriptional antisense regulation. We compared the host response to two clinical isolates and identified distinct immune response networks that are up-regulated in response to each strain, leading to predictions of relative virulence which were confirmed in a mouse infection model. Thus, dual RNA-seq can provide insight into the biology and host-pathogen interactions of a poorly characterized and genetically intractable organism such as Ot.