ABSTRACT Our cells have two genomes, each compartmentalized in the nucleus and mitochondria. The bi-genomic cellular system was established through co-evolution of the early endosymbiotic bacterial genome and the proto-nuclear genome of our ancestral cell over a billion years. Reflecting their long and close relationship, mitochondria and the nucleus actively communicate with each other to coordinate various cellular functions. Such mitonuclear communication is vital to cellular fitness and aging, and increasingly appreciated to be highly sophisticated and complex. However, whereas >1,000 nuclear-encoded proteins directly regulate the mitochondria, no mitochondrial- encoded factors have been known to actively regulate the nucleus. We recently published the first-in-class mitochondrial-encoded peptide (i.e. MOTS-c) that regulates the nuclear genome. Here, we present an unpublished novel mitochondrial-encoded gene that is genetically linked to MOTS-c, which we named MOTS-b. Notably, MOTS- b and MOTS-c interact with each other in the nucleus, determined by co-immunoprecipitation-coupled proteomics (mass spectrometry). Like MOTS-c, the nuclear translocation of MOTS-b appears to be regulated as an adaptive response. For instance, MOTS-b and MOTS-c both dynamically translocate to the nucleus in a temporally coordinated manner upon monocyte differentiation. Further, MOTS-b is enriched in purified nuclear chromatin samples and can directly bind DNA based on our in vitro evolution studies to identify specificMOTS-b-targeted nucleotide sequences. At the functional level, MOTS-b treatment regulates cellular proliferation and metabolism, which again is consistent with MOTS-c. Here, we propose to characterize and validate the nuclear role of MOTS-b. The overarching hypothesis of this proposal is that MOTS-b is a novel mitochondrial-encoded gene that translocates to the nucleus and directly regulates adaptive gene expression in coordination with MOTS-c. First, we will characterize the molecular and cellular mechanisms of MOTS-b using a multipronged approach including mutagenesis, co-immunoprecipitation, proximity-labeling assisted proteomics, and DNA-binding assays. We will map the functional peptide domains of MOTS-b pertinent to DNA binding and peptide interaction, which we hypothesize to be important for its nuclear role. We will also determine the cellular context/event that triggers MOTS-b to translocate to the nucleus. Then, we will determine the MOTS-b-induced transcriptome by global unbiased RNA-seq with and without stress, which will be complemented by genome-wide mapping of MOTS-b-bound chromatin sites (ChIP-seq) that are within open chromatin (ATAC-seq). We will then screen candidate genes that mediate the effects of MOTS-b on cellular proliferation and metabolism. Understanding the contributions of regulators encoded in the mitochondrial genome will provide a more comprehensive genomic perspective with added biological significance. If successful, ...