SUMMARY Mitochondria have numerous signaling pathways for conveying stress to the rest of a cell. Similar to pathogens that release pathogen-associated molecular patterns (PAMPs), mitochondria release novel damage-associated molecular patterns (DAMPs), including lipids, peptides, and mitochondrial DNA (mtDNA), that indicate mitochondrial stress. Mitochondrial double-stranded RNA (mtdsRNA) is a new class of DAMPs that is generated when the noncoding strand in mtRNA is not degraded efficiently and accumulates, allowing base-pairing with the coding strand. Under normal conditions, the helicase SUV3 unwinds the mtRNAs and polynucleotide phosphorylase (PNPase)ndegrades them. However, knockdown of SUV3 results in the accumulation of mtdsRNAs within mitochondria, and knockdown of PNPase leads to the release of the mtdsRNAs into the cytosol. Once in the cytosol, the mtdsRNAs are sensed by dsRNA sensors MDA5 and RIG-I, leading to the induction of the type I interferon pathway. The export of mtdsRNA is likely important as mtdsRNAs have been identified in the cytosol of patients with mutations in PNPT1, encoding PNPase, and in diseases including cancer, cardiac disease, alcohol-associated liver disease, and autoimmune diseases. The hypothesis that mtdsRNAs represent a new biomarker for mitochondrial dysfunction will be tested. As this is a new pathway, there is a critical gap in understanding the molecular rules and mechanisms by which mtdsRNAs cross the mitochondrial inner and outer membranes for cytosolic export. Our study goals are contained within three independent, but thematically connected, specific aims. In Aim 1, mtdsRNAs that are exported from mitochondria will be characterized with respect to size and sequence specificity. In addition, RNA modifying enzymes will be tested to determine which components are essential for the generation of mtdsRNAs. Aim 2 will focus on identification of outer and inner membrane channels and the role of PNPase in the trafficking of mtdsRNAs out of mitochondria. The third aim will define physiologic parameters that lead to the generation of mtdsRNAs and determination of the cytosolic dsRNA sensors that become activated during this process. Because mutations in PNPase lead to mitochondrial disease, mutants will be characterized to determine whether steps in the degradation and/or export of mtdsRNAs can be separated. Our study team has been characterizing PNPase and its function in mitochondria extensively. Unique model systems available for our work include a mouse model in which floxxed PNPT1 can be removed by the Cre recombinase, and mouse embryonic fibroblasts derived from this model. Results from this proposal will define the pathway for mtdsRNA trafficking out of mitochondria in detail and provide a platform for understanding how mutations in PNPase contribute to disease. Long-term, these studies may lead to establishing mtdsRNA as a new biomarker for mitochondrial dysfunction.