Project Summary The goal of this proposal is to investigate retrotransposons as a significant source of immunogenic molecules in aging and diseased human tissues. This is an important but understudied area of human genomic research. I have pioneered methods to detect retrotransposons from genome sequences to determine their role in the human brain and in cancer. In this application, I will build on my earlier success to develop other innovative methods and apply them to the study of immunological impact of retrotransposons. Specifically, this proposal is to discover the origins of retrotransposon-derived molecules (RDMs) and the functional consequences of RDMS in pathogenesis to provide a mechanistic basis for novel therapeutics or biomarkers. Retrotransposons have been recognized as important to evolutionary processes and to the causes of Mendelian disorders; however, we propose a new perspective on these endogenous retroelements, suggesting that these normally suppressed domesticated agents are reactivated, producing RDMs that may be considered as foreign molecules, thus causing immunologic responses mimicking their ancient exogenous identities, especially in aging and diseased tissue. Increasing evidence, including our own work, shows that with aging, there is an increasing rate of somatic retrotransposon mobilization and other mutations, suggesting a corresponding increased load of various forms of RDMs (e.g., cytosolic ss/dsRNA, cDNA, and neo-peptides) contributing to pathophysiology. In particular, we focus on scrutinizing retrotransposon-derived RNA that produces neo- peptides caused by altered splicing (e.g., exonization or gene-retrotransposon fusion) or by mutations in protein-coding sequences of retrotransposons. Building upon my expertise in retrotransposons, somatic mutations, and splicing using a genomic and single-cell approach, this study will build a methodological framework to reveal the landscape of age-dependent retrotransposon activity and the abundance of RDMS in various human tissues as the foundation upon which to achieve further findings in this nascent field of inquiry. It will also characterize RDMs associated with molecular/pathological/clinical parameters and underlying mechanisms. This project will support my long-term research goal of defining the role of retrotransposons in human health and disease to provide the basis for effective therapeutics to improve human life and health.