Summary This supplement application seeks two years of support for Miss Angelina Li for post-baccalaureate training in the Zha laboratory. Miss Li is expected to graduate with a B.S. from Barnard College in May 2024 with an outstanding academic record (see transcript). She is a U.S. citizen and a first-generation college graduate of her family. She plans to pursue Ph.D. training and become an independent investigator. The mentored research experience supported by this supplement would give her firsthand experience in biomedical research and develop critical thinking and project design skills as well as molecular biology, bioinformatics, and computation knowledge necessary for successful Ph.D. studies. The parental grant focuses on DNA-dependent protein kinase (DNA-PK), a DNA repair factor with a newly identified role in RNA metabolism and a cancer therapy target. It will use genetic, cell biology, and single- molecule approaches to dissect the role of DNA-PK during lymphoma and leukemia genesis and therapy. It has three aims - 1) analyze RNA vs. DNA binding by KU and DNA-PK; 2) characterize the impact of acute KU- depletion on RNA metabolism in human cells, 3) the physiological functions of the C-terminal region (CTR) and tail of Ku80 in normal hematopoiesis and malignant transformation. The supplement project focuses on Aim 2: Characterize the acute impact of KU deletion in human cells with an AID-Degron system, especially Aim 2.2: Characterize the impact of KU depletion on other RNA metabolism in human cells. Briefly, our preliminary data showed that inducing KU degradation in human cells (but not mice cells) causes lethality accompanied by a robust interferon (IFN) response mediated by the MAVS-dependent double-stranded RNA (dsRNA) sensing pathway (see below). Given that KU binds to dsRNA and is very abundant in human cells, we hypothesize that KU sequesters structured dsRNA to suppress innate immune responses. We will test this hypothesis in this supplementary grant by Aim 1: Determine the role of KU on RNA metabolisms in human cells. Aim 1.1: Characterize the cellular response (including IFN) to KU degradation in human cells. Aim 1.2: Systematically characterize the dsRNA partners of KU in human cells via irCLIP data analyses. Aim 2: Characterize the evolution of KU protein in human cells that facilitates its RNA-dependent function. Aim 2.1: Determine the sequence and structural changes in human KU that facilitate RNA binding. Aim 2.2: Identify the transcription and post-transcriptional mechanism that enables high KU expression in human cells. The completion of the supplement will not only provide a valuable training experience for Miss Li but will also identify the previously unknown functions of KU in human cells and address the long-standing question of why KU is essential in human cells and why KU expression is 100 folds high in human cells. Both questions will be critical for the DNA-PK kinase inhibitors developed for cancer therapy, si...