UC San Diego

RNA-binding proteins (RBPs) play integral roles in mediating all cellular functions through RNA processing. Given the importance of post-transcriptional gene regulation in maintaining cellular function, and the role that RNA processing dysregulation plays in disease causation, it is of critical importance to understand the underlying biology of RNA-RBP interactions. At the same time, engineered RBP paradigms offer tremendous potential to increase our ability to both further probe and manipulate the transcriptome. In this dissertation, we aim to explore both natural and synthetic RBP systems and the role they play on cellular function. We begin by describing molecular tools developed to perform site-directed RNA editing using CRISPR/Cas technologies and natural adenosine-deaminase enzymes. We demonstrate efficacy of our new platform, as well as perform a systematic evaluation of similar editing platforms in both on- and off-target efficiency. We then describe a novel and simplified sequencing-based platform (STAMP) that demonstrates how RNA editing enzymes can be leveraged to characterize global RNA-RBP interactions both at bulk and single-cell levels. Next, to explore the regulatory roles of a specific RBP in disease modification, we profile the functional landscape of ataxin-2 (ATXN2) in spinal cord harvested from a mouse model of amyotrophic lateral sclerosis (ALS) using RNA sequencing and enhanced CLIP. In doing so, we uncover hundreds of differentially expressed transcripts of moderate fold-change affected by ataxin-2 knockout, the majority of which occur in a dose-dependent manner and can be explained by direct ataxin-2 binding. We also discover a subset of genes whose signatures can be reversed in the context of ALS with ataxin-2 deletion, corresponding to suppression of neuroinflammatory pathways and promotion of neuronal processes, supporting the notion that depletion of ataxin-2 levels may be protective against progression of ALS pathogenesis. Lastly, we describe efforts to further characterize regulatory roles of ataxin-2 in disease susceptibility using newly engineered human stem cell models.