SUBCELLULAR AND QUANTITATIVE PERSPECTIVES OF RNA PROTEIN INTERACTIONS

Gene expression in higher eukaryotic cells involves carefully orchestrated interactions between RNA-binding proteins (RBPs) and RNAs. Understanding the rules that govern the interactions between RBPs and RNAs is a central question in biology, and increasingly, in medicine. This thesis investigates the cellular RNA- RBP interactions in two ways. First, we develop colocalization CLIP, a method that combines CrossLinking and ImmunoPrecipitation (CLIP) with proximity labeling, to explore in-depth the subcellular RNA interactions of the RNA-binding protein HuR. Using this new method, we uncover HuR’s unique binding preferences in the nucleus, cytoplasm, and stress granule, during mock and arsenite stress conditions. Second, using available datasets that describe RBP binding in vitro and in cells, we devise two quantitative metrics to directly compare RBP binding behaviors: inherent specificity and mutational sensitivity. These new metrics provide a new quantitative framework to characterize the binding behaviors of an RBP.