High-Throughput Measurements of RNA-Mediated Regulation of Gene Expression and Oncogenesis

Project Summary Dysregulation of RNA-binding protein (RBP)-RNA interactions impacts the coordination of RNA fate with severe implications for human health. RBPs interact directly with RNA processing machinery to modulate RNA localization, degradation, and translation independently of transcriptional regulation. Disruptions in RBPs or

their RNA binding sites can lead to fluctuations in oncogene or tumor-suppressor gene expression and malignant changes in cellular homeostasis. Understanding the molecular functions of RBPs in regulating gene expression is then imperative for predicting metastatic progression in cancers with RBP mutant gene

signatures. Recent work has led to the discovery of thousands of human RBPs, but much less has been done to systematically describe the function that each protein and its individual domains have on regulating their RNA partners. Additionally, the functional organization of RBPs beyond their distinct and generally

well-characterized RNA-binding domains (RBDs) is not fully understood. Until recently, it was assumed that RBPs were effectively non-modular outside of RBDs; however, current studies suggest that RBPs may be composed of separable ‘effector’ domains that interact with processing machinery and promote regulatory

activity. I hypothesize that most RBPs contain distinct effector domains that post-transcriptionally regulate protein abundance through interactions with cellular machinery that controls RNA decay or translation. The Bintu lab has developed a high-throughput recruitment assay to recruit 80 amino acid protein portions, or

tiles, to a synthetic DNA reporter gene. I propose to extend this methodology to identify functional effector domains within human RBPs, where they also may be more sensitive to oncogenic mutation. First, I will use a high-throughput assay I recently developed to recruit tens of thousands of protein tiles to a synthetic reporter

RNA, evaluate their effects on downstream protein expression, and determine the mechanism by which they regulate RNA levels. This will allow me to identify annotated oncogenic mutations that overlap effector domains and likely cause disease by disrupting RBP regulatory activity. Second, it is currently difficult to predict how

loss of RBP activity affects a single bound transcript, largely because RBPs bind at different positions and in different copy numbers on each of their multiple partner RNAs. I will quantitatively compare how variation in RBP and effector domain positioning and occupancy along transcripts affects gene expression and eventual

different cancer phenotypes. Finally, I will conduct a mechanistic investigation into METTL3, a known RNA-modifying enzyme whose overexpression is believed to drive the development of multiple cancers. I will determine its RNA regulatory potential independent of modification writing and query the direct correlation

between RNA modifications and overall expression. This work will be the first to systematically identify functional domains in RBPs and will increase understanding of how dysregulation of RBP function leads to cancer through altered interactions with crucial cellular machinery.