High-throughput Analysis of Alternative Splicing Regulatory Networks that Control Cell Fate

Advances in high-throughput profiling technologies have revolutionized our view of transcriptome complexity and regulation. Alternative splicing (AS) is increasingly recognized as a widespread mechanism for gene regulation and is responsible for greatly expanding transcriptome and proteome diversity in metazoans. It is often controlled in a precise spatiotemporal manner by an intricate RNA-protein network. Given the extensive roles of AS in development and disease, understanding the molecular mechanisms underlying AS regulation is of critical importance. The main focus of my thesis is to develop and apply high-throughput strategies to systematically discover AS networks that control cell fate, as well as the factors and mechanisms that regulate these AS networks. By combining high-throughput RNA sequencing and splicing code analyses, I describe the identification of the muscleblind-like RNA binding proteins, MBNL1 and MBNL2, as conserved negative regulators of a large program of alternative exons that are differentially spliced between embryonic stem cells (ESCs)/induced pluripotent stem cells (iPSCs) and diverse non-ESCs/tissues. My results from this study provided the first evidence that AS regulatory networks play a central role in the core circuitry required for ESC pluripotency and iPSC reprogramming. Next, I present the development of a new multiplexed functional genomics screen whereby thousands of gene perturbations can be simultaneously assayed for effects on dozens of endogenous AS and gene expression events of interest. Using this system, I revealed a large network of novel positive and negative factors from multiple gene layers that regulate distinct subsets of AS events linked to ESC pluripotency, neural differentiation, and transitions in reprogramming. In summary, these studies have significantly broadened our knowledge of the global regulatory networks that determine cell fate decisions, and provided powerful tools for large-scale investigation of the regulation of AS and other RNA metabolic pathways in diverse cell types.Ph.D.2017-11-30 00:00:0