Molecular studies of regulatory RNA structure and function [electronic resource]

Deciphering the molecular basis of pathway control inside cell remains one of the biggest challenges in basic and medical research. Recent data has demonstrated that the transcriptional complexity of the genome is significantly more complex than originally anticipated. These results and more focused studies have demonstrated that RNA molecules are critical for many cellular pathways and can be drivers of disease, but the full characterization of how RNA exerts its control over such pathways remains far from resolved. In living cells RNAs rarely act alone, frequently partnering with RNA binding proteins (RBPs) that modulate the structure, localization, and function of their RNA targets. With the advent of deep sequencing, the cellular repertoire of RNAs has become vast, while methods to study and characterize their functions have trailed the pace of discovery. Researchers have been able to describe many of the functions that protein-coding genes control, due to the decades of research devoted to tool development. This is far from the case for RNA, where its complexity and expression creates a bottleneck toward fully understanding its physical properties and how it interacts with other cellular biomolecules to control biology. Here we present novel tools to investigate fundamental principals of RNA biology and use them to uncover new roles for RNAs and RBPs inside living cells. First, Fully Automated and Standardized individual nucleotide Crosslinking and Immunoprecipitation (FAST-iCLIP) was developed to improve the speed and accuracy of the standard CLIP method. CLIP reveals where RBPs directly interact with RNAs in vivo, however it previously represented a critical bottleneck for the entire RNA biology community due lack of easy experimental and computational implantation. Second, we developed in vivo CLICK Selective 2' Hydroxyl Acylation and Profiling Experiment (icSHAPE), which provides a global measurement of RNA secondary structure at nucleobase resolution. These strategies provide the groundwork for global and direct measurements of uncharacterized RNA-protein interactions and RNA structures. Application of these methods to unanswered questions was the next critical challenge. The genome encodes 37 DEAD box RNA helicase proteins, however the rules of their RNA target specificity and mechanisms of action remain largely unknown. We found that DDX21, an RNA helicase previously know only to function in the nucleolus, operates across distinct compartments of the nucleus to control both transcriptional and post-transcriptional processed all governing the synthesis of fully functioning ribosomes. Subsequently we characterized a RNA-based mechanism that cells use to globally control the transcriptional output of the genome. We show that the 7SK small nuclear RNA (snRNA) has a regulatory role at promoters, enhancers, and newly identified super enhancer elements. Importantly, 7SK is limits enhancer RNA initiation and synthesis and does so in a novel snRNP majorly composed of the BAF chromatin remodeling complex. Overall, this thesis introduces new RNA-centric technologies and uses them to discover novel principals of gene expression networks. Insights from these studies are applicable to human disease as DDX-family members and components of the BAF complex are known to be mutated in cancer and neural disease such as autism, possibly leading to new avenues for therapeutic stratifies.

Advisor: Chang Howard Y. (Howard Yuan-Hao)
Khavari Paul A. primary advisor. Thesis advisor
Sarnow P. (Peter) advisor. Thesis advisor
Wysocka Joanna