UC Irvine

Traditional gene regulation models focus on protein-coding genes within the paradigm of DNA → mRNA → protein. However, recent insights have shifted this perspective, highlighting the crucial regulatory roles of noncoding regions of the genome. Protein-coding regions constitute only about 1.5% of the genome, while many noncoding RNAs (ncRNAs) are transcribed from other regulatory elements and play significant roles in regulating complex biological processes such as development, differentiation, and metabolism. This emerging understanding underscores ncRNAs as vital molecular players, necessitating advanced tools to study their dynamic expression and activity.The first three chapters of this thesis present recent advancements in metabolic labeling techniques to profile RNA expression dynamics to help elucidate novel RNA activity and dynamics. Metabolic labeling involves incorporating chemically modified nucleosides into nascent RNA, enabling detailed tracking of RNA synthesis, decay, and transient expression through pulse-chase experiments. This addresses the limitations of bulk RNA sequencing and single-cell RNA sequencing (scRNA-seq) methods, which are limited in their abilities to accurately capture RNA dynamics and cell-type-specific gene expression. The approaches outlined in this thesis provide cell-specificity to traditional metabolic labeling approaches to capture RNA activity and dynamics for a cell type of interest within complex cellular contexts. Chapter 4 explored the non-canonical regulatory roles of ncRNAs, focusing on the development of synthetic riboswitches to control mRNA translation. Riboswitches regulate gene expression through ligand-binding aptamer domains. The thesis details the creation of photoriboswitches using in vitro SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to evolve RNA aptamers that bind to photo-labile ligands. Since typical SELEX methodologies cannot efficiently ensure the selection of gene-regulating riboswitches, we sought to generate a more efficient regulatory riboswitch selection platform. Therefore, we took this evolved RNA pool after in vitro SELEX for a modified ribosome display pathway, called Capture the FLAG, to select functional mRNA translational regulators from our evolved RNA pool. Overall, this thesis advances our understanding of ncRNA functions and introduces novel methodologies for profiling and manipulating RNA dynamics. The integration of metabolic labeling and synthetic riboswitches provides powerful tools for studying RNA biology, enhancing our ability to investigate cellular processes and develop therapeutic strategies.