A Pipeline for Discovery and Determination of Functional Tertiary Structures in Large RNAs

Date of Award

Fall 2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular Biophysics and Biochemistry

First Advisor

Pyle, Anna Marie

Abstract

RNAs can adopt complex folded motifs and higher-order three-dimensional (3-D) structures that play a key role in various cellular processes. It has recently become clear that large RNAs (>200 nucleotides in length) contain regions of higher-order structures; however, identifying them remains challenging. To unambiguously elucidate their mechanism and effectively target these RNAs, we must understand the interplay between their higher-order structure and biological function. In this dissertation, I describe my efforts towards developing techniques to discover and visualize RNA structure in diverse systems ranging from ribozymes to long non-coding RNAs (lncRNAs) and viral RNA genomes.First, I developed a high-throughput sequencing-based approach to systemically identify compact tertiary structures using the lanthanide metal terbium (Tb3+). With this approach (Tb-seq), I scanned for stable structural modules and potential riboregulatory motifs in self-splicing group II introns, human RNase P, HCV genomic RNA, and SARS-CoV-2 genomic RNA. Tb-seq reveals sites in which RNA forms sharp, stable turn motifs that result in complex arrangements of nucleotides in 3-D space, thereby providing a powerful new approach for pinpointing regions of complex RNA structure that are potentially associated with RNA functional elements. Next, to directly visualize the molecular level details of these discovered tertiary structures, I used electron cryo-microscopy (Cryo-EM). I studied the molecular architecture of the self-splicing group IIB intron aI5γ from Saccharomyces cerevisiae. This intron has undergone extensive biochemical characterization over the last several decades revealing, it as a model system for understanding RNA folding and catalysis. However, its sub-nanometer resolution structure remains unknown. Here, I report a 3.6 Å structure of this ribozyme, observing for the first time the intricate structural interplay and extensive network of tertiary interactions that regulate the tight folding and catalytic activity. Given its broad applicability, the cryo-EM pipeline can be readily adapted to understand the molecular basis in a diverse set of large RNA systems. Finally, I integrated these two approaches as a systematic workflow to thoroughly characterize the structural architecture of the lncRNA Pnky, known to play a key role in neurogenesis. I discovered that Pnky exhibits a high degree of structural organization, including an RNA pseudoknot and several long-range tertiary interaction sites. Preliminary cryo-EM demonstrates that Pnky adopts a compact and globular architecture to regulate neuronal stem cell differentiation. These findings provide a roadmap of candidate riboregulatory motifs for targeted functional investigation. Taken together, the approaches described in this thesis will serve as a systematic pipeline for discovering and visualizing higher-order structures in complex RNA systems.

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