"Development of techniques to characterize RNA motifs throughout the tr" by Michelle H. Moon

Development of techniques to characterize RNA motifs throughout the transcriptome

Date of Award

Fall 2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Simon, Matthew

Abstract

RNAs have shifted from being regarded simply as a messenger between DNA and proteins, but rather as key regulators of diverse biological processes within the cell. Most of our drug discovery efforts have focused on targeting pathogenic proteins, while their corresponding mRNAs have largely been overlooked as targets. Similar to proteins, many RNAs are able to fold into complex structures that mediate their functions, and it has been shown that such functions can be perturbed by targeting RNA structures, making them attractive therapeutic targets. Although the field has made great strides in identifying RNA binders, we still lack the ability to reliably develop drugs towards the clinic, for which we require a deeper understanding of RNAs throughout the transcriptome. Here I describe my work in applying metabolic labeling to introduce reactive s4U sites to enable characterization of RNA structures and interactions transcriptome-wide.Given the relationship between RNA structure and function, the ability to accurately predict the 3D structure is essential in identifying which elements are responsible for their functions and ultimately what can be targeted to perturb said functions. While secondary structure probing has been instrumental in revealing the existence of structural motifs throughout the transcriptome, we require tertiary information in order to really feature the properties that define complex RNA elements. Using s4U as a chemical handle, I looked to extend an approach termed multiplexed hydroxyl cleavage analysis with paired-end sequencing (MOHCA-seq) towards the transcriptome to interrogate through-space interactions. I show that EDTA groups can be conjugated to s4U RNAs in order to direct hydroxyl radical cleavage. Although downstream steps require further optimization, this work presents an opportunity to map out complex globular structures across cellular RNAs and ultimately identify functional and druggable motifs. Our limited understanding on the properties that define RNA molecular recognition has made designing specific inhibitors difficult to do. Characterization of RNA interactions in a more cellular context has become essential for determining the druggability of RNAs. To this end, I have developed a disulfide tethering screen that uses metabolic labeling to introduce tetherable sites that stabilize and allow for characterization of transcriptome-wide RNA-small molecule interactions. In vitro work shows that mCPBA oxidation effectively renders available s4U sites unreactive without affecting existing disulfide tethers. Experiments with thiol competitors demonstrate the reversibility of the tethers and therefore the tunability of the screen towards identifying binders with varying affinities. When applied to cellular RNA, the expected trends are observed and indicate the existence of structural elements throughout the transcriptome, including a motif within the mitochondrially encoded cytochrome C oxidase I, MT-CO1. Follow-up studies confirm that the identified region within MT-CO1 displays structural features and exhibits differential binding. Altogether, this screen grants us the ability to study RNA-small molecule interactions so that we can begin to dissect what makes RNA pockets druggable and what properties drive specificity. I also describe my contribution in studying the role of a SINE-VNTR-Alu (SVA) retrotransposon insertion in X-linked Dystonia Parkinsonism (XDP), a rare, but severe, neurodegenerative disorder. While the exact mechanism of the disease is unknown, SVA has been shown to induce changes within the TAF1 gene. The studies described here point to the transcription of a SVA-dependent truncated TAF1 that also partially retains intron 32 (xTAF1). This transcript is found to be stabilized with translation potential, and therefore presents a possible XDP molecular phenotype. The antisera developed to validate the existence of the truncated protein product shows xTAF1 specificity, indicating potential applicability to examine levels within patient cell lines.

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