Abstract

Adenosine-to-inosine (A-to-I) RNA editing is a widespread post-transcriptional modification with established roles in transcript stability, protein recoding, and immune regulation. Despite its biological importance, our ability to interrogate A-to-I editing in situ has been severely limited by existing technologies that lack spatial resolution or require RNA extraction, thereby erasing the very context in which these editing events occur. This dissertation aims to address these limitations by developing and applying new tools to visualize, quantify, and explore the biological significance of A-to-I editing and RNA localization in fixed and live cells. In Chapter 2, we introduce Endonuclease V Immunostaining Assay (EndoVIA), which enables spatial mapping of inosine-containing RNAs with nanoscale resolution. This platform uncovers cell type-specific differences in editing localization and establishes the foundation for linking A-to-I editing with RNA trafficking and subcellular function. In Chapter 3, we present EndoVIA 2.0, an optimized workflow compatible with formalin-fixed paraffin-embedded (FFPE) tissues, allowing in situ detection of A-to-I editing in archival clinical samples. This method reveals editing heterogeneity across tissues and enables single-cell analysis of editing landscapes in disease contexts such as cancer. Building on the need to track RNA dynamics over time, Chapter 4 describes a photoaffinity-based strategy for covalently labeling fluorescent RNA aptamers in live cells. This approach provides temporal control and improved RNA tracking, enabling time-resolved imaging of RNA localization. Covalent labeling also opens new avenues for enriching and identifying RNA:protein interactions of spatially localized RNAs. Finally, in Chapter 5, we reflect on the broader implications of these technologies for advancing RNA imaging, investigating the spatial biology of RNA editing, and repurposing other naturally occurring proteins and enzymes for mapping the epitranscriptome. Collectively, this work establishes a versatile toolkit for studying A-to-I editing in its biological context and paves the way for new insights into how RNA modifications regulate cellular function, tissue heterogeneity, and disease progression.

Committee Chair

Jennifer Heemstra

Committee Members

Hani Zaher; José Sáenz; Meredith Jackrel; Monika Raj

Degree

Doctor of Philosophy (PhD)

Author's Department

Biology & Biomedical Sciences (Biochemistry)

Author's School

Graduate School of Arts and Sciences

Document Type

Dissertation

Date of Award

8-7-2025

Language

English (en)

Download

Available for download on Wednesday, August 14, 2030

Included in

Biology Commons

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