ORCID

https://orcid.org/0009-0006-8129-4689

Date of Award

Fall 2024

Language

English

Embargo Period

12-1-2024

Document Type

Master's Thesis

Degree Name

Master of Science (MS)

College/School/Department

Department of Biological Sciences

Program

Biology

First Advisor

Andy Berglund

Committee Members

Andy Berglund, Hannah Shorrock, Kaalak Reddy

Keywords

Spinocerebellar Ataxias, Neurodegenerative Disorders, Microsatellite Repeat Expansion Disorders, SHAPE-MaP, Polyglutamine Diseases, RNA Structure, CAG Repeat Expansion, RNA Biology, Cell Models

Subject Categories

Amino Acids, Peptides, and Proteins | Bioinformatics | Biological Factors | Biology | Biotechnology | Cell Biology | Chemicals and Drugs | Computational Biology | Genetics | Genomics | Life Sciences | Molecular Genetics

Abstract

Spinocerebellar ataxias (SCAs) are a diverse group of over 40 genetically heterogeneous neurodegenerative disorders, many of which are caused by a trinucleotide CAG repeat expansion in the coding region of specific genes. These expansions lead to the production of polyglutamine (polyQ) tracts that interfere with normal protein function, triggering cellular dysfunction and contributing to disease pathogenesis. The most well-known of these SCAs, such as SCA1, SCA2, and SCA3, exhibit progressive neurodegeneration, yet the precise mechanisms through which these mutations cause disease remain poorly understood.

A significant challenge in studying CAG repeat expansion disorders lies in the complexity of the disease mechanisms, compounded by the lack of models that allow for the simultaneous investigation of various SCAs in a disease-independent, mechanism-agnostic context. To address this gap, we developed two cell lines that recapitulate the key transcriptional hallmarks of CAG repeat SCAs. One cell line stably expresses 60 CAG repeats, while the other utilizes an inducible system to control the expression of CAG repeats, offering a dynamic model to study transcriptional regulation and its effects on cellular physiology.

While the molecular mechanisms underlying these disorders are still not fully understood, there is even less knowledge about the in vivo structural dynamics of CAG repeat RNA. It remains unclear whether the RNA structure itself contributes to disease progression and, moreover, whether RNA-targeted therapeutics can be developed. To bridge this knowledge gap, we employed SHAPE-MaP (Selective 2'-Hydroxyl Acylation analyzed by Primer Extension and Mutational Profiling) experiments to investigate the RNA secondary structure of CAG repeats in a cellular environment. This work represents, to our knowledge, the first study of CAG repeat RNA in a cellular context using SHAPE-MaP, providing novel insights into the in cell behavior of these repeats.

We also explored the effect of a small molecule, previously identified as a selective modulator of CAG RNA levels. Our findings revealed that CAG repeat RNA adopts more flexible structures in a cell-free environment compared to the cellular environment, where molecular chaperones or RNA-binding proteins likely stabilize the RNA, limiting flexibility. Notably, we observed that in the cell-free condition, CAG repeats form multiple hairpin structures, while in the cellular context, only one predominant long hairpin structure was present, indicating that the cellular environment plays a crucial role in stabilizing the RNA structure. This suggests that in the absence of stabilizing factors, CAG repeats may adopt more conformations, which could contribute to their pathological behavior.

This study provides the first direct evidence of CAG repeat RNA's structural behavior in a cellular environment, offering valuable insights into the molecular dynamics of CAG repeats in SCAs. These findings could ultimately contribute to the development of therapeutic strategies targeting CAG repeat RNA, focusing on its structural stabilization or the modulation of RNA-protein interactions.

License

Creative Commons Attribution 4.0 International License
This work is licensed under a Creative Commons Attribution 4.0 International License.

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