Sunday, May 11, 2025
5/11/2025
High-Resolution Mapping of Subcellular RNA Dynamics Using Photocatalytic Proximity Labeling - Project Abstract RNA is a key functional biomolecule across all domains of life, and intracellular movement of different transcripts is a widespread strategy employed by cells to regulate biological events, including development, metabolism, cell migration, and neurological function. Given this broad role, it is unsurprising that dysregulated RNA localization is also linked to a host of diseases, including autism, fragile X syndrome, Alzheimer’s disease, Huntington’s disease, and several types of cancer. Despite the critical biological importance of intracellular RNA transport, our understanding of the molecular mechanisms, scale, and impact of these events is significantly limited and confined by the inherent shortcomings in currently available methods for mapping subcellular RNA movement. High-throughput tracking of transcript distribution is vital to understanding how RNA contributes to cellular function and causes disease, and one of the most effective approaches for achieving these goals employs proximity labeling, whereby a catalyst is embedded into different subcellular locations to biotinylate nearby molecules. Engineered biotin ligases and peroxidases have shown utility in these applications, but these techniques also suffer from poor spatial and temporal control over labeling and exhibit in vivo toxicity. To overcome these limitations, the proposed research will leverage the MacMillan group’s photocatalytic proximity labeling approach. In particular, this “micromapping” (µMap) method utilizes an iridium photocatalyst to activate nearby diazirines and label biomolecules of interest. In contrast to enzymatic approaches, this method utilizes a non-toxic blue light trigger, providing high spatiotemporal control over labeling. In addition, activated diazirines are very short-lived (T1/2 ~1 ns) and quenched by water, resulting in a small labeling radius (~2 nm) to generate high-resolution maps of biomolecule localization and interaction networks. Building off these exciting results, this proposal seeks to leverage this photocatalytic approach toward high-resolution mapping of subcellular RNA localization and trafficking. Together, this method will enable researchers to map the intracellular transcriptome with higher spatial resolution, in turn providing better understanding of how these events contribute to cellular function. In addition, these experiments will help elucidate disease-causing mechanisms related to RNA transport, and facilitate identification of new diagnostic and therapeutic targets. Lastly, the methods developed here can be applied to other biological questions surrounding RNA trafficking, including epitranscriptomic modifications, RNA splicing, and metabolism/turnover, in turn providing an impactful technology that is of broad utility to the RNA and cell biology community.
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