Mechanistic Interrogation of Long Non-coding RNAs using Photocatalytic Proximity Labeling and CRISPR Phenotypic Profiling - Project Abstract Over 90% of the human genome is transcribed into RNA, yet only ~1-2% represents protein-coding genes. Long non-coding RNAs (lncRNAs) make up the majority of these transcripts and have vital roles in chromatin organization, gene expression control, and metabolic regulation. Despite their abundance, our understanding of lncRNA function and regulation significantly lags behind that of proteins, leaving the vast majority of lncRNAs understudied or completely uncharacterized. This knowledge gap not only limits our understanding of basic cellular processes but also hinders our ability to therapeutically target RNA or design synthetic transcripts as biomedicines. Significant technical challenges underlie this gap surrounding lncRNAs, and few tools exist for directly studying their function. To address these problems, this proposal aims to develop and apply experimental platforms to mechanistically understand lncRNA function by both mapping their interactomes in cellulo and elucidating their structure-function relationships through CRISPR-based perturbation profiling. In Aim 1 I will apply technology I have developed in my postdoc to directly map native lncRNA interactions with protein, RNA and genomic sites using in situ hybridization photoproximity labeling. The platform will be first validated using known lncRNAs and subsequently applied to probe uncharacterized oncogenic transcripts in breast cancer, as well as mapping the cellular interactions of prime editing guide RNAs for genetic engineering. In Aim 2 I will develop and apply CRISPR-based techniques for mechanistic profiling and functional discovery of lncRNAs using phenotypic screening. This aim will directly explore the phenotypic contributions of oncogenic lncRNAs with unknown mechanisms by measuring their influence on global expression levels using Perturb-seq single- cell knockout. Additionally, pooled CRISPR knockout screening will be employed to identify novel lncRNAs that influence cell migration and metastasis in vivo. Lastly, a novel approach combining minimization by iterative size- exclusion and recombination (MISER) with CRISPR mutational scanning will classify key domains and residues in lncRNAs, providing fundamental insights into their structural biochemistry and regulatory roles in cell behavior. Overall, this research proposal aims to bridge the gap in our understanding of lncRNAs by developing innovative tools for mapping interactomes and discovering functional mechanisms. Directly applying these tools will enable systematic testing of hypotheses regarding lncRNA function and provide fundamental measurements of their effects on global gene expression, cell growth, and motility. If successful, these platforms will deepen our understanding of RNA function and significantly expand our capabilities for both targeting pathologic RNAs with small molecules as well as enable future design and engineering of therapeutic transcripts and RNA biologics.
