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Exploring the Role of Long Non-Coding RNA in Neural Stem Cell Reactivation from Quiescence: A Functional Genomics Approach https://doi.org/10.17863/CAM.108724 Judge, Leia Brand, Andrea Given the proposed role of long non-coding RNA (lncRNA) in fine-tuning large-scale genomic responses to stimuli and cell-fate transitions, previous members of the Brand lab compared genome-wide changes in DNA polymerase II occupancy at lncRNA loci during Drosophila melanogaster neural stem cell (NSC) reactivation from quiescence. Through doing this, ten novel quiescence-associated lncRNAs were identified. One such lncRNA, pron, was found to be strongly upregulated in reactivating NSCs compared to quiescent NSCs. pron is localised to multiple nuclear puncta, suggesting that it might act in trans to regulate gene expression during reactivation from quiescence. Using a mutant line expressing a truncated, non-functional lncRNA molecule, pron was subsequently found to regulate the timing of NSC reactivation from quiescence. Building on these findings, the work in this thesis aims to deepen our understanding of the role of lncRNA molecules in regulating NSC quiescence and reactivation in Drosophila melanogaster and to specifically understand how the candidate lncRNA pron regulates gene expression during this process. I characterised the expression and potential function of NSC-associated lncRNAs and further built upon our understanding of the diverse roles played by lncRNA in vivo. Specifically, using traditional genetic techniques I confirmed that pron acts in trans to regulate the timing of NSC reactivation from quiescence. Given its nuclear localisation, I sought to better understand the molecular function of pron during reactivation at the genomic level and characterise the role of potential target genes. To do this, I used RNA-DamID to identify in vivo targets of pron. I correlated this genome-wide binding data with differential gene expression and chromatin accessibility, using a combination of single-cell RNA sequencing and chromatin accessibility DamID, to better understand the role of pron in vivo. I also identified specific RNA-binding protein and DNA-RNA triplex-forming sequences within pron and used these to inform pron function. In pron mutant NSCs, I identified genome-wide changes in the expression and accessibility of genes associated with Hippo signalling, ribosome biogenesis, mitochondrial energetics and cell cycle regulation—key processes implicated in NSC reactivation. This work demonstrates that pron acts in trans to regulate the expression of a suite of genes involved in orchestrating NSC reactivation, strengthening the evidence for functional roles for lncRNAs in vivo. Positionally orthologous, or syntenic, transcripts of pron were identified in several species, including humans (PROX1-AS1) and mice (Prox1os), indicating a potential evolutionarily conserved function of CR31386, and suggesting the potential importance of lncRNAs in integrating genomic signals during significant cellular events. University of Cambridge 2023-03-01 Thesis Doctoral Doctor of Philosophy (PhD) eng https://www.repository.cam.ac.uk/handle/1810/368575 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/094c3e63-79c0-4192-bffd-3059c60d086e/download 17f4094abb9fbfcc0d1247ab54257419 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/a7893d98-2103-475b-b1dd-d19f60a4a737/download 87eda9de84448d1f82354d60eee3eb5f https://www.rioxx.net/licenses/all-rights-reserved/ Drosophila Long non-coding RNA Neural stem cell Neurogenesis