Insights into RNA metabolism in Drosophila nervous system development and virus infection

Messenger RNA (mRNA) is an integrated component of core cellular function. mRNA holds the capacity to relay genetic information as templates, localise to different spatial compartments within a cell, and bind combinatorially to effector molecules, such as RNA-binding proteins (RBPs). Post-transcriptional control of mRNA metabolism builds on transcriptional cues to provide a highly complex and context-specific network of gene expression patterns. In this thesis, I describe two cases illustrating the unique importance of RNA metabolism in different aspects of biological processes.
Chapter 3 details experiments in which I explored post-transcriptional regulation as a critical player of neurogenesis. In Drosophila, a conserved RBP Syncrip (Syp) controls neural stem cell behaviour and progeny fate. I identified the mRNA targets of Syp, and found an enrichment of binding to transcripts encoding transcription factors and RBPs, which are key regulatory nucleic acid binding proteins. Syp targets contained many transcripts that have isoforms with long 3’ untranslated regions. This illustrates the role of Syp to influence the expression of key regulatory proteins through cis-regulatory elements in their transcripts. I then functionally characterised the post-transcriptional regulation of prospero (pros) and e93, Syp targets as examples of the importance of Syp in regulating various aspects of neural development.
Chapter 4 expands on the breadth of post-transcriptional regulation in the nervous system. In collaboration with Dr Joshua Titlow, Dr Ana Palanca and Maria Kiourlappou, I applied a highly sensitive single-molecule fluorescence in situ hybridisation (smFISH) screen to profile transcription, mRNA accumulation and translation of 200 genes in various larval nervous system cell types. In the process, we demonstrate the common lack of correlation (discordance) between the distribution of mRNA and the encoded protein, a manifestation of post-transcriptional control. Our results suggest that mRNA stability may be a common mechanism to achieve cell-type specific protein expression, and that localised mRNAs are frequently observed in synaptic compartments. Our survey provides new insight into gene expression control in the brain and raises the need to improve further our knowledge of mRNA biology for understanding neurodevelopment and neural functions.
Chapter 5 describes the utility of the same single-molecule and high-resolution microscopy methods used in Chapter 4 to dissect RNA biology of virus infection. In response to the current COVID-19 pandemic, I used smFISH to detect and profile replication kinetics of SARS-CoV-2 RNA during the first hours of the infection. This result represents the first detailed molecular characterisation of SARS-CoV-2 RNA dynamics and provides new insight into the variable susceptibility of individual cells to support virus replication. Furthermore, I show that the B.1.1.7 Variant of Concern (VOC) displays delayed replication kinetics than the ancestral strain, and discuss possible implications for its enhanced dissemination in the human population. Collectively, my thesis emphasises the diverse and unique role of RNA biology in nervous system development and virus infection. I anticipate that this thesis will be a valuable resource for future works to improve our understanding of post-transcriptional control in a diverse range of fields.

Keywords: Syp
neuroblast
SARS-CoV-2
Imp
smFISH
COVID-19
Advisor: Davis, I