Characterising functions of long non-coding RNAs in Drosophila embryogenesis
An appreciation of the complexity of the mammalian transcriptome has expanded our understanding of eukaryotic genome regulation with the discoveries of functional non-coding ribonucleic acids (RNAs). In recent years, the number of studies in the field of long non-coding RNA (lncRNA) biology has increased dramatically. Transcriptomic analyses of the eukaryotic genome revealed that the genome is pervasively transcribed and contains a vast number of lncRNA transcripts, most with unknown functions. Although relatively little is known about lncRNAs in general, a few have been shown to function in the regulation of gene expression during development and have been associated with a number of diseases. The aim of my dissertation is to investigate the impact of lncRNAs loss of function during embryogenesis of Drosophila melanogaster. I chose this model organism for its well documented developmental stages and the plethora of established tools available to facilitate genetic studies. Twenty-two lncRNA candidates were chosen based on their conservation at the sequence level and similar expression profiles across 5 Drosophila species, suggesting their potential for biological importance. The CRISPR/Cas9 system was used to generate lncRNA mutants and their requirement for development and the phenotypic consequences of losing each lncRNA was investigated. 13 lncRNA mutants were generated and two of them, lncRNA-9 and lncRNA-3 respectively, were required for viability, with homozygous mutants showing full lethality. Majority of the lncRNA-9 null mutant embryos were found to be unable to complete embryonic development and whereas lncRNA-3 null mutants had a pupal lethal phenotype. None of the lncRNA mutants were found to be required for fertility. I characterised the sub-cellular localization of lncRNA-9 during embryogenesis using a combination of RNA Fluorescence In Situ Hybridization (RNA-FISH) and Immunofluorescence (IF) approaches. An analysis of the transcriptome of lncRNA-9 mutants, in comparison to controls, was carried out to discover the genes that were mis-regulated and responsible for the observed lethality. Our investigation of lncRNA-9 revealed a nuclear lncRNA that is expressed in neurons and preliminary results from GO analysis revealed a loss of lncRNA-9 resulted in a reduction in the expression of neuronal genes involved in chemical synaptic transmission activities. Further characterization of lncRNA-9 will allow us to understand how lncRNAs contributes to various neural circuits and the overall signalling in the Drosophila connectome.