Elucidating novel roles for post-transcriptional regulation of gene expression in neural stem cell biology

The brain develops from a relatively small number of neural stem cells whose highly regulated divisions lead to a large and diverse population of neurons. The major focus of the field has been on transcriptional regulation of protein expression, to direct this important process. The precise expression of different transcription factors and regulators determines the behaviour of the stem cell throughout development. In parallel to transcriptional regulation, RNA-binding proteins regulate the processing and fate of their target mRNAs, controlling when and where the encoded proteins are expressed, known as post-transcriptional regulation. In Drosophila, two conserved RNA-binding proteins IGF II mRNA-binding protein (Imp) and Syncrip (Syp) are known to act in neural stem cells to specify progeny fate, stem cell shrinkage and termination of division (Liu et al., 2015; Yang et al., 2017a). This thesis examines the importance of post-transcriptional regulation in controlling neural stem cell behaviour, illustrated by two key examples, myc and prospero. In order to understand the action of Imp and Syp, I identified their RNA binding targets in the brain by RNA immunoprecipitation and sequencing, with analysis in collaboration with Dr Aino Järvelin. I found a large number of targets, and an enrichment of binding to transcripts encoding transcription factors and RNA-binding proteins. This illustrates the broad roles of Imp and Syp, and their influence on key regulatory proteins. I then designed a gene-by-gene approach to quantitatively and functionally characterise the post-transcriptional regulation of different targets. I particularly focused on the regulation of neural stem cell size, proliferation rate and termination. I showed that the proto-oncogene myc is a target of both Imp and Syp. Myc is known to regulate cell size and cell cycle progression and the human homolog, c-myc, is frequently dysregulated in brain cancers. I was interested in the function of Myc in regulating neural stem cell size and proliferation. I used single molecule fluorescent in situ hybridisation and immunofluorescence in the intact Drosophila larval brain to examine the regulation of myc by Imp and Syp. This method allowed precise measurement of the levels of myc transcription, cytoplasmic mRNA and protein in individual cells within the brain. I found that Imp stabilises myc mRNA, increasing its half life and Myc protein levels. I examined the phenotype of Myc overexpression and found an increase in neural stem cell size and division rate. Myc may be the key Imp target regulating these important aspects of neural stem cell behaviour. I then examined the regulation of prospero (pros) by Syp, building on work by Dr Lu Yang in the Davis lab. The key regulatory transcription factor, pros, is a target of both Imp and Syp, and is stabilised through Syp binding to a long mRNA isoform with a huge 15 kb extended 3â UTR. The extended isoform of pros is highly stable in the wild type, and is destabilised by loss of Syncrip. I used clonal analysis and endogenous deletions to begin to dissect the functional roles of the different pros isoforms. I uncovered a novel unannotated pros transcript, and a complex network of expression and regulation. The large numbers of Imp and Syp targets identified in this thesis, many with known functions in neurogenesis, show the breadth of post-transcriptional regulation in the brain. The examples of myc and pros illustrate the importance of precise post- transcriptional regulation of two key transcription factors in neural stem cells. We expect that many further cases of post-transcriptional regulation will be uncovered, with far-reaching consequences for our understanding of neurodevelopment.