Anatomy and Neuroscience - Theses
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ItemRibosomal protein depletion in the Drosophila haematopoietic compartment alters cell fate determination( 2020)Ribosomes are essential components of the translational machinery, required for cells to effectively double their protein content in order to undergo cell division. Despite the obvious need for ribosomes, due to the high energetic cost, ribosome biogenesis is tightly regulated by numerous pathways in response to cellular and extracellular signalling cues. Predictably, dysregulation of ribosome levels strongly correlates with disease. Increased ribosome biogenesis is observed in many cancers and decreased ribosome biogenesis underlies a class of developmental disorders collectively termed ribosomopathies. Somewhat surprisingly, given the ubiquitous need for ribosomes, loss of ribosomal proteins has also been associated with tissue overgrowth in both human disease and model organisms, including zebrafish and Drosophila. In the latter, reported mechanisms for overgrowth due to global reduction of ribosomal proteins comprise extrinsic effects, whereby a smaller hormone-secreting gland delays animals in their growth phase to result in larger wings and eyes. Many ribosomopathies are associated with lineage-specific defects, with ribosomal protein loss linked with haematopoietic compartment pathologies, particularly lineage depletion and anaemia. Although lineage depletion phenotypes are well established to arise from apoptosis induced by nucleolar stress, there is also an increased incidence of cancer associated with these ribosomopathies for which mechanisms remain unclear. This thesis has taken advantage of the powerful genetic tools available in Drosophila to achieve tissue-specific depletion, in the larval hematopoietic compartment (the lymph gland), of the two ribosomal proteins most commonly lost in the ribosomopathy Diamond Blackfan Anaemia (DBA), S19a (RpS19a) or S24 (RpS24). In contrast to the haematopoietic lineage depletion observed in human disease, we report cell-intrinsic overproliferation and tissue overgrowth following depletion of either RP. However, despite gross phenotypic similarities, depletion of RpS19a and RpS24 resulted in distinct cell death and differentiation defects. Moreover, while RpS24 resulted in the expected decrease in mature ribosomes, RpS19a depletion resulted in an inexplicable increase in ribosomes. Transcriptome and proteome analyses of RP-depleted lymph glands revealed upregulation of metabolic and signalling pathways; however, factors involved in transcription and translation were disproportionately increased at the protein level, consistent with altered translation. Although certain proteins were elevated after knockdown of either RP, some were specifically increased by RpS19a or RpS24 depletion. Of the 8 candidates tested for capacity of co-depletion to suppress overgrowth defects only 5 (STAT, TOP1, Osa, TCTP and Cdk12) suppressed the RpS24 lymph gland phenotype, suggesting the requirement for increased abundance of these factors in overgrowth. In the case of RpS19a, although depletion of dHEATR1 and TCTP supressed overgrowth, only depletion of Osa, a key component of the SWI/SNF chromatin remodelling complex, suppressed overgrowth through a restoration of progenitors. The increased Osa observed in RpS19a and RpS24 knockdown is, therefore, required for progenitor depletion and lymph gland overgrowth. Further studies are required to determine whether differential translation of Osa, and the other candidates, contributes to lymph gland overgrowth due to depletion of RpS19a or RpS24.