Evolutionary and functional analysis of non-canonical ribosomal protein paralogues in the Drosophila germline

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The central dogma of biology states that "DNA makes RNA makes protein". There has been much research into the regulation of gene expression at the transcriptional level and it is known that the action of RNA polymerase II is highly regulated, however, there has been far less research into the regulation of gene expression at the translational level. Protein synthesis is essential for cell proliferation so, unsurprisingly, the production of sufficient functional ribosomes is hugely important for development, and a variety of human diseases known as ribosomopathies result from mutations in ribosomal proteins or biogenesis factors. The current explanation for the pathogenesis of both of these diseases is that reduced expression levels of specific ribosomal proteins or biogenesis factors leads to fewer ribosomes being assembled, resulting in a lower rate of translation, which is insufficient for cell proliferation. However, this cannot explain the difference in phenotypes caused by mutations in different ribosomal proteins. Accumulating evidence indicates that intrinsic regulation of the translational apparatus provides an additional layer of gene expression control. Such regulation can be achieved in a variety of ways, including the incorporation of ribosomal protein paralogues, posttranscriptional modifications of ribosomal RNAs, and the activity of ribosome-associated factors. An increasing number of examples of "specialised ribosomes" have been described from plants to humans, however, the extent and function of ribosomal heterogeneity is still poorly understood, as research is made difficult due to the lethality of most ribosomal protein knock-downs. The germline does not impose these constraints and is therefore an ideal tissue for research.During an in vivo RNAi screen in Drosophila germline stem cells (GSCs), many factors were identified as being required for specific aspects of GSC development, including ribosome biogenesis factors and ribosomal protein paralogues. The goal of this project is to focus on the role of ribosomal protein paralogues in the germline. Many paralogues were shown to be enriched in undifferentiated germ cells (including RpL22-like, RpS19b, RpS5b, RpL37b, and RpS10a). In addition, germline knockdown of individual ribosomal subunits generally leads to germ cell loss, however, unpublished results indicate that germline knockdown of some individual ribosome protein paralogues (such as RpL22 and RpL22-like) does not affect cell survival, but instead blocks stem cell differentiation. These results support the idea that heterogeneity in ribosome composition underlies key aspects of fate transitions. As germline knockouts are not lethal, I should be able to generate knockout mutants of most ribosomal protein paralogues using CRISPR/Cas9 technology. I will characterise the developmental defects of these mutants through a combination of genetics and immunofluorescent imaging, to understand whether these paralogues are required for fertility, viability or stem cell maintenance. In parallel, genomic-tagged loci will be used to provide a detailed developmental understanding of the expression and localisation of different paralogues. Finally, I will investigate whether these ribosomal protein paralogues are assembled into ribosomes. It is also important to consider possible non-ribosomal roles - some ribosomal proteins are already known to have non-ribosomal roles, for example RpL22 interacts with histone H1 and is associated with the formation of heterochromatin. If the paralogues are indeed assembled into ribosomes, I will investigate whether the translatomes of these ribosomes differ from those of ribosomes not containing the paralogues. Based on this analysis, I aim to uncover the molecular difference between translational machineries, and propose a consolidated model of how heterogeneity in ribosome composition modulates fate transitions.