Regulation of epithelial stem cells in health and disease  

Supervised by Dr David Doupé, Department of Biosciences, Durham University  

Project background: 

Pseudouridine is the most common modified nucleoside - a nitrogenous base plus a sugar - across eukaryota, prokaryota, and archaea, the three kingdoms of life (McKenney, Rubio and Alfonzo, 2017). It is prevalent in rRNA, tRNA and snRNA, which are non-coding genetic material (Vandivier and Gregory, 2017); however, this also means its presence, and any modifications made, affects many areas of a cell. It is formed when uracil isomerises such that the ribose binds to uracil in a different way, meaning more hydrogen bonds can be formed. This provides it with unique structural properties, which affect the folding of tRNAs and rRNA, and it may also affect mRNA coding potential (Vandivier and Gregory, 2017). The biological processes involved in immune cells - including activation, development and migration - and stem cell regulation are affected by RNA modifications. This means changes made to RNA are related to disease prevalence.  

Pseudouridine synthase genes convert specific uridines into pseudouridines, and the presence of these genes has been linked to a higher incidence of cancer in mice and humans (Haruehanroengra et al., 2020). In a similar way, both mice and flies have been used to model how intestinal stem cells function, as mammalian intestines share similar structures. While there are some differences between mammals and Drosophila, such as lack of villi in Drosophila, many other features are the same, such as how dynamic cell turnover occurs (Jiang and Edgar, 2012) and compartmentalisation of epithelia to perform specific localised functions. These need to be maintained through regulation of stem cells by certain genes, or else specific diseases can arise, in Drosophila and mammals (Li and Jasper, 2016). The diseases in humans potentially linked to PUS genes include types of cancer such as colorectal cancer as well as gastrointestinal diseases like Crohn's disease and ulcerative colitis (Rodell, Robalin and Martinez, 2024). This demonstrates why the impact of PUS genes must be studied in the gut. 

Objectives: 

The main focus will be to answer the question “Do pseudouridine synthases regulate Drosophila intestinal stem cells and homeostasis?” To answer this question two main objectives will need to be followed: 

- Characterising the expression of pseudouridine synthases in the Drosophila gut 

- Manipulating the expression of pseudouridine synthases in specific gut cells to assess the effects on stem cells and tissue homeostasis 

Drosophila are the model organism used for this because many genes found in Drosophila, including  pseudouridine synthase genes, are homologous to those involved in human evolution, development, and cell regulation (Tolwinski, 2017), as well as the low cost and rapid regeneration time associated with fruit flies. 

Methodology: 

To achieve this goal of investigating these genes, a range of techniques will need to be used to assess the expression of the different genes in the gut. These specifically include RT-qPCR and types of microscopy. RT-qPCR is an acronym standing for reverse transcription-quantitative polymerase chain reaction. It is a process often involving the enzymes reverse transcriptase to produce cDNA, and then DNA polymerase to amplify the product  (ThermoFisher Scientific, 2019). This allows for specific mRNA to be detected and quantified (Ho-Pun-Cheung et al., 2009), as gene expression can be analysed.  

Once results have been produced from those methods, we will use cell-type specific RNAi - RNA inference - to knock down the expression of each PUS gene. RNAi identifies specific mRNA and enzymatically destroys them, leading translation of that mRNA. This prevents the associated protein from being produced (Gonzalez-Alegre, 2010). To observe the effects on overall gut structure and stem cell proliferation that the lack of this protein causes, confocal microscopy is used. In order to be able to view the cells through confocal microscopy, we will have to stain any tissues being studied with markers of different cell types, often with fluorescent markers. The markers mean particular structures can be seen deep within tissues with high resolution. Confocal microscopy can produce 3D reconstructions of imaged samples (Elliott, 2020), and with the fluorescence, the proliferation of specific cells to be observed. This can give an indication as to what function of the gene which was knocked down was.  

As a result, the key practical training involved in this research is drosophila genetics, confocal microscopy, and molecular biology.  

Overall, this will establish whether the Drosophila gut may be a good model to explore PUS gene function with relevance to roles in mammalian tissues and cancer. This could potentially lead to further exploration and applications in medical settings in the future. 

References:  

Elliott, A.D. (2020). Confocal Microscopy: Principles and Modern Practices. Current Protocols in Cytometry, [online] 92(1), p.e68.  

 Gonzalez-Alegre, P. (2010). RNA Interference. Encyclopedia of Movement Disorders, Science Direct [online] pp. 47-49.  

 Haruehanroengra, P., Zheng, Y.Y., Zhou, Y., Huang, Y. and Sheng, J. (2020). RNA modifications and cancer. RNA Biology, pp.1–16.  

Ho-Pun-Cheung, A., Bascoul-Mollevi, C., Assenat, E., Boissière-Michot, F., Bibeau, F., Cellier, D., Ychou, M. and Lopez-Crapez, E. (2009). Reverse transcription-quantitative polymerase chain reaction: description of a RIN-based algorithm for accurate data normalization. BMC Molecular Biology, [online] 10, p.31.  

Jiang, H. and Edgar, B.A. (2012). Intestinal stem cell function in Drosophila and mice. Current Opinion in Genetics & Development, 22(4), pp.354–360.  

Li, H. and Jasper, H. (2016). Gastrointestinal stem cells in health and disease: from flies to humans. Disease Models & Mechanisms, [online] 9(5), pp.487–499.  

 McKenney, K.M., Rubio, M.A.T. and Alfonzo, J.D. (2017). The Evolution of Substrate Specificity by tRNA Modification Enzymes. The Enzymes, 41, pp.51–88.  

Rodell, R., Robalin, N. and Martinez, N.M. (2024). Why U matters: detection and functions of pseudouridine modifications in mRNAs. Trends in Biochemical Sciences, 49(1), pp.12–27. 

ThermoFisher Scientific (2019). Basic Principles of RT-qPCR | Thermo Fisher Scientific - UK. [online] Thermofisher.com. Available at: https://www.thermofisher.com/uk/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/spotlight-articles/basic-principles-rt-qpcr.html.  

Tolwinski, N. (2017). Introduction: Drosophila—A Model System for Developmental Biology. Journal of Developmental Biology, 5(3), p.9.  

Vandivier, L.E. and Gregory, B.D. (2017). Reading the Epitranscriptome. The Enzymes, 41, pp.269–298.