Ribosome synthesis plays a major role in cellular metabolism, consuming some ~80% of total transcription. Probably because of these high metabolic demands, ribosome synthesis is tightly linked to cell growth and division and numerous human diseases have been linked to defects in ribosome synthesis. A complex, multistep pathway generates the mature rRNAs from a polycistronic precursor, involving >200 proteins and >100 small nucleolar RNAs. Most analyses on eukaryotic ribosome synthesis have focussed on budding yeast, but increasing data for mammalian cells reveal both fundamental similarities and potentially significant differences in the systems.
During the immune response in mammals a key step is the activation of long-lived, dormant, memory T-cells. Following stimulation, T-cells undergo a period of dramatic metabolic activity followed by very rapid cell division, with doubling times as short as 6 hours. The rapid expansion of the T-cell population is crucial in the immune response, particularly during infection, since numbers of pathogens will generally also be rapidly increasing. This exceptional growth rate should necessitate a level of ribosome synthesis far higher than that observed in almost any other mammalian cell type. Initial results from the Zamoyska lab (ref. 1) indicate that this is indeed the case. In this project, we aim to quantify ribosome synthesis and identify feature and factors that drive very high efficiency in activated T-cells.
David Tollervey is an expert in RNA biology and will take the lead in supervising the RNA-based aspect of the project. Rose Zamoyska is a leader in T-cell biology and will take the lead for analyses in T-cells.
The student will 1) Determine the rates of ribosome synthesis during T-cell activation. 2) Characterise the pre-ribosome associated proteome. 3) Identify RNA binding sites for selected, novel or differentially associated proteins. 4) Test the requirements for these proteins in T-cell activation and ribosome synthesis.
The Tollervey group recently developed the approach of total RNA-associated protein purification (TRAPP) (ref. 2). This is based on in vivo UV crosslinking of protein-RNA complexes and denaturing purification, combined with quantitation using SILAC metabolic labelling. TRAPP allows the rapid, proteome-wide identification of RNA-binding proteins and quantitation of changes under conditions of nutrient shift, stress or developmental progression. For selected proteins, this will be followed up by CRAC analyses, which identify the precise binding site within the RNA, a technique in routine use in the lab (see ref. 3).
The student will acquire expertise in a range of biochemical, cell culture, RNA sequencing and proteomics techniques, which will be of value for many other future analyses.
This might include link to your lab web site, any additional funding information and joint supervision details. Information on how to apply will be added by the Graduate School.
The Tollervey group will take the lead in the biochemical purification of RNA-protein interactions, RNA sequencing and proteomics. The Zamoyska group will generate quiescent and time courses of activated mouse T-cells. Both groups will collaborate in generating mutant cell lines for functional testing in T-cells of components identified and characterised in TRAPP and CRAC analyses.
Websites: https://www.wcb.ed.ac.uk/research/tollervey http://zamoyska.bio.ed.ac.uk/
1) Tan TCJ, Knight J, Sbarrato T, Dudek K, Willis AE, Zamoyska R (2017) Suboptimal T-cell receptor signaling compromises protein translation, ribosome biogenesis, and proliferation of mouse CD8 T cells. Proc Natl Acad Sci USA, 114, E6117-E6126. PMCID: PMC5544288
2) Shchepachev, V., Bresson, S., Spanos, C., Petfalski, P., Fischer, L., Rappsilber, J. and Tollervey, D. (2019) Defining the RNA Interactome by Total RNA-Associated Protein Purification. Mol. Sys. Biol. 15, e8689. PMCID: PMC6452921
3) Winz, M.-L., Peil, L., Turowski, T.W., Rappsilber, J. and Tollervey, D. (2019) Molecular interactions between Hel2 and RNA supporting ribosome-associated quality control. Nature Comm. 10.1038/s41467-019-08382-z. PMCID: PMC6362110