Functional interactions between RNAs and proteins underpin gene expression and defects in RNA-protein interactions form the basis of numerous human diseases. RNA binding proteins have important functions at all steps in gene expression, including transcription, RNA processing and mRNA translation, as well as in viral defence mechanisms.
David Tollervey is an expert in RNA biology and his group has very recently developed the approach of total RNA-associated protein purification (TRAPP). This is based on in vivo UV crosslinking of protein-RNA complexes and denaturing purification, combined with quantitation using SILAC metabolic labelling (Ref. 1). TRAPP allows the rapid, proteome-wide identification of RNA-binding proteins and quantitation of changes under conditions of nutrient shift, stress or developmental progression. This technique relies on tandem MS-MS to generate fragments similar to the identification of other sites of protein modification. The related iTRAPP technique (Tollervey; bioRxiv 2018) allows the mapping of precise sites of protein-RNA interaction with amino acid resolution.
Tony Ly is an expert in quantitative proteomics and his group has developed important mass spectrometry techniques to characterise the dynamics of alterations in the total proteome and in protein modifications during changes in cell state (see Ref. 2)
The project aims to apply these approaches to follow changes in RNA-protein interactions during biologically important transitions. To further develop kinetic analyses, we are initially following stress responses in yeast. Our recent work identified nuclear RNA degradation as an actively regulated step in gene expression (Ref. 3). We anticipate that changes in nuclear RNA degradation pathways will play important roles in other situations that require large scale reprogramming of gene expression, such as developmental steps in metazoans. Interactome changes and the signalling pathways involved will be identified using TRAPP. Specific changes in the RNA interactions of proteins identified as being of likely functional importance will be characterised by crosslinking and RNA sequencing (see Ref. 3). This will be pursued during the initial stages of the PhD project.
Subsequently, the student will apply the insights gained from analyses yeast to studies using human cells. Defects in RNA-binding proteins underpin a large number of genetic diseases, but in most cases the links between the specific molecular defect and the systemic effects in affected cells remain unclear. This will be addressed using TRAPP, with particular emphasis on changes that occur during differentiation in a model system for neuronal development.
Work involved: The student will apply, and help further develop, techniques for the characterisation and quantitation of changes in RNA-protein interactions, and actively participate in the analysis of the resulting proteomic and sequence data. During this project the student will acquire expertise in a range of cutting-edge techniques.
1) Shchepachev, V., Bresson, S., Spanos, C., Petfalski, P., Fischer, L., Rappsilber, J. and Tollervey, D. (2018) Defining the RNA interactome by Total RNA-Associated Protein Purification. bioRxiv, doi: https://doi.org/10.1101/436253
2) Ly, T., Whigham, A., Clarke, R., Brenes-Murillo, A.J., Estes, B., Madhessian, D., Lundberg, E., Wadsworth, P., Lamond, A.I. (2017). Proteomic analysis of cell cycle progression in asynchronous cultures, including mitotic subphases, using PRIMMUS. eLife 6, e27574.
3) Bresson, S., Tuck, A., Staneva, D. and Tollervey, D. (2017) Nuclear RNA decay pathways aid rapid remodeling of gene expression in yeast. Mol. Cell, 65, 787-800.
If you wish to apply for this project, please go to this link.