Investigating the role of mRNA capping in T-cells

Lead Research Organisation: University of Dundee
Department Name: School of Life Sciences

Abstract

T-cells are cells of the adaptive immune system, vital for clearing infections. Deregulation of T-cell function leads to a plethora of diseases. Following antigen stimulation and activation of T- cells, significant upregulation in gene expression is observed, including upregulation of the mRNA capping enzymes. This project will ask the question: "What is the role of the mRNA capping methyltransferases in transcription in T-cells and what impact do they have on T-cell function?" Mice with conditional deletions of the cap methyltransferases in T-cells will be utilized in this study, allowing for a wider physiological understanding of the role of mRNA capping. Furthermore, in addition to establishing what the direct effect of cap methyltransferase deletion is in T-cells, I also aim to study how the diminished recruitment of cap binding proteins affects downstream signalling pathways. Only a small number of cap binding proteins have been discovered and characterised at this time, and most of these remain undefined in function within cells of the immune system. Deletion of RNMT and CMTR1 in T-cells leads to reduced proliferative ability and apoptosis. It is therefore my aim to also investigate whether there are any important T-cell-specific proteins that bind to the mRNA cap that would explain this phenotype.
To determine the role of the mRNA cap methyltransferases in transcription I will employ immunological assays, RNA sequencing, qPCR, chromatin immunoprecipitations and thermal shift mass spectrometry. In order to explore potential cap binding proteins in T-cells, I will utilize sepharose resins modified with various mRNA cap structures in pulldowns, followed by advanced label-free mass spectrometric analysis. Both aspects of this project will apply statistical testing, computational analysis utilizing next-generation sequencing packages and proteomic analysis that will produce big data-sets. This project will define whether gene-specific mRNA capping enzyme activity determines the transcription of specific genes in T-cells and will identify cap binding partners, which may be useful information in the treatment of immune disorders.
1.Explain interdisciplinary interface:
This project will utilize experimental techniques, such as RNA sequencing and mass spectrometry that generate "Big Data" set which will require extensive computational analysis and quantification. I will receive training and utilize multiple bioinformatics packages to be able to determine changes in gene expression upon RNMT and CMTR1 deletion in CD8 T-cells. The outcome of this project may result in an improved understanding of how gene expression is regulated in T-cells and may have a wider impact on the fields of immunology and medicine. The project has biology-chemistry and computational aspects to it.

2.Does project require significant amount of quantitative skills? YES

3.Does project require significant amount of whole organism physiology skills? YES

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