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mRNA cap regulation and function in CD8 T cells

Periodic Reporting for period 1 - TCAPS (mRNA cap regulation and function in CD8 T cells)

Reporting period: 2018-05-01 to 2019-10-31

T cells are vital for our response to infection, directly killing infected cells and stimulating other cells of
the immune system. Major health problems world-wide involve deregulated T cells, including lymphomas, leukaemias, auto-immune disorders and organ transplant rejection. Damaging deficiencies of T cells are found in old-age. T cells also form a major defence against the accumulation of cancer cells. Current research efforts aim to enhance the T cell response to cancer and furthermore to engineer T cells which target particular tumour cells. To develop therapeutic approaches which target or utilise T cells, it is important that we understand how they function. The aim of the TCAPs project is to understand how T cells respond to activation during an infection or in response to tumours by increasing their size and contents.

It is important for our society to understand how T cells function because of the burden of T cell pathologies. Diseases in which T cell pathologies are the driver occur in the very young, e.g. lymphomas, leukaemias, throughout life, e.g. auto-immune disorders and organ transplant rejection, and in old age, e.g. T cell deficiencies. In addition, the current efforts to engineer T cells which target cancer cells are showing great promise. If we can contribute to efforts to determine how T cells function, we may provide therapeutic opportunities to prevent some pathologies and to treat others.

The objective of this TCAPs project is to determine the contribution of a structure called the mRNA cap to T cell function. Our preliminary data had indicated that regulation of the mRNA cap is critical for the physiology of T cells.
1. We will determine how the mRNA cap is regulated in T cells following activation
2. We will determine the role of the mRNA cap in naïve T cells which circulate in the blood, surveying for signs of infection
3. We will determine the role of the mRNA cap in activated T cells, following interaction with infected cells

mRNA cap regulation is likely to be operational throughout mammalian physiology and therefore TCAPS will contribute significantly to our understanding of regulated gene expression in mammals.
We have established the production and growth of T cells in our laboratory. We have developed experiments that allow us to look at how mRNA caps are made in T cells including analysing the enzymes which make the caps. These are depicted in the attached picture. For experimental purposes we have developed a system to inhibit certain genes of the mRNA capping system. We inhibit capping enzymes in T cells and then see how the T cell responds. This gives us a good idea of the function of the capping enzymes in T cells. We analyse how fast T cells grow and proliferate.

We are interested in which proteins are present in T cells because that indicates whether they are functioning normally, or whether specific parts of the cell are damaged. We perform proteomic experiments which analyse the protein content of the cell using a mass spectrometer in an unbiased approach. We also know a lot about T cells from the work of other researchers and therefore we sometimes analyse the behaviour of particular proteins in the cell. We also analyse the RNA content of T cells, because RNA is the template for protein production. When we take all this data together, we will be able to determine the role of the different capping enzymes present in the cell.
For the progress of the TCAPs project, it was advantageous to develop a methodology to detect the mRNA cap structures present in cells. Although protocols currently exist to analyse cellular mRNA cap structures, they typically involve numerous processing steps, which may result in the loss of particular cap structures and enhanced detection of others. In collaboration with Prof Mike Ferguson, University of Dundee, we developed a mRNA cap quantitation method which we called CAP-MAP. In this CAP-MAP method we used minimal processing steps in conjunction with state-of-the-art chromatography and mass spectrometry to detect and quantitate guanosine cap structures. Quantification of the cap structures in the CAP-MAP method was made possible by the use of chemical standards provided by the Edward Darzynkiewicz group. This CAP-MAP technology is proving to be advantageous for our T cell research and for collaborative projects in many other physiological systems.
The mRNA cap and capping enzymes