Periodic Reporting for period 3 - TCAPS (mRNA cap regulation and function in CD8 T cells) Reporting period: 2021-05-01 to 2022-04-30 Summary of the context and overall objectives of the project 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. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far 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.We have determined that the cap methyltransferases, RNMT and CMTR1, are upregulated following T cell activation and are required for the expression of specific subsets of genes and for proliferation during differentiation. RNMT and CMTR1 dependent genes are overlap by about 50% but also include distinct subsets.RNMT dependent genes increase ribosome production during T cell activation. RNMT was presumed to be essential for all genes to be expressed, but we demonstrated that its major target genes are ribosomal protein genes, ribosome processing genes, ribosomal RNA transcription and processing factors. The mRNA produced from these genes is controlled by LARP1, a RNA cap binding protein which stabilises the mRNA to which it binds. In addition RNMT controls the production of snoRNA which guides ribosomal RNA processing; some of these RNAs are found in LARP1 target genes which explains how they are controlled. We found that RNMT is required for proliferation during the early stages of T cell activation, prior to differentiation. This involves RNMT regulation of the cytokine IL7.CMTR1 controls some of the same genes as RNMT but also a distinct subset of genes/proteins as well. The CMTR1 KO revealled that CMTR1 does not control proliferation as early as RNMT and is only required for specific T cell lineages. This is a potentially important finding from a therapeutic perspective; CMTR1 inhibitors may be important for controlling which subsets of T cells are produced which influences the response to cancer, infection and other pathological conditions. We have found that CMTR1 is controlling several stages of gene regulation including splicing and translation initiation. We are using biochemical methods and advanced RNA sequencing to determine the mechanism by which CMTR1 controls gene regulation. Such information may be useful in the development of therapeutic agents to interfere or direct T cell function. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) 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.Many of the RNA and protein analyses carried out are routine (RNA seq, ribo-seq), whereas others are more specialised, including the analysis of LARP1-interacting RNAs and the analysis of methylation of ribosomal RNA. It has been challenging to perform these techniques on the very small amount of material present in T cell populations. We have detailed our adapted methodologies in our publications. The mRNA cap and capping enzymes
