Periodic Reporting for period 2 - TCAPS (mRNA cap regulation and function in CD8 T cells)
Reporting period: 2019-11-01 to 2021-04-30
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 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. Current work on RNMT involves looking at
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.
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.