RNA processing mechanisms control lymphocyte development and activation

One of the well recognised features of ageing is an increased susceptibility to infection that is caused by a decline in the function of the immune system. Our immune system forms during foetal development and the early neonatal period and is made up of many types of cells including the white blood cells. These are continuously replenished throughout the life-course from specialised stem cells called haematopoietic stem cells that are found in the bone marrow. The white blood cells can be further divided into lymphocytes and non lymphocytes. Amongst the lymphocytes are B cells which produce antibodies and specialised cells that kill virally infected cells. A class of lymphocyte that develops in an organ called the thymus is called the T cell and this cell co-ordinates the function of antibody producing and killer lymphocytes. One important feature of lymphocytes is that they hold a memory of previous encounters with infections. This has been used to create vaccines which make us immune to diseases without having to suffer the disease. While vaccines work well in young individuals they work poorly, or not at all, in the elderly. Because of the importance of lymphocytes they have been much studied and we have plenty of information on the different stages of development the cells go through as they mature and become stimulated by encounter with antigen (the technical term immunologists use for anything that stimulates the immune system). We know much about the signalling processes that take place inside the cell when receptors on the surface of lymphocytes are triggered. We also know much about a class of genes that encode proteins, called transcription factors, which act as on/off switches for genes in the DNA. Transcription factors convert the information encoded in the DNA sequence of genes into a message, called mRNA, which specifies the order in which amino acids are incorporated into proteins. Our project is aimed at understanding how the mRNA is regulated subsequent to its production. We suspect special proteins, called mRNA binding proteins, physically interact with the mRNA and control how long it remains in the cell and the rate at which it can give rise to new proteins. This type of regulation is called post-transcriptional control and is far less well understood. Our study will ask what the function of these mRNA binding proteins is in lymphocytes and will reveal how they work. Our gaol is to understand how mRNA binding proteins interact with signalling pathways and ultimately with transcription factors to control lymphocytes and the immune response.

We propose that post-transcriptional control mediated by RNA binding proteins (RBPs) is a dominant force in the regulation of lymphocyte development and activation. As such it will be important during the ageing process. We will examine this hypothesis using lines of mice with conditional alleles of three zfp36 family members which are inactivated by Cre during T lymphocyte development. Furthermore, we will create mice in which ROD1 and/or PTB function can be regulated at different stages of B and T cell development. We will seek to understand how these RBP are regulated by signal transduction pathways downstream of antigen receptors. For studies of immunity we will employ model antigens and infectious organisms to study the responses of young and old mice. This will be complemented by in vitro studies of lymphocyte differentiation. To identify the specific targets of RBPs, purified naïve and memory lymphocytes will be exposed to UV light to crosslink RNA to protein and specific antibodies used to purify RNA-RBP complexes. The recovered RNA will be identified by deep-sequencing of cDNA. This will be combined with the study of specific splicing patterns using splice junction arrays; the turnover of mRNA using thiouridine labelling methods that allow the labelling of newly synthesized RNA; and the analysis of mRNA association with translating ribosomes. Using informatics approaches we will integrate the knowledge obtained from these experiments on normal lymphocytes with data obtained on RNA handling in lymphocytes in the absence of specific RBPs. These large datasets will be used to generate regulatory maps describing post-transcriptional control. Together, these studies will provide a detailed picture of the global regulation of mRNA handling at the post-transcriptional level in primary cells from animals of different ages.

Our project will address fundamental aspects of how gene expression and genome function is controlled. By studying RNA binding proteins and post-transcriptional control we are focussing on an area of this regulation that has, to date, not been intensively studied. Through long-term planning and investment we have positioned ourselves to have the concepts and tools that will allow us to deliver ground breaking insights into post-transcriptional control. One important aspect of the work is the use of highly purified non-transformed cells to generate genome-wide data sets on splicing, RNA turnover and the interaction of RNA binding proteins with RNA. This will be a valuable resource for modelling studies which integrate different data sets. The postdoctoral scientists working on this project will be trained to combine in vivo and high-throughput data generation/analysis skills which will equip them to become leaders in 21st century bioscience. Our project will bring new investigators into the study of healthy ageing. Its outputs will be relevant to the work of other research councils through cross council initiatives such as Lifelong Health and Wellbeing. Our knowledge may also prove to be of interest to biotech and pharma who seek to develop interventions that affect health and the quality of life. Groups at the Novartis Institutes for Biomedical Research in Basel have developed low molecular weight inhibitors of the RNA binding protein HuR (Nat. Chem. Biol. 2007: 508-15) and are pursuing other targeted approaches to modify RNA decay (J. Biomol. Screening 2010:609-622). The impact of our work may also affect third sector organisations such as charities. Our project will also facilitate greater integration between a BBSRC institute, the University of Cambridge and the LMB. These stronger links will allow for greater efficiency in the take-up of new technologies such as the adoption of transgenic mouse technologies in the Smith lab. At the LMB the data arising from the application of iCLIP to lymphocytes complements the main focus of the Ule lab on the RNA binding proteins in neurons. We anticipate appreciation the similarities and differences between these systems will lead to a greater understanding of post-transcriptional control and uncover generic principles relevant to other cell systems. Ageing leads to the altered homeostasis of the immune system; loss of the ability to distinguish self from non-self, and vaccine failure in the elderly. Manipulation of the immune system has an impressive track-record in bringing benefits on a global scale but better understanding of the molecular mechanisms of immune function during ageing, such as those propose here, are required for future enhancement of lives. Furthermore, understanding the basic science underpinning the immune response will allow better education about the benefits of vaccination. Emerging concepts place the immune system as an important player in the maintenance of organ and barrier integrity and as a regulator of metabolism, in part though the elaboration of cytokines but also through the regulation of the gut microbiota which are increasingly being recognized as important to obesity and for healthy ageing. It is likely that our study will have implications for stem cell biology and the emerging field of regenerative medicine by improving our understanding of how cell identity is established and maintained. Overexpressing a few genes allows cells to become reprogrammed and it is likely additional factors will be found that regulate this. Modulating mRNAs post-transcriptionally offers a strategy for altering the expression of endogenous genes and may contribute to our ability to produce vectors for gene expression that are effective and safe. Great investment is currently being made in RNA based therapeutics. These approaches are promising but face challenges which require understanding of post-transcriptional regulation.