RNA-binding proteins and control of mRNA metabolism in the regulation of adaptive immunity in humans

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Post-transcriptional gene regulation can determine mRNA splicing, stability, translation and sub-cellular localization, thereby potentially impacting all cellular functions. In the immune system, the importance of post-transcriptional control in regulating the differentiation and functions of T lymphocytes is becoming increasingly clear, although many questions remain to be addressed. RNA-binding proteins (RBPs) have been variably involved in immune modulation by regulating target mRNA stability and translation. Understanding mechanisms of post-transcriptional regulation of gene expression is particularly important for immune cells, in which the expression of effector molecules such as cytokines must increase rapidly in response to danger signals, but it also must be quickly turned off to avoid excessive inflammation and tissue damage. Indeed, many mRNAs encoding for cytokines and other immune-relevant genes are unstable and/ or kept in a translationally silenced state until needed. However, there are still major gaps in our knowledge regarding the mechanisms of action of RBPs and their functional role in immune cells, especially in humans. Most importantly, the level of cross-interaction, cooperation or redundancy of the different RBPs in regulating the metabolism of specific mRNAs remains almost completely unknown. With this project we will therefore fill these critical knowledge gaps by investigating the following Specific Aims:- Aim 1: To determine the importance of the different RBPs in the differentiation and function of human T lymphocytes. This aim will tackle two major questions: i) what is the impact of mRNA regulation mediated by RBPs specifically in the human system, and ii) to what extent the different family members of a given RBP family have unique and/ or redundant functions. These questions will be addressed primarily by taking advantage of recently optimized systems to perform CRISPR-Cas9-mediated gene deletion in primary human T lymphocytes and by assessing the functional effects of such deletions.- Aim 2: In depth mechanistic analysis of selected RBPs in human T lymphocytes. In this aim we will use a combination of ‘omics’ and targeted approaches to establish the binding profile of selected RPBs in human T cells and their effect on mRNA stability and translation.Overall, we will integrate a comprehensive description of the functional role of selected RBPs in modulating aspects of T lymphocyte biology with a detailed mechanistic analysis of the role of these RBPs in regulating mRNA metabolism during cell activation, differentiation and proliferation. By combining our expertise in the regulation of immune responses by RBPs and in human T lymphocyte biology we aim at unravelling general paradigms linking RBP expression to the regulation of complex cellular responses in human T lymphocytes.