Exploring unique and redundant functions of m6A-recognizing RNA-binding proteins in T cells

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RNA-binding proteins (RBPs) constitute an essential layer of gene regulation. Recent work from our and other labs involved the fast-expanding field of "epitranscriptomic" in T cell biology. In this concept, writer proteins modify specific adenosines in the mRNA with methyl groups, while reader proteins bind these marks and change the half-life or translation efficiency of the tagged mRNA. However, additional functions have also been involved, as also nuclear reader proteins can recognize m6A marks in nascent mRNA and induce changes in histones modification at the transcribed gene. m6A marks are present on many mRNAs including those of the key regulators Cish, Socs1 and Socs3 or Tnf, Ripk1 and Orai1, and m6A-regulated processes include thymocyte development, cytokine signaling, T cell homeostasis and T cell receptor (TCR) activation-induced cell death. While m6A loss-of-function has been studied extensively in T cells, many questions revolving around m6A reader proteins are still unanswered. In the proposed project we will address these questions: Which m6A recognizing RBPs are responsible for the phenotypes observed upon loss of m6A? How do they impact on T helper cell differentiation? How do prototypic YTH domain containing RBPs differ in their interaction with the transcriptome, do they only recognize m6A? Do these YTH domain containing RBPs execute redundant or non-redundant functions, and which molecular mechanisms do they involve? Specifically, we propose to dissect the roles of all T cell-expressed YTH-domain containing proteins including Ythdc1, Ythdf1, Ythdf2 and Ythdf3. Our preliminary data show that Ythdc1 exerts non-redundant control over thymocyte differentiation, while results from T cell-specific inactivation of Ythdf2 suggest redundant functions in T cell homeostasis. In our work program we will first focus on Ythdc1 and address in aim 1 the mRNA targets and epigenetic changes as well as molecular mechanisms that can explain Ythdc1 function in thymocyte development. In aim 2, we will focus on non-redundant and redundant functions of Ythdf1, Ythdf2 and Ythdf3 using individual and combined T cell-specific inactivation of their floxed alleles. In these mice we will study T cell homeostasis and differentiation or experimentally induced immune responses in vivo. Finally, in aim 3, we will compare crosslinking and immunoprecipitation data of m6A modification (miCLIP) and interactions of Ythdc1 as well as Ythdf1, 2 and 3 proteins with the transcriptome and ask whether these factors have binding preferences that go beyond the recognition of m6A. Together, our analyses will provide a comprehensive phenotypic investigation and uncover novel molecular mechanisms underlying YTH protein function in T cell development, homeostasis and immune responses.