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

RNA结合蛋白（RBP）是基因调控的重要组成部分。我们和其他实验室最近的工作涉及T细胞生物学中快速扩展的“epitranscriptomic”领域。在这个概念中，写入器蛋白用甲基修饰mRNA中的特定腺苷，而读取器蛋白结合这些标记并改变标记mRNA的半衰期或翻译效率。然而，还涉及其他功能，因为核阅读器蛋白也可以识别新生mRNA中的m6A标记，并诱导转录基因组蛋白修饰的变化。m6A标记存在于许多mRNA上，包括关键调节因子Cish、Socs1和Socs3或Tnf、Ripk1和Orai 1的mRNA，m6A调节的过程包括胸腺细胞发育、细胞因子信号传导、T细胞稳态和T细胞受体（TCR）激活诱导的细胞死亡。 虽然m6A功能丧失已经在T细胞中被广泛研究，但围绕m6A阅读器蛋白的许多问题仍然没有答案。在拟议的项目中，我们将解决这些问题：哪些m6A识别RBP负责m6A丢失后观察到的表型？它们如何影响T辅助细胞分化？含有RBP的原型YTH结构域在与转录组的相互作用中有何不同，它们是否仅识别m6A？这些含有YTH结构域的RBP是否执行冗余或非冗余功能，以及它们涉及哪些分子机制？ 具体来说，我们建议解剖的所有T细胞表达的YTH结构域包含蛋白质，包括Ythdc1，Ythdf1，Ythdf2和Ythdf3的作用。我们的初步数据表明，Ythdc1发挥非冗余控制胸腺细胞分化，而从T细胞特异性失活Ythdf2的结果表明冗余功能的T细胞稳态。在我们的工作计划中，我们将首先关注Ythdc 1，并在目标1中解决mRNA靶点和表观遗传变化以及可以解释Ythdc 1在胸腺细胞发育中功能的分子机制。在目标2中，我们将专注于Ythdf1，Ythdf2和Ythdf3的非冗余和冗余功能，使用单独和组合的T细胞特异性失活其floxed等位基因。在这些小鼠中，我们将研究T细胞稳态和分化或实验诱导的体内免疫应答。最后，在目标3中，我们将比较m6A修饰（miCLIP）的交联和免疫沉淀数据以及Ythdc1和Ythdf1，2和3蛋白与转录组的相互作用，并询问这些因子是否具有超越m6A识别的结合偏好。总之，我们的分析将提供一个全面的表型调查，并揭示新的分子机制YTH蛋白在T细胞发育，稳态和免疫反应的功能。

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.