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到T细胞生物学中快速发展的“表转录组学”领域。在这个概念中，写蛋白用甲基修饰mRNA中的特定腺苷，而读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，其他功能也参与其中，如核解读蛋白可以识别新生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，包括关键调控因子Cish、Socs1和Socs3或Tnf、Ripk1和Orai1， m6A调控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功能丧失在T细胞中已被广泛研究，但围绕m6A解读蛋白的许多问题仍未得到解答。在提议的项目中，我们将解决这些问题：哪些识别m6A的rbp负责在m6A丢失时观察到的表型？它们如何影响辅助性T细胞分化？包含rbp的原型YTH结构域与转录组的相互作用有何不同，它们是否只识别m6A？这些含有YTH结构域的rbp是否执行冗余或非冗余功能，它们涉及哪些分子机制？具体来说，我们建议剖析所有T细胞表达的含有Ythdc1、Ythdf1、Ythdf2和Ythdf3的yth结构域蛋白的作用。我们的初步数据显示Ythdc1对胸腺细胞分化具有非冗余控制，而T细胞特异性灭活Ythdf2的结果表明在T细胞稳态中具有冗余功能。在我们的工作计划中，我们将首先关注Ythdc1，并在目标1中讨论mRNA靶点和表观遗传变化以及可以解释Ythdc1在胸腺细胞发育中的功能的分子机制。在目标2中，我们将重点研究Ythdf1， Ythdf2和Ythdf3的非冗余和冗余功能，通过单独和联合T细胞特异性失活其固定等位基因。在这些小鼠中，我们将研究体内T细胞稳态和分化或实验诱导的免疫反应。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，我们的分析将提供全面的表型研究，并揭示YTH蛋白在T细胞发育、体内平衡和免疫反应中功能的新分子机制。

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.