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

RNA结合蛋白(RBPs)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细胞生物学中迅速扩大的“表位转录”领域。在这个概念中，编写者蛋白用甲基修饰mRNA中的特定腺苷，而读取器蛋白结合这些标记并改变标记的mRNA的半衰期或翻译效率。然而，也参与了额外的功能，因为核阅读器蛋白也可以识别新生mRNA中的m6A标记，并在转录的基因诱导组蛋白修饰的变化。M6A标记存在于许多mRNAs上，包括关键调控因子CISH、SOCS1和SOCS3或肿瘤坏死因子Ripk1和Orai1的M6A标记，M6A调控的过程包括胸腺细胞发育、细胞因子信号转导、T细胞动态平衡和T细胞受体(TCR)激活诱导的细胞死亡。虽然在T细胞中已经对m6A功能丧失进行了广泛的研究，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：哪些识别M6A的限制性商业惯例对丢失M6A后观察到的表型负责？它们对辅助性T细胞分化有何影响？含有限制性商业惯例的原型YTH结构域在与转录组的相互作用方面有什么不同，它们只识别m6A吗？这些含有限制性商业惯例的YTH结构域是执行冗余功能还是非冗余功能，它们涉及哪些分子机制？具体地说，我们建议剖析所有T细胞表达的包含Ythdc1、Ythdf1、Ythdf2和Ythdf3结构域的蛋白的作用。我们的初步数据显示，Ythdc1对胸腺细胞分化实施非冗余控制，而T细胞特异性Ythdf2失活的结果表明，Ythdc1在T细胞动态平衡中具有冗余功能。在我们的工作计划中，我们首先将重点放在Ythdc1上，并在目标1中解决可以解释Ythdc1在胸腺细胞发育中功能的信使核糖核酸靶标和表观遗传学变化以及分子机制。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，方法是单独和联合灭活它们的T细胞特异性等位基因。在这些小鼠中，我们将研究T细胞的动态平衡和分化，或者在体内实验诱导免疫反应。最后，在目标3中，我们将比较m6A修饰的交联和免疫沉淀数据(MiCLIP)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出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研究，并揭示YTH蛋白在T细胞发育、动态平衡和免疫反应中潜在的新的分子机制。

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.