Regulatory T cells impose specific translatome changes in effector CD4 T cells

项目总结/摘要 调节性T细胞（Treg）是免疫细胞的一个独特亚群，在正常维持免疫系统中起关键作用。 免疫稳态事实上，Treg稳态或功能的变化高度涉及多种免疫应答。 自身免疫性疾病的设置以及在癌症中，这表明了解Treg的临床重要性 生物学尽管数十年来对TGFAP的研究，TGFAP如何抑制不必要的免疫反应的分子基础， 答复不明确。这一障碍主要来自于“太多”的机制， 功能在这里，我们提出了一个新的范式的功能，与中心假设， 在靶效应CD4 T细胞（Teff）中在"翻译"水平上施加"核心"抑制程序， 蛋白质是从以RNA形式编码的遗传信息生成的过程。虽然 蛋白质是赋予大多数生物功能的生物分子，令人惊讶的是， 大量的mRNA被称为转录组，通常是选择用来解释 免疫反应的机制。我们认为，这种差距是由于缺乏精确的工具来询问 翻译组（在给定时刻翻译的所有mRNA转录物）。我们 重新利用RiboTag遗传工具，在核心核糖体蛋白中引入表位标签来研究 通过翻译核糖体亲和纯化（TRAP），在原代免疫细胞的翻译组中表达。利用这个工具，我们 我建议就我们的初步意见采取后续行动，即确实有必要和足够的时间来监管 Teff细胞在体外和体内的总蛋白质合成输出。在目标1中，我们将审问 在Treg遭遇期间核糖体结合mRNA转录物的全基因组变化。然后我们将描述 在特定的mRNA转录物中的顺式调节元件（mRNA分子内的序列）， 生物信息学分析，以了解这种特异性的机制。在Aim2中，我们将探索 假设与核糖体（核糖蛋白质组）本身相关的反式作用蛋白质因子处于动态 在Treg遭遇时进行调节。使用相同的遗传工具，允许直接核糖体免疫沉淀， 我们将使用质谱分析来询问这种结合因子的整个蛋白质组。划定 特定的mRNA如何在时间和空间上被选择成为功能性蛋白质分子背后的逻辑 可能会彻底改变我们对免疫中基因表达控制的看法。

Project Summary/Abstract Regulatory T cells (Treg) are a distinct subset of immune cells that play a critical role in properly maintaining immune homeostasis. Indeed, changes in Treg homeostasis or function is highly implicated in a variety of autoimmune diseases settings as well as in cancer, suggesting clinical importance of understanding Treg biology. Despite decades of research on Tregs, the molecular basis of how Tregs suppress unwanted immune responses are unclear. The hurdle mainly comes from ‘too many’ mechanisms proposed as to how Tregs function. Here, we propose a new paradigm for the function of Tregs with the central hypothesis that Tregs impose a ‘core’ suppressive program in target effector CD4 T cells (Teff) at the level of ‘translation,’ the process where proteins are generated from the genetic information encoded in the format of RNA. Although proteins are the biomolecules that confer most biological function, it is surprising that the steady-state abundance of mRNA known as the transcriptome is usually the end-point analysis chosen to explain mechanisms of immune responses. We believe this gap comes from the lack of precise tools to interrogate the translatome (all the mRNA transcripts that are translated in a given moment) in primary immune cells. We repurposed the RiboTag genetic tool that introduces an epitope-tag in a core ribosomal protein to study the translatome of primary immune cells by translating ribosome affinity purification (TRAP). Using this tool, we propose to follow up on our preliminary observation that indeed Tregs are necessary and sufficient to regulate the overall protein synthetic output in Teff cells both in vitro and in vivo. In Aim1, we will interrogate the genome-wide changes in ribosome-bound mRNA transcripts during Treg encounter. We will then characterize cis-regulatory elements (sequence within the mRNA molecule) in the specific mRNA transcripts using bioinformatics analysis to understand the mechanism of such specificity. In Aim2, we will explore the hypothesis that trans-acting protein factors associated with the ribosome (riboproteome) itself is under dynamic regulation during Treg encounter. Using the same genetic tool that allows direct ribosome immunoprecipitation, we will interrogate the entire proteome of such bound factors using mass spectrometry analysis. Delineation of the logic behind how specific mRNAs are chosen in time and space to become functional protein molecules may revolutionize the way we view gene expression control in immunity.