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

调节性 T 细胞在效应 CD4 T 细胞中施加特定的翻译组变化

• 批准号: 9908473
• 负责人: Lomon So
• 金额: $ 2.89万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-03 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9908473
• 关键词: Affinity Chromatography; Antigen-Presenting Cells; Autoantigens; Autoimmune Diseases; Bioinformatics; Biological Assay; Biological Process; Biology; CD4 Positive T Lymphocytes; Cells; Cellular Structures; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Critical Pathways; DNA; DNA Sequence Alteration; Data; Development; Epitopes; Eragrostis; Event; FOXP3 gene; Gene Expression; Gene Expression Regulation; Genetic; Homeostasis; Human; Immune; Immune Tolerance; Immune response; Immunity; Immunoprecipitation; In Vitro; Link; Logic; Luciferases; Malignant Neoplasms; Mass Spectrum Analysis; Mature T-Lymphocyte; Mediating; Messenger RNA; Modeling; Molecular; Molecular Biology Techniques; Mutagenesis; Output; Play; Proteins; Proteome; RNA; Regulation; Regulatory Element; Regulatory T-Lymphocyte; Reporter; Research; RiboTag; Ribosomal Proteins; Ribosomes; Role; Signal Pathway; Specificity; Syndrome; Time; Transcript; Transgenic Mice; Translating; Translation Process Protein; Translations; Untranslated Regions; base; biological systems; effector T cell; follow-up; genetic information; genome-wide; immune function; in vivo; mouse model; novel; programs; recruit; stem; tool; transcriptome; transcriptomics; translation factor; translatome

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.

项目概要/摘要 调节性 T 细胞 (Treg) 是免疫细胞的一个独特子集，在正确维持免疫细胞功能方面发挥着关键作用。 事实上，Treg 稳态或功能的变化与多种免疫稳态密切相关。 自身免疫性疾病以及癌症，表明了解 Treg 的临床重要性 尽管对 Tregs 进行了数十年的研究，但 Tregs 如何抑制不需要的免疫的分子基础仍然存在。 障碍主要来自于关于 Tregs 如何发挥作用的“太多”机制。 在这里，我们提出了 Tregs 功能的新范式，其中心假设是 Tregs。 靶效应 CD4 T 细胞 (Teff) 在“翻译”水平上的“核心”抑制程序 从以 RNA 格式编码的遗传信息生成蛋白质的过程。 蛋白质是赋予大多数生物功能的生物分子，令人惊讶的是稳态 称为转录组的 mRNA 丰度通常是用来解释的终点分析 我们认为这种差距是由于缺乏精确的工具来探究免疫反应的机制。 原代免疫细胞中的翻译组（在给定时刻翻译的所有 mRNA 转录本）。 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 我们使用该工具通过翻译核糖体亲和纯化（TRAP）对原代免疫细胞进行翻译。 建议跟进我们的初步观察，即Tregs确实是调节的必要且充分的 在 Aim1 中，我们将询问 Teff 细胞在体外和体内的总体蛋白质合成输出。 然后我们将描述 Treg 相遇期间核糖体结合 mRNA 转录本的全基因组变化。 使用特定 mRNA 转录物中的顺式调控元件（mRNA 分子内的序列） 在 Aim2 中，我们将通过生物信息学分析来了解这种特异性的机制。 假设与核糖体（核糖蛋白质组）本身相关的反式作用蛋白因子处于动态状态 使用允许直接核糖体免疫沉淀的相同遗传工具， 我们将使用质谱分析来询问此类结合因子的整个蛋白质组。 特定 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看待免疫中基因表达控制的方式。