RNA-binding proteins and control of mRNA metabolism in the regulation of adaptive immunity in humans

转录后基因调控可以决定mRNA的剪接、稳定性、翻译和亚细胞定位，从而潜在地影响所有细胞功能。在免疫系统中，转录后控制在调节T淋巴细胞分化和功能中的重要性越来越清楚，尽管仍有许多问题有待解决。rna结合蛋白（rbp）通过调节靶mRNA的稳定性和翻译参与免疫调节。了解基因表达的转录后调控机制对免疫细胞尤为重要，在免疫细胞中，细胞因子等效应分子的表达必须迅速增加以响应危险信号，但也必须迅速关闭以避免过度炎症和组织损伤。事实上，许多编码细胞因子和其他免疫相关基因的mrna在需要之前是不稳定的和/或保持在翻译沉默状态。然而，关于rbp的作用机制及其在免疫细胞中的功能作用，特别是在人类中的作用，我们的知识仍然存在重大差距。最重要的是，不同rbp在调节特定mrna代谢中的交叉相互作用、合作或冗余水平几乎完全未知。因此，在这个项目中，我们将通过研究以下具体目标来填补这些关键的知识空白：目标1：确定不同rbp在人类T淋巴细胞分化和功能中的重要性。这一目标将解决两个主要问题：1)RBP在人体系统中介导的mRNA调控的影响是什么；2)给定RBP家族的不同家族成员在多大程度上具有独特和/或冗余功能。这些问题将主要通过利用最近优化的系统在原代人T淋巴细胞中执行crispr - cas9介导的基因缺失，并通过评估这种缺失的功能影响来解决。-目的2：深入分析人T淋巴细胞中选定的rbp的机制。在这个目标中，我们将使用“组学”和靶向方法的结合来建立选定的rbp在人类T细胞中的结合谱及其对mRNA稳定性和翻译的影响。总的来说，我们将综合描述选定的rbp在调节T淋巴细胞生物学方面的功能作用，并详细分析这些rbp在细胞活化、分化和增殖过程中调节mRNA代谢的作用。通过结合我们在RBP免疫反应调节和人类T淋巴细胞生物学方面的专业知识，我们旨在揭示将RBP表达与人类T淋巴细胞复杂细胞反应调节联系起来的一般范例。

Post-transcriptional gene regulation can determine mRNA splicing, stability, translation and sub-cellular localization, thereby potentially impacting all cellular functions. In the immune system, the importance of post-transcriptional control in regulating the differentiation and functions of T lymphocytes is becoming increasingly clear, although many questions remain to be addressed. RNA-binding proteins (RBPs) have been variably involved in immune modulation by regulating target mRNA stability and translation. Understanding mechanisms of post-transcriptional regulation of gene expression is particularly important for immune cells, in which the expression of effector molecules such as cytokines must increase rapidly in response to danger signals, but it also must be quickly turned off to avoid excessive inflammation and tissue damage. Indeed, many mRNAs encoding for cytokines and other immune-relevant genes are unstable and/ or kept in a translationally silenced state until needed. However, there are still major gaps in our knowledge regarding the mechanisms of action of RBPs and their functional role in immune cells, especially in humans. Most importantly, the level of cross-interaction, cooperation or redundancy of the different RBPs in regulating the metabolism of specific mRNAs remains almost completely unknown. With this project we will therefore fill these critical knowledge gaps by investigating the following Specific Aims:- Aim 1: To determine the importance of the different RBPs in the differentiation and function of human T lymphocytes. This aim will tackle two major questions: i) what is the impact of mRNA regulation mediated by RBPs specifically in the human system, and ii) to what extent the different family members of a given RBP family have unique and/ or redundant functions. These questions will be addressed primarily by taking advantage of recently optimized systems to perform CRISPR-Cas9-mediated gene deletion in primary human T lymphocytes and by assessing the functional effects of such deletions.- Aim 2: In depth mechanistic analysis of selected RBPs in human T lymphocytes. In this aim we will use a combination of ‘omics’ and targeted approaches to establish the binding profile of selected RPBs in human T cells and their effect on mRNA stability and translation.Overall, we will integrate a comprehensive description of the functional role of selected RBPs in modulating aspects of T lymphocyte biology with a detailed mechanistic analysis of the role of these RBPs in regulating mRNA metabolism during cell activation, differentiation and proliferation. By combining our expertise in the regulation of immune responses by RBPs and in human T lymphocyte biology we aim at unravelling general paradigms linking RBP expression to the regulation of complex cellular responses in human T lymphocytes.