Identification and characterization of RNA modifications with immune-modulatory properties acting on Toll-like receptors

作用于 Toll 样受体的具有免疫调节特性的 RNA 修饰的鉴定和表征

• 批准号: 404931941
• 负责人: Professor Dr. Alexander Dalpke
• 金额: --
• 依托单位: Abteilung Medizinische Mikrobiologie und Hygiene
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/404931941?language=en
• 关键词: Identification; characterization; RNA; modifications; immune

Self/foreign discrimination by the innate immune system depends on recognition receptors which identify molecular patterns as associated to pathogens. Among others, this group includes endosomal toll-like receptors, among which TLR7, 8 and 13 recognize and discriminate mammalian from microbial, potentially pathogen-associated, RNA. One of the discriminatory principles is the recognition of endogenous RNA modifications. Our previous work has identified Gm, a naturally occurring ribose-methylation within tRNA that not only avoided TLR7 recognition but within a defined sequence context acted as antagonist thus suppressing TLR7-mediated interferon responses even in the presence of otherwise stimulatory RNA. The present project aims at the identification of further modifications that affect TLR-mediated immune responses. Subfractionated tRNA preparations showing differential activity in an ELISA based immunostimulation assay serve as starting point for the isolation of single, non-stimulatory tRNA species. Within such tRNAs, the relevant modifications can be identified via synthesis and testing of so-called modivariants, which are tRNAs carrying only a single or a subset of the naturally occurring modifications. This approach recently yielded a new non-stimulatory modification. This modification, namely Tm, displays a ribose methylation but, despite this similarity, is significantly less active than Gm. We therefore propose to vary systematically the nucleobase structure of a ribose-methylated nucleoside within oligoribonucleotides, in order to delineate which atomic details govern the dampening effect on TLR7-mediated interferon response to RNA. To approach physiological effects, we will analyze expression levels of relevant methyltransferases, and stimuli changing the latter. With an eye to aspects of innate immune responses in different biological systems, including immune-evasion during infection and induction of autoimmunity, we also propose to use and generate knock-outs that lack ribose-methylating enzymatic activity in order to study the biological impact of RNA modifications in vivo.

先天免疫系统的自体/外来辨别取决于识别受体，其识别与病原体相关的分子模式。除其他外，该组包括内体 Toll 样受体，其中 TLR7、8 和 13 识别并区分哺乳动物和可能与病原体相关的微生物 RNA。歧视性原则之一是识别内源性 RNA 修饰。我们之前的工作已经鉴定出 Gm，一种 tRNA 内天然存在的核糖甲基化，它不仅避免了 TLR7 识别，而且在确定的序列背景下充当拮抗剂，从而抑制 TLR7 介导的干扰素反应，即使在存在其他刺激性 RNA 的情况下也是如此。本项目旨在确定影响 TLR 介导的免疫反应的进一步修饰。在基于 ELISA 的免疫刺激测定中显示出不同活性的亚分级 tRNA 制剂可作为分离单一非刺激性 tRNA 种类的起点。在此类 tRNA 中，可以通过合成和测试所谓的修饰变体来鉴定相关修饰，修饰变体是仅携带天然存在修饰的单个或子集的 tRNA。这种方法最近产生了一种新的非刺激性修改。这种修饰（即 Tm）表现出核糖甲基化，但尽管有这种相似性，但其活性明显低于 Gm。因此，我们建议系统地改变寡核糖核苷酸内核糖甲基化核苷的核碱基结构，以描绘哪些原子细节控制对 TLR7 介导的干扰素对 RNA 反应的抑制作用。为了研究生理效应，我们将分析相关甲基转移酶的表达水平以及改变后者的刺激。着眼于不同生物系统中先天免疫反应的各个方面，包括感染期间的免疫逃避和自身免疫的诱导，我们还建议使用和生成缺乏核糖甲基化酶活性的敲除，以研究体内RNA修饰的生物学影响。