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

先天免疫系统的自我/异体辨别取决于识别受体，该受体识别与病原体相关的分子模式。其中，这个组包括内体Toll样受体，其中TLR7、8和13识别和区分哺乳动物和微生物，潜在的病原体相关的RNA。歧视性原则之一是承认内源性RNA修饰。本项目旨在确定影响TLR介导的免疫反应的进一步修饰。在基于酶联免疫吸附试验的免疫刺激试验中显示不同活性的亚分离tRNA制剂可作为分离单个非刺激性tRNA物种的起点。在这样的tRNA中，可以通过合成和测试所谓的变异体来识别相关的修饰，这些变异体是仅携带自然发生的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饰。这种修饰，即TM，表现出核糖甲基化，但尽管有这种相似之处，其活性明显低于GM。为了探讨生理效应，我们将分析相关甲基转移酶的表达水平，以及刺激改变后者。

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.