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

先天免疫系统的自我/外来区分依赖于识别与病原体相关的分子模式的识别受体。其中，这一组包括内体toll样受体，其中TLR7、8和13识别和区分哺乳动物和微生物的潜在病原体相关RNA。其中一个区别原则是内源性RNA修饰的识别。我们之前的工作已经确定了Gm，一种在tRNA中自然发生的核糖甲基化，它不仅避免了TLR7的识别，而且在特定的序列背景下作为拮抗剂，从而抑制TLR7介导的干扰素反应，即使在存在其他刺激RNA的情况下。目前的项目旨在确定影响tlr介导的免疫反应的进一步修饰。在基于ELISA的免疫刺激实验中，显示出差异活性的减法tRNA制剂可作为分离单一、非刺激性tRNA物种的起点。在这些trna中，相关的修饰可以通过所谓的修饰变异体的合成和测试来识别，这些修饰变异体是仅携带单一或自然发生修饰的子集的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，显示了核糖甲基化，但尽管这种相似性，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中核糖甲基化核苷的核碱基结构，以描述哪些原子细节控制tlr7介导的干扰素对RNA反应的抑制作用。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，以及改变后者的刺激。着眼于不同生物系统中的先天免疫反应，包括感染期间的免疫逃避和自身免疫的诱导，我们还建议使用和产生缺乏核糖甲基化酶活性的敲除，以研究RNA修饰在体内的生物学影响。

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.