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

先天性免疫系统的自身/外来识别依赖于识别与病原体相关的分子模式的识别受体。其中，该组包括内体toll样受体，其中TLR 7、8和13识别并区分哺乳动物与微生物、潜在病原体相关的RNA。区分原则之一是识别内源性RNA修饰。我们先前的工作已经鉴定了Gm，其是tRNA内天然存在的核糖甲基化，其不仅避免了TLR 7识别，而且在限定的序列背景下充当拮抗剂，从而抑制TLR 7介导的干扰素应答，即使在存在其他刺激性RNA的情况下也是如此。本项目旨在鉴定影响TLR介导的免疫应答的进一步修饰。在基于ELISA的免疫刺激测定中显示差异活性的亚分级tRNA制备物用作分离单一非刺激性tRNA种类的起始点。在这样的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，所述修饰变体是仅携带单个或天然存在的修饰的子集的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，显示核糖甲基化，但尽管有这种相似性，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的核糖甲基化核苷的核碱基结构，以描绘哪些原子细节控制TLR 7介导的干扰素对RNA的应答的抑制作用。为了接近生理效应，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。着眼于在不同的生物系统中的先天免疫反应的方面，包括在感染和诱导自身免疫过程中的免疫逃避，我们还建议使用和产生敲除缺乏核糖甲基化酶活性，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.