Discovery and characterization of RNA modifications in a bacterial model pathogen

细菌模型系统至关重要地帮助我们了解天然 rRNA 和 tRNA 修饰以及同源修饰酶的多样性和范围。然而，与真核生物中的发现相反，细菌中 mRNA 和非编码 RNA 的功能相关修饰仍然未知。该项目的目的是首次研究细菌模型病原体沙门氏菌中的 RNA 修饰。我们将 RIP-seq 和 CLIP-seq 技术应用于假尿苷合酶的初步研究表明，这些酶可能在体内结合多种 mRNA 和 sRNA。此外，我们还开发了一种基于 RNA 的亲和层析方法，用于纯化 sRNA 和相关蛋白质复合物，其数量适合生化分析。使用新描述的基于RNA-seq的方法（称为pseudouridine-seq），我们的目标是获得沙门氏菌中pseudouridine修饰的全转录组图谱。类似地，将确定各种细菌生长条件下转录组假尿苷谱的变化，并评估与靶mRNA或蛋白质稳定性的可能相关性。我们将获得 TruD（一种可能靶向许多 mRNA 和 sRNA 的假尿苷合酶）与新型 RNA 配体的共晶结构，以便更好地理解原子水平上的底物识别。此外，纯化的 sRNA 上可能的修饰将通过 LC-MS 确定，映射到特定位置，然后通过已建立的分子技术和报告系统研究功能相关性。为了找到负责这些修饰的蛋白质，我们将检查与纯化的 sRNA 相关的蛋白质。我们期望这个项目能够开创对重要肠道病原体中功能性 RNA 修饰范围的新认识。

Bacterial model systems have crucially informed our understanding of the variety and scope of natural rRNA and tRNA modifications as well as the cognate modifying enzymes. Contrary to discoveries in eukaryotes, however, functionally relevant modifications of mRNAs and non-coding RNAs have remained unknown in bacteria. The aim of the project is to for the first time investigate RNA modifications in the bacterial model pathogen Salmonella. Our preliminary studies applying RIP-seq and CLIP-seq techniques to pseudouridine synthases have shown that these enzymes may bind a plethora of mRNA and sRNA species in vivo. Furthermore, we have developed an RNA-based affinity chromatography method to purify sRNAs and associated protein complexes in quantities suitable for biochemical analyses. Using a newly described RNA-seq based method called pseudouridine-seq, we aim to obtain a transcriptome-wide map of pseudouridine modification in Salmonella. Changes in transcriptomic pseudouridine profile under various bacterial growth conditions will similarly be determined and possible correlations to target mRNA or protein stability assessed. We will obtain co-crystal structures of TruD (a pseudouridine synthase that may target many mRNAs and sRNAs) with novel RNA ligands for a better understanding of substrate recognition on the atomic level. Additionally, possible modifications on purified sRNAs will be determined by LC-MS, mapped to specific locations and thereafter functional relevance investigated by established molecular techniques and reporter systems. To arrive at proteins responsible for these modifications we will examine the proteins found associated with the purified sRNA. We expect this project to pioneer a new understanding of the scope of functional RNA modifications in the important group of enteric pathogens.