Discovery and characterization of RNA modifications in a bacterial model pathogen

细菌模型系统对我们理解天然rRNA和tRNA修饰的种类和范围以及同源修饰酶具有至关重要的作用。然而，与真核生物中的发现相反，在细菌中，mRNAs和非编码RNAs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的RNA修饰。我们将RIP-SEQ和CLIP-SEQ技术应用于假尿苷合成酶的初步研究表明，这些酶可能在体内结合了大量的mRNA和sRNA物种。此外，我们还开发了一种基于RNA的亲和层析方法，以纯化适合生化分析的sRNA和相关蛋白复合体。使用一种新描述的基于RNA-seq的方法称为假尿苷-seq，我们的目标是获得沙门氏菌中假尿苷修饰的转录组范围的图谱。在不同细菌生长条件下转录转录假尿苷图谱的变化将同样被确定，并评估与目标mRNA或蛋白质稳定性的可能相关性。为了在原子水平上更好地理解底物识别，我们将获得Trud(一种可能靶向许多mRNAs和sRNAs的伪尿苷合成酶)的共晶结构。此外，对纯化的sRNA的可能的修饰将通过LC-MS确定，并被映射到特定的位置，然后通过已建立的分子技术和报告系统来研究功能相关性。为了得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。我们期望这一项目能够对重要的肠道病原体的功能性RNA修饰的范围有一个新的理解。

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.