Discovery and characterization of RNA modifications in a bacterial model pathogen

细菌模型系统对我们了解天然rRNA和tRNA修饰以及同源修饰酶的种类和范围至关重要。然而，与真核生物的发现相反，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的RNA修饰。我们应用RIP-seq和CLIP-seq技术对假尿嘧啶合成酶进行的初步研究表明，这些酶可能在体内结合过多的mRNA和sRNA物种。此外，我们已经开发了一种基于rna的亲和层析方法来纯化sRNAs和相关蛋白复合物，其数量适合生化分析。使用一种新描述的基于RNA-seq的方法，称为伪尿嘧啶-seq，我们的目标是获得沙门氏菌伪尿嘧啶修饰的转录组全图谱。在不同细菌生长条件下，转录组伪尿嘧啶谱的变化也将被确定，并可能与靶mRNA或蛋白稳定性相关。我们将用新的RNA配体获得TruD（一种伪尿嘧啶合成酶，可以靶向许多mrna和sRNAs）的共晶结构，以便更好地理解在原子水平上的底物识别。此外，纯化的sRNAs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。我们希望这个项目能够开拓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.