Discovery and characterization of RNA modifications in a bacterial model pathogen

细菌模型系统至关重要地告知我们的理解的种类和范围的天然rRNA和tRNA修饰以及同源修饰酶。然而，与真核生物中的发现相反，mRNA和非编码RNA的功能相关修饰在细菌中仍然未知。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技术应用于假尿苷激酶的初步研究表明，这些酶可以在体内结合过多的mRNA和sRNA种类。此外，我们还开发了一种基于RNA的亲和层析方法，以纯化sRNA和相关的蛋白质复合物，其数量适合于生化分析。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地图。将类似地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，将通过LC-MS确定纯化sRNA上的可能修饰，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，我们将研究与纯化的sRNA相关的蛋白质。我们希望这个项目开拓了一个新的理解的范围内的功能性RNA修饰的重要组肠道病原体。

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.