Discovery and characterization of RNA modifications in a bacterial model pathogen

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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.