Identification and functional characterization of pseudouridine in mRNAs and non-coding RNAs of the bacterial human pathogen Campylobacter jejuni
Biochemistry
Metabolism, Biochemistry and Genetics of Microorganisms
Final Report Abstract
More than 100 different RNA modifications have been described in all kingdoms of life. While most modifications are found in abundant housekeeping RNAs such as rRNA, tRNA, and snRNAs, recent genome-wide approaches have also revealed modifications in eukaryotic and archaeal mRNAs. Here, we aimed to study RNA modifications in the pathogenic Epsilonproteobacterium Campylobacter jejuni, currently the most common cause of bacterial gastroenteritis in humans, with a focus on pseudouridine (Ψ). This universally conserved modified RNA nucleoside is a uridine isomer that is posttranscriptionally generated by pseudouridine synthases (PUS) and is the most abundant modification in tRNAs and rRNAs. Using Pseudo-seq, a novel method of global Ψ profiling based on deep sequencing of reverse transcription stops at chemically modified Ψ sites, this modification was recently reported to be present in human and yeast mRNAs, yet its functions in mRNAs are still unclear. Even less is known about Ψ in bacterial RNAs. In addition, there are hints that PUS enzymes could have additional functions beyond modification of RNA and, e.g., act as RNA chaperones and impact gene expression. To better understand the potential roles of Ψ and PUS enzymes in bacteria, we first performed a transcriptome-wide mapping of Ψ using Pseudo-seq in C. jejuni and to study conserved functions of PUS enzymes, also in the related gastric pathogen H. pylori. This approach allowed for the first time a genome-wide identification and mapping of Ψ sites in tRNAs and rRNAs of the two pathogens. Moreover, by comparing the Ψ profiles in Pseudoseq data of wild-type and ΔPUS strains, we further validated the tRNA substrates of these PUS enzymes. Several sites were further confirmed using primer extension analysis in CMC assays. From our global Ψ analyses emerged that, in C. jejuni, tRNAGlu is modified by the PUS enzyme TruD at position 13. This tRNA is only present in one copy in the C. jejuni genome. Intriguingly, deletion of truD in this pathogen showed a severe growth defect compared to its parental wild-type strain. Complementation with a catalytically inactive CjTruD (D85N) recovered the ΔtruD growth defect, indicating that TruD might have an additional function in C. jejuni. Moreover, complementation of ΔtruD with TruD from H. pylori restored the growth phenotype, although HpTruD was not able to modify the C. jejuni tRNAGlu. This further indicates a function of TruD beyond its role in generation of Ψ. In our ongoing work we are examining gene expression changes in truD mutant strains using RNA-seq and ribosome profiling analysis and aim to identify TruD targets that mediate the growth phenotype in C. jejuni. Collectively, our study allowed to map Ψ for the first time in two pathogenic Epsilonproteobacteria and our findings suggest an emerging role of the PUS enzyme TruD that is independent of its tRNA modifying function.
