Project Details
Projekt
Identification and functional characterization of pseudouridine in mRNAs and non-coding RNAs of the bacterial human pathogen Campylobacter jejuni
Applicant
Professorin Dr. Cynthia Mira Sharma
Subject Area
Biological and Biomimetic Chemistry
Biochemistry
Metabolism, Biochemistry and Genetics of Microorganisms
Biochemistry
Metabolism, Biochemistry and Genetics of Microorganisms
Term
from 2015 to 2019
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 277446585
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. Modifications in bacterial mRNAs have not yet been reported. Here, we aim to study RNA modifications in Campylobacter jejuni, currently the most common cause of bacterial gastroenteritis in humans, with a focus on pseudouridine (PU). This universally-conserved modified RNA nucleoside is an isomer of uridine posttranscriptionally generated by pseudouridine synthases (PUS), and is the most abundant modification in tRNA and rRNA. Using Pseudo-seq, a novel method of global PU profiling based on deep sequencing of reverse transcription stops at chemically-modified PU sites, PU was recently reported in human and yeast mRNAs, yet its functions are still enigmatic. An increase in PU under stress conditions indicated it might modulate RNA stability, structure, or even coding potential, since artificially-introduced PU residues can mediate nonsense suppression in yeast. Using unbiased genomics approaches, we aim to globally profile PU in transcripts of the emerging food-borne pathogen C. jejuni and investigate the presence and function of this modification in bacterial mRNAs. Our previous RNA-seq-based transcriptome analysis revealed a compact transcriptional output and many regulatory RNAs in C. jejuni, indicating posttranscriptional regulation is an important layer of gene expression control. Using co-immunoprecipitation combined with RNA-seq (RIP-seq) to globally study RNA substrates of the tRNA-modifying PUS TruB, we could both enrich for tRNAs and identify several mRNAs as potential TruB substrates. Our first Pseudo-seq of C. jejuni wildtype (WT) RNA successfully detected PU in tRNA and rRNA and revealed several candidate mRNAs with potential PU sites. By combining RIP-seq of PUS enzymes with Pseudo-seq of WT and PUS mutant strains grown under routine and stress conditions, we will provide a global map of PU sites and PUS consensus motifs for C. jejuni. The PU sites will be validated by primer extension, in vitro binding studies of RNA targets and PUS enzymes, and detection of PU of in vitro-synthesized RNAs following treatment with purified PUS enzymes by thin-layer chromatography and mass spectrometry. Copper free click-chemistry will be used to attach biotin or fluorophores to PU in either in vitro-modified or total RNA to allow for labeling and purification of modified RNA. Using biochemical, molecular biology, and genetics methods, we will investigate the functions of PU in bacterial mRNAs. Potential changes in RNA stability, structure, and/or coding potential by PU for selected candidate mRNAs will be assessed. The study of PU in C. jejuni will provide insight into potential posttranscriptional regulation by RNA modifications and their function, also in eukaryotes.
DFG Programme
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