Yannick Noah Frommeyer (Hannover / DE), Nicolas Oswaldo Gomez (Brunswick / DE), Matthias Preusse (Brunswick / DE), Alejandro Arce Rodriguez (Brunswick / DE), Kerstin Neubauer (Brunswick / DE), Benedikt Kennepohl (Hannover / DE), Julius Friedrich Witte (Hannover / DE), Jelena Erdmann (Hannover / DE; Brunswick / DE), Andreas Pich (Hannover / DE), Heike Bähre (Hannover / DE), Daniel P. Depledge (Hannover / DE), Susanne Häußler (Hannover / DE; Brunswick / DE)
Introduction
Transfer RNAs (tRNAs) are crucial adapter molecules facilitating protein synthesis by decoding mRNA sequences. They harbor a diverse range of conserved chemical modifications, collectively known as the tRNA epitranscriptome, which play pivotal roles in maintaining translational accuracy and structural integrity. Perturbations in tRNA modification levels can disrupt protein homeostasis and impact bacterial virulence phenotypes on a global scale. [1], reviewed in [2].
Aim of the study
Here, we elucidated the biosynthesis of 5-carboxymethoxyuridine (xo5U) tRNA modifications in the opportunistic human pathogen P. aeruginosa. New insights into the nature and dynamics of post-transcriptional tRNA modification promise to expand our current understanding of gene regulation mechanisms shaping bacterial behavior with potential implications for antimicrobial therapy development.
Results
In the first part, we identified genes responsible for the biogenesis and characterized the epitranscriptomic landscape of xo5U modifications in P. aeruginosa tRNA using LC-MS/MS and direct tRNA nanopore-sequencing. Mutant strains lacking xo5U modifications exhibited altered virulence and secondary metabolite production, highlighting the significance of these modifications in bacterial physiology. Furthermore, we applied a multi-omics approach to unravel the molecular basis for xo5U tRNA modification-dependent translational regulation.
Key methods used in this study
- Targeted LC-MS/MS
- Direct tRNA nanopore-sequencing (nano-tRNAseq [3])
- Transcriptomics/Translatomics/Proteomics analyses
- Phenotypic characterization assays (Virulence, secondary metabolite quantification, bacterial growth monitoring)
References
[1] Y. H. Chionh et al., "TRNA-mediated codon-biased translation in mycobacterial hypoxic persistence," Nature Communications, doi: 10.1038/ncomms13302.
[2] V. de Crécy-Lagard et al., "Functions of Bacterial tRNA Modifications: From Ubiquity to Diversity," Trends in Microbiology, doi: 10.1016/j.tim.2020.06.010.
[3] M. C. Lucas et al., "Quantitative analysis of tRNA abundance and modifications by nanopore RNA sequencing.," Nature biotechnology, 2023, doi: 10.1038/s41587-023-01743-6.