RNA modifications digest Summer 2025
Bimonthly newsletter: RNA modifications and related topics, mostly in bacteria. Summer 2025, first issue!
🧬 1 — tRNA Modifications
tRNA Modification Profiling Reveals Epitranscriptome Regulatory Networks in Pseudomonas aeruginosa
Jingjing Sun, Junzhou Wu, Yifeng Yuan, Leon Fan, Wei Lin Patrina Chua, et al.
DOI: https://doi.org/10.1093/nar/gkaf696
Developed a high-throughput LC–MS/MS platform to map tRNA modifications across 5,746 P. aeruginosa knockouts, yielding 200,000+ data points. Validated known and discovered novel tRNA-modifying enzymes, linking modification patterns to S-adenosylmethionine metabolism and stress pathways.
Key insight: Scalable workflow to link tRNA epitranscriptome dynamics with bacterial gene networks and translational control.
5-Methyluridine Is Ubiquitous in Pseudomonas aeruginosa tRNA and Modulates Antimicrobial Resistance and Virulence
Jurairat Chittrakanwong, Ruixi Chen, Junzhou Wu, Michael S. Demott, et al.
DOI: https://doi.org/10.1016/j.jmb.2025.169020
TrmA installs m⁵U54 in all tRNAs. Loss of TrmA boosts polymyxin resistance, increases T3SS gene expression, and elevates macrophage responses without altering growth or morphology.
Key insight: A conserved tRNA methylation directly tunes bacterial virulence and antibiotic resistance.
Are Bacterial Processes Dependent on Global Ribosome Pausing Affected by tRNA Modification Defects?
Valérie de Crécy-Lagard, Zeynep Baharoglu, Yifeng Yuan, Grégory Boël, et al.
DOI: https://doi.org/10.1016/j.jmb.2025.169107
Proposes that tRNA modification defects alter translation kinetics, disrupting motility, iron homeostasis, and leader peptide attenuation pathways. Some phenotypes result from single-modification loss, others from combined defects.
Key insight: Missing tRNA modifications can rewire bacterial physiology through translation speed control.
tRNA Hydroxylation Is an Epitranscriptomic Modulator of Metabolic States Affecting Pseudomonas aeruginosa Pathogenicity
Yannick N. Frommeyer, Nicolas O. Gomez, Matthias Preusse, Alejandro Arce-Rodriguez, et al.
DOI: https://doi.org/10.1093/nar/gkaf719
Loss of TrhPO-dependent xo⁵U₃₄ hydroxylation reroutes metabolism, increasing aromatic amino acids and phenazines, reducing virulence in infection models.
Key insight: A single wobble tRNA modification acts as a metabolic switch influencing pathogenicity.
Queuosine Is Incorporated into Precursor tRNA Before Splicing
Wei Guo, Igor Kaczmarczyk, Kevin Kopietz, Florian Flegler, et al.
DOI: https://doi.org/10.1038/s41467-025-62220-z
Shows queuosine modification occurs on intron-containing pre-tRNAᴛʸʳ before splicing, confirmed by cryo-EM and multi-species experiments.
Key insight: Challenges the dogma of modification occurring only after tRNA splicing.
Post-Transcriptional Regulation of the MiaA Prenyl Transferase by CsrA and CsrB in E. coli
Joseph I. Aubee, Kinlyn Williams, Alexandria Adigun, Olufolakemi Olusanya, et al.
DOI: https://doi.org/10.3390/ijms26136068
MiaA (i⁶A37 tRNA modification) is post-transcriptionally regulated by CsrA and CsrB. Affects decoding fidelity, stress response, and virulence gene expression.
Key insight: Global RNA regulators control tRNA modification enzymes for adaptive translation.
Key RNA-Binding Domains in the La Protein Establish tRNA Modification Levels in Trypanosoma brucei
Lankani Gunaratne, Henry Moore, Nicholas Albaum, Ananth Casius, et al.
DOI: https://doi.org/10.1093/nar/gkaf594
La protein domains control queuosine levels at Q₃₄ in multiple tRNAs, also affecting other modifications via enzyme interactions.
Key insight: RNA-binding proteins can coordinate multiple tRNA modification pathways.
Natural Human tRNAᴬˡᵃ Anticodon Variants Mistranslate the Genetic Code
Rasangi Tennakoon, Teija M. I. Bily, Farah Hasan, Kyle S. Hoffman, Patrick O'Donoghue
DOI: http://www.rnajournal.org/cgi/doi/10.1261/rna.080450.125
Rare tRNAᴬˡᵃ anticodon variants misread codons, causing alanine misincorporation and protein synthesis defects.
Key insight: Natural human tRNA variants can disrupt translation fidelity.
Coupling tRNA^Gly Gene Redundancy with S. aureus Cell Wall Integrity and Virulence
Adamantia Kouvela, Jose R. Jaramillo Ponce, Nikoleta Giarimoglou, Johana Chicher, et al.
DOI: https://doi.org/10.1093/nar/gkaf599
Deleting a proteinogenic tRNA^Gly gene impairs cell wall integrity, increases antibiotic sensitivity, and reduces biofilms without affecting growth.
Key insight: tRNA gene redundancy supports bacterial cell wall robustness.
Loss of s²U tRNA Modification Induces Antibiotic Tolerance in Yersinia pseudotuberculosis
Katherine L. Cotten, Abigail McShane, Peter C. Dedon, Thomas J. Begley, Kimberly M. Davis
DOI: https://doi.org/10.1101/2025.07.01.662621
Loss of 2-thiouridine increases tolerance to multiple antibiotics, linking tRNA sulfur modification to persistence phenotypes.
Key insight: A single tRNA sulfur modification can drive tolerance to diverse drugs.
🧪 2 — rRNA Modifications
Methylation of Cytidine 1407 Increases the Lifetimes of the A-Site States in E. coli 16S rRNA
Stefan Hilber, Alessandro Marotto, Christoph Mitteregger, Martin Tollinger, Christoph Kreutz
DOI: https://doi.org/10.1021/jacs.5c06523
C5-methylation of C1407 slows interconversion between ground and excited states of the 16S rRNA A-site loop by ~1.6×, stabilizing codon–anticodon recognition and improving translational fidelity.
Key insight: A single methyl group can act as a kinetic regulator of ribosome decoding accuracy.
First-in-Class Inhibitors Targeting Pathogen-Associated 16S rRNA Methyltransferase NpmA
Debayan Dey, Benjamin E. Deprez, Natalia Zelinskaya, Jose M. Castro, William M. Wuest, Graeme L. Conn
DOI: https://doi.org/10.1021/acsinfecdis.5c00297
Structure-guided design identified three small molecules binding a unique Y-shaped NpmA pocket on the 30S subunit, blocking m¹A1408 installation and restoring aminoglycoside efficacy.
Key insight: Exploiting methyltransferase–ribosome interfaces offers new antibiotic strategies.
Distant Ribose 2′-O-Methylation of 23S rRNA Helix 69 Regulates Capreomycin Binding
Suparno Nandi, Debayan Dey, Pooja Srinivas, Christine M. Dunham, Graeme L. Conn
DOI: https://doi.org/10.1093/nar/gkaf618
Methylation at C2144 triggers allosteric changes in helix 69, opening the capreomycin pocket and stabilizing antibiotic-binding conformation.
Key insight: Distant rRNA modifications can pre-order antibiotic sites for drug sensitivity.
Post-Transcriptional Modifications of Large Subunit rRNA Assembly Intermediates in DbpA-Inactive E. coli
Luis A. Gracia Mazuca, Jonathon E. Mohl, Samuel S. Cho, Eda Koculi
DOI: https://doi.org/10.1021/acs.biochem.5c00034
Loss of DbpA helicase activity stalls 50S assembly and reduces late-stage 2′-O-methylation, pseudouridylation, and base methylation in functional centers.
Key insight: RNA helicases enable proper rRNA modification during ribosome maturation.
Bacterial Ribosome Heterogeneity Facilitates Rapid Stress Response
Yi-Lin Shen, Lei Xu, Ying Zhou, Bang-Ce Ye
DOI: https://doi.org/10.1128/jb.00058-25
Highlights chemical modification (methylation, pseudouridylation) as a driver of ribosome heterogeneity, tuning translation under stress and impacting antibiotic resistance.
Key insight: Ribosome diversity via rRNA modification is a conserved bacterial adaptation strategy.
Stress-Dependent m⁵C rRNA Dynamics in E. coli
Leonardo Vásquez-Camus, Sebastian Riquelme-Barrios, Kirsten Jung
DOI: https://doi.org/10.1101/2025.06.13.659538
m⁵C-Rol-LAMP reveals rRNA methylation shifts during heat shock and oxidative stress, showing dynamic regulation of specific sites.
Key insight: rRNA methylation is a flexible layer of translational control.
🧬💬 3 — mRNA
A-to-I mRNA Editing in Bacteria Affects Protein Sequence and Function
Liron Didi, Ofir Fargeon, Liam Aspit, Eyal Elias, et al.
DOI: https://doi.org/10.1093/nar/gkaf584
A-to-I editing in E. coli hokB toxin mRNA changes tyrosine to cysteine, enabling disulfide bond formation and boosting toxin activity. Conserved in pathogens.
Key insight: First example of bacterial mRNA editing altering protein chemistry.
🧪🩺 4 — Epitranscriptomics in Disease & Diagnostics
RNA N-Glycosylation Enables Immune Evasion and Efferocytosis
Vincent R. Graziano, Jennifer Porat, Marie Dominique Ah Kioon, et al.
DOI: https://doi.org/10.1038/s41586-025-09310-6
Glycosylation of acp³U in small RNAs masks them from immune sensing; removing glycans triggers type I interferon responses.
Key insight: RNA glycosylation acts as a self-tolerance shield.
Modifications of Microbiome-Derived cfRNA Discriminate Colorectal Cancer
Cheng-Wei Ju, Ruitu Lyu, Han Li, Jiangbo Wei, et al.
DOI: https://doi.org/10.1038/s41587-025-02731-8
LIME-seq maps cfRNA modifications (m¹A, m⁵C, Ψ, m¹G, i⁶A) from human and microbiome sources; patterns distinguish cancer from controls.
Key insight: cfRNA epitranscriptomic signatures offer high-accuracy liquid biopsy biomarkers.
⚙️💊 5 — Ribosome Structure, Function & Antibiotic Interactions
Extensive Natural Variation in Bacterial Ribosomal Drug-Binding Sites
Ekemezie et al.
DOI: https://doi.org/10.1016/j.celrep.2025.115878
Evolutionary analysis shows widespread natural variation in rRNA antibiotic-binding residues, affecting intrinsic drug resistance/sensitivity.
Key insight: Drug-binding site diversity enables lineage-specific antibiotic strategies.
🧫🦠 6 — Miscellaneous & Host–Pathogen RNA Biology
Phage tRNAs: Decoding the Enigma
Daan F. van den Berg, Stan J.J. Brouns
DOI: https://doi.org/10.1016/j.tim.2025.05.009
Phage-encoded tRNAs aid codon optimization, evade host defenses, and often carry their own modifications.
Key insight: Phages actively control tRNA modification to fine-tune infection.
Ribonuclease Activity Undermines Immune Sensing of Naked exRNA
Mauricio Castellano, Valentina Blanco, Marco Li Calzi, et al.
DOI: https://doi.org/10.1016/j.xgen.2025.100874
RNase inhibition unmasks immunostimulatory potential of naked exRNA, triggering TLR and cytosolic sensor activation.
Key insight: RNase activity normally hides potent immune signals from extracellular RNA.
A Scalable Gut Epithelial Organoid Model Maps Shigella flexneri Colonization
Maria Letizia Di Martino, Laura Jenniches, Anjeela Bhetwal, et al.
DOI: https://doi.org/10.1038/s41588-025-02218-x
Organoid + TraDIS screen finds >100 chromosomal genes key for epithelial colonization; tRNA-modifying enzymes (MnmE/G) regulate T3SS expression.
Key insight: Post-transcriptional tRNA modification shapes bacterial virulence programs.
Escherichia coli with a 57-Codon Genetic Code
Wesley E. Robertson, Fabian B. H. Rehm, Martin Spinck, et al.
DOI: https://doi.org/10.1126/science.ady4368
Synthetic E. coli genome removes 7 codons; strain viable but slower growing.
Key insight: Recoded bacteria open routes for virus resistance and novel translation chemistries.
Extracellular Exosomal RNAs Are Glyco-Modified
Sunny Sharma, Xinfu Jiao, Jun Yang, Kelvin Y. Kwan, Megerditch Kiledjian
DOI: https://doi.org/10.1038/s41556-025-01682-1
Exosomal glycoRNAs resist degradation, load selectively into vesicles, and transfer between cells.
Key insight: RNA glycosylation may control selective RNA export and intercellular signaling.