Nanopore t(our)RNAment - deciphering tRNA modification landscapes and circuits

• Applicants: Professor Dr. Christoph Dieterich; Professorin Dr. Ann Elizabeth Ehrenhofer-Murray
• Subject Area: Biochemistry ; Bioinformatics and Theoretical Biology ; Cell Biology
• Term: since 2026
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 572737469

Transfer RNAs (tRNAs) play a central role in protein translation by decoding mRNA codons and delivering corresponding amino acids to the ribosome. Their function relies heavily on chemical modifications, which influence tRNA folding, stability, aminoacylation, and codon–anticodon recognition. Over 150 modifications have been identified, with each tRNA typically modified at 5–15 positions. These modifications vary in complexity—from simple chemical changes (e.g., methylation, deamination) to elaborate enzymatic pathways (e.g., queuosine, mcm5S2U). Most are evolutionarily conserved, underscoring their importance for living organisms. Diseases caused by defective tRNA modification are called “tRNA modopathies”. Studying tRNA modifications and the enzymes responsible for them is vital for understanding “tRNA modopathies”. To date, 54 such enzymes and their partners have been linked to human diseases, including neurological disorders, kidney dysfunction, mitochondrial diseases, and cancer. For example, mitochondrial tRNA mutations contribute to MELAS and MERRF syndromes. Mutations in NSUN2, a tRNA methyltransferase for m⁵C, cause intellectual disability and microcephaly. Additionally, modified suppressor tRNAs hold therapeutic potential for correcting pathogenic nonsense mutations. Thus, tRNA modification profiling is critical for understanding and treating various human diseases. The goal of this project is to obtain a tRNA transcriptome-wide view of the tRNA modification landscape in the experimental organisms Schizosaccharomyces pombe and in human cells HCT116 and hIPSC. Methodologically, we will obtain this information by performing direct RNA sequencing of tRNAs ex cellulo using Oxford Nanopore Technology (ONT). We and others have shown that beyond standard RNA bases (G, A, U, C), also modified bases/ nucleosides can be reliably detected by Nanopore sequencing. In the absence of dedicated modification callers, this is done by employing ONT base-calling software using current traces. Comparison of error readings from tRNAs in the modified versus the unmodified state, or anomaly detection in signal traces, pinpoints tRNA positions that have altered properties in the two samples (KO vs. WT), which is interpreted as a change in tRNA modification. This, in principle, detects the modification itself, but also cross-modification cross-talks. Hence, we will employ this strategy on a tRNA transcriptome-wide scale to identify tRNA modification circuits. We will look at the entire tRNA complement, nuclear and mitochondrial, as well as unspliced (i.e. intron-containing) tRNAs. Given the relatively small size of the S. pombe genome, studies in yeast provide an effective platform for investigating tRNA modifications, serving here as a testbed for approaches that will be extended to human cells, a strategy that has proven successful across many areas of genetics and molecular biology.

• DFG Programme: Research Grants