Translatomics for tRNA studies
Recent Publications Harnessing the Power of Translatomics
Every week we provide a digest of a small number of recent interesting papers in the field of translatomics.
In this week’s Sunday papers,
- Pierce et al. find that prime editing can be used for generating sup-tRNAs that can efficiently read through the disease-causing PTC.
- Kochavi et al. find that RNA004 chemistry of Oxford Nanopore Technologies is capable of reliably assessing the tRNA abundances and modifications.
- White et al. introduces Oxford Nanopore sequencing of intact aminoacylated tRNAs.
Prime editing-installed suppressor tRNAs for disease-agnostic genome editing
Nature, 2025.
Pierce, S.E., Erwood, S., Oye, K., An, M., Krasnow, N., Zhang, E., Raguram, A., Seelig, D., Osborn, M.J. and Liu, D.R.
Nonsense mutations account for roughly 24% of pathogenic alleles in the ClinVar database. Although suppressor tRNAs (sup-tRNAs) can treat diseases caused by premature stop codons, current approaches require lifelong delivery and yield only modest benefits. To address these limitations, the authors developed a disease-agnostic strategy: Prime Editing-based tRNA Rescue Technology (PERT) – which permanently converts endogenous tRNAs into optimized sup-tRNAs.
By screening thousands of variants across all 418 human tRNAs, the researchers identified a subset with the strongest TAG-targeting suppression potential. In human cell models carrying nonsense mutations that cause Batten disease, Tay-Sachs disease, and Niemann-Pick type C1, installation of a single optimized sup-tRNA using the same prime editor restored 20–70% of normal enzyme activity. Additional experiments confirmed that these engineered sup-tRNAs efficiently read through most clinically relevant TAG premature termination codons listed in ClinVar.
The team further validated PERT in vivo by co-delivering the editing machinery with a GFP reporter harboring a nonsense mutation. In healthy mice and in a Hurler syndrome model in which a TAG mutation disrupts the IDUA gene, treatment restored ~25% of GFP expression and achieved ~6% of normal IDUA activity, sufficient to produce near-complete rescue of disease pathology. A major advantage of PERT is its minimal impact on global gene regulation. RNA-seq, qPCR, mass spectrometry, and differential expression analyses showed that converting an endogenous tRNA into a sup-tRNA did not significantly alter transcriptome or proteome profiles (apart from the expected rescue of the reporter), or disrupt overall tRNA abundances.
Exploiting nanopore sequencing advances for tRNA sequencing of human cancer model
NAR Cancer, 2025.
Kochavi, A., Velds, A., Suzuki, M., Akichika, S., Suzuki, T., Beijersbergen, R.L. and Agami, R.
The authors evaluated a novel nanopore-based tRNA sequencing method using the new Oxford Nanopore RNA004 chemistry combined with updated Dorado base-calling to chart tRNA abundance and modifications in human cancer models. They found that this platform reliably detects variation in tRNA expression across different cancer cell lines and under external stress, achieving high reproducibility between biological replicates.
Beyond quantifying tRNA levels, the study demonstrated that analysis of base-calling error rates can reveal known tRNA modifications, as exemplified by detection of the cancer-associated wybutosine modification on tRNA-Phe, and that the updated modification-calling feature of the pipeline can systematically predict common tRNA modifications.
These advances represent a major step forward: by enabling simultaneous measurement of both tRNA abundance and modification status, RNA004 nanopore tRNA-seq holds the potential to illuminate how the tRNAome, the full complement of tRNAs and their chemical modifications, shapes translational regulation in cancer. The authors also note limitations and challenges with modification calling, but argue that these technical hurdles can be addressed, opening a path for broader application.
Overall, this work provides compelling evidence that deep, modification-aware tRNA-seq can transform our understanding of translational control in human disease, offering a scalable method to map tRNA landscapes and their translational consequences in cancer and beyond.
Nanopore sequencing of intact aminoacylated tRNAs
Nature Communications, 2025.
White, L.K., Radakovic, A., Sajek, M.P., Dobson, K., Riemondy, K.A., Del Pozo, S., Szostak, J.W. and Hesselberth, J.R.
The authors introduce a method called aa-tRNA-seq, which for the first time enables simultaneous measurement of tRNA sequence, chemical modifications, and aminoacylation (charge) levels in a single high-throughput run. The core innovation is a chemical ligation workflow that attaches an adaptor around the amino acid on charged tRNAs, followed by nanopore direct-RNA sequencing.
Using synthetic tRNAs charged with each of the 20 standard amino acids, the researchers demonstrated that each aminoacylated tRNA produces distinct distortions in the nanopore current signal (reduced ionic current and increased dwell time at a defined region), allowing the construction of a machine-learning (RNN) model capable of accurately distinguishing charged vs. uncharged tRNAs and inferring the identity of the attached amino acid.
Applying aa-tRNA-seq to budding yeast, they observed changes in tRNA aminoacylation and abundance under conditions of nutrient limitation and tRNA hypomodification, confirming known effects and uncovering previously unrecognized regulation.
Thus, aa-tRNA-seq represents a major advance for translation biology: by capturing tRNA identity, modification status, and charging in a single read, the method opens the door to studying how environmental conditions, stress, or modification‐enzyme loss influence the tRNA aminoacylation landscape, and consequently the efficiency and fidelity of protein synthesis.
