Protein production in cells depends on transfer RNAs, or tRNAs, which deliver amino acids to the ribosome based on instructions encoded in messenger RNA. While tRNAs are essential for translation, it has been difficult to measure how they behave during active protein synthesis, especially under different cellular conditions.

Researchers at the Centre for Genomic Regulation in Spain have developed a new method called tRIBO-seq to address this challenge. This technique uses nanopore sequencing to directly analyze tRNAs that are actively engaged with ribosomes, providing a detailed view of their abundance, chemical modifications, and fragmentation patterns.

Traditional approaches often measure total tRNA levels in a cell, but this does not always reflect what is happening during translation. By focusing specifically on ribosome associated tRNAs, the researchers were able to capture a more accurate picture of how protein synthesis is regulated in real time.

Experimental design and benchmarking of tRIBO-seq

(A) Schematic overview of the tRIBO-seq workflow. Translationally active ribosomes are isolated from cell lysates pre-treated with cycloheximide (CHX) to preserve ribosome-mRNA-tRNA complexes. Following nuclease digestion, ribosomes are purified using the RiboLace probe (‘RsP’) followed by RNA elution. The resulting RNA is used for obtaining ribo-tRNAs and mRNA footprints (Ribo-mRNAs), which can be sequenced using Ribo-seq. (B) Schematic representation of the experimental design: total tRNAs isolated from whole-cell lysates (total-tRNAs, captured using Nano-tRNAseq), and ribosome-associated tRNAs obtained from pulldown fractions (Ribo-tRNAs captured using tRIBO-seq). (C) Left: Principal component analysis (PCA) of tRNA abundances reveals clear separation between total and ribosome-associated tRNAs. Right: Volcano plot showing differential tRNA abundance when comparing ribo-tRNAs and total-tRNAs from HEK293T cell cultures (n = 2 independent biological replicates per condition). Significance was defined as p < 0.05 with an absolute log2 fold-change ≥ 1. Full-length reads were used for pairwise analysis (see also Table S2). (D) Scatterplots depicting the replicability of tRNA abundances between independent biological replicates of HEK293T cultures, for ribo-tRNAs (left) and total-tRNAs (middle). Right: comparison between ribo-tRNA and total-tRNAs. 

Using tRIBO-seq, the team examined how cells respond to different types of stress. They found that tRNA behavior changes depending on the stress condition. For example, viral infection and nutrient deprivation altered the abundance of specific ribosome associated tRNAs, while methionine starvation led to a loss of certain chemical modifications. In contrast, arsenite exposure caused significant fragmentation of selected tRNAs without changing their overall levels or modification patterns.

These findings highlight that tRNAs are not simply passive carriers of amino acids. Instead, they play an active role in regulating translation, with their abundance, structure, and modifications adapting to different cellular environments.

By combining nanopore sequencing with a targeted approach to capture active tRNAs, tRIBO-seq provides a powerful tool for studying the dynamics of protein synthesis. The method also opens new opportunities to explore how translation is altered in disease, stress responses, and therapeutic contexts.

As interest in RNA biology continues to grow, techniques that complement RNA sequencing by focusing on translation will be important for understanding how gene expression is controlled at multiple levels.

Availability – All code used in this work is available at: https://github.com/novoalab/tRIBO-seq