CRG and IMMAGINA Develop tRIBO-seq Method to Track Ribosome tRNAs in Vitro

Researchers at the Centre for Genomic Regulation in Barcelona, in collaboration with the Italian company IMMAGINA Biotechnology, have introduced a nanopore-based sequencing method named tRIBO-seq to isolate and profile transfer RNAs actively involved in protein translation, according to a study published in Nature Methods doi.orgNature Methodsdoi.orgOpen source ↗. Conventional workflows require roughly a week of cell cultivation along with specialized equipment, whereas the reported approach uses a single dish of cells and takes about a day and a half to deliver results from a single experiment doi.orgNature Methodsdoi.orgOpen source ↗.

Transfer RNAs deliver specific amino acids to translating ribosomes based on codons within messenger RNA. Standard sequencing protocols measure the total tRNA content across the entire cell, which does not always reflect the active pool. The tRIBO-seq method isolates translating ribosomes from cell cultures and directly reads individual ribosome-bound tRNAs, recording abundance, chemical modifications, and fragmentation profiles in one run doi.orgNature Methodsdoi.orgOpen source ↗.

Cellular Stress Impacts

Using tRIBO-seq in cultured cells, the authors observed that active ribosome-associated tRNAs frequently diverge from the total tRNA pool when cells undergo stress doi.orgNature Methodsdoi.orgOpen source ↗. For example, leucine starvation caused specific tRNAs to accumulate on the ribosome rather than disappear doi.orgNature Methodsdoi.orgOpen source ↗. The data also revealed distinct stress-dependent dynamics: viral infection altered ribo-tRNA quantities, methionine starvation triggered extensive loss of methyl modifications, and sodium arsenite exposure caused selective fragmentation in specific subsets of ribo-tRNAs without shifting overall quantities or modification profiles doi.orgNature Methodsdoi.orgOpen source ↗.

Implications for Engineered tRNAs

Roughly one in 10 genetic disease mutations creates a premature stop codon that aborts protein synthesis, and engineered suppressor tRNAs are being investigated in cell lines and mouse models to restore full-length proteins without editing patient DNA doi.orgNature Methodsdoi.orgOpen source ↗. By determining whether designed molecules actually enter functioning ribosomes in adequate quantities, the authors reported that the new sequencing pipeline could help screen candidate tRNA therapeutics for genetic conditions doi.orgNature Methodsdoi.orgOpen source ↗.