CRG's tRIBO-seq nanopore method reads tRNAs on active ribosomes and shows how stress changes them

Researchers at the Centre for Genomic Regulation (CRG) in Barcelona, together with the Italian company IMMAGINA Biotechnology, developed tRIBO-seq, a nanopore-based method that selectively captures transfer RNAs (tRNAs) from actively translating ribosomes, according to a paper in Nature Methods doi.orgNature Methodsdoi.orgOpen source ↗. From a single experiment the method returns tRNA abundance, chemical modifications and fragmentation doi.orgNature Methodsdoi.orgOpen source ↗. The study was led by ICREA Research Professor Eva Novoa; the first author is Mie Monti.

Why ribosome-associated tRNAs matter

During translation, tRNAs deliver specific amino acids to the ribosome in line with the sequence encoded by the messenger RNA doi.orgNature Methodsdoi.orgOpen source ↗. Their contribution to the modulation of translation remains poorly understood, mainly because there were no methods to characterize tRNA abundance and modifications from actively translating ribosomes doi.orgNature Methodsdoi.orgOpen source ↗.

Until now, tRNAs were usually studied by measuring all of them in a cell at once, on the assumption that this reflects the molecules doing the work. Monti said the new results show this is not necessarily the case, particularly under stress.

The method fishes out ribosomes that are busy making proteins and then reads the tRNAs inside one by one by nanopore sequencing. According to the CRG, it needs a single dish of cells and about a day and a half, while conventional methods require about a week and specialist equipment.

What changes under stress

Using tRIBO-seq, the authors found that ribosome-associated tRNAs (ribo-tRNAs), but often not total tRNAs, are significantly altered under stress doi.orgNature Methodsdoi.orgOpen source ↗. The changes depended strongly on the type of stress doi.orgNature Methodsdoi.orgOpen source ↗.

Viral infection and leucine deprivation changed ribo-tRNA abundances doi.orgNature Methodsdoi.orgOpen source ↗. In leucine-starved cells the team saw tRNAs accumulating on the ribosome, although a loss of tRNAs had been expected, Monti said. Methionine starvation caused a dramatic loss of methyl-based tRNA modifications doi.orgNature Methodsdoi.orgOpen source ↗.

Arsenite exposure altered neither tRNA abundances nor modification patterns. Instead, it caused major fragmentation in selected subsets of ribo-tRNAs doi.orgNature Methodsdoi.orgOpen source ↗.

The authors describe tRIBO-seq as a robust and reproducible approach for mapping the tRNA landscape, covering abundance, modification and fragmentation in both total and actively translating tRNA populations doi.orgNature Methodsdoi.orgOpen source ↗.