Selective profiling of translationally active tRNAs and their dynamics under stress

ABSTRACT:

During translation, transfer RNAs (tRNAs) deliver specific amino acids to the ribosome in a coordinated manner with the sequence encoded by the mRNA. Despite their central role in protein synthesis, the precise contribution of tRNAs to the modulation of translation remains poorly understood, primarily due to lack of methods to characterise tRNA abundances and their modifications from actively translating ribosomes. Here we develop RiboNano-tRNAseq, a novel method to quantify native ribosome-associated tRNAs (ribo-tRNAs) from actively translating ribosomes, achieved by coupling tag-free pulldown of active ribosomes with native tRNA nanopore sequencing. Applying RiboNano-tRNAseq, we investigate how the ribosome-associated tRNAome varies under diverse stress conditions. Our work reveals highly tailored tRNA responses depending on the stress type. For example, under leucine starvation, we observe enrichment of a subset of tRNALeu isoacceptors in ribo-tRNAs. By contrast, methionine starvation leads to a decrease in methyl-based tRNA modifications, such  as m1A or m2,2G, reflecting the decreased availability of the methyl donor S-adenosylmethionine (SAM). Strikingly, arsenite starvation does not affect tRNA modification profiles, but instead causes a drastic fragmentation of tRNAs, predominantly within ribosome-associated tRNAs. Altogether, our work findings demonstrate that RiboNano-tRNAseq enables comprehensive characterization of the tRNAome and its dynamics by simultaneously capturing tRNA abundance, modification and fragmentation patterns, both in total and actively translating tRNA pools.

INSTRUMENT(S): Liquid Chromatography MS - positive - reverse phase

PROVIDER: MTBLS12806 | MetaboLights | 2026-06-25

REPOSITORIES: MetaboLights