Quantitative analysis of tRNA abundance and modifications by nanopore RNA sequencing

Lucas, Morghan C.; Pryszcz, Leszek Piotr, 1985-; Medina, Rebeca; Milenkovic, Ivan; Camacho, Noelia; Marchand, Virginie; Motorin, Yuri; Ribas de Pouplana, Lluís; Novoa, Eva Maria

doi:http://dx.doi.org/10.1038/s41587-023-01743-6

Quantitative analysis of tRNA abundance and modifications by nanopore RNA sequencing

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Lucas MC, Pryszcz LP, Medina R, Milenkovic I, Camacho N, Marchand V, et al. Quantitative analysis of tRNA abundance and modifications by nanopore RNA sequencing. Nat Biotechnol. 2024 Jan;42(1):72-86. DOI: 10.1038/s41587-023-01743-6

Abstract

Transfer RNAs (tRNAs) play a central role in protein translation. Studying them has been difficult in part because a simple method to simultaneously quantify their abundance and chemical modifications is lacking. Here we introduce Nano-tRNAseq, a nanopore-based approach to sequence native tRNA populations that provides quantitative estimates of both tRNA abundances and modification dynamics in a single experiment. We show that default nanopore sequencing settings discard the vast majority of tRNA reads, leading to poor sequencing yields and biased representations of tRNA abundances based on their transcript length. Re-processing of raw nanopore current intensity signals leads to a 12-fold increase in the number of recovered tRNA reads and enables recapitulation of accurate tRNA abundances. We then apply Nano-tRNAseq to Saccharomyces cerevisiae tRNA populations, revealing crosstalks and interdependencies between different tRNA modification types within the same molecule and changes in tRNA populations in response to oxidative stress.