Detection of queuosine and queuosine precursors in tRNAs by direct RNA sequencing
Queuosine (Q) is a complex tRNA modification found at position 34 of four tRNAs with a GUN anticodon, and it regulates the translational efficiency and fidelity of the respective codons that differ at the Wobble position. In bacteria, the biosynthesis of Q involves two precursors, preQ0 and preQ1, whereas eukaryotes directly obtain Q from bacterial sources. The study of queuosine has been challenging due to the limited availability of high-throughput methods for its detection and analysis. Here, we have employed direct RNA sequencing using nanopore technology to detect the modification of tRNAs with Q and Q precursors. These modifications were detected with high accuracy on synthetic tRNAs as well as on tRNAs extracted from Schizosaccharomyces pombe and Escherichia coli by comparing unmodified to modified tRNAs using the tool JACUSA2. Furthermore, we present an improved protocol for the alignment of raw sequence reads that gives high specificity and recall for tRNAs ex cellulo that, by nature, carry multiple modifications. Altogether, our results show that such 7-deazaguanine-derivatives are readily detectable using direct RNA sequencing. This advancement opens up new possibilities for investigating these modifications in native tRNAs, furthering our understanding of their biological function. Overall design: To investigate the detection of Q by direct RNA nanopore sequencing, in vitro-transcribed tRNAAsp, tRNAAsn, tRNAHis, and tRNATyr with or without Q-modification were subjected to direct RNA sequencing (nanopore). Total tRNA purified from S. pombe, either unmodified or Q-modified in vivo, was sequenced, and the experiment was conducted with three biological samples per condition. Additionally, total tRNA from E. coli wt, queA?, queF?, and tgt? was subject to sequencing to investigate the detection of Q and Q precursors preQ1 and preQ0. We then analyzed the error rates of each samples using data generated from nanopore sequencing using JACUSA2.