Chemical manipulation of m<sup>1</sup>A mediates its detection in human tRNA
Kinga Pajdzik, Ruitu Lyu, Xiaoyang Dou, Chang Ye, Li-Sheng Zhang, Qing Dai, Chuan He
Abstract
N 1 -methyl adenosine (m 1 A) is a widespread RNA modification present in tRNA, rRNA, and mRNA. m 1 A modification sites in tRNAs are evolutionarily conserved and its formation on tRNA is catalyzed by methyltransferase TRMT61A and TRMT6 complex. m 1 A promotes translation initiation and elongation. Due to its positive charge under physiological conditions, m 1 A can notably modulate RNA structure. It also blocks Watson–Crick–Franklin base-pairing and causes mutation and truncation during reverse transcription. Several misincorporation-based high-throughput sequencing methods have been developed to sequence m 1 A. In this study, we introduce a reduction-based m 1 A sequencing (red-m 1 A-seq). We report that NaBH 4 reduction of m 1 A can improve the mutation and readthrough rates using commercially available RT enzymes to give a better positive signature, while alkaline-catalyzed Dimroth rearrangement can efficiently convert m 1 A to m 6 A to provide good controls, allowing the detection of m 1 A with higher sensitivity and accuracy. We applied red-m 1 A-seq to sequence human small RNA, and we not only detected all the previously reported tRNA m 1 A sites, but also new m 1 A sites in mt-tRNA Asn-GTT and 5.8S rRNA.