NGS-based analysis of i6A and ms2i6A modification levels on tRNATyr in Schizosaccharomyces pombe and Escherichia coli

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. Previous studies have shown that the Q34 modification enhances m5C38 formation in tRNAAsp by DNMT2 in Schizosaccharomyces pombe, an evolutionarily conserved phenomenon also observed in other organisms. Notably, in tRNATyr, the relative positions of Q34 and i6A (in S. pombe) or ms2i6A37 (in Escherichia coli) within the anticodon loop mirror the spatial relationship between Q34 and m5C38 in tRNAAsp. This structural similarity prompted us to investigate whether Q34 and i6A/ms2i6A37 might participate in a comparable modification circuit, where the presence of abasence of Q34 influences the formation of i6A or ms2i6A. To address this question, we adapted an approach in which tRNATyr molecules were reverse-transcribed using an RT-active DNA polymerase variant (RT-KTq I614Y), followed by PCR amplification and next genetation sequencing (NGS). This method generates higher error rates at i6A/ms2i6A37-modified positions compared with canonical A37 residues, enabling sensitive detection of these modifications. We furthur validated the approach using calibration samples created by mixing tRNA containing or lacking i6A/ms2i6A37, establishing a quantitative relationship between RT-derived error rates and the relative abundance of these modification. Overall design: For E. coli, ms2i6A37 levels in tRNATyr were analyzed, including wild-type BW25113 (containing Q34 in tRNATyr), ?tgt (containing G34), ?queA (containing preQ1?34), ?queF (containing preQ0?34), ?miaA (containing Q34 and unmodified A37, used as a negative control), and ?miaA ?tgt (containing G34 and unmodified A37, also used as a negative control). All E. coli strains were cultivated at 37°C or 42°C to mid-log phase before RNA extraction. Calibration samples were generated by mixing small RNAs from the ?miaA strain, which lacks ms2i6A, with small RNAs from the wild-type strain, which contains ms2i6A at an unknown absolute level. These mixtures, consisting of 12.5%, 25%, 50%, and 75% wild-type RNA, were used to establish a relationship between RT-derived error rates and relative modification abundance. For Schizosaccharomyces pombe, i6A37 levels in tRNATyr were examined in wild-type cells grown with queuine supplementation, resulting in Q34-containing tRNATyr, and without queuine supplementation, producing G34-containing tRNATyr. In addition, tit1? mutant cells were analyzed under the same supplementation conditions; tit1? produces tRNATyr containing Q34 and unmodified A37 when supplemented with queuine, and G34 with unmodified A37 when cultivated without queuine. These tit1? samples served as negative controls for i6A37 detection. For calibration, mixtures of RNA isolated from wild type cells and from the tit1? strain grown without queuine supplementation were prepared, enabling the concersion of the obserced error rates into the relative i6A37 modificaton level with respect to the wild type.