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

ABSTRACT: 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.

ORGANISM(S): Escherichia coli Schizosaccharomyces pombe

PROVIDER: GSE311025 | GEO | 2026/02/16

REPOSITORIES: GEO

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