Site-specific m¹A Installation by the MTR1 Ribozyme and Modulation of Mitochondrial mRNA Translation in vitro

RNA modifications play essential roles in regulating RNA structure, stability, and translation. Among these, m¹A is a functionally important modification that influences Watson-Crick base pairing and translational fidelity. However, approaches for site-specific installation and func tional interrogation of m¹A in RNA remain limited. Here, we characterize the catalytic properties of the methyltransferase ribozyme MTR1 with a focus on donor compatibility, sequence-dependent kinetics, and activity on structured RNA substrates. We show that MTR1 catalyzes alkyl transfer from O⁶-methylguanine- derived donors to adenosine residues in RNA and accommodates fluorophore-conjugated donors for covalent RNA labeling. Target-site mutation and reverse transcription-based analyses further validate the adenosine-dependent specificity of MTR1-mediated transfer. Comparative kinetic analyses under mildly acidic (pH 6.0) and alkaline (pH 7.5) conditions reveal that pH is a key determinant of MTR1 catalytic efficiency across sequence contexts. Although local sequence context influences reaction rates, pH 6.0 substantially enhances catalytic activity across tested substrates, enabling efficient modification of sequences with limited activity under pH 7.5 conditions. These findings indicate that sequence preference primarily affects reaction kinetics rather than modification capability under mildly acidic conditions, expanding the practical sequence compatibility of MTR1-mediated RNA modification. We further demonstrate that MTR1 enables site-specific m¹A modification in structured mitochondrial mRNAs, including mouse COX2 and human ATP8 transcripts, and that these modifications reduce translation in a cell-free mitochondrial system. Together, these results define the donor compatibility, sequence-dependent kinetics, and substrate scope of MTR1 and provide insights into ribozyme-mediated RNA alkylation chemistry.