MjNep1 mis-methylates pseudouridine containing substrates corresponding to tRNA T-arms at position 55. (A) Chemical structure of N1-methyl pseudouridine. (B) Secondary structure and sequences of minimal substrates for MjNep1 derived from the small subunit rRNA helix 35 (Wurm et al. 2010). Nucleotides that are similar between these substrates and T-arm sequences of archaeal tRNAs are highlighted in red. (C) Secondary structure of H. volcanii tRNATrp with the modified nucleotide positions indicated and sequence similarities to MjNep1 substrates highlighted in red. The T-arm sequences and secondary structures of H. volcanii and M. jannaschii tRNATrp are virtually identical. (D) Methylation activity of MjNep1 toward 17 mer RNA substrates resembling the T-arm of M. jannaschii tRNATrp with pseudoruridines at position 54 (top), 55 (middle), or both (bottom) analyzed by MALDI mass spectrometry. Only the RNAs with pseudouridine at positions 55 or 54 and 55 are methylated after 5-min reaction time at 65°C. (E) Identification of the methylation site in the RNA substrate containing two pseudouridine residues at positions corresponding to Ψ54 and Ψ55 in full-length tRNAs by partial acidic hydrolysis coupled to MALDI mass spectrometry of the reaction product. Shown are the sections of the spectrum for two indicated 5′ fragments.
