RNA-methyltransferase activity of Mja_1640. (A) Methylation activity of Mja_1640 toward T-arm substrates containing no pseudouridine (top), pseudouridine at position 55 (second from top), pseudouridine at position 54 (second from bottom), or pseudouridine at positions 54 and 55 (bottom). In the last two cases a complete methylation is observed after a 5-min reaction time as indicated by the shift in the mass spectrum by 14 Da. (B) Identity of the modified nucleotide. HPLC chromatograms of nucleoside mixtures after digestion and dephosphorylation of the 17 mer RNA substrate containing pseudouridine at position 54. The HPLC chromatogram of the unreacted substrate RNA contains only peaks corresponding to Ψ, C, U, G, and A (red trace), whereas upon methylation by Mja_1640 the Ψ peak disappears, and a novel peak appears with a retention time typical for N1-methyl pseudouridine (black trace). A reference for commercially obtained N1-methyl pseudouridine is shown on top. (C) Mass spectra of the T-arm substrate containing two pseudouridines after methylation with Mja_1640 and limited acidic hydrolysis. Shown are the sections of the spectrum for two indicated 5′- fragments indicative of methylation at the pseudouridine corresponding to position 54 in full-length tRNAs. (D) Secondary structure for a 74 mer RNA substrate resembling unmodified M. jannaschii tRNATrp with a single pseudoruridine at position 54. HPLC traces for unreacted (red) and Mja_1640-treated 74 mer RNA after digestion and dephosphorylation and after a 1-h reaction time (black). A novel peak corresponding to N1-methyl pseudouridine appears following incubation with Mja_1640 and SAM.
