Fig. 5
From: CO2-sensitive tRNA modification associated with human mitochondrial disease
In vitro reconstitution of t6A37 on mt-tRNAs and impairment of t6A37 by pathogenic point mutations associated with mitochondrial diseases. a In vitro formation of t6A37 on mt-tRNAThr transcripts with or without YRDC and OSGEPL1 in the presence of Thr, ATP, and bicarbonate. After the reaction, mt-tRNAThr was digested with RNase T1 and analyzed by LC/MS. XICs generated by integration of multiply-charged negative ions of A37-containing fragments of mt-tRNAThr harboring A37 (top) and t6A37 (bottom) (Supplementary Table 1). Intensity fractions (%) of modified or unmodified fragments are denoted. N.D., not detected. b In vitro formation of t6A37 on mt-tRNAIle transcript or native mt tRNAIle isolated from OSGEPL1 KO#1 cells. XICs generated by integration of multiply-charged negative ions of A37-containing fragments of mt-tRNAIle harboring A37 (top) and t6A37 (bottom) (Supplementary Table 1). Intensity fractions (%) of modified or unmodified fragments are denoted. c Efficiency of t6A37 formation in mt-tRNA mutants. The numbering of each mutant corresponds to that in Fig. 1b. Bars corresponding to severe and mild reduction in t6A37 formation are colored in blue and green, respectively. d G4296A in mt-tRNAIle (left panel) is a unique pathogenic mutation that promotes t6A37 formation. XICs show A37-containing fragments of WT and G4296A mutant mt-tRNAIle harboring A37 (black) and t6A37 (red). m/z values of each fragment are listed in Supplementary Table 1. e Impairment of t6A37 in mt-tRNAThr with A15923G mutation isolated from the myoblasts of a patient with MERRF-like symptoms. Anticodon stem-loop sequences of WT (left) and A15923G mutant (right) mt-tRNAThr. Positions of RNase T1 and RNase A digestion are indicated by arrowheads. m3C32 in the A15923G mutant is partially converted to C32 (Supplementary Fig. 5b). XICs generated by integration of multiply-charged negative ions of A37-containing fragments of mt-tRNAThr harboring A37 (upper panels) and t6A37 (lower panels) (Supplementary Table 1) from WT (left panels) and A15923G myoblasts (right panels). Intensity fractions (%) of modified or unmodified fragments are indicated
