Fig. 4. Temporal placement of modified nucleosides in tRNAPheGAA.
Cells were grown in unlabeled DMEM D0422 (supplemented with unlabeled uridine and adenine) for 7 days. At T = 0 the medium was exchanged to DMEM D0422 supplemented with labeled uridine and adenine. Cells were harvested after set time points. tRNAPhe was purified and analyzed by LC-MS/MS. Modifications are plotted next to their location in the D-, TΨC- or anticodon loop. Plotted on the y-axis is the abundance of modification in new transcripts normalized to the respective nucleoside originating from unlabeled medium before experiment initiation (T = 0). The experiment was done in n = 3 biol. replicates for time points 2, 4, and 48 h and in n = 6 biol. replicates for time points 0, 6, and 24 h. Symbols reflect the mean and error bars reflect standard deviation.