Fig. 4

Characterization of QTGAL and QTMAN in human culture cells (A and B) Complete loss of galQ (A) or manQ (B) in cytoplasmic tRNATyr or tRNAAsp isolated from QTGAL KO or QTMAN KO cells, respectively. XICs show Q-containing fragments (top) and galQ- or manQ-containing fragments (bottom) of tRNATyr or tRNAAsp isolated from WT, KO#1, and KO#2. Judging from the peak intensities, WT tRNATyr has 98% galQ34 and 2% Q34, while WT tRNAAsp is fully modified by manQ34. (C) Loss of galQ and manQ in QTGAL KO and QTMAN KO cells. XICs show proton adducts of Q (upper) and galQ or manQ (lower) in total nucleosides from QTGAL KO#1 or QTMAN KO#1. (D) Northern blotting of cytoplasmic tRNAs in the KO cells. Relative steady-state levels of cytoplasmic tRNATyr (left) and tRNAAsp (right) in WT HEK293T, QTGAL KO cells, or QTMAN KO cells, normalized against a loading control (5S rRNA). Each data point plotted on the bar graph shows mean ± SD (n = 3). (E) Subcellular localization of QTGAL and QTMAN. Immunofluorescence detection of the N-terminal FLAG-tagged QTGAL (upper) and QTMAN (lower) transiently expressed in HeLa cells. The green signal shows immunofluorescence detected by anti-FLAG antibody (left). Nuclei (blue) are stained by DAPI (middle). Merged images are shown on the right. Scale bars, 10 μm. (F) Nucleoside analysis of Q, galQ, and manQ in total RNA from D. rerio, C. elegans, and D. melanogaster. See also Figure S2.