Fig. 6. QTRT1 and QTRT2 are necessary for Q34 in mt-tRNAs.

a Gene structure of the human QTRT1 (upper) and QTRT2 (lower) with mutation sites introduced by the CRISPR/Cas9 system. The target sequence of the single-guide RNA (sgRNA) is underlined. The protospacer adjacent motif (PAM) sequence is boxed. Insertion and deletion are indicated by magenta letters and dashed lines, respectively. Electropherogram of Sanger sequence is shown in each KO cell. b XICs of Q nucleoside of human mt-tRNAs for Asp, His, Asn, and Tyr isolated from WT (top), QTRT1 KO (middle), and QTRT2 KO cells (bottom). c Boxplot of fold change in the ribosomal A-site codon occupancy of mitoribosome from QTRT2 KO versus WT HEK293T cells. Codon occupancy is calculated by average of biological replicates (n = 2). Each box shows the first quartile, median, and third quartile. The whiskers represent the 1.5× interquartile ranges. *P < 1.0 × 10⁻³ value was calculated by two-sided Wilcoxon rank-sum test. d Proteome stress of QTRT2 KO measured by the firefly luciferase (Fluc)-based thermal stability sensor fused with enhanced green fluorescent protein (EGFP). The Fluc sensor with no mutation (No mut.), single- (R188Q) or double-(R188Q/R261Q) mutation was introduced to QTRT2 KO or WT HEK293T cells. The aggregation rate was calculated by dividing the number of sensor protein aggregates by the area of EGFP fluorescence in each microscopic images. Horizontal lines in the scattered dot plot represent the first quartile, median, and third quartile from the bottom. P value were calculated by two-sided Wilcoxon rank-sum test. Source data are provided as a Source Data file.