Fig. 1: Consequences of loss of mitochondrial translation termination factors.
a Comparison of mtRF1a (top, red colors) and mtRF1 (bottom, blue colors). The domains I–IV of the mitochondrial release factors are shown in boxes. Corresponding sequences of the decoding motifs in domain II (α5 helix: green; PxT motif: blue) and the GGQ motif in the peptidyl tRNA hydrolase (PTH) domain III are indicated. b CRISPR/Cas9-mediated knockout of mtRF1 and mtRF1a. Mitochondrial lysates (10 µg for mtRF1a and 75 µg for mtRF1) were separated by SDS-PAGE followed by western blotting and immunodetection using the indicated antibodies. c Ablation of mitochondrial release factors affects cellular growth. Cells (7.5 × 104) were seeded on day 0. Cell growth was monitored by cell counts for the indicated time points in HEK293 wild-type cells (WT; gray) and in mtRF1- (mtRF1−/−, blue) and mtRF1a-deficient cells (mtRF1a−/−; red). (n > 6 biological replicates; mean ± SEM; significance for day 4 was calculated by two-sample one-tailed Student’s t-test and defined as ***p ≤ 0.001). d Elevated reactive oxygen species (ROS) production upon loss of mtRF1 and mtRF1a. ROS production was monitored by FACS using MitoSox Red. Relative ROS production in WT is indicated as dashed line (100%) and individual data points are shown as circles. Statistical analysis was carried out as two-sample one-tailed Student’s t-test with n = 3 biologically independent samples and shown as mean ± SEM. Significance was defined as *p ≤ 0.05 ; **p ≤ 0.01 . Source data are provided as a Source Data file.
