Fig. 1: Loss of MTG3 leads to disturbed small subunit assembly.

a Confirmation of MTG3 knock out in two cell lines generated using CRISPR/Cas9 technology. Isolated mitochondria (10 µg) from wildtype and Mtg3^−/− cell lines (cl.1 and cl.2) were analyzed by western blotting with antibodies as indicated. Similar results were obtained in n ≥ 3 biologically independent experiments. b Schematic representation of the genomic locus of the generated Mtg3^−/− cell line (cl.1) in comparison to the wild type sequence. The guide RNA targets exon 1 of the MTG3 gene, which encodes for a 648 aa protein. A two bp deletion and a four bp insertion in the two alleles of the Mtg3^−/− cl.1 lead to premature stop codons and truncated proteins (65 aa). c Ablation of MTG3 reduces growth rate. Equal amounts of wild type and Mtg3^−/− cells were seeded in three biologically independent experiments on day 0 and counted at the indicated time points (n = 3; mean ± SEM). Significance was calculated by two-sample one-tailed Student’s t-test and defined as **p ≤ 0.01. d Translation of mtDNA-encoded proteins is disturbed upon loss of MTG3. Mitochondrial translation in wild type, Mtg3^−/− and Mtg3^−/− cells inducibly expressing MTG3^FLAG was analyzed via [³⁵S]Methionine de novo incorporation and subsequently visualized via autoradiography and with indicated antibodies. The signal in Mtg3^−/− using MTG3 antibody represents unspecific binding of the antibody in whole cell lysates as we confirmed several times the loss of MTG3 in isolated mitochondria. Similar results were obtained in n ≥ 3 biologically independent experiments. e, f MTG3 loss leads to reduced mtSSU MRP level. Steady state analysis of MRPs, assembly factors, and translation-related proteins in the Mtg3^−/− cells in comparison to wild type cells. Isolated mitochondria were analyzed via western blotting with indicated antibodies (e) and protein levels in Mtg3^−/− were quantified relative to wild type control (f). SDHA was used as a loading control. Statistical analysis was performed as two-sample one-tailed Student’s t-test with n ≥ 3 biologically independent samples shown as mean ± SEM (individual data points are shown as circles). Significance was defined as *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. g, h Effect of MTG3 loss on rRNA and mRNA stability. g RNA isolated from Mtg3^−/− and wild type cells was subjected to northern blotting using indicated probes (MT-RNR1: 12S rRNA; MT-RNR2: 16S rRNA; MT-CO1: mRNA encoding for COX1). 18S-rRNA was used as loading control. h Quantification of RNA signals in Mtg3^−/− from (g) relative to wild type signals. Statistical analysis was performed as two-sample one-tailed Student’s t-test with n = 3 biologically independent samples shown as mean ± SEM (individual data points are shown as circles). Significance was defined as ***p ≤ 0.001. i mtSSU and monosome levels are severely reduced in Mtg3^−/− cells. Isolated mitoplasts (500 µg) were lysed and subjected to sucrose density gradient centrifugation. Fractions (1-16) were collected and analyzed via western blotting with antibodies against MRPs and assembly factors as indicated. Input = 10% of total. Similar results were obtained in n ≥ 3 biologically independent experiments.