Fig. 5: Deletion in N-terminus affects MTG3 functionality.
From: Coupling of ribosome biogenesis and translation initiation in human mitochondria
a ΔN-MTG3FLAG lacking 10 conserved aa in the N-terminus can only partially restore mitochondrial translation. Translation of mtDNA-encoded proteins in wild type, Mtg3−/− and Mtg3−/− cell lines expressing full length (MTG3FLAG) or mutated MTG3 (ΔN-MTG3FLAG), respectively, were analyzed using [35S]Methionine de novo incorporation and visualized via autoradiography and western blotting. GAPDH was used as a loading control. Similar results were obtained in n ≥ 3 biologically independent experiments. b ΔN-MTG3FLAG does not co-purify any MRPs. FLAG-immunoprecipitation was performed with wild type cells, wild type cells expressing MTG3FLAG and Mtg3−/− cells expressing MTG3FLAG or ΔN-MTG3FLAG, respectively. Samples were analyzed using western blotting and antibodies as indicated (total = 3%, eluate = 100%). Similar results were obtained in n ≥ 3 biologically independent experiments. c, d Monosome level can be restored upon ΔN-MTG3FLAG expression. Mitoplasts (500 µg) were isolated from wild type, Mtg3−/− and Mtg3−/− cell lines expressing full length (MTG3FLAG) or ΔN-MTG3FLAG, respectively. Mitoribosomal complexes were separated via sucrose density gradient centrifugation and collected fractions (1-16) were analyzed via western blotting with indicated antibodies against MRPs and assembly factors (c) or northern blotting with probes against 12S rRNA (MT-RNR1) and 16S rRNA (MT-RNR2) (d). Similar results were obtained in n ≥ 3 biologically independent experiments. e Abundance of mt-mRNAs bound to monosomes is similar in the Mtg3−/− + ΔN-MTG3FLAG cell line in comparison to wild type MTG3FLAG. Fraction 11 from sucrose gradients from (c) were used to isolate RNA and perform NanoString analysis. Level of mt-mRNAs were normalized to 16S-rRNA and RNA abundance bound to monosomes in the Mtg3−/− + ΔN-MTG3FLAG cell line was calculated relative to Mtg3−/− + MTG3FLAG cell line (n = 3 biologically independent samples shown as mean ± SEM; individual data points are shown as circles). Statistical analysis was performed as two-sample one-tailed Student’s t-test. Significance was defined as *p ≤ 0.05, **p ≤ 0.01.
