Figure 7. m³C₃₂ unbalancing in mt-tRNA^Ser(UCN) and mt-tRNA^Thr affects translation dynamics and respiratory complex assembly.

(A) Radioactive pulse labeling of de novo mitochondrial protein synthesis. Flp-In TREx293, PANC-1, HAP1 METTL8 WT and METTL8 KO cell lines were labeled with [³⁵S] methionine after cytoplasmic protein synthesis was blocked by emetine dihydrochloride. Total lysates were separated by Tricine-SDS-PAGE and stained with Coomassie Blue as a loading control (left panel). Corresponding phosphorimaging quantification is shown (right panel).Means ± SED of two independent measurements are plotted.

(B) Mitochondrial lysates from METTL8 WT, METTL8 KO, and METTL8 overexpressing (WT_OE) Flp-In TREx293 cell lines were separated by blue native (BN) page and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis (left panel). Assembly of the complex I and II submodules is shown to the side of the panel. Right: listed proteins were not detectable in LC-MS/MS analysis and are mapped to their related module. Additional proteins undetectable in Flp-In TREx METTL8 KO are highlighted in red.

(C) Amino acid overrepresentation at positions relative to the P site in Flp-In TREx293 METTL8 KO cells. Horizontal red line indicates significant overrepresentation.

(D) Amino acid overrepresentation at positions relative to the P site in Flp-In TREx293 cells overexpressing METTL8. Horizontal red line indicates significant overrepresentation.

(E) Ribosomal redistribution in E, P, and A site of Flp-In TREx293 METTL8 KO cells based on the codon. Red dots represent mt-tRNA^Ser(UCN) codons, mt-tRNA^Thr codons are highlighted in blue. Significant overrepresented codons are underlined.

(F) Ribosomal redistribution in E, P, and A site of Flp-In TREx293 cells overexpressing METTL8 based on the codon. Red dots represent mt-tRNA^Ser(UCN) codons, mt-tRNA^Thr codons are highlighted in blue. Significant overrepresented codons are underlined.

(G) Ribosome stalling in either the P site or A site of Flp-In TREx293 METTL8 KO cells based on the UCN codon and downstream nucleotides. Top: UCN codons with overrepresented downstream nucleotides in the P site. Bottom: UCN codons with overrepresented downstream stream nucleotides in the Asite. Red dots represent mt-tRNA^Ser(UCN) codons. Significantly enriched codons are underlined.

(H) Ribosome stalling of Flp-In TREx293 cells overexpressing METTL8 based on the ACN codon and downstream nucleotides in the A site. Blue dots represent mt-tRNA^Thr codons. Significantly enriched codons are underlined.