The mitochondrial methylation potential gates early and late ribosome assembly

S-adenosylmethionine (SAM) is the principal methyl group donor of the cell, required to modify various molecules, including DNA, RNA and proteins. Inside mitochondria, SAM is associated with several steps of mitochondrial gene expression. However, the exact role and significance of these modifications remains debated. Here, we depleted mitochondria of SAM, constitutively in mouse embryonic fibroblasts or progressively in mouse skeletal muscle. Our data reveal that mitochondrial SAM is crucial for both early and late stages of mitochondrial gene expression. Direct long-read RNA sequencing demonstrated that the mitochondrial ribosomal gene cluster is particularly sensitive to perturbed mitochondrial methylation potential, leading to the accumulation of unprocessed RNA precursors. Stable isotope labelling of amino acids in culture (SILAC) followed by mass spectrometry on ribosome fractions shows that these precursors are associated with processing and ribosome assembly factors. Finally, structural analysis by cryogenic electron microscopy (Cryo-EM) revealed that mitochondrial ribosome assembly is adversely affected by the absence of mitochondrial SAM and that methylation is integral for correct peptidyl transferase centre assembly of the large ribosome subunit. Our data thus identifies two critical steps for methylation during mitochondrial gene expression.