The human mitochondrial genome encodes thirteen core subunits of the oxidative phosphorylation system, and defects in mitochondrial gene expression lead to severe neuromuscular disorders. However, the mechanisms of mitochondrial gene expression remain poorly understood due to a lack of experimental approaches to analyze these processes. Here, we present an in vitro system to silence translation in purified mitochondria. In vitro import of chemically synthesized precursor-morpholino hybrids allows us to target translation of individual mitochondrial mRNAs. By applying this approach, we conclude that the bicistronic, overlapping ATP8/ATP6 transcript is translated through a single ribosome/mRNA engagement. We show that recruitment of COX1 assembly factors to translating ribosomes depends on nascent chain formation. By defining mRNA-specific interactomes for COX1 and COX2, we reveal an unexpected function of the cytosolic oncofetal IGF2BP1, an RNA-binding protein, in mitochondrial translation. Our data provide insight into mitochondrial translation and innovative strategies to investigate mitochondrial gene expression.

Modified nucleotides in tRNAs are important determinants of folding, structure and function. Here we identify METTL8 as a mitochondrial matrix protein and active RNA methyltransferase responsible for installing m³C₃₂ in the human mitochondrial (mt-)tRNA^Thr and mt-tRNA^Ser(UCN). METTL8 crosslinks to the anticodon stem loop (ASL) of many mt-tRNAs in cells, raising the question of how methylation target specificity is achieved. Dissection of mt-tRNA recognition elements revealed U₃₄G₃₅ and t⁶A₃₇/(ms²)i⁶A₃₇, present concomitantly only in the ASLs of the two substrate mt-tRNAs, as key determinants for METTL8-mediated methylation of C₃₂. Several lines of evidence demonstrate the influence of U₃₄, G₃₅, and the m³C₃₂ and t⁶A₃₇/(ms²)i⁶A₃₇ modifications in mt-tRNA^Thr/Ser(UCN) on the structure of these mt-tRNAs. Although mt-tRNA^Thr/Ser(UCN) lacking METTL8-mediated m³C₃₂ are efficiently aminoacylated and associate with mitochondrial ribosomes, mitochondrial translation is mildly impaired by lack of METTL8. Together these results define the cellular targets of METTL8 and shed new light on the role of m³C₃₂ within mt-tRNAs.

Ribosome biogenesis requires auxiliary factors to promote folding and assembly of ribosomal proteins and RNA. Particularly, maturation of the peptidyl transferase center (PTC) is mediated by conserved GTPases, but the molecular basis is poorly understood. Here, we define the mechanism of GTPase-driven maturation of the human mitochondrial large ribosomal subunit (mtLSU) using endogenous complex purification, in vitro reconstitution and cryo-EM. Structures of transient native mtLSU assembly intermediates that accumulate in GTPBP6-deficient cells reveal how the biogenesis factors GTPBP5, MTERF4 and NSUN4 facilitate PTC folding. Addition of recombinant GTPBP6 reconstitutes late mtLSU biogenesis in vitro and shows that GTPBP6 triggers a molecular switch and progression to a near-mature PTC state. Additionally, cryo-EM analysis of GTPBP6-treated mature mitochondrial ribosomes reveals the structural basis for the dual-role of GTPBP6 in ribosome biogenesis and recycling. Together, these results provide a framework for understanding step-wise PTC folding as a critical conserved quality control checkpoint.

The human mitochondrial translation apparatus, which synthesizes the core subunits of the oxidative phosphorylation system, is of central interest as mutations in several genes encoding for mitoribosomal proteins or translation factors cause severe human diseases. Little is known, how this complex machinery assembles from nuclear-encoded protein components and mitochondrial-encoded RNAs, and which ancillary factors are required to form a functional mitoribosome. We have characterized the human Obg protein GTPBP10, which associates specifically with the mitoribosomal large subunit at a late maturation state. Defining its interactome, we have shown that GTPBP10 is in a complex with other mtLSU biogenesis factors including mitochondrial RNA granule components, the 16S rRNA module and late mtLSU assembly factors such as MALSU1, SMCR7L, MTERF4 and NSUN4. GTPBP10 deficiency leads to a drastic reduction in 55S monosome formation resulting in defective mtDNA-expression and in a decrease in cell growth. Our results suggest that GTPBP10 is a ribosome biogenesis factor of the mtLSU required for late stages of maturation.

Cytochrome c oxidase of the mitochondrial oxidative phosphorylation system reduces molecular oxygen with redox equivalent-derived electrons. The conserved mitochondrial-encoded COX1- and COX2-subunits are the heme- and copper-center containing core subunits that catalyze water formation. COX1 and COX2 initially follow independent biogenesis pathways creating assembly modules with subunit-specific, chaperone-like assembly factors that assist in redox centers formation. Here, we find that COX16, a protein required for cytochrome c oxidase assembly, interacts specifically with newly synthesized COX2 and its copper center-forming metallochaperones SCO1, SCO2, and COA6. The recruitment of SCO1 to the COX2-module is COX16- dependent and patient-mimicking mutations in SCO1 affect interaction with COX16. These findings implicate COX16 in Cu_A-site formation. Surprisingly, COX16 is also found in COX1-containing assembly intermediates and COX2 recruitment to COX1. We conclude that COX16 participates in merging the COX1 and COX2 assembly lines.

Translation and ribosome biogenesis in mitochondria require auxiliary factors that ensure rapid and accurate synthesis of mitochondrial proteins. Defects in translation are associated with oxidative phosphorylation deficiency and cause severe human diseases, but the exact roles of mitochondrial translation-associated factors are not known. Here we identify the functions of GTPBP6, a homolog of the bacterial ribosome-recycling factor HflX, in human mitochondria. Similarly to HflX, GTPBP6 facilitates the dissociation of ribosomes in vitro and in vivo. In contrast to HflX, GTPBP6 is also required for the assembly of mitochondrial ribosomes. GTPBP6 ablation leads to accumulation of late assembly intermediate(s) of the large ribosomal subunit containing ribosome biogenesis factors MTERF4, NSUN4, MALSU1 and the GTPases GTPBP5, GTPBP7 and GTPBP10. Our data show that GTPBP6 has a dual function acting in ribosome recycling and biogenesis. These findings contribute to our understanding of large ribosomal subunit assembly as well as ribosome recycling pathway in mitochondria.