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Biogenesis of mammalian mitochondrial ribosomes requires a concerted maturation of both the small (SSU) and large subunit (LSU). We demonstrate here that the m⁵C methyltransferase NSUN4, which forms a complex with MTERF4, is essential in mitochondrial ribosomal biogenesis as mitochondrial translation is abolished in conditional Nsun4 mouse knockouts. Deep sequencing of bisulfite-treated RNA shows that NSUN4 methylates cytosine 911 in 12S rRNA (m5C911) of the SSU. Surprisingly, NSUN4 does not need MTERF4 to generate this modification. Instead, the NSUN4/MTERF4 complex is required to assemble the SSU and LSU to form a monosome. NSUN4 is thus a dual function protein, which on the one hand is needed for 12S rRNA methylation and, on the other hand interacts with MTERF4 to facilitate monosome assembly. The presented data suggest that NSUN4 has a key role in controlling a final step in ribosome biogenesis to ensure that only the mature SSU and LSU are assembled. Mitochondria perform a number of essential functions in the cell, including synthesis of ATP via the oxidative phosphorylation (OXPHOS) system. Normal mitochondrial function requires coordinated expression of two genomes: mitochondria's own genome (mtDNA), which encodes 13 respiratory chain subunits with essential structural and functional roles for the OXPHOS system, and the nuclear genome encoding the remaining ∼80 subunits. The mtDNA-encoded polypeptides are synthesized on mitochondrial ribosomes (mitoribosomes) located in the mitochondrial matrix. Biogenesis, maintenance and regulation of the complex mitochondrial translation apparatus are poorly understood despite its fundamental importance for cellular energy homeostasis. Here, we show that inactivation of the Nsun4 gene, encoding a mitochondrial m⁵C-methyltransferase, causes embryonic lethality, whereas tissue-specific disruption of Nsun4 in the heart causes cardiomyopathy with mitochondrial dysfunction. By performing sequencing of bisulfite-treated RNA we report that NSUN4 methylates C911 in 12S rRNA of the small ribosomal subunit. Surprisingly, NSUN4 can on its own perform this rRNA modification, whereas interaction with its partner protein MTERF4 is required for assembly of functional ribosomes. NSUN4 thus has dual roles in ribosome maturation and performs an important final quality control step to ensure that only mature mitoribosomal subunits are assembled into functional ribosomes.

Replication of the mammalian mitochondrial DNA (mtDNA) is dependent on the minimal replisome, consisting of the heterotrimeric mtDNA polymerase (POLG), the hexameric DNA helicase TWINKLE and the tetrameric single-stranded DNA-binding protein (mtSSB). TWINKLE has been shown to unwind DNA during the replication process and many disease-causing mutations have been mapped to its gene. Patients carrying Twinkle mutations develop multiple deletions of mtDNA, deficient respiratory chain function and neuromuscular symptoms. Despite its importance in human disease, it has been unclear whether TWINKLE is the only replicative DNA helicase in mammalian mitochondria. Furthermore, a substantial portion of mtDNA replication events is prematurely terminated at the end of mitochondrial control region (D-loop) and it is unknown whether TWINKLE also has a role in this abortive replication. Here, we present a conditional mouse knockout for Twinkle and demonstrate that TWINKLE is essential for mouse embryonic development and thus is the only replicative DNA helicase in mammalian mitochondria. Conditional knockout of Twinkle results in severe and rapid mtDNA depletion in heart and skeletal muscle. No replication intermediates or deleted mtDNA molecules are observed after Twinkle knockout, suggesting that TWINKLE once loaded is very processive. We also demonstrate that TWINKLE is essential for nascent H-strand synthesis in the D-loop, thus showing that there is no separate DNA helicase responsible for replication of this region. Our data thus suggest that the relative levels of abortive D-loop synthesis versus complete mtDNA replication are regulated and may provide a mechanism to control progression to complete mtDNA replication.

Mitochondrial dynamics is an essential physiological process controlling mitochondrial content mixing and mobility to ensure proper function and localization of mitochondria at intracellular sites of high-energy demand. Intriguingly, for yet unknown reasons, severe impairment of mitochondrial fusion drastically affects mtDNA copy number. To decipher the link between mitochondrial dynamics and mtDNA maintenance, we studied mouse embryonic fibroblasts (MEFs) and mouse cardiomyocytes with disruption of mitochondrial fusion. Super-resolution microscopy revealed that loss of outer mitochondrial membrane (OMM) fusion, but not inner mitochondrial membrane (IMM) fusion, leads to nucleoid clustering. Remarkably, fluorescence in situ hybridization (FISH), bromouridine labeling in MEFs and assessment of mitochondrial transcription in tissue homogenates revealed that abolished OMM fusion does not affect transcription. Furthermore, the profound mtDNA depletion in mouse hearts lacking OMM fusion is not caused by defective integrity or increased mutagenesis of mtDNA, but instead we show that mitochondrial fusion is necessary to maintain the stoichiometry of the protein components of the mtDNA replisome. OMM fusion is necessary for proliferating MEFs to recover from mtDNA depletion and for the marked increase of mtDNA copy number during postnatal heart development. Our findings thus link OMM fusion to replication and distribution of mtDNA. Mammalian mitochondria contain multiple copies of the mitochondrial genome (mtDNA), which encodes genes that are essential for the oxidative phosphorylation system. An important feature of mtDNA is that it is evenly distributed throughout the mitochondrial network. Dynamin-related GTPase proteins help control the size and shape of mitochondria by fusion and fission events and are intimately linked to maintenance and distribution of mtDNA. Certain human mutations in mitofusin 2 (MFN2) and optic atrophy protein 1 (OPA1) cause disease phenotypes, such as peripheral neuropathy and optic atrophy, which are often also associated with mtDNA depletion. However, the mechanism whereby MFNs and OPA1 are involved in maintenance of mtDNA is unclear. In this study, we demonstrate that rapid mtDNA synthesis in proliferating tissue-culture cells or cardiomyocytes during post-natal heart development requires mitochondrial fusion. However, the absence of mitochondrial fusion in mouse heart is not associated with mtDNA integrity defects but instead affects the replication of mtDNA. These findings provide direct evidence for the importance of mitochondrial fusion in maintaining mtDNA replication.