Cytochrome c oxidase biogenesis – from translation to early assembly of the core subunit COX1

Mitochondria are the powerhouses of the cell as they produce the majority of ATP with their oxidative phosphorylation (OXPHOS) machinery. The OXPHOS system is composed of the F1Fo ATP synthase and four mitochondrial respiratory chain complexes, the terminal enzyme of which is the cytochrome c oxidase (complex IV) that transfers electrons to oxygen, generating water. Complex IV comprises of 14 structural subunits of dual genetic origin: while the three core subunits are mitochondrial encoded, the remaining constituents are encoded by the nuclear genome. Hence, the assembly of complex IV requires the coordination of two spatially separated gene expression machinery. Recent efforts elucidated an increasing number of proteins involved in mitochondrial gene expression, which are linked to complex IV assembly. Additionally, several COX1 biogenesis factors have been intensively biochemically investigated and an increasing number of structural snapshots shed light on the organization of macromolecular complexes such as the mitoribosome or the cytochrome c oxidase. Here, we focus on COX1 translation regulation and highlight the advanced understanding of early steps during COX1 assembly and its link to mitochondrial translation regulation

Integrating mitochondrial translation into the cellular context.

Mitochondrial-encoded subunits of the oxidative phosphorylation system assemble with nuclear-encoded subunits into enzymatic complexes. Recent findings showed that mitochondrial translation is linked to other mitochondrial functions, as well as to cellular processes. The supply of mitochondrial- encoded proteins is coordinated by the coupling of mitochondrial protein synthesis with assembly of respiratory chain complexes. MicroRNAs imported from the cytoplasm into mitochondria were, surprisingly, found to act as regulators of mitochondrial translation. In turn, translation in mitochondria controls cellular proliferation, and mitochondrial ribosomal subunits contribute to the cytoplasmic stress response. Thus, translation in mitochondria is apparently integrated into cellular processes

Hierarchical folding of the catalytic core during mitochondrial ribosome biogenesis

Final maturation steps during ribosome biogenesis require the assistance of assembly and quality control factors to ensure the folding of rRNA and proteins into a functional translation machinery. Here we integrate several recent structural snapshots of native large ribosomal subunit intermediates into the complex pathway of mitochondrial ribosome assembly

An in vitro system to silence mitochondrial gene expression

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 mech- anisms of mitochondrial gene expression remain poorly understood due to a lack of experimental ap- proaches 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

Dual function of GTPBP6 in biogenesis and recycling of human mitochondrial ribosomes

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

SAMMSON fosters cancer cell fitness by concertedly enhancing mitochondrial and cytosolic translation

Synchronization of mitochondrial and cytoplasmic translation rates is critical for the maintenance of cellular fitness, with cancer cells being especially vulnerable to translational uncoupling. Although alterations of cytosolic protein synthesis are common in human cancer, compensating mechanisms in mitochondrial translation remain elusive. Here we show that the malignant long non-coding RNA (lncRNA) SAMMSON promotes a balanced increase in ribosomal RNA (rRNA) maturation and protein synthesis in the cytosol and mitochondria by modulating the localization of CARF, an RNA-binding protein that sequesters the exo-ribonuclease XRN2 in the nucleoplasm, which under normal circumstances limits nucleolar rRNA maturation. SAMMSON interferes with XRN2 binding to CARF in the nucleus by favoring the formation of an aberrant cytoplasmic RNA-protein complex containing CARF and p32, a mitochondrial protein required for the processing of the mitochondrial rRNAs. These data highlight how a single oncogenic lncRNA can simultaneously modulate RNA-protein complex formation in two distinct cellular compartments to promote cell growth

Structural basis of GTPase-mediated mitochondrial ribosome biogenesis and recycling

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

Identification of TMEM126A as OXA1L-interacting protein reveals cotranslational quality control in mitochondria

Cellular proteostasis requires transport of polypeptides across membranes. Although defective transport processes trigger cytosolic rescue and quality control mechanisms that clear translocases and membranes from unproductive cargo, proteins that are synthesized within mitochondria are not accessible to these mechanisms. Mitochondrial-encoded proteins are inserted cotranslationally into the inner membrane by the conserved insertase OXA1L. Here, we identify TMEM126A as a OXA1L-interacting protein. TMEM126A associates with mitochondrial ribosomes and translation products. Loss of TMEM126A leads to the destabilization of mitochondrial translation products, triggering an inner membrane quality control process, in which newly synthesized proteins are degraded by the mitochondrial iAAA protease. Our data reveal that TMEM126A cooperates with OXA1L in protein insertion into the membrane. Upon loss of TMEM126A, the cargo-blocked OXA1L insertase complexes undergo proteolytic clearance by the iAAA protease machinery together with its cargo

Hungry Codons Promote Frameshifting in Human Mitochondrial Ribosomes

Human mitochondria are not strict adherents to the universal genetic code, with modifications that include the apparent recoding of two arginine triplets to termination signals. This use of both AGA and AGG occurs rarely in other mammals, and this putative change has long posed a challenging conundrum. A –1 mitoribosome frameshift upstream of the rare codons would necessitate recognition of only the conventional UAA and UAG termination codons. By using a sequence-specific endoribonuclease, we show that the rare arginine codons, presumably in association with other cis elements, promote frameshifting in human mitoribosomes