Friedreich’s ataxia (FA) is a debilitating, multisystemic disease caused by the depletion of frataxin (FXN), a mitochondrial iron-sulfur (Fe-S) cluster biogenesis factor. To understand the cellular pathogenesis of FA, we performed quantitative proteomics in FXN-deficient human cells. Nearly every annotated Fe-S cluster-containing protein was depleted, indicating that as a rule, cluster binding confers stability to Fe-S proteins. We also observed depletion of a small mitoribosomal assembly factor METTL17 and evidence of impaired mitochondrial translation. Using comparative sequence analysis, mutagenesis, biochemistry, and cryoelectron microscopy, we show that METTL17 binds to the mitoribosomal small subunit during late assembly and harbors a previously unrecognized [Fe₄S₄]²⁺ cluster required for its stability. METTL17 overexpression rescued the mitochondrial translation and bioenergetic defects, but not the cellular growth, of FXN-depleted cells. These findings suggest that METTL17 acts as an Fe-S cluster checkpoint, promoting translation of Fe-S cluster-rich oxidative phosphorylation (OXPHOS) proteins only when Fe-S cofactors are replete.

The mitoribosome translates mitochondrial mRNAs and regulates energy conversion that is a signature of aerobic life forms. We present a 2.2 Å resolution structure of human mitoribosome together with validated mitoribosomal RNA (rRNA) modifications, including aminoacylated CP-tRNA^Val. The structure shows how mitoribosomal proteins stabilise binding of mRNA and tRNA helping to align it in the decoding center, whereas the GDP-bound mS29 stabilizes intersubunit communication. Comparison between different states, with respect to tRNA position, allowed us to characterize a non-canonical L1 stalk, and molecular dynamics simulations revealed how it facilitates tRNA transitions in a way that does not require interactions with rRNA. We also report functionally important polyamines that are depleted when cells are subjected to an antibiotic treatment. The structural, biochemical, and computational data illuminate the principal functional components of the translation mechanism in mitochondria and provide a description of the structure and function of the human mitoribosome.

In mammalian mitochondria, mRNAs are cotranscriptionally stabilized by the protein factor LRPPRC (leucine-rich pentatricopeptide repeat-containing protein). Here, we characterize LRPPRC as an mRNA delivery factor and report its cryo-electron microscopy structure in complex with SLIRP (SRA stem-loop-interacting RNA-binding protein), mRNA and the mitoribosome. The structure shows that LRPPRC associates with the mitoribosomal proteins mS39 and the N terminus of mS31 through recognition of the LRPPRC helical repeats. Together, the proteins form a corridor for handoff of the mRNA. The mRNA is directly bound to SLIRP, which also has a stabilizing function for LRPPRC. To delineate the effect of LRPPRC on individual mitochondrial transcripts, we used RNA sequencing, metabolic labeling and mitoribosome profiling, which showed a transcript-specific influence on mRNA translation efficiency, with cytochrome c oxidase subunit 1 and 2 translation being the most affected. Our data suggest that LRPPRC–SLIRP acts in recruitment of mitochondrial mRNAs to modulate their translation. Collectively, the data define LRPPRC–SLIRP as a regulator of the mitochondrial gene expression system.

Protein synthesis in crop plants contributes to the balance of food and fuel on our planet, which influences human metabolic activity and lifespan. Protein synthesis can be regulated with respect to changing environmental cues via the deposition of chemical modifications into rRNA. Here, we present the structure of a plant ribosome from tomato and a quantitative mass spectrometry analysis of its rRNAs. The study reveals fine features of the ribosomal proteins and 71 plant-specific rRNA modifications, and it re-annotates 30 rRNA residues in the available sequence. At the protein level, isoAsp is found in position 137 of uS11, and a zinc finger previously believed to be universal is missing from eL34, suggesting a lower effect of zinc deficiency on protein synthesis in plants. At the rRNA level, the plant ribosome differs markedly from its human counterpart with respect to the spatial distribution of modifications. Thus, it represents an additional layer of gene expression regulation, highlighting the molecular signature of a plant ribosome. The results provide a reference model of a plant ribosome for structural studies and an accurate marker for molecular ecology.

Mitoribosomes are essential for the synthesis and maintenance of bioenergetic proteins. Here we use cryo-electron microscopy to determine a series of the small mitoribosomal subunit (SSU) intermediates in complex with auxiliary factors, revealing a sequential assembly mechanism. The methyltransferase TFB1M binds to partially unfolded rRNA h45 that is promoted by RBFA, while the mRNA channel is blocked. This enables binding of METTL15 that promotes further rRNA maturation and a large conformational change of RBFA. The new conformation allows initiation factor mtIF3 to already occupy the subunit interface during the assembly. Finally, the mitochondria-specific ribosomal protein mS37 (ref. 1) outcompetes RBFA to complete the assembly with the SSU–mS37–mtIF3 complex2 that proceeds towards mtIF2 binding and translation initiation. Our results explain how the action of step-specific factors modulate the dynamic assembly of the SSU, and adaptation of a unique protein, mS37, links the assembly to initiation to establish the catalytic human mitoribosome.

The mitoribosome regulates cellular energy production, and its dysfunction is associated with aging. Inhibition of the mitoribosome can be caused by off-target binding of antimicrobial drugs and was shown to be coupled with a bilateral decreased visual acuity. Previously, we reported mitochondria-specific protein aspects of the mitoribosome, and in this article we present a 2.4-Å resolution structure of the small subunit in a complex with the anti-tuberculosis drug streptomycin that reveals roles of non-protein components. We found iron–sulfur clusters that are coordinated by different mitoribosomal proteins, nicotinamide adenine dinucleotide (NAD) associated with rRNA insertion, and posttranslational modifications. This is the first evidence of inter-protein coordination of iron–sulfur, and the finding of iron–sulfur clusters and NAD as fundamental building blocks of the mitoribosome directly links to mitochondrial disease and aging. We also report details of streptomycin interactions, suggesting that the mitoribosome-bound streptomycin is likely to be in hydrated gem-diol form and can be subjected to other modifications by the cellular milieu. The presented approach of adding antibiotics to cultured cells can be used to define their native structures in a bound form under more physiological conditions, and since streptomycin is a widely used drug for treatment, the newly resolved features can serve as determinants for targeting.

Mitochondrial ribosomes (mitoribosomes) are tethered to the mitochondrial inner membrane to facilitate the cotranslational membrane insertion of the synthesized proteins. We report cryo-electron microscopy structures of human mitoribosomes with nascent polypeptide, bound to the insertase oxidase assembly 1-like (OXA1L) through three distinct contact sites. OXA1L binding is correlated with a series of conformational changes in the mitoribosomal large subunit that catalyze the delivery of newly synthesized polypeptides. The mechanism relies on the folding of mL45 inside the exit tunnel, forming two specific constriction sites that would limit helix formation of the nascent chain. A gap is formed between the exit and the membrane, making the newly synthesized proteins accessible. Our data elucidate the basis by which mitoribosomes interact with the OXA1L insertase to couple protein synthesis and membrane delivery.

Mitoribosomes are specialized protein synthesis machineries in mitochondria. However, how mRNA binds to its dedicated channel, and tRNA moves as the mitoribosomal subunit rotate with respect to each other is not understood. We report models of the translating fungal mitoribosome with mRNA, tRNA and nascent polypeptide, as well as an assembly intermediate. Nicotinamide adenine dinucleotide (NAD) is found in the central protuberance of the large subunit, and the ATPase inhibitory factor 1 (IF1) in the small subunit. The models of the active mitoribosome explain how mRNA binds through a dedicated protein platform on the small subunit, tRNA is translocated with the help of the protein mL108, bridging it with L1 stalk on the large subunit, and nascent polypeptide paths through a newly shaped exit tunnel involving a series of structural rearrangements. An assembly intermediate is modeled with the maturation factor Atp25, providing insight into the biogenesis of the mitoribosomal large subunit and translation regulation.

Translation initiation in human mitochondria relies upon specialized mitoribosomes and initiation factors, mtIF2 and mtIF3, which have diverged from their bacterial counterparts. Here we report two distinct mitochondrial pre-initiation assembly steps involving those factors. Single-particle cryo-EM revealed that in the first step, interactions between mitochondria-specific protein mS37 and mtIF3 keep the small mitoribosomal subunit in a conformation favorable for a subsequent accommodation of mtIF2 in the second step. Combination with fluorescence cross-correlation spectroscopy analyses suggests that mtIF3 promotes complex assembly without mRNA or initiator tRNA binding, where exclusion is achieved by the N-terminal and C-terminal domains of mtIF3. Finally, the association of large mitoribosomal subunit is required for initiator tRNA and leaderless mRNA recruitment to form a stable initiation complex. These data reveal fundamental aspects of mammalian protein synthesis that are specific to mitochondria.

The AAA+ GTPase McrB powers DNA cleavage by the endonuclease McrC. The GTPase itself is activated by McrC. The architecture of the GTPase and nuclease complex, and the mechanism of their activation remained unknown. Here, we report a 3.6 angstrom structure of a GTPase-active and DNA-binding deficient construct of McrBC. Two hexameric rings of McrB are bridged by McrC dimer. McrC interacts asymmetrically with McrB protomers and inserts a stalk into the pore of the ring, reminiscent of the gamma subunit complexed to alpha(3)beta(3) of F-1-ATPase. Activation of the GTPase involves conformational changes of residues essential for hydrolysis. Three consecutive nucleotide-binding pockets are occupied by the GTP analogue 5'-guanylyl imidodiphosphate and the next three by GDP, which is suggestive of sequential GTP hydrolysis.