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The mitochondrial ribosome (mitoribosome) synthesizes 13 protein subunits of the oxidative phosphorylation system encoded by the mitochondrial genome. The mitoribosome is composed of 12S rRNA, 16S rRNA, and 82 mitoribosomal proteins encoded by nuclear genes. To date, variants in 12 genes encoding mitoribosomal proteins are associated with rare monogenic disorders and frequently show combined oxidative phosphorylation deficiency. Here, we describe five unrelated individuals with bi-allelic variants in death-associated protein 3 (DAP3), a nuclear gene encoding mitoribosomal small subunit 29 (MRPS29), with variable clinical presentations ranging from Perrault syndrome (sensorineural hearing loss and ovarian insufficiency) to an early childhood neurometabolic phenotype. Assessment of respiratory-chain function and proteomic profiling of fibroblasts from affected individuals demonstrated reduced MRPS29 protein amounts and, consequently, decreased levels of additional protein components of the mitoribosomal small subunit, as well as an associated combined deficiency of complexes I and IV. Lentiviral transduction of fibroblasts from affected individuals with wild-type DAP3 cDNA increased DAP3 mRNA expression and partially rescued protein levels of MRPS7, MRPS9, and complex I and IV subunits, demonstrating the pathogenicity of the DAP3 variants. Protein modeling suggested that DAP3 disease-associated missense variants can impact ADP binding, and in vitro assays demonstrated that DAP3 variants can consequently reduce both intrinsic and extrinsic apoptotic sensitivity, DAP3 thermal stability, and DAP3 GTPase activity. Our study presents genetic and functional evidence that bi-allelic variants in DAP3 result in a multisystem disorder of combined oxidative phosphorylation deficiency with pleiotropic presentations, consistent with mitochondrial dysfunction., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The phylum Apicomplexa comprises eukaryotic parasites that cause fatal diseases affecting millions of people and animals worldwide. Their mitochondrial genomes have been significantly reduced, leaving only three protein-coding genes and highly fragmented mitoribosomal rRNAs, raising challenging questions about mitoribosome composition, assembly and structure. Our study reveals how Toxoplasma gondii assembles over 40 mt-rRNA fragments using exclusively nuclear-encoded mitoribosomal proteins and three lineage-specific families of RNA-binding proteins. Among these are four proteins from the Apetala2/Ethylene Response Factor (AP2/ERF) family, originally known as transcription factors in plants and Apicomplexa, now repurposed as essential mitoribosome components. Cryo-EM analysis of the mitoribosome structure demonstrates how these AP2 proteins function as RNA binders to maintain mitoribosome integrity. The mitoribosome is also decorated with members of lineage-specific RNA-binding proteins belonging to RAP (RNA-binding domain abundant in Apicomplexa) proteins and HPR (heptatricopeptide repeat) families, highlighting the unique adaptations of these parasites. Solving the molecular puzzle of apicomplexan mitoribosome could inform the development of therapeutic strategies targeting organellar translation., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Ribosome biogenesis is a highly regulated multistep process aided by energy-consuming auxiliary factors. GTPases form the largest class of auxiliary factors used by bacterial, cytosolic, and mitochondrial ribosomes for their maturation. Mtg3, a circularly permuted YqeH family of GTPase, is implicated in the mitoribosome small subunit biogenesis. However, its precise mechanistic role has yet to be characterized. Mtg3 is likely to bind precursor mitoribosome molecules during subunit maturation in vivo. However, this interaction has yet to be observed with mitoribosomes biochemically. In this study, we delineate the specific conditions necessary for preserving the association of Mtg3 with mitoribosomes on a sucrose density gradient. We show that the C-terminal domain of Mtg3 is required for robust binding to the mitoribosome. Furthermore, point mutants likely to abrogate GTP/GDP binding and GTPase activity compromise protein function in vivo. Surprisingly, the association with the mitoribosome was not compromised in mutants likely to be deficient for nucleotide binding/hydrolysis. Thus, our finding supports a model wherein Mtg3 binds to a precursor mitoribosome through its C-terminus to facilitate a conformational change or validate a folding intermediate driven by the GTP/GDP binding and hydrolysis cycle., Competing Interests: Declaration of competing interest The authors declare no potential conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Mitochondria contain dedicated ribosomes (mitoribosomes), which synthesize the mitochondrial-encoded core components of the oxidative phosphorylation complexes. The RNA and protein components of mitoribosomes are encoded on two different genomes (mitochondrial and nuclear) and are assembled into functional complexes with the help of dedicated factors inside the organelle. Defects in mitoribosome biogenesis are associated with severe human diseases, yet the molecular pathway of mitoribosome assembly remains poorly understood. Here, we applied a multidisciplinary approach combining biochemical isolation and analysis of native mitoribosomal assembly complexes with quantitative mass spectrometry and mathematical modeling to reconstitute the entire assembly pathway of the human mitoribosome. We show that, in contrast to its bacterial and cytosolic counterparts, human mitoribosome biogenesis involves the formation of ribosomal protein-only modules, which then assemble on the appropriate ribosomal RNA moiety in a coordinated fashion. The presence of excess protein-only modules primed for assembly rationalizes how mitochondria cope with the challenge of forming a protein-rich ribonucleoprotein complex of dual genetic origin. This study provides a comprehensive roadmap of mitoribosome biogenesis, from very early to late maturation steps, and highlights the evolutionary divergence from its bacterial ancestor., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Mitoribosome biogenesis is a complex process involving RNA elements encoded in the mitochondrial genome and mitoribosomal proteins typically encoded in the nuclear genome. This process is orchestrated by extra-ribosomal proteins, nucleus-encoded assembly factors, which play roles across all assembly stages to coordinate ribosomal RNA processing and maturation with the sequential association of ribosomal proteins. Both biochemical studies and recent cryo-EM structures of mammalian mitoribosomes have provided insights into their assembly process. In this article, we will briefly outline the current understanding of mammalian mitoribosome biogenesis pathways and the factors involved. Special attention is devoted to the recent identification of iron-sulfur clusters as structural components of the mitoribosome and a small subunit assembly factor, the existence of redox-sensitive cysteines in mitoribosome proteins and assembly factors, and the role they may play as redox sensor units to regulate mitochondrial translation under stress., (© 2024 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The prokaryotic translation elongation factor P (EF-P) and the eukaryotic/archaeal counterparts eIF5A/aIF5A are proteins that serve a crucial role in mitigating ribosomal stalling during the translation of specific sequences, notably those containing consecutive proline residues (1,2). Although mitochondrial DNA-encoded proteins synthesized by mitochondrial ribosomes also contain polyproline stretches, an EF-P/eIF5A mitochondrial counterpart remains unidentified. Here, we show that the missing factor is TACO1, a protein causative of a juvenile form of neurodegenerative Leigh's syndrome associated with cytochrome c oxidase deficiency, until now believed to be a translational activator of COX1 mRNA. By using a combination of metabolic labeling, puromycin release and mitoribosome profiling experiments, we show that TACO1 is required for the rapid synthesis of the polyproline-rich COX1 and COX3 cytochrome c oxidase subunits, while its requirement is negligible for other mitochondrial DNA-encoded proteins. In agreement with a role in translation efficiency regulation, we show that TACO1 cooperates with the N-terminal extension of the large ribosomal subunit bL27m to provide stability to the peptidyl-transferase center during elongation. This study illuminates the translation elongation dynamics within human mitochondria, a TACO1-mediated biological mechanism in place to mitigate mitoribosome stalling at polyproline stretches during protein synthesis, and the pathological implications of its malfunction., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Mitoribosomes are essential for the production of biological energy. The Human Mitoribosomal Small Subunit unit (MRPS) family, responsible for encoding mitochondrial ribosomal small subunits, is actively engaged in protein synthesis within the mitochondria. Intriguingly, MRPS family genes appear to play a role in cancer. A multistep process was employed to establish a risk model associated with MRPS genes, aiming to delineate the immune and pharmacogenomic landscapes in clear cell renal cell carcinoma (ccRCC). MRPScores were computed for individual patients to assess their responsiveness to various treatment modalities and their susceptibility to different therapeutic targets and drugs. While MRPS family genes have been implicated in various cancers as oncogenes, our findings reveal a contrasting tumor suppressor role for MRPS genes in ccRCC. Utilizing an MRPS-related risk model, we observed its excellent prognostic capability in predicting survival outcomes for ccRCC patients. Remarkably, the subgroup with high MRPS-related scores (MRPScore) displayed poorer prognosis but exhibited a more robust response to immunotherapy. Through in silico screening of 2183 drug targets and 1646 compounds, we identified two targets (RRM2 and OPRD1) and eight agents (AZ960, carmustine, lasalocid, SGI-1776, AZD8055_1059, BPD.00008900_1998, MK.8776_2046, and XAV939_1268) with potential therapeutic implications for high-MRPScore patients. Our study represents the pioneering effort in proposing that molecular classification, diagnosis, and treatment strategies can be formulated based on MRPScores. Indeed, a high MRPScore profile appears to elevate the risk of tumor progression and mortality, potentially through its influence on immune regulation. This suggests that the MRPS-related risk model holds promise as a prognostic predictor and may offer novel insights into personalized therapeutic strategies., (© 2024 International Union of Biochemistry and Molecular Biology.)

The mitochondrial ribosome (mitoribosome) is responsible for the synthesis of key oxidative phosphorylation subunits encoded by the mitochondrial genome. Defects in mitoribosomal function therefore can have serious consequences for the bioenergetic capacity of the cell. Mutation of the conserved mitoribosomal mL44 protein has been directly linked to childhood cardiomyopathy and progressive neurophysiology issues. To further explore the functional significance of the mL44 protein in supporting mitochondrial protein synthesis, we have performed a mutagenesis study of the yeast mL44 homolog, the MrpL3/mL44 protein. We specifically investigated the conserved hydrophobic pocket region of the MrpL3/mL44 protein, where the known disease-related residue in the human mL44 protein (L156R) is located. While our findings identify a number of residues in this region critical for MrpL3/mL44's ability to support the assembly of translationally active mitoribosomes, the introduction of the disease-related mutation into the equivalent position in the yeast protein (residue A186) was found to not have a major impact on function. The human and yeast mL44 proteins share many similarities in sequence and structure; however results presented here indicate that these two proteins have diverged somewhat in evolution. Finally, we observed that mutation of the MrpL3/mL44 does not impact the translation of all mitochondrial encoded proteins equally, suggesting the mitochondrial translation system may exhibit a transcript hierarchy and prioritization., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Mitochondrial gene expression relies on mitoribosomes to translate mitochondrial mRNAs. The biogenesis of mitoribosomes is an intricate process involving multiple assembly factors. Among these factors, GTP-binding proteins (GTPBPs) play important roles. In bacterial systems, numerous GTPBPs are required for ribosome subunit maturation, with EngB being a GTPBP involved in the ribosomal large subunit assembly. In this study, we focus on exploring the function of GTPBP8, the human homolog of EngB. We find that ablation of GTPBP8 leads to the inhibition of mitochondrial translation, resulting in significant impairment of oxidative phosphorylation. Structural analysis of mitoribosomes from GTPBP8 knock-out cells shows the accumulation of mitoribosomal large subunit assembly intermediates that are incapable of forming functional monosomes. Furthermore, fPAR-CLIP analysis reveals that GTPBP8 is an RNA-binding protein that interacts specifically with the mitochondrial ribosome large subunit 16 S rRNA. Our study highlights the role of GTPBP8 as a component of the mitochondrial gene expression machinery involved in mitochondrial large subunit maturation., (© 2024. The Author(s).)

Tigecycline is widely used for treating complicated bacterial infections for which there are no effective drugs. It inhibits bacterial protein translation by blocking the ribosomal A-site. However, even though it is also cytotoxic for human cells, the molecular mechanism of its inhibition remains unclear. Here, we present cryo-EM structures of tigecycline-bound human mitochondrial 55S, 39S, cytoplasmic 80S and yeast cytoplasmic 80S ribosomes. We find that at clinically relevant concentrations, tigecycline effectively targets human 55S mitoribosomes, potentially, by hindering A-site tRNA accommodation and by blocking the peptidyl transfer center. In contrast, tigecycline does not bind to human 80S ribosomes under physiological concentrations. However, at high tigecycline concentrations, in addition to blocking the A-site, both human and yeast 80S ribosomes bind tigecycline at another conserved binding site restricting the movement of the L1 stalk. In conclusion, the observed distinct binding properties of tigecycline may guide new pathways for drug design and therapy., (© 2024. The Author(s).)

Human mitochondrial DNA is one of the most simplified cellular genomes and facilitates compartmentalized gene expression. Within the organelle, there is no physical barrier to separate transcription and translation, nor is there evidence that quality control surveillance pathways are active to prevent translation on faulty mRNA transcripts. Mitochondrial ribosomes synthesize 13 hydrophobic proteins that require co-translational insertion into the inner membrane of the organelle. To maintain the integrity of the inner membrane, which is essential for organelle function, requires responsive quality control mechanisms to recognize aberrations in protein synthesis. In this review, we explore how defects in mitochondrial protein synthesis can arise due to the culmination of inherent mistakes that occur throughout the steps of gene expression. In turn, we examine the stepwise series of quality control processes that are needed to eliminate any mistakes that would perturb organelle homeostasis. We aim to provide an integrated view on the quality control mechanisms of mitochondrial protein synthesis and to identify promising avenues for future research., (© The Author(s) 2024. Published by Oxford University Press.)

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., (© 2024. The Author(s).)

DEAD-box helicases are important players in mitochondrial gene expression, which is necessary for mitochondrial respiration. In this study, we characterized Schizosaccharomyces pombe Mss116 (spMss116), a member of the family of DEAD-box RNA helicases. Deletion of spmss116 in a mitochondrial intron-containing background significantly reduced the levels of mitochondrial DNA (mtDNA)-encoded cox1 and cob1 mRNAs and impaired mitochondrial translation, leading to a severe respiratory defect and a loss of cell viability during stationary phase. Deletion of mitochondrial introns restored the levels of cox1 and cob1 mRNAs to wide-type (WT) levels but could not restore mitochondrial translation and respiration in Δspmss116 cells. Furthermore, deletion of spmss116 in both mitochondrial intron-containing and intronless backgrounds impaired mitoribosome assembly and destabilization of mitoribosomal proteins. Our findings suggest that defective mitochondrial translation caused by deletion of spmss116 is most likely due to impaired mitoribosome assembly., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Oxidative phosphorylation (OXPHOS) complexes, encoded by both mitochondrial and nuclear DNA, are essential producers of cellular ATP, but how nuclear and mitochondrial gene expression steps are coordinated to achieve balanced OXPHOS subunit biogenesis remains unresolved. Here, we present a parallel quantitative analysis of the human nuclear and mitochondrial messenger RNA (mt-mRNA) life cycles, including transcript production, processing, ribosome association, and degradation. The kinetic rates of nearly every stage of gene expression differed starkly across compartments. Compared with nuclear mRNAs, mt-mRNAs were produced 1,100-fold more, degraded 7-fold faster, and accumulated to 160-fold higher levels. Quantitative modeling and depletion of mitochondrial factors LRPPRC and FASTKD5 identified critical points of mitochondrial regulatory control, revealing that the mitonuclear expression disparities intrinsically arise from the highly polycistronic nature of human mitochondrial pre-mRNA. We propose that resolving these differences requires a 100-fold slower mitochondrial translation rate, illuminating the mitoribosome as a nexus of mitonuclear co-regulation., Competing Interests: Declaration of interests R.I. is a founder, board member, and shareholder of Cellforma, unrelated to the present work., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Ribosomes across species contain subsets of zinc finger proteins that play structural roles by binding to rRNA. While the majority of these zinc fingers belong to the C2-C2 type, the large subunit protein L36 in bacteria and mitochondria exhibits an atypical C2-CH motif. To comprehend the contribution of each coordinating residue in S. cerevisiae bL36m to mitoribosome assembly and function, we engineered and characterized strains carrying single and double mutations in the zinc coordinating residues. Our findings reveal that although all four residues markedly influence protein stability, C to A mutations in C66 and/or C69 have a more pronounced effect compared to those at C82 and H88. Importantly, protein stability directly correlates with the assembly and function of the mitoribosome and the growth rate of yeast in respiratory conditions. Mass spectrometry analysis of large subunit particles indicates that strains deleted for bL36m or expressing mutant variants have defective assembly of the L7/L12 stalk base, limiting their functional competence. Furthermore, we employed a synthetic bL36m protein collection, including both wild-type and mutant proteins, to elucidate their ability to bind zinc. Our data indicate that mutations in C82 and, particularly, H88 allow for some zinc binding albeit inefficient or unstable, explaining the residual accumulation and activity in mitochondria of bL36m variants carrying mutations in these residues. In conclusion, stable zinc binding by bL36m is essential for optimal mitoribosome assembly and function. MS data are available via ProteomeXchange with identifierPXD046465., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. This research was supported by National Institute of General Medicine (NIGMS) R35 grant GM118141 (to AB)., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The mitochondrial genomes of apicomplexans comprise merely three protein-coding genes, alongside a set of thirty to forty genes encoding small RNAs (sRNAs), many of which exhibit homologies to rRNA from E. coli . The expression status and integration of these short RNAs into ribosomes remains unclear and direct evidence for active ribosomes within apicomplexan mitochondria is still lacking. In this study, we conducted small RNA sequencing on the apicomplexan Toxoplasma gondii to investigate the occurrence and function of mitochondrial sRNAs. To enhance the analysis of sRNA sequencing outcomes, we also re-sequenced the T. gondii mitochondrial genome using an improved organelle enrichment protocol and Nanopore sequencing. It has been established previously that the T. gondii genome comprises 21 sequence blocks that undergo recombination among themselves but that their order is not entirely random. The enhanced coverage of the mitochondrial genome allowed us to characterize block combinations at increased resolution. Employing this refined genome for sRNA mapping, we find that many small RNAs originated from the junction sites between protein-coding blocks and rRNA sequence blocks. Surprisingly, such block border sRNAs were incorporated into polysomes together with canonical rRNA fragments and mRNAs. In conclusion, apicomplexan ribosomes are active within polysomes and are indeed assembled through the integration of sRNAs, including previously undetected sRNAs with merged mRNA-rRNA sequences. Our findings lead to the hypothesis that T. gondii's block-based genome organization enables the dual utilization of mitochondrial sequences as both messenger RNAs and ribosomal RNAs, potentially establishing a link between the regulation of rRNA and mRNA expression., Competing Interests: ST, AH, ND, ZG, SM, PL, SW, CS No competing interests declared, (© 2024, Tetzlaff et al.)

Ribosomes are large macromolecular complexes composed of both proteins and RNA, that require a plethora of factors and post-transcriptional modifications for their biogenesis. In human mitochondria, the ribosomal RNA is post-transcriptionally modified at ten sites. The N4-methylcytidine (m 4 C) methyltransferase, METTL15, modifies the 12S rRNA of the small subunit at position C1486. The enzyme is essential for mitochondrial protein synthesis and assembly of the mitoribosome small subunit, as shown here and by previous studies. Here, we demonstrate that the m 4 C modification is not required for small subunit biogenesis, indicating that the chaperone-like activity of the METTL15 protein itself is an essential component for mitoribosome biogenesis.

Mitochondrial diseases are a group of disorders defined by defects in oxidative phosphorylation caused by nuclear- or mitochondrial-encoded gene mutations. A main cellular phenotype of mitochondrial disease mutations is redox imbalances and inflammatory signaling underlying pathogenic signatures of these patients. One method to rescue this cell death vulnerability is the inhibition of mitochondrial translation using tetracyclines. However, the mechanisms whereby tetracyclines promote cell survival are unknown. Here, we show that tetracyclines inhibit the mitochondrial ribosome and promote survival through suppression of endoplasmic reticulum (ER) stress. Tetracyclines increase mitochondrial levels of the mitoribosome quality control factor MALSU1 (Mitochondrial Assembly of Ribosomal Large Subunit 1) and promote its recruitment to the mitoribosome large subunit, where MALSU1 is necessary for tetracycline-induced survival and suppression of ER stress. Glucose starvation induces ER stress to activate the unfolded protein response and IRE1α-mediated cell death that is inhibited by tetracyclines. These studies establish a new interorganelle communication whereby inhibition of the mitoribosome signals to the ER to promote survival, implicating basic mechanisms of cell survival and treatment of mitochondrial diseases., (© 2023 The Authors.)

Mitochondria contain their own genetic information and a dedicated translation system to express it. The mitochondrial ribosome is assembled from mitochondrial-encoded RNA and nuclear-encoded ribosomal proteins. Assembly is coordinated in the mitochondrial matrix by biogenesis factors that transiently associate with the maturing particle. Here, we present a structural snapshot of a large mitoribosomal subunit assembly intermediate containing 7 biogenesis factors including the GTPases GTPBP7 and GTPBP10. Our structure illustrates how GTPBP10 aids the folding of the ribosomal RNA during the biogenesis process, how this process is related to bacterial ribosome biogenesis, and why mitochondria require two biogenesis factors in contrast to only one in bacteria., (© 2023. The Author(s).)

The human mitochondrial ribosome contains three [2Fe-2S] clusters whose assembly pathway, role, and implications for mitochondrial and metabolic diseases are unknown. Here, structure-function correlation studies show that the clusters play a structural role during mitoribosome assembly. To uncover the assembly pathway, we have examined the effect of silencing the expression of Fe-S cluster biosynthetic and delivery factors on mitoribosome stability. We find that the mitoribosome receives its [2Fe-2S] clusters from the GLRX5-BOLA3 node. Additionally, the assembly of the small subunit depends on the mitoribosome biogenesis factor METTL17, recently reported containing a [4Fe-4S] cluster, which we propose is inserted via the ISCA1-NFU1 node. Consistently, fibroblasts from subjects suffering from 'multiple mitochondrial dysfunction' syndrome due to mutations in BOLA3 or NFU1 display previously unrecognized attenuation of mitochondrial protein synthesis that contributes to their cellular and pathophysiological phenotypes. Finally, we report that, in addition to their structural role, one of the mitoribosomal [2Fe-2S] clusters and the [4Fe-4S] cluster in mitoribosome assembly factor METTL17 sense changes in the redox environment, thus providing a way to regulate organellar protein synthesis accordingly., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Mitochondrial translation occurs on the mitochondrial ribosome, also known as the mitoribosome. The assembly of mitoribosomes is a highly coordinated process. During mitoribosome biogenesis, various assembly factors transiently associate with the nascent ribosome, facilitating the accurate and efficient construction of the mitoribosome. However, the specific factors involved in the assembly process, the precise mechanisms, and the cellular compartments involved in this vital process are not yet fully understood. In this study, we discovered a crucial role for GTP-binding protein 8 (GTPBP8) in the assembly of the mitoribosomal large subunit (mt-LSU) and mitochondrial translation. GTPBP8 is identified as a novel GTPase located in the matrix and peripherally bound to the inner mitochondrial membrane. Importantly, GTPBP8 is specifically associated with the mt-LSU during its assembly. Depletion of GTPBP8 leads to an abnormal accumulation of mt-LSU, indicating that GTPBP8 is critical for proper mt-LSU assembly. Furthermore, the absence of GTPBP8 results in reduced levels of fully assembled 55S monosomes. This impaired assembly leads to compromised mitochondrial translation and, consequently, impaired mitochondrial function. The identification of GTPBP8 as an important player in these processes provides new insights into the molecular mechanisms underlying mitochondrial protein synthesis and its regulation., (© 2023. The Author(s).)

Cellular activities are commonly associated with dynamic proteomic changes at the subcellular level. Although several techniques are available to quantify whole-cell protein turnover dynamics, such measurements often lack sufficient spatial resolution at the subcellular level. Herein, we report the development of prox-SILAC method that combines proximity-dependent protein labeling (APEX2/HRP) with metabolic incorporation of stable isotopes (pulse-SILAC) to map newly synthesized proteins with subcellular spatial resolution. We apply prox-SILAC to investigate proteome dynamics in the mitochondrial matrix and the endoplasmic reticulum (ER) lumen. Our analysis reveals a highly heterogeneous distribution in protein turnover dynamics within macromolecular machineries such as the mitochondrial ribosome and respiratory complexes I-V, thus shedding light on their mechanism of hierarchical assembly. Furthermore, we investigate the dynamic changes of ER proteome when cells are challenged with stress or undergoing stimulated differentiation, identifying subsets of proteins with unique patterns of turnover dynamics, which may play key regulatory roles in alleviating stress or promoting differentiation. We envision that prox-SILAC could be broadly applied to profile protein turnover at various subcellular compartments, under both physiological and pathological conditions., (© 2023. The Author(s).)

Lung cancer has a high fatality rate and incidence rate. At present, the initial and progress mechanism of lung cancer has not been completely elucidated and new therapeutic targets still need to be developed. In this study, the screening process was based on lung cancer expression profile data and survival analysis. Mitochondrial ribosome protein L9 (MRPL9) was upregulated in lung cancer tissues and related to the poor overall survival rate and recurrence-free survival rate of lung cancer patients. Knockdown of MRPL9 inhibited the proliferation, sphere-formation, and migration ability of lung cancer cells. MRPL9 was associated with the c-MYC signaling pathway, and lung cancer patients with high expression of both MRPL9 and MYC had a poor prognosis. Furthermore, c-MYC was associated with the epithelial-mesenchymal transition (EMT) regulatory protein zinc finger E-box binding homeobox 1 (ZEB1) by bioinformatics analysis. The relationship between ZEB1 and c-MYC was further confirmed by interfering with c-MYC expression. MRPL9 is a potential therapeutic target for lung cancer and exerts its biological functions by affecting the transcription factor c-MYC thereby regulating the EMT regulator ZEB1., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier GmbH. All rights reserved.)

The mitochondrial ribosome (mitoribosome) has diverged drastically from its evolutionary progenitor, the bacterial ribosome. Structural and compositional diversity is particularly striking in the phylum Euglenozoa, with an extraordinary protein gain in the mitoribosome of kinetoplastid protists. Here we report an even more complex mitoribosome in diplonemids, the sister-group of kinetoplastids. Affinity pulldown of mitoribosomal complexes from Diplonema papillatum, the diplonemid type species, demonstrates that they have a mass of > 5 MDa, contain as many as 130 integral proteins, and exhibit a protein-to-RNA ratio of 11:1. This unusual composition reflects unprecedented structural reduction of ribosomal RNAs, increased size of canonical mitoribosomal proteins, and accretion of three dozen lineage-specific components. In addition, we identified >50 candidate assembly factors, around half of which contribute to early mitoribosome maturation steps. Because little is known about early assembly stages even in model organisms, our investigation of the diplonemid mitoribosome illuminates this process. Together, our results provide a foundation for understanding how runaway evolutionary divergence shapes both biogenesis and function of a complex molecular machine., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The mitochondrial ribosome (mitoribosome) is a multicomponent machine that has unique structural features. Biogenesis of the human mitoribosome includes correct maturation and folding of the mitochondria-encoded RNA components (12S and 16S mt-rRNAs, and mt-tRNAVal) and their assembly together with 82 nucleus-encoded mitoribosomal proteins. This complex process requires the coordinated action of multiple assembly factors. Recent advances in single-particle cryo-electron microscopy (cryo-EM) have provided detailed insights into the specific functions of several mitoribosome assembly factors and have defined their timing. In this review we summarize mitoribosomal small (mtSSU) and large subunit (mtLSU) biogenesis based on structural findings, and we discuss potential crosstalk between mtSSU and mtLSU assembly pathways as well as coordination between mitoribosome biogenesis and other processes involved in mitochondrial gene expression., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Insulin-degrading enzyme (IDE) is a highly conserved metalloprotease that is mainly localized in the cytosol. Although IDE can degrade insulin and some other low molecular weight substrates efficiently, its ubiquitous expression suggests additional functions supported by experimental findings, such as a role in stress responses and cellular protein homeostasis. The translation of a long full-length IDE transcript has been reported to result in targeting to mitochondria, but the role of IDE in this compartment is unknown. To obtain initial leads on the function of IDE in mitochondria, we used a proximity biotinylation approach to identify proteins interacting with wild-type and protease-dead IDE targeted to the mitochondrial matrix. We find that IDE interacts with multiple mitochondrial ribosomal proteins as well as with proteins involved in the synthesis and assembly of mitochondrial complex I and IV. The mitochondrial interactomes of wild type and mutant IDE are highly similar and do not reveal any likely proteolytic IDE substrates. We speculate that IDE could adopt similar additional non-proteolytic functions in mitochondria as in the cytosol, acting as a chaperone and contributing to protein homeostasis and stress responses.

Background: Circular RNAs (circRNAs) are a large class of mammalian RNAs. Several protein products translated by circRNAs have been reported to be involved in the development of various tissues and systems; however, their physiological functions in male reproduction have yet not been explored., Results: Here, we report an endogenous circRNA (circRsrc1) that encodes a novel 161-amino-acid protein which we named Rsrc1-161aa through circRNA sequencing coupled with mass spectrometry analysis on mouse testicular tissues. Deletion of Rsrc1-161aa in mice impaired male fertility with a significant decrease in sperm count and motility due to dysfunctions of mitochondrial energy metabolism. A series of in vitro rescue experiments revealed that circRsrc1 regulates mitochondrial functions via its encoded protein Rsrc1-161aa. Mechanistically, Rsrc1-161aa directly interacts with mitochondrial protein C1qbp and enhances its binding activity to mitochondrial mRNAs, thereby regulating the assembly of mitochondrial ribosomes and affecting the translation of oxidative phosphorylation (OXPHOS) proteins and mitochondrial energy metabolism., Conclusions: Our studies reveal that Rsrc1-161aa protein encoded by circRsrc1 regulates mitochondrial ribosome assembly and translation during spermatogenesis, thereby affecting male fertility., (© 2023. The Author(s).)

Ribosomopathies are rare congenital disorders associated with defective ribosome biogenesis due to pathogenic variations in genes that encode proteins related to ribosome function and biogenesis. Defects in ribosome biogenesis result in a nucleolar stress response involving the TP53 tumor suppressor protein and impaired protein synthesis leading to a deregulated translational output. Despite the accepted notion that ribosomes are omnipresent and essential for all cells, most ribosomopathies show tissue-specific phenotypes affecting blood cells, hair, spleen, or skin. On the other hand, defects in mitochondrial ribosome biogenesis are associated with a range of clinical manifestations affecting more than one organ. Intriguingly, the deregulated ribosomal function is also a feature in several human malignancies with a selective upregulation or downregulation of specific ribosome components. Here, we highlight the clinical conditions associated with defective ribosome biogenesis in the nucleus and mitochondria with a description of the affected genes and the implicated pathways, along with a note on the treatment strategies currently available for these disorders., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2022 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Mitochondrial ribosomes synthesize essential components of the oxidative phosphorylation (OXPHOS) system in a tightly regulated process. In the yeast Saccharomyces cerevisiae, mitochondrial mRNAs require specific translational activators, which orchestrate protein synthesis by recognition of their target gene's 5'-untranslated region (UTR). Most of these yeast genes lack orthologues in mammals, and only one such gene-specific translational activator has been proposed in humans-TACO1. The mechanism by which TACO1 acts is unclear because mammalian mitochondrial mRNAs do not have significant 5'-UTRs, and therefore must promote translation by alternative mechanisms. In this study, we examined the role of the TACO1 orthologue in yeast. We found this 29 kDa protein to be a general mitochondrial translation factor, Dpc29, rather than a COX1-specific translational activator. Its activity was necessary for the optimal expression of OXPHOS mtDNA reporters, and mutations within the mitoribosomal large subunit protein gene MRP7 produced a global reduction of mitochondrial translation in dpc29Δ cells, indicative of a general mitochondrial translation factor. Northern-based mitoribosome profiling of dpc29Δ cells showed higher footprint frequencies at the 3' ends of mRNAs, suggesting a role in translation post-initiation. Additionally, human TACO1 expressed at native levels rescued defects in dpc29Δ yeast strains, suggesting that the two proteins perform highly conserved functions., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Eukaryotes contain two sets of genomes: the nuclear genome and the mitochondrial genome. The mitochondrial genome transcripts 13 mRNAs that encode 13 essential proteins for the oxidative phosphorylation complex, 2 rRNAs (12s rRNA and 16s rRNA), and 22 tRNAs. The proper assembly and maturation of the mitochondrial ribosome (mitoribosome) are critical for the translation of the 13 key proteins and the function of the mitochondrion. Human ribosome-binding factor A (hsRBFA) is a mitoribosome assembly factor that binds with helix 28, helix 44 and helix 45 of 12S rRNA and facilitates the transcriptional modification of 12S rRNA during the mitoribosomal biogenesis. Previous research mentioned that the malfunction of hsRBFA will induce the instability of mitoribosomes and affect the function of mitochondria, but the mechanisms underlying the interaction between hsRBFA and 12S rRNA and its influence on mitochondrial function are still unknown. In this study, we found that hsRBFA binds with double strain RNA (dsRNA) through its whole N-terminus (Nt) instead of the KH-like domain alone, which is different from the other homologous. Furthermore, we mapped the key residues that affected the RNA binding and maturation of mitoribosomes in vitro. Finally, we investigated how these residues affect mitochondrial functions in detail and systematically., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Mitochondrial ribosomes (mitoribosomes) synthesize proteins encoded within the mitochondrial genome that are assembled into oxidative phosphorylation complexes. Thus, mitoribosome biogenesis is essential for ATP production and cellular metabolism 1 . Here we used cryo-electron microscopy to determine nine structures of native yeast and human mitoribosomal small subunit assembly intermediates, illuminating the mechanistic basis for how GTPases are used to control early steps of decoding centre formation, how initial rRNA folding and processing events are mediated, and how mitoribosomal proteins have active roles during assembly. Furthermore, this series of intermediates from two species with divergent mitoribosomal architecture uncovers both conserved principles and species-specific adaptations that govern the maturation of mitoribosomal small subunits in eukaryotes. By revealing the dynamic interplay between assembly factors, mitoribosomal proteins and rRNA that are required to generate functional subunits, our structural analysis provides a vignette for how molecular complexity and diversity can evolve in large ribonucleoprotein assemblies., (© 2022. The Author(s).)

Mitochondrial ribosomes are specialized to translate the 13 membrane proteins encoded in the mitochondrial genome, which shapes the oxidative phosphorylation complexes essential for cellular energy metabolism. Despite the importance of mitochondrial translation (MT) control, it is challenging to identify and quantify the mitochondrial-encoded proteins because of their hydrophobic nature and low abundance. Here, we introduce a mass spectrometry-based proteomic method that combines biochemical isolation of mitochondria with pulse stable isotope labeling by amino acids in cell culture. Our method provides the highest protein identification rate with the shortest measurement time among currently available methods, enabling us to quantify 12 of the 13 mitochondrial-encoded proteins. We applied this method to uncover the global picture of (post-)translational regulation of both mitochondrial- and nuclear-encoded subunits of oxidative phosphorylation complexes. We found that inhibition of MT led to degradation of orphan nuclear-encoded subunits that are considered to form subcomplexes with the mitochondrial-encoded subunits. This method should be readily applicable to study MT programs in many contexts, including oxidative stress and mitochondrial disease., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The biogenesis of mitoribosomes is an intricate process that relies on the coordinated synthesis of nuclear-encoded mitoribosomal proteins (MRPs) in the cytosol, their translocation across mitochondrial membranes, the transcription of rRNA molecules in the matrix as well as the assembly of the roughly 80 different constituents of the mitoribosome. Numerous chaperones, translocases, processing peptidases, and assembly factors of the cytosol and in mitochondria support this complex reaction. The budding yeast Saccharomyces cerevisiae served as a powerful model organism to unravel the different steps by which MRPs are imported into mitochondria, fold into their native structures, and assemble into functional ribosomes.In this chapter, we provide established protocols to study these different processes experimentally. In particular, we describe methods to purify mitochondria from yeast cells, to import radiolabeled MRPs into isolated mitochondria, and to elucidate the assembly reaction of MRPs by immunoprecipitation. These protocols and the list of dos and don'ts will enable beginners and experienced scientists to study the import and assembly of MRPs., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Mitoribosome biogenesis is a complex and energetically costly process that involves RNA elements encoded in the mitochondrial genome and mitoribosomal proteins most frequently encoded in the nuclear genome. The process is catalyzed by extra-ribosomal proteins, nucleus-encoded assembly factors that act in all stages of the assembly process to coordinate the processing and maturation of ribosomal RNAs with the hierarchical association of ribosomal proteins. Biochemical studies and recent cryo-EM structures of mammalian mitoribosomes have provided hints regarding their assembly. In this general concept chapter, we will briefly describe the current knowledge, mainly regarding the mammalian mitoribosome biogenesis pathway and factors involved, and will emphasize the biological sources and approaches that have been applied to advance the field., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Faithful expression of the mitochondrial genome is required for the synthesis of the oxidative phosphorylation complexes and cell fitness. In humans, mitochondrial DNA (mtDNA) encodes 13 essential subunits of four oxidative phosphorylation complexes along with tRNAs and rRNAs needed for the translation of these proteins. Protein synthesis occurs on unique ribosomes within the organelle. Over the last decade, the revolution in genetic diagnostics has identified disruptions to the faithful synthesis of these 13 mitochondrial proteins as the largest group of inherited human mitochondrial pathologies. All of the molecular steps required for mitochondrial protein synthesis can be affected, from the genome to protein, including cotranslational quality control. Here, we describe methodologies for the biochemical separation of mitochondrial ribosomes from cultured human cells for RNA and protein analysis. Our method has been optimized to facilitate analysis for low-level sample material and thus does not require prior organelle enrichment., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Mitochondria maintain their own translational machinery that is responsible for the synthesis of essential components of the oxidative phosphorylation system. The mammalian mitochondrial translation system differs significantly from its cytosolic and bacterial counterparts. Here, we describe detailed protocols for efficient in vitro reconstitution of the mammalian mitochondrial translation initiation complex, which can be further used for mechanistic analyses of different aspects of mitochondrial translation., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

To understand the human mitochondrial translation process, tools are required to dissect this system at a global scale. The mechanisms and regulation of translation in mitochondria are different from those in the cytosol, and mitochondrial ribosomes have distinct biochemical properties. In this chapter, we describe in detail the modifications we have made to the ribosome profiling approach to adapt it to the unique characteristics of the human mitochondrial ribosome. This approach maximizes the fraction of mitochondrial ribosomes recovered, providing a snapshot of the mitochondrial translation landscape with minimal bias. We also describe the use of mouse lysate as an internal spike-in control for normalization, allowing quantification of global changes in translation across samples. Finally, we outline the bioinformatic pipelines to process the raw reads and identify mitoribosome A sites in the absence of untranslated regions flanking open reading frames. This method offers a subcodon-resolution time-sensitive global approach to explore the mitochondrial translation process in human cells., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Studies of yeast mitoribosome assembly have been historically hampered by the difficulty of generating mitoribosome protein-coding gene deletion strains with a stable mitochondrial genome. The identification of mitochondrial DNA-stabilizing approaches allows for the generation of a complete set of yeast deletion strains covering all mitoribosome proteins and known assembly factors. These strains can be used to analyze the integrity and assembly state of mitoribosomes by determining the sedimentation profile of these structures by sucrose gradient centrifugation of mitochondrial extracts, coupled to mass spectrometry analysis of mitoribosome composition. Subsequent hierarchical cluster analysis of mitoribosome subassemblies accumulated in mutant strains reveals details regarding the order of protein association during the mitoribosome biogenetic process. These strains also allow the expression of truncated protein variants to probe the role of mitochondrion-specific protein extensions, the relevance of protein cofactors, or the importance of RNA-protein interactions in functional sites of the mitoribosome. In this chapter, we will detail the methodology involved in these studies., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cryogenic milling, or cryomilling, involves the use of liquid nitrogen to lower the temperature of the biological material and/or the milling process. When applied to the study of subcellular or suborganellar structures and processes, it allows for their rapid extraction from whole cells frozen in the physiological state of choice. This approach has proven to be useful for the study of yeast mitochondrial ribosomes. Following cryomilling of 100 mL of yeast culture, conveniently tagged mitochondrial ribosomes can be immunoprecipitated and purified in native conditions. These ribosomes are suitable for the application of downstream approaches. These include mitoribosome profiling to analyze the mitochondrial translatome or mass spectrometry analyses to assess the mitoribosome proteome in normal growth conditions or under stress, as described in this method., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

RNA-binding proteins and mitochondrial ribosomes have been found to be linchpins of mitochondrial gene expression in health and disease. The expanding repertoire of proteins that bind and regulate the mitochondrial transcriptome has necessitated the development of new tools and methods to examine their molecular functions. Next-generation sequencing technologies have advanced the RNA biology field through application of high-throughput methods to study RNA-protein interactions. Here we describe a digital RNase footprinting method to analyze protein and ribosome interactions with mitochondrially encoded transcripts that provides insight into their mechanisms and minimal binding sites. We provide details on RNase digestion and next-generation sequencing, along with computational analyses and visualization of the binding targets within the mitochondrial transcriptome., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The ribosome is among the most complex and ancient cellular macromolecular assemblies that plays a central role in protein biosynthesis in all living cells. Its function of translation of genetic information encoded in messenger RNA into protein molecules also extends to subcellular compartments in eukaryotic cells such as apicoplasts, chloroplasts, and mitochondria. The origin of mitochondria is primarily attributed to an early endosymbiotic event between an alpha-proteobacterium and a primitive (archaeal) eukaryotic cell. The timeline of mitochondrial acquisition, the nature of the host, and their diversification have been studied in great detail and are continually being revised as more genomic and structural data emerge. Recent advancements in high-resolution cryo-EM structure determination have provided architectural details of mitochondrial ribosomes (mitoribosomes) from various species, revealing unprecedented diversifications among them. These structures provide novel insights into the evolution of mitoribosomal structure and function. Here, we present a brief overview of the existing mitoribosomal structures in the context of the eukaryotic evolution tree showing their diversification from their last common ancestor., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Mitochondrial protein biosynthesis is maintained by an interplay between the mitochondrial ribosome (mitoribosome) and a large set of protein interaction partners. This interactome regulates a diverse set of functions, including mitochondrial gene expression, translation, protein quality control, and respiratory chain assembly. Hence, robust methods to biochemically and structurally analyze this molecular machinery are required to understand the sophisticated regulation of mitochondrial protein biosynthesis. In this chapter, we present detailed protocols for immunoprecipitation, sucrose cushions, and linear sucrose gradients to purify and analyze mitoribosomes and their interaction partners., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Mitochondrial protein synthesis is essential for the life of aerobic eukaryotes. Without it, oxidative phosphorylation cannot be coupled. Evolution has shaped a battery of factors and machinery that are key to production of just a handful of critical proteins. In this general concept chapter, we attempt to briefly summarize our current knowledge of the overall process in mitochondria from a variety of species, breaking this down to the four parts of translation: initiation, elongation, termination, and recycling. Where appropriate, we highlight differences between species and emphasize gaps in our understanding. Excitingly, with the current revolution in cryoelectron microscopy and mitochondrial genome editing, it is highly likely that many of these gaps will be resolved in the near future. However, the absence of a faithful in vitro reconstituted system to study mitochondrial translation is still problematic., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Translation in mitochondria is mediated by mitochondrial ribosomes, or mitoribosomes, complex ribonucleoprotein machines with dual genetic origin. Mitoribosomes in trypanosomatid parasites diverged markedly from their bacterial ancestors and other eukaryotic lineages in terms of protein composition, rRNA content, and overall architecture, yet their core functional elements remained conserved. Recent cryo-electron microscopy studies provided atomic models of trypanosomatid large and small mitoribosomal subunits and their precursors, making these parasites the organisms with the best-understood biogenesis of mitoribosomes. The structures revealed molecular mechanisms and players involved in the assembly of mitoribosomes not only in the parasites, but also in eukaryotes in general., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Translation termination requires release factors that read a STOP codon in the decoding center and subsequently facilitate the hydrolysis of the nascent peptide chain from the peptidyl tRNA within the ribosome. In human mitochondria eleven open reading frames terminate in the standard UAA or UAG STOP codon, which can be recognized by mtRF1a, the proposed major mitochondrial release factor. However, two transcripts encoding for COX1 and ND6 terminate in the non-conventional AGA or AGG codon, respectively. How translation termination is achieved in these two cases is not known. We address this long-standing open question by showing that the non-canonical release factor mtRF1 is a specialized release factor that triggers COX1 translation termination, while mtRF1a terminates the majority of other mitochondrial translation events including the non-canonical ND6. Loss of mtRF1 leads to isolated COX deficiency and activates the mitochondrial ribosome-associated quality control accompanied by the degradation of COX1 mRNA to prevent an overload of the ribosome rescue system. Taken together, these results establish the role of mtRF1 in mitochondrial translation, which had been a mystery for decades, and lead to a comprehensive picture of translation termination in human mitochondria., (© 2022. The Author(s).)

Mitoribosomes of green algae display a great structural divergence from their tracheophyte relatives, with fragmentation of both rRNA and proteins as a defining feature. Here, we report a 2.9 Å resolution structure of the mitoribosome from the alga Polytomella magna harbouring a reduced rRNA split into 13 fragments. We found that the rRNA contains a non-canonical reduced form of the 5S, as well as a permutation of the LSU domain I. The mt-5S rRNA is stabilised by mL40 that is also found in mitoribosomes lacking the 5S, which suggests an evolutionary pathway. Through comparison to other ribosomes with fragmented rRNAs, we observe that the pattern is shared across large evolutionary distances, and between cellular compartments, indicating an evolutionary convergence and supporting the concept of a primordial fragmented ribosome. On the protein level, eleven peripherally associated HEAT-repeat proteins are involved in the binding of 3' rRNA termini, and the structure features a prominent pseudo-trimer of one of them (mL116). Finally, in the exit tunnel, mL128 constricts the tunnel width of the vestibular area, and mL105, a homolog of a membrane targeting component mediates contacts with an inner membrane bound insertase. Together, the structural analysis provides insight into the evolution of the ribosomal machinery in mitochondria., (© 2022. The Author(s).)

Ribosome biogenesis is a complicated, multistage process coordinated by ribosome assembly factors. Ribosome binding factor A (RbfA) is a bacterial one, which possesses a single structural type-II KH domain. By this domain, RbfA binds to a 16S rRNA precursor in small ribosomal subunits to promote its 5'-end processing. The human RbfA homolog, mtRbfA, binds to 12S rRNAs in the mitoribosomal small subunits and promotes its critical maturation process, the dimethylation of two highly conserved consecutive adenines, which differs from that of RbfA. However, the structural basis of the mtRbfA-mediated maturation process is poorly understood. Herein, we report the 1 H, 15 N, and 13 C resonance assignments of the KH domain of mtRbfA and its solution structure. The mtRbfA domain adopts essentially the same α1-β1-β2-α2(kinked)-β3 topology as the type-II KH domain. Comparison with the RbfA counterpart showed structural differences in specific regions that function as a putative RNA-binding site. Particularly, the α2 helix of mtRbfA forms a single helix with a moderate kink at the Ser-Ala-Ala sequence, whereas the corresponding α2 helix of RbfA is interrupted by a distinct kink at the Ala-x-Gly sequence, characteristic of bacterial RbfA proteins, to adopt an α2-kink-α3 conformation. Additionally, the region linking α1 and β1 differs considerably in the sequence and structure between RbfA and mtRbfA. These findings suggest some variations of the RNA-binding mode between them and provide a structural basis for mtRbfA function in mitoribosome biogenesis., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Genetic variations in mitoribosomal subunits and mitochondrial transcription factors are related to type 2 diabetes. However, the role of islet mitoribosomes in the development of type 2 diabetes has not been determined. We investigated the effects of the mitoribosomal gene on β-cell function and glucose homeostasis. Mitoribosomal gene expression was analyzed in datasets from the NCBI GEO website (GSE25724, GSE76894, and GSE76895) and the European Nucleotide Archive (ERP017126), which contain the transcriptomes of type 2 diabetic and nondiabetic organ donors. We found deregulation of most mitoribosomal genes in islets from individuals with type 2 diabetes, including partial downregulation of CRIF1. The phenotypes of haploinsufficiency in a single mitoribosomal gene were examined using β-cell-specific Crif1 (Mrpl59) heterozygous-deficient mice. Crif1 beta+/- mice had normal glucose tolerance, but their islets showed a loss of first-phase glucose-stimulated insulin secretion. They also showed increased β-cell mass associated with higher expression of Reg family genes. However, Crif1 beta+/- mice showed earlier islet failure in response to high-fat feeding, which was exacerbated by aging. Haploinsufficiency of a single mitoribosomal gene predisposes rodents to glucose intolerance, which resembles the early stages of type 2 diabetes in humans., (© 2022. The Author(s).)

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 complex 2 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., (© 2022. The Author(s).)