The human mitochondrial translation factor TACO1 alleviates mitoribosome stalling at polyproline stretches.

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.

Importance of conserved hydrophobic pocket region in yeast mitoribosomal mL44 protein for mitotranslation and transcript preference.

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.

GTPBP8 plays a role in mitoribosome formation in human mitochondria.

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.

Mitochondrial ribosome biogenesis and redox sensing.

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.

Structural basis for differential inhibition of eukaryotic ribosomes by tigecycline.

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.

The catalytic activity of methyltransferase METTL15 is dispensable for its role in mitochondrial ribosome biogenesis.

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 (m4C) 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 m4C 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.

Mitoribosome structure with cofactors and modifications reveals mechanism of ligand binding and interactions with L1 stalk.

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-tRNAVal. 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.

The Schizosaccharomyces pombe DEAD-box protein Mss116 is required for mitoribosome assembly and mitochondrial translation.

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.

Characterization of Mitoribosomal Small Subunit unit genes related immune and pharmacogenomic landscapes in renal cell carcinoma.

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.

A kinetic dichotomy between mitochondrial and nuclear gene expression processes.

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.

The zinc finger motif in the mitochondrial large ribosomal subunit protein bL36m is essential for optimal yeast mitoribosome assembly and function.

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.

Small RNAs from mitochondrial genome recombination sites are incorporated into T. gondii mitoribosomes.

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.

Mitochondrial protein synthesis quality control.

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.

Structural insights into the role of GTPBP10 in the RNA maturation of the mitoribosome.

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.

GTPBP8 is required for mitoribosomal biogenesis and mitochondrial translation.

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.

Spatially resolved mapping of proteome turnover dynamics with subcellular precision.

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.

Tetracyclines activate mitoribosome quality control and reduce ER stress to promote cell survival.

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.

BOLA3 and NFU1 link mitoribosome iron-sulfur cluster assembly to multiple mitochondrial dysfunctions syndrome.

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.

Insulin-Degrading Enzyme Interacts with Mitochondrial Ribosomes and Respiratory Chain Proteins.

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.

Identification of a novel therapeutic target for lung cancer: Mitochondrial ribosome protein L9.

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.