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Iron-sulfur clusters bridge mitoribosomal proteins. (A) Each of the two 2Fe-2S clusters is coordinated by mitochondria-specific components (red) from two different proteins. Superposition of 2Fe-2S cluster-binding sites in the human mitoribosome with corresponding sites in E. coli ribosome (PDB ID: 7K00, white) reveals that mitoribosomal protein elements participating in 2Fe-2S coordination compensate for missing rRNA helices. (B) The conserved overall fold of bS18 contributes to Zn 2+ or 2Fe-2S binding in the mitoribosome.
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The mitoribosome regulates cellular energy production, and its dysfunction is associated with aging. Inhibition of the mitoribosome can be caused by off-target binding of antimicrobial drugs and was shown to be coupled with a bilateral decreased visual acuity. Previously, we reported mitochondria-specific protein aspects of the mitoribosome, and in...
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... Fe-S clusters are part of the respiratory complexes I, II and III and their formation via the ISC pathway is critical for the electron transport chain. Moreover, a recent report shows that Fe-S clusters are coordinated as structural features of the mitoribosome suggesting another vital link between the ISC pathway and the mitochondrial functions [71]. The successful introduction of the first LYRM protein within the ISC pathway might have paved the way for their expansion to other key sites of mitochondrial metabolism for tighter control. ...
Core mitochondrial processes such as the electron transport chain, protein translation and the formation of Fe–S clusters (ISC) are of prokaryotic origin and were present in the bacterial ancestor of mitochondria. In animal and fungal models, a family of small Leu-Tyr-Arg motif-containing proteins (LYRMs) uniformly regulates the function of mitochondrial complexes involved in these processes. The action of LYRMs is contingent upon their binding to the acylated form of acyl carrier protein (ACP). This study demonstrates that LYRMs are structurally and evolutionarily related proteins characterized by a core triplet of α-helices. Their widespread distribution across eukaryotes suggests that 12 specialized LYRMs were likely present in the last eukaryotic common ancestor to regulate the assembly and folding of the subunits that are conserved in bacteria but that lack LYRM homologues. The secondary reduction of mitochondria to anoxic environments has rendered the function of LYRMs and their interaction with acylated ACP dispensable. Consequently, these findings strongly suggest that early eukaryotes installed LYRMs in aerobic mitochondria as orchestrated switches, essential for regulating core metabolism and ATP production.
... A notable feature of the cryo-EM map is the presence of continuous tubular densities associated with rRNA that we assigned as polyamines, justified by the electrostatic and hydrogen-bonding interactions at the amine groups ( Supplementary Fig. 5). Thus, in addition to a previously assigned spermine (SPM) 45 , we detected one triamine spermidine (SPD) in the SSU, and a diamine putrescine (PUT) and three SPDs in the LSU. The identified polyamines are bound to negatively charged rRNA where mitochondria specific changes occur, providing required structural support ( Fig. 4; Supplementary Fig. 5). ...
... The PUT is found in the exit tunnel upper region of uL22m hairpin. Finally, the model contains six protein modifications; three ironsulfur clusters, one of which is coordinated by uL10m and mL66, and a molecule of nicotinamide adenosine dinucleotide (NAD) reported previously 26,45,46 (Fig. 1b, Supplementary Table 1, Supplementary Table 2). We also identified a disulfide bond in one of the two redox active cysteine pairs in mS37 (CHCHD1) (Fig. 2) that is oxidized in the intermembrane space during mitochondrial import, representing a quality control mechanism 47 . ...
... A well-resolved density, combined with the mass-spectrometry SILNAS 40,57 approach further allowed us to validate the complete set of the rRNA modifications, including six mitochondria specific, and propose a role in stabilizing the decoding center (Fig. 5, Supplementary Figs. 4 and 5). Close to the decoding center, the residue G899 was recently shown to interact with streptomycin in human mtSSU 45 . Its methylated to m 7 G527 in E.coli ( Supplementary Fig. 11) and this modification partly contributes to streptomycin sensitivity 58 . ...
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 selective impact of CLPXP on mtLSU was surprising, because Perrault syndrome is triggered by mutant HARS2 / LARS2 / ERAL1 all of which are associated with the mtSSU. In addition, the progressive sensorineural deafness due to the chronic administration of aminoglycoside antibiotics is also caused by neurotoxic interference and genetic variance at the mtSSU [193][194][195]. A detailed review of mitoribosomal pathology underlying progressive deafness as in PRLTS was recently compiled [49]. ...
Mitochondrial matrix peptidase CLPP is crucial during cell stress. Its loss causes Perrault syndrome type 3 (PRLTS3) with infertility, neurodegeneration and growth deficit. Its target proteins are disaggregated by CLPX, which also regulates heme biosynthesis via unfolding ALAS enzyme, providing access of pyridoxal-5-phosphate (PLP). Despite efforts in diverse organisms with multiple techniques, CLPXP substrates remain controversial. Here, avoiding recombinant overexpression, we employed complexomics in mitochondria from three mouse tissues to identify endogenous targets. CLPP absence caused accumulation and dispersion of CLPX-VWA8 as AAA+ unfoldases, and of PLPBP. Similar changes and CLPX-VWA8 comigration were evident for mitoribosomal central protuberance clusters, translation factors like GFM1-HARS2, RNA granule components LRPPRC-SLIRP, and enzymes OAT-ALDH18A1. Mitochondrially translated proteins in testis showed reductions to <30% for MTCO1-3, misassembly of complex-IV supercomplex, and accumulated metal-binding assembly factors COX15-SFXN4. Indeed, heavy metal levels were increased for iron, molybdenum, cobalt and manganese. RT-qPCR showed compensatory downregulation only for Clpx mRNA, most accumulated proteins appeared transcriptionally upregulated. Immunoblots validated VWA8, MRPL38, MRPL18, GFM1 and OAT accumulation. Coimmunoprecipitation confirmed CLPX binding to MRPL38, GFM1 and OAT, so excess CLPX and PLP may affect their activity. Our data elucidate mechanistically the mitochondrial translation fidelity deficits, which underlie progressive hearing impairment in PRLTS3.
... Generally, most of these antibiotic-ribosome structures are reported at resolutions ranging from 2.5 to 3.5 Å, with a few recent studies obtaining resolutions below 2.5 Å (refs. [7][8][9][10][11] and, to date, only one study better than 2 Å (ref. 12) (Supplementary Table 1). ...
... A big difference between the two structures is the interaction of ring II of Str with a second Mg 2+ ion in the mitoribosome that is not present in bacterial ribosomes, probably due to differences in uS12. Such differences represent attractive areas for future development of streptomycin derivatives that exhibit fewer side-effects due to interaction with human mitoribosomes 11 . In many instances, our analysis included more than one antibiotic member from the same family, such as for the tetracyclines, orthosomycins, lincosamides and pleuromutilins (Fig. 4d-l). ...
... For the antibiotics analyzed in this study, the ribosomal binding sites are highly conserved, suggesting that the interactions observed here on E. coli ribosomes are likely to be conserved on ribosomes from other organisms. This is also supported by the high similarity between the structures determined here of streptomycin, lincomycin and spectinomycin on the E. coli ribosome with recent sub-2.5 Å structures on the human mitochondrial (Fig. 4c) 11 , L. monocytogenes (Fig. 4i) 47 and E. faecalis ribosomes (Extended Data Fig. 9a-c) 8 , respectively. A systematic structural analysis of antibiotic-ribosome complexes from diverse species will be required to investigate this hypothesis further. ...
The ribosome is a major target for clinically used antibiotics, but multidrug resistant pathogenic bacteria are making our current arsenal of antimicrobials obsolete. Here we present cryo-electron-microscopy structures of 17 distinct compounds from six different antibiotic classes bound to the bacterial ribosome at resolutions ranging from 1.6 to 2.2 Å. The improved resolution enables a precise description of antibiotic–ribosome interactions, encompassing solvent networks that mediate multiple additional interactions between the drugs and their target. Our results reveal a high structural conservation in the binding mode between antibiotics with the same scaffold, including ordered water molecules. Water molecules are visualized within the antibiotic binding sites that are preordered, become ordered in the presence of the drug and that are physically displaced on drug binding. Insight into RNA–ligand interactions will facilitate development of new antimicrobial agents, as well as other RNA-targeting therapies.
... The position of the two known ototoxic variants is indicated with red, bold font. (B) Inter-subunit face of the SSU as observed in the 2.2-Å cryo-EM structure of the human mito-ribosome (Itoh et al., 2021;Itoh et al., 2022). 12S mt-rRNA, red ribbon; SSU r-proteins, pink surface. ...
... At the same time, the availability of near-atomic resolution structures of mammalian mito-ribosome structures caught in the process of translation finally permits an assessment of their disruptive potential on mt-RNA structure. In particular, the recent 2.2-Å cryo-EM structure of the human mito-ribosome, generated by the Amunts laboratory, has the highest resolution ever achieved for a mito-ribosome and resulted in an extremely well-refined model (Itoh et al., 2021;Itoh et al., 2022), which permits the structural dissection of mt-rRNA variants with unprecedented detail. The new theoretical framework, together with the latest structural tools, was used here to structurally dissect the large collection of mt-rRNA variants reportedly associated with deafness, aiming at ascertaining which base changes could lead to non-silent phenotypes. ...
... 05 the single-stranded stretch linking h3 and the functionally important central pseudoknot (helices h1 and h2) to h19 (Supplementary Figure S1A) (Brink et al., 1993;Poot et al., 1996;Poot et al., 1998). In the 2.2-Å mito-ribosomal structure (Itoh et al., 2021;Itoh et al., 2022), the O6 of 283G (m.930G) has been modeled 2.39 Å away from Arg 47 of MRPS12/uS12m ( Figure 3A and Supplementary Figure S1A), whose equivalent position in the bacterial ribosome would be near sites of fidelity mutations ( Figure 2A). However, the two residues are likely quite disordered, as judged by the lack of clear density covering them (Supplementary Figure S1C). ...
The last few years have witnessed dramatic advances in our understanding of the structure and function of the mammalian mito-ribosome. At the same time, the first attempts to elucidate the effects of mito-ribosomal fidelity (decoding accuracy) in disease have been made. Hence, the time is right to push an important frontier in our understanding of mitochondrial genetics, that is, the elucidation of the phenotypic effects of mtDNA variants affecting the functioning of the mito-ribosome. Here, we have assessed the structural and functional role of 93 mitochondrial (mt-) rRNA variants thought to be associated with deafness, including those located at non-conserved positions. Our analysis has used the structural description of the human mito-ribosome of the highest quality currently available, together with a new understanding of the phenotypic manifestation of mito-ribosomal-associated variants. Basically, any base change capable of inducing a fidelity phenotype may be considered non-silent. Under this light, out of 92 previously reported mt-rRNA variants thought to be associated with deafness, we found that 49 were potentially non-silent. We also dismissed a large number of reportedly pathogenic mtDNA variants, 41, as polymorphisms. These results drastically update our view on the implication of the primary sequence of mt-rRNA in the etiology of deafness and mitochondrial disease in general. Our data sheds much-needed light on the question of how mt-rRNA variants located at non-conserved positions may lead to mitochondrial disease and, most notably, provide evidence of the effect of haplotype context in the manifestation of some mt-rRNA variants.
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.
Iron-sulfur (Fe-S) clusters are an essential and ubiquitous class of protein-bound prosthetic centers that are involved in a broad range of biological processes (e.g. respiration, photosynthesis, DNA replication and repair and gene regulation) performing a wide range of functions including electron transfer, enzyme catalysis, and sensing. In a general manner, Fe-S clusters can gain or lose electrons through redox reactions, and are highly sensitive to oxidation, notably by small molecules such as oxygen and nitric oxide. The [2Fe-2S] and [4Fe-4S] clusters, the most common Fe-S cofactors, are typically coordinated by four amino acid side chains from the protein, usually cysteine thiolates, but other residues (e.g. histidine, aspartic acid) can also be found. While diversity in cluster coordination ensures the functional variety of the Fe-S clusters, the lack of conserved motifs makes new Fe-S protein identification challenging especially when the Fe-S cluster is also shared between two proteins as observed in several dimeric transcriptional regulators and in the mitoribosome. Thanks to the recent development of in cellulo, in vitro, and in silico approaches, new Fe-S proteins are still regularly identified, highlighting the functional diversity of this class of proteins. In this review, we will present three main functions of the Fe-S clusters and explain the difficulties encountered to identify Fe-S proteins and methods that have been employed to overcome these issues.
Friedreich’s ataxia (FA) is a debilitating, multisystemic disease caused by a depletion of frataxin (FXN), a mitochondrial iron-sulfur (Fe-S) cluster biogenesis factor. To understand the cellular pathogenesis of FA, we performed quantitative proteomics in FXN-deficient human cells. Nearly every annotated Fe-S cluster-containing protein was depleted, indicating that as a rule, cluster binding confers stability to Fe-S proteins. We also observed depletion of a small mitoribosomal assembly factor METTL17 and evidence of impaired mitochondrial translation. Using comparative sequence analysis, mutagenesis, biochemistry and cryoelectron microscopy we show that METTL17 binds to the mitoribosomal small subunit during late assembly and harbors a previously unrecognized [Fe4S4]²⁺ cluster required for its stability. METTL17 overexpression rescued the mitochondrial translation and bioenergetic defects, but not the cellular growth, of FXN-depleted cells. These findings suggest that METTL17 acts as an Fe-S cluster checkpoint: promoting translation of Fe-S cluster rich OXPHOS proteins only when Fe-S cofactors are replete.
Schizosaccharomyces pombe (fission yeast) is an attractive model for mitochondrial research. The organism resembles human cells in terms of mitochondrial inheritance, mitochondrial transport, sugar metabolism, mitogenome structure and dependence of viability on the mitogenome (the petite ‐negative phenotype). Transcriptions of these genomes produce only a few polycistronic transcripts, which then undergo processing as per the tRNA punctuation model. In general, the machinery for mitochondrial gene expression is structurally and functionally conserved between fission yeast and humans. Furthermore, molecular research on S. pombe is supported by a considerable number of experimental techniques and database resources. Owing to these advantages, fission yeast has significantly contributed to biomedical and fundamental research. Here, we review the current state of knowledge regarding S. pombe mitochondrial gene expression, and emphasise the pertinence of fission yeast as both a model and tool, especially for studies on mitochondrial translation.
The mitochondrial matrix peptidase CLPP is crucial during cell stress. Its loss causes Perrault syndrome type 3 (PRLTS3) with infertility, neurodegeneration, and a growth deficit. Its target proteins are disaggregated by CLPX, which also regulates heme biosynthesis via unfolding ALAS enzymes, providing access for pyridoxal-5′-phosphate (PLP). Despite efforts in diverse organisms with multiple techniques, CLPXP substrates remain controversial. Here, avoiding recombinant overexpression, we employed complexomics in mitochondria from three mouse tissues to identify endogenous targets. A CLPP absence caused the accumulation and dispersion of CLPX-VWA8 as AAA+ unfoldases, and of PLPBP. Similar changes and CLPX-VWA8 co-migration were evident for mitoribosomal central protuberance clusters, translation factors like GFM1-HARS2, the RNA granule components LRPPRC-SLIRP, and enzymes OAT-ALDH18A1. Mitochondrially translated proteins in testes showed reductions to <30% for MTCO1-3, the mis-assembly of the complex IV supercomplex, and accumulated metal-binding assembly factors COX15-SFXN4. Indeed, heavy metal levels were increased for iron, molybdenum, cobalt, and manganese. RT-qPCR showed compensatory downregulation only for Clpx mRNA; most accumulated proteins appeared transcriptionally upregulated. Immunoblots validated VWA8, MRPL38, MRPL18, GFM1, and OAT accumulation. Co-immunoprecipitation confirmed CLPX binding to MRPL38, GFM1, and OAT, so excess CLPX and PLP may affect their activity. Our data mechanistically elucidate the mitochondrial translation fidelity deficits which underlie progressive hearing impairment in PRLTS3.
