An in vitro system to silence mitochondrial gene expression.

The human mitochondrial genome encodes thirteen core subunits of the oxidative phosphorylation system, and defects in mitochondrial gene expression lead to severe neuromuscular disorders. However, the mechanisms of mitochondrial gene expression remain poorly understood due to a lack of experimental approaches to analyze these processes. Here, we present an in vitro system to silence translation in purified mitochondria. In vitro import of chemically synthesized precursor-morpholino hybrids allows us to target translation of individual mitochondrial mRNAs. By applying this approach, we conclude that the bicistronic, overlapping ATP8/ATP6 transcript is translated through a single ribosome/mRNA engagement. We show that recruitment of COX1 assembly factors to translating ribosomes depends on nascent chain formation. By defining mRNA-specific interactomes for COX1 and COX2, we reveal an unexpected function of the cytosolic oncofetal IGF2BP1, an RNA-binding protein, in mitochondrial translation. Our data provide insight into mitochondrial translation and innovative strategies to investigate mitochondrial gene expression.

Mitochondrial Protein Synthesis Adapts to Influx of Nuclear-Encoded Protein.

Mitochondrial ribosomes translate membrane integral core subunits of the oxidative phosphorylation system encoded by mtDNA. These translation products associate with nuclear-encoded, imported proteins to form enzyme complexes that produce ATP. Here, we show that human mitochondrial ribosomes display translational plasticity to cope with the supply of imported nuclear-encoded subunits. Ribosomes expressing mitochondrial-encoded COX1 mRNA selectively engage with cytochrome c oxidase assembly factors in the inner membrane. Assembly defects of the cytochrome c oxidase arrest mitochondrial translation in a ribosome nascent chain complex with a partially membrane-inserted COX1 translation product. This complex represents a primed state of the translation product that can be retrieved for assembly. These findings establish a mammalian translational plasticity pathway in mitochondria that enables adaptation of mitochondrial protein synthesis to the influx of nuclear-encoded subunits.

Molecular Wiring of a Mitochondrial Translational Feedback Loop.

The mitochondrial oxidative phosphorylation system comprises complexes assembled from subunits derived from mitochondrial and nuclear gene expression. Both genetic systems are coordinated by feedback loops, which control the synthesis of specific mitochondrial encoded subunits. Here, we studied how this occurs in the case of cytochrome b, a key subunit of mitochondrial complex III. Our data suggest the presence of a molecular rheostat consisting of two translational activators, Cbp3-Cbp6 and Cbs1, which operates at the mitoribosomal tunnel exit to connect translational output with assembly efficiency. When Cbp3-Cbp6 is engaged in assembly of cytochrome b, Cbs1 binds to the tunnel exit to sequester the cytochrome b-encoding mRNA, repressing its translation. After mediating complex III assembly, binding of Cbp3-Cbp6 to the tunnel exit replaces Cbs1 and the bound mRNA to permit cytochrome b synthesis. Collectively, the data indicate the molecular wiring of a feedback loop to regulate synthesis of a mitochondrial encoded protein.

Structural Insights into the Mechanism of Mitoribosomal Large Subunit Biogenesis.

In contrast to the bacterial translation machinery, mitoribosomes and mitochondrial translation factors are highly divergent in terms of composition and architecture. There is increasing evidence that the biogenesis of mitoribosomes is an intricate pathway, involving many assembly factors. To better understand this process, we investigated native assembly intermediates of the mitoribosomal large subunit from the human parasite Trypanosoma brucei using cryo-electron microscopy. We identify 28 assembly factors, 6 of which are homologous to bacterial and eukaryotic ribosome assembly factors. They interact with the partially folded rRNA by specifically recognizing functionally important regions such as the peptidyltransferase center. The architectural and compositional comparison of the assembly intermediates indicates a stepwise modular assembly process, during which the rRNA folds toward its mature state. During the process, several conserved GTPases and a helicase form highly intertwined interaction networks that stabilize distinct assembly intermediates. The presented structures provide general insights into mitoribosomal maturation.

Insights into mitoribosomal biogenesis from recent structural studies.

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.

Widespread use of unconventional targeting signals in mitochondrial ribosome proteins.

Mitochondrial ribosomes are complex molecular machines indispensable for respiration. Their assembly involves the import of several dozens of mitochondrial ribosomal proteins (MRPs), encoded in the nuclear genome, into the mitochondrial matrix. Proteomic and structural data as well as computational predictions indicate that up to 25% of yeast MRPs do not have a conventional N-terminal mitochondrial targeting signal (MTS). We experimentally characterized a set of 15 yeast MRPs in vivo and found that five use internal MTSs. Further analysis of a conserved model MRP, Mrp17/bS6m, revealed the identity of the internal targeting signal. Similar to conventional MTS-containing proteins, the internal sequence mediates binding to TOM complexes. The entire sequence of Mrp17 contains positive charges mediating translocation. The fact that these sequence properties could not be reliably predicted by standard methods shows that mitochondrial protein targeting is more versatile than expected. We hypothesize that structural constraints imposed by ribosome assembly interfaces may have disfavored N-terminal presequences and driven the evolution of internal targeting signals in MRPs.

Facioscapulohumeral Muscular Dystrophy is Associated With Altered Myoblast Proteome Dynamics.

Proteomic studies in facioscapulohumeral muscular dystrophy (FSHD) could offer new insight into disease mechanisms underpinned by post-transcriptional processes. We used stable isotope (deuterium oxide; D2O) labeling and peptide mass spectrometry to investigate the abundance and turnover rates of proteins in cultured muscle cells from two individuals affected by FSHD and their unaffected siblings (UASb). We measured the abundance of 4420 proteins and the turnover rate of 2324 proteins in each (n = 4) myoblast sample. FSHD myoblasts exhibited a greater abundance but slower turnover rate of subunits of mitochondrial respiratory complexes and mitochondrial ribosomal proteins, which may indicate an accumulation of "older" less viable mitochondrial proteins in myoblasts from individuals affected by FSHD. Treatment with a 2'-O-methoxyethyl modified antisense oligonucleotide targeting exon 3 of the double homeobox 4 (DUX4) transcript tended to reverse mitochondrial protein dysregulation in FSHD myoblasts, indicating the effect on mitochondrial proteins may be a DUX4-dependent mechanism. Our results highlight the importance of post-transcriptional processes and protein turnover in FSHD pathology and provide a resource for the FSHD research community to explore this burgeoning aspect of FSHD.

Nucleolar targeting in an early-branching eukaryote suggests a general mechanism for ribosome protein sorting.

The compartmentalised eukaryotic cell demands accurate targeting of proteins to the organelles in which they function, whether membrane-bound (like the nucleus) or non-membrane-bound (like the nucleolus). Nucleolar targeting relies on positively charged localisation signals and has received rejuvenated interest since the widespread recognition of liquid-liquid phase separation (LLPS) as a mechanism contributing to nucleolus formation. Here, we exploit a new genome-wide analysis of protein localisation in the early-branching eukaryote Trypanosoma brucei to analyse general nucleolar protein properties. T. brucei nucleolar proteins have similar properties to those in common model eukaryotes, specifically basic amino acids. Using protein truncations and addition of candidate targeting sequences to proteins, we show both homopolymer runs and distributed basic amino acids give nucleolar partition, further aided by a nuclear localisation signal (NLS). These findings are consistent with phase separation models of nucleolar formation and physical protein properties being a major contributing mechanism for eukaryotic nucleolar targeting, conserved from the last eukaryotic common ancestor. Importantly, cytoplasmic ribosome proteins, unlike mitochondrial ribosome proteins, have more basic residues - pointing to adaptation of physicochemical properties to assist segregation.

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.

A novel protein encoded by circRsrc1 regulates mitochondrial ribosome assembly and translation during spermatogenesis.

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.

Stress signaling and cellular proliferation reverse the effects of mitochondrial mistranslation.

Translation fidelity is crucial for prokaryotes and eukaryotic nuclear-encoded proteins; however, little is known about the role of mistranslation in mitochondria and its potential effects on metabolism. We generated yeast and mouse models with error-prone and hyper-accurate mitochondrial translation, and found that translation rate is more important than translational accuracy for cell function in mammals. Specifically, we found that mitochondrial mistranslation causes reduced overall mitochondrial translation and respiratory complex assembly rates. In mammals, this effect is compensated for by increased mitochondrial protein stability and upregulation of the citric acid cycle. Moreover, this induced mitochondrial stress signaling, which enables the recovery of mitochondrial translation via mitochondrial biogenesis, telomerase expression, and cell proliferation, and thereby normalizes metabolism. Conversely, we show that increased fidelity of mitochondrial translation reduces the rate of protein synthesis without eliciting a mitochondrial stress response. Consequently, the rate of translation cannot be recovered and this leads to dilated cardiomyopathy in mice. In summary, our findings reveal mammalian-specific signaling pathways that respond to changes in the fidelity of mitochondrial protein synthesis and affect metabolism.

Defective kernel 66 encodes a GTPase essential for kernel development in maize.

The mitochondrion is a semi-autonomous organelle that provides energy for cell activities through oxidative phosphorylation. In this study, we identified a defective kernel 66 (dek66)-mutant maize with defective kernels. We characterized a candidate gene, DEK66, encoding a ribosomal assembly factor located in mitochondria and possessing GTPase activity (which belongs to the ribosome biogenesis GTPase A family). In the dek66 mutant, impairment of mitochondrial structure and function led to the accumulation of reactive oxygen species and promoted programmed cell death in endosperm cells. Furthermore, the transcript levels of most of the key genes associated with nutrient storage, mitochondrial respiratory chain complex, and mitochondrial ribosomes in the dek66 mutant were significantly altered. Collectively, the results suggest that DEK66 is essential for the development of maize kernels by affecting mitochondrial function. This study provides a reference for understanding the impact of a mitochondrial ribosomal assembly factor in maize kernel development.

Structure of the mature kinetoplastids mitoribosome and insights into its large subunit biogenesis.

Kinetoplastids are unicellular eukaryotic parasites responsible for such human pathologies as Chagas disease, sleeping sickness, and leishmaniasis. They have a single large mitochondrion, essential for the parasite survival. In kinetoplastid mitochondria, most of the molecular machineries and gene expression processes have significantly diverged and specialized, with an extreme example being their mitochondrial ribosomes. These large complexes are in charge of translating the few essential mRNAs encoded by mitochondrial genomes. Structural studies performed in Trypanosoma brucei already highlighted the numerous peculiarities of these mitoribosomes and the maturation of their small subunit. However, several important aspects mainly related to the large subunit (LSU) remain elusive, such as the structure and maturation of its ribosomal RNA. Here we present a cryo-electron microscopy study of the protozoans Leishmania tarentolae and Trypanosoma cruzi mitoribosomes. For both species, we obtained the structure of their mature mitoribosomes, complete rRNA of the LSU, as well as previously unidentified ribosomal proteins. In addition, we introduce the structure of an LSU assembly intermediate in the presence of 16 identified maturation factors. These maturation factors act on both the intersubunit and the solvent sides of the LSU, where they refold and chemically modify the rRNA and prevent early translation before full maturation of the LSU.

Maintaining mitochondrial ribosome function: The role of ribosome rescue and recycling factors.

The universally conserved process of protein biosynthesis is crucial for maintaining cellular homoeostasis and in eukaryotes, mitochondrial translation is essential for aerobic energy production. Mitochondrial ribosomes (mitoribosomes) are highly specialized to synthesize 13 core subunits of the oxidative phosphorylation (OXPHOS) complexes. Although the mitochondrial translation machinery traces its origin from a bacterial ancestor, it has acquired substantial differences within this endosymbiotic environment. The cycle of mitoribosome function proceeds through the conserved canonical steps of initiation, elongation, termination and mitoribosome recycling. However, when mitoribosomes operate in the context of limited translation factors or on aberrant mRNAs, they can become stalled and activation of rescue mechanisms is required. This review summarizes recent advances in the understanding of protein biosynthesis in mitochondria, focusing especially on the mechanistic and physiological details of translation termination, and mitoribosome recycling and rescue.

Single-cell genomics unveils a canonical origin of the diverse mitochondrial genomes of euglenozoans.

BACKGROUND: The supergroup Euglenozoa unites heterotrophic flagellates from three major clades, kinetoplastids, diplonemids, and euglenids, each of which exhibits extremely divergent mitochondrial characteristics. Mitochondrial genomes (mtDNAs) of euglenids comprise multiple linear chromosomes carrying single genes, whereas mitochondrial chromosomes are circular non-catenated in diplonemids, but circular and catenated in kinetoplastids. In diplonemids and kinetoplastids, mitochondrial mRNAs require extensive and diverse editing and/or trans-splicing to produce mature transcripts. All known euglenozoan mtDNAs exhibit extremely short mitochondrial small (rns) and large (rnl) subunit rRNA genes, and absence of tRNA genes. How these features evolved from an ancestral bacteria-like circular mitochondrial genome remains unanswered. RESULTS: We sequenced and assembled 20 euglenozoan single-cell amplified genomes (SAGs). In our phylogenetic and phylogenomic analyses, three SAGs were placed within kinetoplastids, 14 within diplonemids, one (EU2) within euglenids, and two SAGs with nearly identical small subunit rRNA gene (18S) sequences (EU17/18) branched as either a basal lineage of euglenids, or as a sister to all euglenozoans. Near-complete mitochondrial genomes were identified in EU2 and EU17/18. Surprisingly, both EU2 and EU17/18 mitochondrial contigs contained multiple genes and one tRNA gene. Furthermore, EU17/18 mtDNA possessed several features unique among euglenozoans including full-length rns and rnl genes, six mitoribosomal genes, and nad11, all likely on a single chromosome. CONCLUSIONS: Our data strongly suggest that EU17/18 is an early-branching euglenozoan with numerous ancestral mitochondrial features. Collectively these data contribute to untangling the early evolution of euglenozoan mitochondria.

Genetic ablation of the mitoribosome in the malaria parasite Plasmodium falciparum sensitizes it to antimalarials that target mitochondrial functions.

The mitochondrion of malaria parasites contains several clinically validated drug targets. Within Plasmodium spp., the causative agents of malaria, the mitochondrial DNA (mtDNA) is only 6 kb long, being the smallest mitochondrial genome among all eukaryotes. The mtDNA encodes only three proteins of the mitochondrial electron transport chain and ∼27 small, fragmented rRNA genes having lengths of 22-195 nucleotides. The rRNA fragments are thought to form a mitochondrial ribosome (mitoribosome), together with ribosomal proteins imported from the cytosol. The mitoribosome of Plasmodium falciparum is essential for maintenance of the mitochondrial membrane potential and parasite viability. However, the role of the mitoribosome in sustaining the metabolic status of the parasite mitochondrion remains unclear. The small ribosomal subunit in P. falciparum has 14 annotated mitoribosomal proteins, and employing a CRISPR/Cas9-based conditional knockdown tool, here we verified the location and tested the essentiality of three candidates (PfmtRPS12, PfmtRPS17, and PfmtRPS18). Using immuno-EM, we provide evidence that the P. falciparum mitoribosome is closely associated with the mitochondrial inner membrane. Upon knockdown of the mitoribosome, parasites became hypersensitive to inhibitors targeting mitochondrial Complex III (bc1), dihydroorotate dehydrogenase (DHOD), and the F1F0-ATP synthase complex. Furthermore, the mitoribosome knockdown blocked the pyrimidine biosynthesis pathway and reduced the cellular pool of pyrimidine nucleotides. These results suggest that disruption of the P. falciparum mitoribosome compromises the metabolic capacity of the mitochondrion, rendering the parasite hypersensitive to a panel of inhibitors that target mitochondrial functions.

Pathogenic variants in MRPL44 cause infantile cardiomyopathy due to a mitochondrial translation defect.

Cardiac dysfunction is a common phenotypic manifestation of primary mitochondrial disease with multiple nuclear and mitochondrial DNA pathogenic variants as a cause, including disorders of mitochondrial translation. To date, five patients have been described with pathogenic variants in MRPL44, encoding the ml44 protein which is part of the large subunit of the mitochondrial ribosome (mitoribosome). Three presented as infants with hypertrophic cardiomyopathy, mild lactic acidosis, and easy fatigue and muscle weakness, whereas two presented in adolescence with myopathy and neurological symptoms. We describe two infants who presented with cardiomyopathy from the neonatal period, failure to thrive, hypoglycemia and in one infant lactic acidosis. A decompensation of the cardiac function in the first year resulted in demise. Exome sequencing identified compound heterozygous variants in the MRPL44 gene including the known pathogenic variant c.467 T > G and two novel pathogenic variants. We document a combined respiratory chain enzyme deficiency with emphasis on complex I and IV, affecting heart muscle tissue more than skeletal muscle or fibroblasts. We show this to be caused by reduced mitochondrial DNA encoded protein synthesis affecting all subunits, and resulting in dysfunction of complex I and IV assembly. The degree of oxidative phosphorylation dysfunction correlated with the impairment of mitochondrial protein synthesis due to different pathogenic variants. These functional studies allow for improved understanding of the pathogenesis of MRPL44-associated mitochondrial disorder.

CLPP coordinates mitoribosomal assembly through the regulation of ERAL1 levels.

Despite being one of the most studied proteases in bacteria, very little is known about the role of ClpXP in mitochondria. We now present evidence that mammalian CLPP has an essential role in determining the rate of mitochondrial protein synthesis by regulating the level of mitoribosome assembly. Through a proteomic approach and the use of a catalytically inactive CLPP, we produced the first comprehensive list of possible mammalian ClpXP substrates involved in the regulation of mitochondrial translation, oxidative phosphorylation, and a number of metabolic pathways. We further show that the defect in mitoribosomal assembly is a consequence of the accumulation of ERAL1, a putative 12S rRNA chaperone, and novel ClpXP substrate. The presented data suggest that the timely removal of ERAL1 from the small ribosomal subunit is essential for the efficient maturation of the mitoribosome and a normal rate of mitochondrial translation.

Biallelic Mutations in MRPS34 Lead to Instability of the Small Mitoribosomal Subunit and Leigh Syndrome.

The synthesis of all 13 mitochondrial DNA (mtDNA)-encoded protein subunits of the human oxidative phosphorylation (OXPHOS) system is carried out by mitochondrial ribosomes (mitoribosomes). Defects in the stability of mitoribosomal proteins or mitoribosome assembly impair mitochondrial protein translation, causing combined OXPHOS enzyme deficiency and clinical disease. Here we report four autosomal-recessive pathogenic mutations in the gene encoding the small mitoribosomal subunit protein, MRPS34, in six subjects from four unrelated families with Leigh syndrome and combined OXPHOS defects. Whole-exome sequencing was used to independently identify all variants. Two splice-site mutations were identified, including homozygous c.321+1G>T in a subject of Italian ancestry and homozygous c.322-10G>A in affected sibling pairs from two unrelated families of Puerto Rican descent. In addition, compound heterozygous MRPS34 mutations were identified in a proband of French ancestry; a missense (c.37G>A [p.Glu13Lys]) and a nonsense (c.94C>T [p.Gln32∗]) variant. We demonstrated that these mutations reduce MRPS34 protein levels and the synthesis of OXPHOS subunits encoded by mtDNA. Examination of the mitoribosome profile and quantitative proteomics showed that the mitochondrial translation defect was caused by destabilization of the small mitoribosomal subunit and impaired monosome assembly. Lentiviral-mediated expression of wild-type MRPS34 rescued the defect in mitochondrial translation observed in skin fibroblasts from affected subjects, confirming the pathogenicity of MRPS34 mutations. Our data establish that MRPS34 is required for normal function of the mitoribosome in humans and furthermore demonstrate the power of quantitative proteomic analysis to identify signatures of defects in specific cellular pathways in fibroblasts from subjects with inherited disease.

Abnormal Expression of Mitochondrial Ribosomal Proteins and Their Encoding Genes with Cell Apoptosis and Diseases.

Mammalian mitochondrial ribosomes translate 13 proteins encoded by mitochondrial genes, all of which play roles in the mitochondrial respiratory chain. After a long period of reconstruction, mitochondrial ribosomes are the most protein-rich ribosomes. Mitochondrial ribosomal proteins (MRPs) are encoded by nuclear genes, synthesized in the cytoplasm and then, transported to the mitochondria to be assembled into mitochondrial ribosomes. MRPs not only play a role in mitochondrial oxidative phosphorylation (OXPHOS). Moreover, they participate in the regulation of cell state as apoptosis inducing factors. Abnormal expressions of MRPs will lead to mitochondrial metabolism disorder, cell dysfunction, etc. Many researches have demonstrated the abnormal expression of MRPs in various tumors. This paper reviews the basic structure of mitochondrial ribosome, focuses on the structure and function of MRPs, and their relationships with cell apoptosis and diseases. It provides a reference for the study of the function of MRPs and the disease diagnosis and treatment.