Purification of Mitochondrial Ribosomal Complexes from Trypanosoma cruzi and Leishmania tarentolae for Cryo-EM Analysis.

Kinetoplastids are unicellular eukaryotic parasites responsible for human pathologies such as Chagas disease, sleeping sickness or Leishmaniasis, caused by Trypanosoma cruzi, Trypanosoma brucei, and various Leishmania spp., respectively. They harbor a single large mitochondrion that is essential for the survival of the parasite. Interestingly, most of the mitochondrial gene expression machineries and processes present significant differences from their nuclear and cytosolic counterparts. A striking example concerns their mitochondrial ribosomes, in charge of translating the few essential mRNAs encoded by mitochondrial genomes. Here, we present a detailed protocol including the specific procedures to isolate mitochondria from two species of kinetoplastids, T. cruzi and L. tarentolae, by differential centrifugations. Then, we detail the protocol to purify mitochondrial ribosomal complexes from these two species of parasites (including ribosomal maturating complexes) by a sucrose gradient approach. Finally, we describe how to prepare cryo-electron microscopy (cryo-EM) grids from these two sorts of samples. This protocol will be useful for further studies aiming at analyzing mitochondrial translation regulation.

Decoding the impact of disease-causing mutations in an essential aminoacyl-tRNA synthetase.

Aminoacyl-tRNA synthetases are housekeeping enzymes that catalyze the specific attachment of amino acids onto cognate tRNAs, providing building blocks for ribosomal protein synthesis. Owing to the absolutely essential nature of these enzymes, the possibility that mutations in their sequence could be the underlying cause of diseases had not been foreseen. However, we are learning of patients bearing familial mutations in aminoacyl-tRNA synthetases at an exponential rate. In a recent issue of JBC, Jin et al. analyzed the impact of two such mutations in the very special bifunctional human glutamyl-prolyl-tRNA synthetase and convincingly decode how these mutations elicit the integrated stress response.

Peculiarities of aminoacyl-tRNA synthetases from trypanosomatids.

Trypanosomatid parasites are responsible for various human diseases, such as sleeping sickness, animal trypanosomiasis, or cutaneous and visceral leishmaniases. The few available drugs to fight related parasitic infections are often toxic and present poor efficiency and specificity, and thus, finding new molecular targets is imperative. Aminoacyl-tRNA synthetases (aaRSs) are essential components of the translational machinery as they catalyze the specific attachment of an amino acid onto cognate tRNA(s). In trypanosomatids, one gene encodes both cytosolic- and mitochondrial-targeted aaRSs, with only three exceptions. We identify here a unique specific feature of aaRSs from trypanosomatids, which is that most of them harbor distinct insertion and/or extension sequences. Among the 26 identified aaRSs in the trypanosome Leishmania tarentolae, 14 contain an additional domain or a terminal extension, confirmed in mature mRNAs by direct cDNA nanopore sequencing. Moreover, these RNA-Seq data led us to address the question of aaRS dual localization and to determine splice-site locations and the 5'-UTR lengths for each mature aaRS-encoding mRNA. Altogether, our results provided evidence for at least one specific mechanism responsible for mitochondrial addressing of some L. tarentolae aaRSs. We propose that these newly identified features of trypanosomatid aaRSs could be developed as relevant drug targets to combat the diseases caused by these parasites.

Phenotypic diversity of brain MRI patterns in mitochondrial aminoacyl-tRNA synthetase mutations.

BACKGROUND AND PURPOSE: Mitochondrial aminoacyl-tRNA synthetases-encoded by ARS2 genes-are evolutionarily conserved enzymes that catalyse the attachment of amino acids to their cognate tRNAs, ensuring the accuracy of the mitochondrial translation process. ARS2 gene mutations are associated with a wide range of clinical presentations affecting the CNS. METHODS: Two senior neuroradiologists analysed brain MRI of 25 patients (age range: 3 d-25 yrs.; 11 males; 14 females) with biallelic pathogenic variants of 11 ARS2 genes in a retrospective study conducted between 2002 and 2019. RESULTS: Though several combinations of brain MRI anomalies were highly suggestive of specific aetiologies (DARS2, EARS2, AARS2 and RARS2 mutations), our study detected no MRI pattern common to all patients. Stroke-like lesions were associated with pathogenic SARS2 and FARS2 variants. We also report early onset cerebellar atrophy and calcifications in AARS2 mutations, early white matter involvement in RARS2 mutations, and absent involvement of thalami in EARS2 mutations. Finally, our findings show that normal brain MRI results do not exclude the presence of ARS2 mutations: 5 patients with normal MRI images were carriers of pathogenic IARS2, YARS2, and FARS2 variants. CONCLUSION: Our study extends the spectrum of brain MRI anomalies associated with pathogenic ARS2 variants and suggests ARS2 mutations are largely underdiagnosed.

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.

When a common biological role does not imply common disease outcomes: Disparate pathology linked to human mitochondrial aminoacyl-tRNA synthetases.

Mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs) are essential components of the mitochondrial translation machinery. The correlation of mitochondrial disorders with mutations in these enzymes has raised the interest of the scientific community over the past several years. Most surprising has been the wide-ranging presentation of clinical manifestations in patients with mt-aaRS mutations, despite the enzymes' common biochemical role. Even among cases where a common physiological system is affected, phenotypes, severity, and age of onset varies depending on which mt-aaRS is mutated. Here, we review work done thus far and propose a categorization of diseases based on tissue specificity that highlights emerging patterns. We further discuss multiple in vitro and in cellulo efforts to characterize the behavior of WT and mutant mt-aaRSs that have shaped hypotheses about the molecular causes of these pathologies. Much remains to do in order to complete our understanding of these proteins. We expect that futher work is likely to result in the discovery of new roles for the mt-aaRSs in addition to their fundamental function in mitochondrial translation, informing the development of treatment strategies and diagnoses.

How to fold and protect mitochondrial ribosomal RNA with fewer guanines.

Mammalian mitochondrial ribosomes evolved from bacterial ribosomes by reduction of ribosomal RNAs, increase of ribosomal protein content, and loss of guanine nucleotides. Guanine is the base most sensitive to oxidative damage. By systematically comparing high-quality, small ribosomal subunit RNA sequence alignments and solved 3D ribosome structures from mammalian mitochondria and bacteria, we deduce rules for folding a complex RNA with the remaining guanines shielded from solvent. Almost all conserved guanines in both bacterial and mammalian mitochondrial ribosomal RNA form guanine-specific, local or long-range, RNA-RNA or RNA-protein interactions. Many solvent-exposed guanines conserved in bacteria are replaced in mammalian mitochondria by bases less sensitive to oxidation. New guanines, conserved only in the mitochondrial alignment, are strategically positioned at solvent inaccessible sites to stabilize the ribosomal RNA structure. New mitochondrial proteins substitute for truncated RNA helices, maintain mutual spatial orientations of helices, compensate for lost RNA-RNA interactions, reduce solvent accessibility of bases, and replace guanines conserved in bacteria by forming specific amino acid-RNA interactions.

Three human aminoacyl-tRNA synthetases have distinct sub-mitochondrial localizations that are unaffected by disease-associated mutations.

Human mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs) are key enzymes in the mitochondrial protein translation system and catalyze the charging of amino acids on their cognate tRNAs. Mutations in their nuclear genes are associated with pathologies having a broad spectrum of clinical phenotypes, but with no clear molecular mechanism(s). For example, mutations in the nuclear genes encoding mt-AspRS and mt-ArgRS are correlated with the moderate neurodegenerative disorder leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) and with the severe neurodevelopmental disorder pontocerebellar hypoplasia type 6 (PCH6), respectively. Previous studies have shown no or only minor impacts of these mutations on the canonical properties of these enzymes, indicating that the role of the mt-aaRSs in protein synthesis is mostly not affected by these mutations, but their effects on the mitochondrial localizations of aaRSs remain unclear. Here, we demonstrate that three human aaRSs, mt-AspRS, mt-ArgRS, and LysRS, each have a specific sub-mitochondrial distribution, with mt-ArgRS being exclusively localized in the membrane, LysRS exclusively in the soluble fraction, and mt-AspRS being present in both. Chemical treatments revealed that mt-AspRs is anchored in the mitochondrial membrane through electrostatic interactions, whereas mt-ArgRS uses hydrophobic interactions. We also report that novel mutations in mt-AspRS and mt-ArgRS genes from individuals with LBSL and PCH6, respectively, had no significant impact on the mitochondrial localizations of mt-AspRS and mt-ArgRS. The variable sub-mitochondrial locations for these three mt-aaRSs strongly suggest the existence of additional enzyme properties, requiring further investigation to unravel the mechanisms underlying the two neurodegenerative disorders.

Recent Advances in Mitochondrial Aminoacyl-tRNA Synthetases and Disease.

Dysfunctions in mitochondria - the powerhouses of the cell - lead to several human pathologies. Because mitochondria integrate nuclear and mitochondrial genetic systems, they are richly intertwined with cellular activities. The nucleus-encoded mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs) are key components of the mitochondrial translation apparatus. Mutations in these enzymes predominantly affect the central nervous system (CNS) but also target other organs. Comparable mutations in mt-aaRSs can lead to vastly diverse diseases, occurring at different stages in life, and within different tissues; this represents a confounding issue. With newer information available, we propose that the pleiotropy and tissue-specificity of mt-aaRS-associated diseases result from the molecular integration of mitochondrial translation events within the cell; namely, through specific crosstalk between the cellular program and the energy demands of the cell. We place particular focus on neuronal cells.

MiSynPat: An integrated knowledge base linking clinical, genetic, and structural data for disease-causing mutations in human mitochondrial aminoacyl-tRNA synthetases.

Numerous mutations in each of the mitochondrial aminoacyl-tRNA synthetases (aaRSs) have been implicated in human diseases. The mutations are autosomal and recessive and lead mainly to neurological disorders, although with pleiotropic effects. The processes and interactions that drive the etiology of the disorders associated with mitochondrial aaRSs (mt-aaRSs) are far from understood. The complexity of the clinical, genetic, and structural data requires concerted, interdisciplinary efforts to understand the molecular biology of these disorders. Toward this goal, we designed MiSynPat, a comprehensive knowledge base together with an ergonomic Web server designed to organize and access all pertinent information (sequences, multiple sequence alignments, structures, disease descriptions, mutation characteristics, original literature) on the disease-linked human mt-aaRSs. With MiSynPat, a user can also evaluate the impact of a possible mutation on sequence-conservation-structure in order to foster the links between basic and clinical researchers and to facilitate future diagnosis. The proposed integrated view, coupled with research on disease-related mt-aaRSs, will help to reveal new functions for these enzymes and to open new vistas in the molecular biology of the cell. The purpose of MiSynPat, freely available at http://misynpat.org, is to constitute a reference and a converging resource for scientists and clinicians.

Two proteomic methodologies for defining N-termini of mature human mitochondrial aminoacyl-tRNA synthetases.

Human mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs) are encoded in the nucleus, synthesized in the cytosol and targeted for importation into mitochondria by a N-terminal mitochondrial targeting sequence. This targeting sequence is presumably cleaved upon entry into the mitochondria, following a process still not fully deciphered in human, despite essential roles for the mitochondrial biogenesis. Maturation processes are indeed essential both for the release of a functional enzyme and to route correctly the protein within mitochondria. The absence of consensus sequences for cleavage sites and the discovery of possible multiple proteolytic steps render predictions of N-termini difficult. Further, the knowledge of the cleavages is key for the design of protein constructions compatible with efficient production in bacterial strains. Finally, full comprehension becomes essential because a growing number of mutations are found in genes coding for mt-aaRS. In the present study, we take advantage of proteomic methodological developments and identified, in mitochondria, three N-termini for the human mitochondrial aspartyl-tRNA synthetase. This first description of the co-existence of different forms opens new perspectives in the biological understanding of this enzyme. Those methods are extended to the whole set of human mt-aaRSs and methodological advice are provided for further investigations.

Neurodegenerative disease-associated mutants of a human mitochondrial aminoacyl-tRNA synthetase present individual molecular signatures.

Mutations in human mitochondrial aminoacyl-tRNA synthetases are associated with a variety of neurodegenerative disorders. The effects of these mutations on the structure and function of the enzymes remain to be established. Here, we investigate six mutants of the aspartyl-tRNA synthetase correlated with leukoencephalopathies. Our integrated strategy, combining an ensemble of biochemical and biophysical approaches, reveals that mutants are diversely affected with respect to their solubility in cellular extracts and stability in solution, but not in architecture. Mutations with mild effects on solubility occur in patients as allelic combinations whereas those with strong effects on solubility or on aminoacylation are necessarily associated with a partially functional allele. The fact that all mutations show individual molecular and cellular signatures and affect amino acids only conserved in mammals, points towards an alternative function besides aminoacylation.

Mutations of human NARS2, encoding the mitochondrial asparaginyl-tRNA synthetase, cause nonsyndromic deafness and Leigh syndrome.

Here we demonstrate association of variants in the mitochondrial asparaginyl-tRNA synthetase NARS2 with human hearing loss and Leigh syndrome. A homozygous missense mutation ([c.637G>T; p.Val213Phe]) is the underlying cause of nonsyndromic hearing loss (DFNB94) and compound heterozygous mutations ([c.969T>A; p.Tyr323*] + [c.1142A>G; p.Asn381Ser]) result in mitochondrial respiratory chain deficiency and Leigh syndrome, which is a neurodegenerative disease characterized by symmetric, bilateral lesions in the basal ganglia, thalamus, and brain stem. The severity of the genetic lesions and their effects on NARS2 protein structure cosegregate with the phenotype. A hypothetical truncated NARS2 protein, secondary to the Leigh syndrome mutation p.Tyr323* is not detectable and p.Asn381Ser further decreases NARS2 protein levels in patient fibroblasts. p.Asn381Ser also disrupts dimerization of NARS2, while the hearing loss p.Val213Phe variant has no effect on NARS2 oligomerization. Additionally we demonstrate decreased steady-state levels of mt-tRNAAsn in fibroblasts from the Leigh syndrome patients. In these cells we show that a decrease in oxygen consumption rates (OCR) and electron transport chain (ETC) activity can be rescued by overexpression of wild type NARS2. However, overexpression of the hearing loss associated p.Val213Phe mutant protein in these fibroblasts cannot complement the OCR and ETC defects. Our findings establish lesions in NARS2 as a new cause for nonsyndromic hearing loss and Leigh syndrome.

Idiosyncrasies in decoding mitochondrial genomes.

Mitochondria originate from the α-proteobacterial domain of life. Since this unique event occurred, mitochondrial genomes of protozoans, fungi, plants and metazoans have highly derived and diverged away from the common ancestral DNA. These resulting genomes highly differ from one another, but all present-day mitochondrial DNAs have a very reduced coding capacity. Strikingly however, ATP production coupled to electron transport and translation of mitochondrial proteins are the two common functions retained in all mitochondrial DNAs. Paradoxically, most components essential for these two functions are now expressed from nuclear genes. Understanding how mitochondrial translation evolved in various eukaryotic models is essential to acquire new knowledge of mitochondrial genome expression. In this review, we provide a thorough analysis of the idiosyncrasies of mitochondrial translation as they occur between organisms. We address this by looking at mitochondrial codon usage and tRNA content. Then, we look at the aminoacyl-tRNA-forming enzymes in terms of peculiarities, dual origin, and alternate function(s). Finally we give examples of the atypical structural properties of mitochondrial tRNAs found in some organisms and the resulting adaptive tRNA-protein partnership.

Pathogenic implications of human mitochondrial aminoacyl-tRNA synthetases.

Mitochondria are considered as the powerhouse of eukaryotic cells. They host several central metabolic processes fueling the oxidative phosphorylation pathway (OXPHOS) that produces ATP from its precursors ADP and inorganic phosphate Pi (PPi). The respiratory chain complexes responsible for the OXPHOS pathway are formed from complementary sets of protein subunits encoded by the nuclear genome and the mitochondrial genome, respectively. The expression of the mitochondrial genome requires a specific and fully active translation machinery from which aminoacyl-tRNA synthetases (aaRSs) are key actors. Whilst the macromolecules involved in mammalian mitochondrial translation have been under investigation for many years, there has been an explosion of interest in human mitochondrial aaRSs (mt-aaRSs) since the discovery of a large (and growing) number of mutations in these genes that are linked to a variety of neurodegenerative disorders. Herein we will review the present knowledge on mt-aaRSs in terms of their biogenesis, their connection to mitochondrial respiration, i.e., the respiratory chain (RC) complexes, and to the mitochondrial translation machinery. The pathology-related mutations detected so far are described, with special attention given to their impact on mt-aaRSs biogenesis, functioning, and/or subsequent activities. The collected data to date shed light on the diverse routes that are linking primary molecular possible impact of a mutation to its phenotypic expression. It is envisioned that a variety of mechanisms, inside and outside the translation machinery, would play a role on the heterogeneous manifestations of mitochondrial disorders.

Released selective pressure on a structural domain gives new insights on the functional relaxation of mitochondrial aspartyl-tRNA synthetase.

Mammalian mitochondrial aminoacyl-tRNA synthetases are nuclear-encoded enzymes that are essential for mitochondrial protein synthesis. Due to an endosymbiotic origin of the mitochondria, many of them share structural domains with homologous bacterial enzymes of same specificity. This is also the case for human mitochondrial aspartyl-tRNA synthetase (AspRS) that shares the so-called bacterial insertion domain with bacterial homologs. The function of this domain in the mitochondrial proteins is unclear. Here, we show by bioinformatic analyses that the sequences coding for the bacterial insertion domain are less conserved in opisthokont and protist than in bacteria and viridiplantae. The divergence suggests a loss of evolutionary pressure on this domain for non-plant mitochondrial AspRSs. This discovery is further connected with the herein described occurrence of alternatively spliced transcripts of the mRNAs coding for some mammalian mitochondrial AspRSs. Interestingly, the spliced transcripts alternately lack one of the four exons that code for the bacterial insertion domain. Although we showed that the human alternative transcript is present in all tested tissues; co-exists with the full-length form, possesses 5'- and 3'-UTRs, a poly-A tail and is bound to polysomes, we were unable to detect the corresponding protein. The relaxed selective pressure combined with the occurrence of alternative splicing, involving a single structural sub-domain, favors the hypothesis of the loss of function of this domain for AspRSs of mitochondrial location. This evolutionary divergence is in line with other characteristics, established for the human mt-AspRS, that indicate a functional relaxation of non-viridiplantae mt-AspRSs when compared to bacterial and plant ones, despite their common ancestry.

Thermodynamic properties distinguish human mitochondrial aspartyl-tRNA synthetase from bacterial homolog with same 3D architecture.

In the mammalian mitochondrial translation apparatus, the proteins and their partner RNAs are coded by two genomes. The proteins are nuclear-encoded and resemble their homologs, whereas the RNAs coming from the rapidly evolving mitochondrial genome have lost critical structural information. This raises the question of molecular adaptation of these proteins to their peculiar partner RNAs. The crystal structure of the homodimeric bacterial-type human mitochondrial aspartyl-tRNA synthetase (DRS) confirmed a 3D architecture close to that of Escherichia coli DRS. However, the mitochondrial enzyme distinguishes by an enlarged catalytic groove, a more electropositive surface potential and an alternate interaction network at the subunits interface. It also presented a thermal stability reduced by as much as 12°C. Isothermal titration calorimetry analyses revealed that the affinity of the mitochondrial enzyme for cognate and non-cognate tRNAs is one order of magnitude higher, but with different enthalpy and entropy contributions. They further indicated that both enzymes bind an adenylate analog by a cooperative allosteric mechanism with different thermodynamic contributions. The larger flexibility of the mitochondrial synthetase with respect to the bacterial enzyme, in combination with a preserved architecture, may represent an evolutionary process, allowing nuclear-encoded proteins to cooperate with degenerated organelle RNAs.