Mitochondrial DNA Translation Research Articles

Abstract 7062: CIP2A enhances mitochondrial respiratory electron transport chain complexes and induces lung cancer in vivo

Abstract Identification of oncogenes that can induce cancer in vivo is critical to elucidation of tumorigenesis and development of targeted therapies. Here, we reported for the first time that forced ectopic expression of CIP2A, the cancerous inhibitor of protein phosphatase 2A (PP2A), in lung tissues induced lung adenocarcinoma in mice. CIP2A upregulated the transcription and translation of mitochondrial DNA (mtDNA), and increases the enzymatic activity of mitochondrial electron transport chain (ETC) complex IV and V, thus enhancing the function of mitochondria and promoting the level of oxidative phosphorylation in tumors. CIP2A inhibited the binding of PP2A-B56α to FOXO3a, leading to the enhancement of the phosphorylation level of FOXO3a. The phosphorylated FOXO3a then translocated into the mitochondrion, and acted as a transcription factor to regulate CIP2A-mediated metabolic reprogramming in cancer cells. We found that oxidative phosphorylation inhibitors effectively inhibited CIP2A-induced cell proliferation. In non-small cell lung cancer, CIP2A level in tumor tissues was positively associated with the level of mtDNA. Our results indicated that CIP2A is able to induce lung adenocarcinoma in mice and has a critical role in mitochondrial oxidative phosphorylation, and represents a promising therapeutic target for non-small cell lung cancer. Competing interests: The authors declare no competing interests. Citation Format: Fu-Ying Yang, Di Wang, Li-Jun liang, Gui-Zhen Wang, Guang-Biao Zhou. CIP2A enhances mitochondrial respiratory electron transport chain complexes and induces lung cancer in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7062.

Sls1 and Mtf2 mediate the assembly of the Mrh5C complex required for activation of cox1 mRNA translation

Mitochondrial translation depends on mRNA-specific activators. In Schizosaccharomyces pombe, DEAD-box protein Mrh5, pentatricopeptide repeat (PPR) protein Ppr4, Mtf2, and Sls1 form a stable complex (designated Mrh5C) required for translation of mitochondrial DNA (mtDNA)‐encoded cox1 mRNA, the largest subunit of the cytochrome c oxidase complex. However, how Mrh5C is formed and what role Mrh5C plays in cox1 mRNA translation have not been reported. To address these questions, we investigated the role of individual Mrh5C subunits in the assembly and function of Mrh5C. Our results revealed that Mtf2 and Sls1 form a subcomplex that serves as a scaffold to bring Mrh5 and Ppr4 together. Mrh5C binds to the small subunit of the mitoribosome (mtSSU), but each subunit could not bind to the mtSSU independently. Importantly, Mrh5C is required for the association of cox1 mRNA with the mtSSU. Finally, we investigated the importance of the signature DEAD-box in Mrh5. We found that the DEAD-box of Mrh5 is required for the association of Mrh5C and cox1 mRNA with the mtSSU. Unexpectedly, this motif is also required for the interaction of Mrh5 with other Mrh5C subunits. Altogether, our results suggest that Mrh5 and Ppr4 cooperate in activating the translation of cox1 mRNA. Our results also suggest that Mrh5C activates the translation of cox1 mRNA by promoting the recruitment of cox1 mRNA to the mtSSU.

Assessing the Role of Post-Translational Modifications of Mitochondrial RNA Polymerase.

The mitochondrial proteome is subject to abundant post-translational modifications, including lysine acetylation and phosphorylation of serine, threonine, and tyrosine. The biological function of the majority of these protein modifications is unknown. Proteins required for the transcription and translation of mitochondrial DNA (mtDNA) are subject to modification. This suggests that reversible post-translational modifications may serve as a regulatory mechanism for mitochondrial gene transcription, akin to mechanisms controlling nuclear gene expression. We set out to determine whether acetylation or phosphorylation controls the function of mitochondrial RNA polymerase (POLRMT). Mass spectrometry was used to identify post-translational modifications on POLRMT. We analyzed three POLRMT modification sites (lysine 402, threonine 315, threonine 993) found in distinct structural regions. Amino acid point mutants that mimic the modified and unmodified forms of POLRMT were employed to measure the effect of acetylation or phosphorylation on the promoter binding ability of POLRMT in vitro. We found a slight decrease in binding affinity for the phosphomimic at threonine 315. We did not identify large changes in viability, mtDNA content, or mitochondrial transcript level upon overexpression of POLRMT modification mimics in HeLa cells. Our results suggest minimal biological impact of the POLRMT post-translational modifications studied in our system.

Role of Glycogen Mobilization and Mitochondrial Bioenergetics in Platelet Function, Hemostasis, and Thrombosis

Platelets are one of the most metabolically active cells in the bloodstream. They show metabolic flexibility, switching between glycolysis and oxidative phosphorylation (OxPhos) depending on oxygen tension and the availability of substrates. Platelets also have metabolizable glycogen granules that contribute to the energy pool upon activation. Our goal was to investigate platelet energy metabolism under normal physiological conditions to better understand the relative roles of the major ATP-generating processes (glycolysis, OxPhos, and glycogenolysis). To address the role of glycogen, we used 2 glycogen phosphorylase inhibitors and tested their effects on standard platelet functions e.g., secretion, aggregation, clot contraction, and thrombus formation under shear. We also measured the glycogen levels in resting and activated platelets and the presence of inhibitors. Consistent with previous studies, glycogen was utilized when platelets are activated with a ~25% decrease upon thrombin-mediated activation. Both inhibitors blocked the use of glycogen and led to a 3-fold increase in glycogen compared to controls. These data suggest the active turnover of glycogen in platelets. Our inhibitor studies indicated that glycogen is dispensable for low-energy processes like aggregation but contributed to secretion, clot contraction, and thrombus formation under shear. The inhibition was rescued by either increasing external glucose or bypassing glycolysis with added pyruvate. This study shows that the glycogen granules are metabolically active and contribute to the energy pool upon platelet activation; this pool is important for high-energy platelet functions like clot contraction. Deletion of platelet glucose transporters (GLUT1 and GLUT3) revealed an essential role of glucose metabolism in hemostasis in vivo. Glucose can power platelets via glycolysis alone or in combination with OxPhos (in the mitochondria). We sought to probe the relative importance of platelet mitochondrial bioenergetics in hemostasis and thrombosis. Previously used mitochondrial inhibitors (antimycin, oligomycin) are toxic and cannot be used for in vivo studies, thus we developed two novel mouse models with altered mitochondrial function using a platelet-specific deletion of TFAM and QPC. TFAM (Transcription Factor A Mitochondrial), is essential for the maintenance, transcription, and translation of mitochondrial DNA. Its deletion is expected to disrupt platelet mitochondrial DNA, which encodes 13 subunits of OxPhos. QPC is a subunit of ubiquinol-cytochrome c reductase complex III. Its deletion is expected to disrupt Complex III, which is part of the platelet mitochondrial respiratory chain. We confirmed TFAM deletion using Western blot analysis. Using the Seahorse Analyzer, we confirmed that deleting TFAM disrupted platelet mitochondrial bioenergetics in both resting and thrombin-stimulated platelets as seen as a ~40% decrease in Oxygen Consumption Rate (OCR). We also observed a significant decrease in ATP production indicating the contribution of OxPhos to ATP generation. Aggregation was not affected but in vitro clot contraction and thrombus formation under shear were defective (~58% increase in lag time, ~30% increase in AUC, and ~60% decrease in surface area coverage). Both the KO animals showed an increased tail-bleeding time, an increased occlusion time in the FeCl3 carotid injury model, and delayed hemostasis in a jugular puncture injury model with significantly higher rebleeding indicating that mitochondrial bioenergetics is important for clot stability. Using two novel mouse models with dysfunctional mitochondrial bioenergetics, we show that OxPhos is dispensable for low-energy demanding platelet functions such as aggregation but is important for secretion, clot contraction, hemostasis, and thrombosis (Table 1). Platelets show considerable plasticity in energy metabolism using both glycolysis and oxidative phosphorylation. Using mouse models and inhibitors, we show the relative importance of these energy-producing processes and the fuels and metabolic pathway choices made by platelets at different functional stages. Improving our understanding of platelet metabolism and the relative contributions of each pathway in the future could lead to a better understanding of how metabolic disorders can affect thrombotic risk in diabetic and obese patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Non-canonical phosphoglycerate dehydrogenase activity promotes liver cancer growth via mitochondrial translation and respiratory metabolism.

Phosphoglycerate dehydrogenase (PHGDH) is a key serine biosynthesis enzyme whose aberrant expression promotes various types of tumors. Recently, PHGDH has been found to have some non-canonical functions beyond serine biosynthesis, but its specific mechanisms in tumorigenesis remain unclear. Here, we show that PHGDH localizes to the inner mitochondrial membrane and promotes the translation of mitochondrial DNA (mtDNA)-encoded proteins in liver cancer cells. Mechanistically, we demonstrate that mitochondrial PHGDH directly interacts with adenine nucleotide translocase 2 (ANT2) and then recruits mitochondrial elongation factor G2 (mtEFG2) to promote mitochondrial ribosome recycling efficiency, thereby promoting mtDNA-encoded protein expression and subsequent mitochondrial respiration. Moreover, we show that treatment with a mitochondrial translation inhibitor or depletion of mtEFG2 diminishes PHGDH-mediated tumor growth. Collectively, our findings uncover a previously unappreciated function of PHGDH in tumorigenesis acting via promotion of mitochondrial translation and bioenergetics.

MTERF3 contributes to MPP+-induced mitochondrial dysfunction in SH-SY5Y cells.

Parkinson's disease (PD) is a neurodegenerative disorder causing severe social and economic burdens. The origin of PD has been usually attributed to mitochondrial dysfunction. To this end, mitochondrial transcription regulators become attractive subjects for understanding PD pathogenesis. Previously, we found that the expression of mitochondrial transcription termination factor 3 (MTERF3) was reduced in MPP+-induced mice model of PD. In the present study, we probe the function of MTERF3 and its role in MPP+-induced cellular model of PD. Initially, we observe that MTERF3 expression is also reduced in MPP+-induced cellular model of PD, which can be mainly attributed to the increase of MTERF3 degradation. Next, we examine the effect of MTERF3 knockdown and overexpression on the replication, transcription, and translation of mitochondrial DNA (mtDNA). We show that knockdown and overexpression of MTERF3 have opposite effects on mtDNA transcript level but similar effects on mtDNA expression level, in line with MTERF3's dual roles in mtDNA transcription and translation. In addition, we examine the effect of MTERF3 knockdown and overexpression on mitochondrial function with and without MPP+ treatment, and find that MTERF3 seems to play a generally protective role in MPP+-induced mitochondrial dysfunction. Together, this work suggests a regulatory role of MTERF3 in MPP+-induced cellular model of PD and may provide clues in designing novel therapeutics against PD.

Doxycycline sensitizes renal cell carcinoma to chemotherapy by preferentially inhibiting mitochondrial translation

ObjectivesThe anti-cancer activity of doxycycline has been reported in many cancers but not renal cell carcinoma (RCC). This study aimed to determine the efficacy of doxycycline alone and in combination with paclitaxel and analyze the underlying mechanism in RCC.MethodsProliferation, colony formation and apoptosis assays were performed in RCC cell lines after drug treatments. An RCC xenograft mouse model was generated, and tumor growth was monitored. Mechanistic studies focused on mitochondrial translation and functions.ResultsDoxycycline at clinically achievable concentrations inhibited proliferation and colony formation and induced apoptosis in RCC cell lines. In normal kidney cells, doxycycline at the same concentrations either had no effect or was less effective. The combination index value demonstrated that doxycycline and paclitaxel were synergistic in vitro. Consistently, this combination therapy was significantly more effective than the monotherapy in RCC xenograft mice without causing significant toxicity. Mechanistic studies revealed that doxycycline acts on RCC cells via preferentially inhibiting mitochondrial DNA translation, thereby disrupting multiple mitochondrial complexes and impairing mitochondrial respiration.ConclusionsDoxycycline is a useful addition to the treatment strategy for RCC. Our work also highlights the therapeutic value of mitochondrial translation inhibition in sensitizing RCC to chemotherapy.

Anti-tumor activity of tigecycline: a review

Tigecycline is a novel glycylcycline antibacterial drug, which shows both antibiotic function and anti-tumor activity. This review summarizes the single and combined use of tigecycline for tumor treatment and the underpinning mechanisms. As an inhibitor for mitochondrial DNA translation, tigecycline affects the proliferation, migration, and invasion of tumor cells mainly through inhibiting mitochondrial protein synthesis and inducing mitochondrial dysfunction. Although the effect of tigecycline monotherapy is controversial, the efficacy of combined use of tigecycline is satisfactory. Therefore, it is important to explore the molecular mechanisms underpinning the anti-tumor activity of tigecycline, with the aim to use it as a cheap and effective new anti-tumor drug.

TRMU deficiency: A broad clinical spectrum responsive to cysteine supplementation

TRMU is a nuclear gene crucial for mitochondrial DNA translation by encoding tRNA 5-methylaminomethyl-2-thiouridylate methyltransferase, which thiolates mitochondrial tRNA. Biallelic pathogenic variants in TRMU are associated with transient infantile liver failure. Other less common presentations such as Leigh syndrome, myopathy, and cardiomyopathy have been reported. Recent studies suggested that provision of exogenous L-cysteine or N-acetylcysteine may ameliorate the effects of disease-causing variants and improve the natural history of the disease. Here, we report six infants with biallelic TRMU variants, including four previously unpublished patients, all treated with exogenous cysteine. We highlight the first report of an affected patient undergoing orthotopic liver transplantation, the long-term effects of cysteine supplementation, and the ability of the initial presentation to mimic multiple inborn errors of metabolism. We propose that TRMU deficiency should be suspected in all children presenting with persistent lactic acidosis and hypoglycemia, and that combined N-acetylcysteine and L-cysteine supplementation should be considered prior to molecular diagnosis, as this is a low-risk approach that may increase survival and mitigate the severity of the disease course.

Tigecycline-induced inhibition of mitochondrial DNA translation may cause lethal mitochondrial dysfunction in humans

BackgroundA 65-year-old patient developed an unexplained and ultimately lethal metabolic acidosis under prolonged treatment with tigecycline. Tigecycline is known to have a selective inhibitory effect on eukaryotic mitochondrial translation. The underlying molecular mechanisms of the metabolic acidosis in this patient were explored. MethodsOxidative phosphorylation system (OXPHOS) analysis, blue native polyacrylamide gel electrophoresis followed by in-gel activity staining in mitochondria, molecular analysis of mitochondrial DNA (mtDNA) for genomic rearrangements and sequencing of the rRNA genes was performed on the subject's skeletal muscle. ResultsOXPHOS analysis revealed a combined deficiency of the complexes I, III, IV and V, with a preserved function of complex II (encoded by nuclear DNA), thus demonstrating a defective mtDNA translation. There were no known underlying mitochondrial genetic defects. The patient had a (m.1391T>A) variant within the 12SrRNA gene in heteroplasmy (50–60%). ConclusionsThis patient developed an ultimately lethal mitochondrial toxicity while receiving prolonged treatment with tigecycline, which was caused by a defective translation of the mtDNA. Tigecycline is known to suppress eukaryotic mitochondrial DNA translation, but until now this effect has been considered to be clinically insignificant. The observations in this patient suggest a clinically significant mitochondrial toxicity of tigecycline in this patient, and warrant further investigation.

Novel mutation in mitochondrial Elongation Factor EF-Tu associated to dysplastic leukoencephalopathy and defective mitochondrial DNA translation

The mitochondrial Elongation Factor Tu (EF-Tu), encoded by the TUFM gene, is a highly conserved GTPase, which is part of the mitochondrial protein translation machinery. In its activated form it delivers the aminoacyl-tRNAs to the A site of the mitochondrial ribosome. We report here on a baby girl with severe infantile macrocystic leukodystrophy with micropolygyria and a combined defect of complexes I and IV in muscle biopsy, caused by a novel mutation identified in TUFM. Using human mutant cells and the yeast model, we demonstrate the pathological role of the novel variant. Moreover, results of a molecular modeling study suggest that the mutant is inactive in mitochondrial polypeptide chain elongation, probably as a consequence of its reduced ability to bind mitochondrial aa-tRNAs. Four patients have so far been described with mutations in TUFM, and, following the first description of the disease in a single patient, we describe similar clinical and neuroradiological features in an additional patient.

MTERF4 regulates the mitochondrial dysfunction induced by MPP+ in SH-SY5Y cells

Mitochondrial transcription termination factor 4, MTERF4, a member of the MTERF family, has been implicated in the regulation of mitochondrial translation by targeting NSUN4 to the large mitochondrial ribosome. Here, we found a novel role for MTERF4 in regulating mitochondrial dysfunction induced by MPP+. We observed that knockdown of MTERF4 in SH-SY5Y cells resulted in increased mitochondrial DNA transcription levels and decreased mitochondrial DNA translation levels. In addition, after treatment with 2 mM MPP+ for 24 h, the expression levels of MTERF4 were decreased compared to wide-type SH-SY5Y cells. Moreover, after exposure to 2 mM MPP+ for 24 h, knockdown of MTERF4 in SH-SY5Y cells worsened the mitochondrial dysfunction induced by MPP+, including increased reactive oxygen species, accumulated cleaved PARP-1, decreased mitochondrial membrane potential and depressed mitochondrial complexes. Furthermore, overexpression of MTERF4 in SH-SY5Y cells partially alleviated the mitochondrial dysfunction induced by MPP+. Based on these findings, we suggest that the main function of MTERF4 is regulating mtDNA expression, and it is the crucial factor in the mechanism of mitochondrial dysfunction in SH-SY5Y cells induced by MPP+. MTERF4 probably is the triggering of the pathogenesis of Parkinson's disease induced by environmental toxin.

PP05.8 – 2528: Early-onset mild type leukoencephalopathy caused by a homozygous EARS2 mutation

Objective Mitochondrial diseases are a heterogeneous group of disorders resulting from primary dysfunction of the respiratory chain due to both nuclear and mitochondrial DNA mutations. Defective translation of mitochondrial DNA-encoded proteins, caused by mutations in either the mitochondrial or nuclear genomes, often manifest as severe infantile leukoencephalopathy. EARS2 is one of the newest members of nuclear mitochondrial disorders characterized by disturbed mitochondrial translation. This gene encodes the mitochondrial aminoacyl-tRNA synthetase specific for glutamate which is involved in mitochondrial DNA translation. Methods A patient is presented with an early onset, mild leukoencephalopathy which is caused by mutations in a single gene; EARS2. Results We report a 16 month-old girl, who was presented with a history of failure to walk. She achieved head control at 2 months and succeeded in sitting without support at 10 months. She was able to stand with support however she couldn't walk. Neurological findings were normal except failure to walk. Brain MRI revealed T2 and Fluid Attenuated Inversion Recovery (FLAIR) hyperintensity of the thalami, nucleus caudatus, putamen, midbrain, pons and cerebellar white matter. Periventricular and supraventricular white matter, corpus callosum and periaqueductal grey matter had T2 and FLAIR hyperintensity. Notably, periventricular rim, bilateral capsula interna and corona radiata were spared and had normal T2 signal. We sequenced the exons, intron-exon boundaries and promotor region of the EARS2 gene. We found homozygous mutation of the gene. Both of the maternal and paternal allele harboured a c.322C>T transition, predicting a p.R108W change. Conclusion We present a mild phenotype of EARS2 mutation, in addition to the reported cases of rapidly progressive disease with severe CNS involvement, which indicates particular mild phenotypes may remain under-diagnosed due to vague symptoms.

Mitochondrial disease—an important cause of end-stage renal failure

Kidneys are highly aerobic organs. They receive roughly a quarter of the cardiac output and contain a high density of mitochondria, particularly in the cortical tubules, which are required to produce adenosine triphosphate (ATP) in sufficient quantity to power the re-uptake of over 98 % of the filtered load. Given the dependence of renal function on aerobic metabolism, it is not surprising that impairment of normal mitochondrial function-due to insults such as ischaemia, drug toxicity and genetic mitochondrial disease-can lead to kidney failure. In this edition of Pediatric Nephrology, D'Aco and colleagues (doi: 10.1007/s00467-012-2354-y ) describe a patient who developed end-stage renal failure caused by a pathogenic mutation (m.586G > A) in the gene encoding the mitochondrial tRNA for phenylalanine, which adversely affects the translation of mitochondrial DNA. The pathogenicity of this mutation was confirmed in cybrid studies using fibroblasts obtained from the patient. In light of this report, m.586G > A should now be added to the rapidly expanding list of mitochondrial and nuclear gene mutations causing mitochondrial disease with renal involvement. Furthermore, mitochondrial disease should be considered as an underlying aetiology in cases of unexplained renal failure, particularly in the context of a multisystem disorder. Renal replacement therapy is an option for patients with mitochondrial disease, but life expectancy even with this therapy may be limited by co-morbidities.

Leukoencephalopathy with thalamus and brainstem involvement and high lactate ‘LTBL’ caused by EARS2 mutations

In the large group of genetically undetermined infantile-onset mitochondrial encephalopathies, multiple defects of mitochondrial DNA-related respiratory-chain complexes constitute a frequent biochemical signature. In order to identify responsible genes, we used exome-next-generation sequencing in a selected cohort of patients with this biochemical signature. In an isolated patient, we found two mutant alleles for EARS2, the gene encoding mitochondrial glutamyl-tRNA synthetase. The brain magnetic resonance imaging of this patient was hallmarked by extensive symmetrical cerebral white matter abnormalities sparing the periventricular rim and symmetrical signal abnormalities of the thalami, midbrain, pons, medulla oblongata and cerebellar white matter. Proton magnetic resonance spectroscopy showed increased lactate. We matched this magnetic resonance imaging pattern with that of a cohort of 11 previously selected unrelated cases. We found mutations in the EARS2 gene in all. Subsequent detailed clinical and magnetic resonance imaging based phenotyping revealed two distinct groups: mild and severe. All 12 patients shared an infantile onset and rapidly progressive disease with severe magnetic resonance imaging abnormalities and increased lactate in body fluids and proton magnetic resonance spectroscopy. Patients in the 'mild' group partially recovered and regained milestones in the following years with striking magnetic resonance imaging improvement and declining lactate levels, whereas those of the 'severe' group were characterized by clinical stagnation, brain atrophy on magnetic resonance imaging and persistent lactate increases. This new neurological disease, early-onset leukoencephalopathy with thalamus and brainstem involvement and high lactate, is hallmarked by unique magnetic resonance imaging features, defined by a peculiar biphasic clinical course and caused by mutations in a single gene, EARS2, expanding the list of medically relevant defects of mitochondrial DNA translation.

Next‐generation Sequencing Facilitates the Diagnosis in a Child With Twinkle Mutations Causing Cholestatic Liver Failure

Next‐generation Sequencing Facilitates the Diagnosis in a Child With Twinkle Mutations Causing Cholestatic Liver Failure

Spinal Cord Calcification in an Early-Onset Progressive Leukoencephalopathy

Spinal cord calcifications are an unusual finding in pediatric neurology. We here describe a young child who presented severe psychomotor delay, tetraplegia, deafness, and anemia. Neuroradiological investigations revealed severe leukodystrophy and unusual calcifications in the cerebral white matter and all along the medullary pathways. Common infectious and metabolic diseases were ruled out. A mild reduction in the activity of several respiratory chain complexes was documented on muscle biopsy. Of interest, we found an intronic variant in DARS2, a gene involved in mitochondrial DNA translation, responsible for the syndrome of leukoencephalopathy with brainstem and spinal cord involvement and high brain lactate. In our opinion, our case, and probably 2 previously reported Japanese siblings with a picture very similar to that of our patient, could represent a new, progressive leukoencephalomyelopathy.

Deleterious Mutation in the Mitochondrial Arginyl–Transfer RNA Synthetase Gene Is Associated with Pontocerebellar Hypoplasia

Homozygosity mapping was performed in a consanguineous Sephardic Jewish family with three patients who presented with severe infantile encephalopathy associated with pontocerebellar hypoplasia and multiple mitochondrial respiratory-chain defects. This resulted in the identification of an intronic mutation in RARS2, the gene encoding mitochondrial arginine-transfer RNA (tRNA) synthetase. The mutation was associated with the production of an abnormally short RARS2 transcript and a marked reduction of the mitochondrial tRNA(Arg) transcript in the patients' fibroblasts. We speculate that missplicing mutations in mitochondrial aminoacyl-tRNA synthethase genes preferentially affect the brain because of a tissue-specific vulnerability of the splicing machinery.

Infantile Encephalopathy and Defective Mitochondrial DNA Translation in Patients with Mutations of Mitochondrial Elongation Factors EFG1 and EFTu

Mitochondrial protein translation is a complex process performed within mitochondria by an apparatus composed of mitochondrial DNA (mtDNA)-encoded RNAs and nuclear DNA-encoded proteins. Although the latter by far outnumber the former, the vast majority of mitochondrial translation defects in humans have been associated with mutations in RNA-encoding mtDNA genes, whereas mutations in protein-encoding nuclear genes have been identified in a handful of cases. Genetic investigation involving patients with defective mitochondrial translation led us to the discovery of novel mutations in the mitochondrial elongation factor G1 (EFG1) in one affected baby and, for the first time, in the mitochondrial elongation factor Tu (EFTu) in another one. Both patients were affected by severe lactic acidosis and rapidly progressive, fatal encephalopathy. The EFG1-mutant patient had early-onset Leigh syndrome, whereas the EFTu-mutant patient had severe infantile macrocystic leukodystrophy with micropolygyria. Structural modeling enabled us to make predictions about the effects of the mutations at the molecular level. Yeast and mammalian cell systems proved the pathogenic role of the mutant alleles by functional complementation in vivo. Nuclear-gene abnormalities causing mitochondrial translation defects represent a new, potentially broad field of mitochondrial medicine. Investigation of these defects is important to expand the molecular characterization of mitochondrial disorders and also may contribute to the elucidation of the complex control mechanisms, which regulate this fundamental pathway of mtDNA homeostasis.

Mitochondrial disorders.

Mitochondria, the organelles devoted to energy production, have unique genetic features. They possess their own genome encoding several subunits of the respiratory chain, the majority of which are encoded by nuclear DNA, as well as factors involved in replication, transcription and translation of mitochondrial DNA. In the past few years, molecular lesions of mitochondrial DNA have been reported with increasing frequency as a source of human disorders. Several mutations of mitochondrial DNA, either as sporadic large scale rearrangements (deletions, duplications) or maternally-inherited point mutations, have been associated with well defined clinical syndromes. Furthermore, because of the nuclear DNA contribution to the synthesis of respiratory chain enzymes, phenotypes transmitted as Mendelian traits have also been identified and associated with qualitative (multiple deletions) and quantitative (depletion) lesions of the mitochondrial genome. The clinical manifestations of mitochondrial DNA mutations are extremely heterogeneous, ranging from myopathies, encephalomyopathies, cardiopathies, to complex multisystem syndromes. Clinical, morphological, biochemical and molecular genetic data are necessary for diagnosis. The recent advances in genetic studies provide both diagnostic tools and new pathogenetic insights into this rapidly expanding area of human pathology.