Mitochondrial Polymerase Research Articles

Evolution of plant phage-type RNA polymerases: the genome of the basal angiosperm Nuphar advena encodes two mitochondrial and one plastid phage-type RNA polymerases

BackgroundIn mono- and eudicotyledonous plants, a small nuclear gene family (RpoT, RNA polymerase of the T3/T7 type) encodes mitochondrial as well as chloroplast RNA polymerases homologous to the T-odd bacteriophage enzymes. RpoT genes from angiosperms are well characterized, whereas data from deeper branching plant species are limited to the moss Physcomitrella and the spikemoss Selaginella. To further elucidate the molecular evolution of the RpoT polymerases in the plant kingdom and to get more insight into the potential importance of having more than one phage-type RNA polymerase (RNAP) available, we searched for the respective genes in the basal angiosperm Nuphar advena.ResultsBy screening a set of BAC library filters, three RpoT genes were identified. Both genomic gene sequences and full-length cDNAs were determined. The NaRpoT mRNAs specify putative polypeptides of 996, 990 and 985 amino acids, respectively. All three genes comprise 19 exons and 18 introns, conserved in their positions with those known from RpoT genes of other land plants. The encoded proteins show a high degree of conservation at the amino acid sequence level, including all functional crucial regions and residues known from the phage T7 RNAP. The N-terminal transit peptides of two of the encoded polymerases, NaRpoTm1 and NaRpoTm2, conferred targeting of green fluorescent protein (GFP) exclusively to mitochondria, whereas the third polymerase, NaRpoTp, was targeted to chloroplasts. Remarkably, translation of NaRpoTp mRNA has to be initiated at a CUG codon to generate a functional plastid transit peptide. Thus, besides AGAMOUS in Arabidopsis and the Nicotiana RpoTp gene, N. advena RpoTp provides another example for a plant mRNA that is exclusively translated from a non-AUG codon. In contrast to the RpoT of the lycophyte Selaginella and those of the moss Physcomitrella, which are according to phylogenetic analyses in sister positions to all other phage-type polymerases of angiosperms, the Nuphar RpoTs clustered with the well separated clades of mitochondrial (NaRpoTm1 and NaRpoTm2) and plastid (NaRpoTp) polymerases.ConclusionsNuphar advena encodes two mitochondrial and one plastid phage-type RNAP. Identification of a plastid-localized phage-type RNAP in this basal angiosperm, orthologous to all other RpoTp enzymes of flowering plants, suggests that the duplication event giving rise to a nuclear gene-encoded plastid RNA polymerase, not present in lycopods, took place after the split of lycopods from all other tracheophytes. A dual-targeted mitochondrial and plastididal RNA polymerase (RpoTmp), as present in eudicots but not monocots, was not detected in Nuphar suggesting that its occurrence is an evolutionary novelty of eudicotyledonous plants like Arabidopsis.

Nucleoside reverse transcriptase inhibitor toxicity and mitochondrial DNA

Importance of the field: HIV/AIDS is a worldwide epidemic. While there remains no cure for the HIV-1 infection, nucleoside reverse transcriptase inhibitors (NRTIs) have helped transform the HIV-1 infection from a lethal disease into a chronic illness. Though NRTIs inhibit HIV-1 replication, they exhibit side effects in human tissues that appear to result from NRTI inhibition of human mitochondrial polymerase γ (pol γ).Areas covered in this review: This review discusses the current knowledge of NRTI-induced toxicity, specifically the inhibition of pol γ and the mitochondrial toxicity from incorporation of NRTIs into mitochondrial DNA. Details are discussed about general mechanisms of NRTI toxicity and how specific tissue toxicities in mitochondria relate to clinical manifestation.What the reader will gain: A detailed knowledge of the mitochondrial toxicity resulting from NRTI-inclusive therapies and related tissue toxicities are provided. This review presents both the molecular effects of NRTI usage on mitochondrial genetic homeostasis and energy metabolism as well as the clinical manifestations associated with NRTI toxicities.Take home message: NRTIs remain a critical component of current HIV-1 treatment regimens. Future NRTIs should provide higher specificity for HIV-RT and lower incorporation by pol γ to minimize mitochondrial toxicity. Alternatively, therapeutic interventions to prevent or alleviate mitochondrial toxicity should be addressed.

POLG, but not PEO1, is a frequent cause of cerebellar ataxia in Central Europe

Nuclear genes, in particular mitochondrial polymerase gamma (POLG) and PEO1, have been increasingly recognized to cause mitochondrial diseases. Both genes assume a complementary role as part of the mitochondrial DNA (mtDNA) replication fork and, accordingly, seem to present with largely overlapping phenotypical spectra. We assessed the frequency and phenotypic spectrum of PEO1 compared to POLG mutations in a cohort of 80 patients with cerebellar ataxia for which common repeat expansion diseases had been excluded. Patients were selected to present additional features previously described for PEO1 mutations, namely early age of onset, progressive external ophthalmoplegia (PEO), or epilepsy. Whereas PEO1 mutations were not found in our cohort, POLG frequently caused ataxia with PEO (47%), psychiatric comorbidities (20%) and, more rarely, with epilepsy (14%). Thus, PEO1 is rare in Central Europe even in those patients displaying characteristic phenotypic features. In contrast, POLG is rather common in Central European ataxia patients. It should be particularly considered in ataxia patients with PEO, psychiatric comorbidities, and/or sensory neuropathy, even if characteristic mitochondrial extra-CNS features are absent.

Mitochondrial DNA depletion and its correlation with TFAM, TFB1M, TFB2M and POLG in human diffusely infiltrating astrocytomas

Mitochondrial DNA (mtDNA) alterations and their clinical and pathological implications have been analyzed in several human malignancies. A marked decrease in mtDNA copy number along with the increase in malignancy was observed in diffusely infiltrating astrocytomas (24 WHO grade II, 18 grade III, and 78 grade IV or GBM) compared to non-neoplastic brain tissues, being mostly depleted in GBM. Although high relative gene expression levels of mtDNA replication regulators (mitochondrial polymerase catalytic subunit ( POLG), transcription factors A ( TFAM), B1 ( TFB1M) and B2 ( TFB2M)) were detected, it cannot successfully revert the mtDNA depletion observed in our samples. On the other hand, a strong correlation among the expression levels of mitochondrial transcription factors corroborates with the TFAM role in the direct control of TFB1M and TFB2M during initiation of mtDNA replication. POLG expression was related to decreased mtDNA copy number, and its overexpression associated with TFAM expression levels also have an impact on long-term survival among GBM patients, interpreted as a potential predictive factor for better prognosis.

Thalamus lesions in chronic and acute seizure disorders

Transient signal changes in the pulvinar have been described following status epilepticus. However, we observed persistent thalamus changes after seizures. The purpose of this study was to characterize thalamus changes in patients with seizure disorders and to correlate imaging findings with clinical features. We searched among 5,500 magnetic resonance imaging (MRI) exams performed in patients with seizures and identified 43 patients. The MRI scans of these patients were reviewed and correlated with clinical data. We identified four patterns of thalamus lesions: (a) fluid attenuated inversion recovery-hyperintense pulvinar lesions (20 patients), as known from status epilepticus. Ten patients in this group had a status epilepticus. Among the remaining patients, three had frequent seizures and seven had sporadic seizures. Twelve patients had follow-up exams for a median of 11months. The lesions had persisted in 11/12 cases in the last available exam and were reversible in one case only. In seven cases, cone-shaped thalamus atrophy resulted, (b) linear defects in the medial and anterior thalamus (five patients), accompanied by atrophy of the mamillary body and the fornix in patients with chronic epilepsy, (c) extensive bilateral thalamus lesions in two patients with a syndrome caused by mutation in the mitochondrial polymerase gamma, and (d) other thalamus lesions not associated with the seizure disorder (16 patients). The spectrum of thalamus lesions in patients with seizure disorders is wider than previously reported. Postictal pulvinar lesions can persist and may result in thalamic atrophy. Linear defects in the anterior thalamus are associated with limbic system atrophy.

POLG1 p.R722H mutation associated with multiple mtDNA deletions and a neurological phenotype

BackgroundThe c.2447G>A (p.R722H) mutation in the gene POLG1 of the catalytic subunit of human mitochondrial polymerase gamma has been previously found in a few occasions but its pathogenicity has remained uncertain. We set out to ascertain its contribution to neuromuscular disease.MethodsProbands from two families with probable mitochondrial disease were examined clinically, muscle and buccal epithelial DNA were analyzed for mtDNA deletions, and the POLG1, POLG2, ANT1 and Twinkle genes were sequenced.ResultsAn adult proband presented with progressive external ophthalmoplegia, sensorineural hearing impairment, diabetes mellitus, dysphagia, a limb myopathy and dementia. Brain MRI showed central and cortical atrophy, and 18F-deoxyglucose PET revealed reduced glucose uptake. Histochemical analysis of muscle disclosed ragged red fibers and cytochrome c oxidase-negative fibers. Electron microscopy showed subsarcolemmal aggregates of morphologically normal mitochondria. Multiple mtDNA deletions were found in the muscle, and sequencing of the POLG1 gene revealed a homozygous c.2447G>A (p.R722H) mutation. His two siblings were also homozygous with respect to the p.R722H mutation and presented with dementia and sensorineural hearing impairment. In another family the p.R722H mutation was found as compound heterozygosity with the common p.W748S mutation in two siblings with mental retardation, ptosis, epilepsy and psychiatric symptoms. The estimated carrier frequency of the p.R722H mutation was 1:135 in the Finnish population. No mutations in POLG2, ANT1 and Twinkle genes were found. Analysis of the POLG1 sequence by homology modeling supported the notion that the p.R722H mutation is pathogenic.ConclusionsThe recessive c.2447G>A (p.R722H) mutation in the linker region of the POLG1 gene is pathogenic for multiple mtDNA deletions in muscle and is associated with a late-onset neurological phenotype as a homozygous state. The onset of the disease can be earlier in compound heterozygotes.

Mitochondrial DNA variation in natural populations of Japanese larch (Larix kaempferi)

AbstractLevels of genetic variation within and among natural populations of Japanese larch [Larix kaempferi (Lamb.) Carrière] were evaluated by Restriction Fragment Length Polymorphism (RFLP) analysis of mitochondrial DNA (mtDNA) obtained from 209 individuals representing 14 natural populations distributed across most of the species’ natural range in central Japan. Polymorphisms were screened by two RFLP techniques: Southern hybridization of mitochondrial gene probes and Polymerase Chain Reaction (PCR)-RFLP analysis of sequence-tagged sites. However, obvious polymorphism was only detected around the coxIII gene, following digestion with the EcoRI restriction enzyme, in the Southern hybridization analysis. Five haplotypes were identified across the 14 populations, with 11 populations exhibiting little or no genetic variation. Within-population genetic variation was low (HS = 0.158), while population differentiation was substantial (a standardized genetic differentiation measure, G’ST = 0.581). The northernmost, marginal population on Mt. Manokamidake made a significant contribution to the total detected genetic variation. The mtDNA variation displayed geographic structure despite the restricted natural distribution of Japanese larch. Results of this study should be useful for establishing criteria for the genetic conservation and management of natural populations of the species.

A patient with typical clinical features of mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) but without an obvious genetic cause: a case report

IntroductionThere are currently 23 missense point mutations and one 4 basepair deletion spanning different mitochondrial genes associated with mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS). The spectrum of mitochondrial DNA mutations in Arab patients with MELAS is largely unknown.Case presentationA standard clinical examination was carried out on a 34-year-old Saudi woman showing clinical features of MELAS. Fresh frozen muscle tissue was subjected to enzyme histochemical analysis. DNA was extracted from her leukocytes and muscle tissue, and the full mitochondrial genome was screened for base substitution mutations and deletions. Additionally, we screened the polymerase gamma-1 nuclear gene for mutations. The patient was negative for the most common m.3243 A>G MELAS mutation. Sequencing the full mitochondrial genome did not reveal any known or potentially pathogenic sequence changes. The polymerase gamma-1 gene was also free from mutations.ConclusionThe clinical picture described here typically fits that observed in patients with MELAS or mitochondrial stroke-like events, but mutations in recognized genes (mitochondrial DNA and polymerase gamma-1 gene) were absent. We report the case of a patient with typical clinical features of MELAS, but without an obvious genetic cause.

Mitochondrial tRNAs as light strand replication origins: Similarity between anticodon loops and the loop of the light strand replication origin predicts initiation of DNA replication

Stem-loop hairpins formed by mitochondrial light strand replication origins (OL) and by heavy strand DNA coding for tRNAs that form OL-like structures initiate mitochondrial replication. The loops are recognized by one of the two active sites of the vertebrate mitochondrial gamma polymerase, which are homologous to the active sites of class II amino-acyl tRNA synthetases. Therefore, the polymerase site recognizing the OL loop could recognize tRNA anticodon loops and sequence similarity between anticodon and OL loops should predict initiation of DNA replication at tRNAs. Strengths of genome-wide deamination gradients starting at tRNA genes estimate extents by which replication starts at that tRNA. Deaminations (A-->G and C-->T) occur proportionally to time spent single stranded by heavy strand DNA during mitochondrial light strand replication. Results show that deamination gradients starting at tRNAs are proportional to sequence similarity between OL and tRNA loops: most for anticodon-, least D-, intermediate for TpsiC-loops, paralleling tRNA synthetase recognition interactions with these tRNA loops. Structural and sequence similarities with regular OLs predict OL function, loop similarity is dominant in most tRNAs. Analyses of sequence similarity and structure independently substantiate that DNA sequences coding for mitochondrial tRNAs sometimes function as alternative OLs. Pathogenic mutations in anticodon loops increase similarity with the human OL loop, non-pathogenic polymorphisms do not. Similarity/homology alignment hypotheses are experimentally testable in this system.

Digenic mutations in severe myoclonic epilepsy of infancy

The clinical features of severe myoclonic epilepsy of infancy (SMEI) resemble those of mitochondrial diseases, although most patients have the sodium channel (SCN1A) mutation. We describe a patient with SMEI and enlarged muscle mitochondria associated with mutations in mitochondrial polymerase gamma 1 (POLG1) and SCN1A. Due to increased risk of valproate-induced liver failure in patients with POLG1 mutations, we recommend POLG1 gene analysis for SMEI patients before valproate administration.

Ketogenic Diet in Alpers-Huttenlocher Syndrome

We report on a young girl with Alpers-Huttenlocher syndrome, as confirmed by mitochondrial polymerase gamma sequencing, who was treated with the classic (4 parts fat:1 part each of carbohydrate and protein) ketogenic diet after she presented with epilepsia partialis continua. She improved clinically, and her electroencephalogram improved dramatically. This is the first detailed report on the efficacy of the ketogenic diet in treating the epileptic encephalopathy of Alpers-Huttenlocher syndrome. We present a literature review of the utility of a ketogenic diet in mitochondrial disorders, and speculations as to why the diet may be helpful in Alpers-Huttenlocher syndrome.

Mitochondrial DNA level, but not active replicase, is essential for Caenorhabditis elegans development

A number of studies showed that the development and the lifespan of Caenorhabditis elegans is dependent on mitochondrial function. In this study, we addressed the role of mitochondrial DNA levels and mtDNA maintenance in development of C. elegans by analyzing deletion mutants for mitochondrial polymerase gamma (polg-1(ok1548)). Surprisingly, even though previous studies in other model organisms showed necessity of polymerase gamma for embryonic development, homozygous polg-1(ok1548) mutants had normal development and reached adulthood without any morphological defects. However, polg-1 deficient animals have a seriously compromised gonadal function as a result of severe mitochondrial depletion, leading to sterility and shortened lifespan. Our results indicate that the gonad is the primary site of mtDNA replication, whilst the mtDNA of adult somatic tissues mainly stems from the developing embryo. Furthermore, we show that the mtDNA copy number shows great plasticity as it can be almost tripled as a response to the environmental stimuli. Finally, we show that the mtDNA copy number is an essential limiting factor for the worm development and therefore, a number of mechanisms set to maintain mtDNA levels exist, ensuring a normal development of C. elegans even in the absence of the mitochondrial replicase.

Polyneuropathy and hepatopathy as an infantile manifestation of a POLG1 gene defect

Aims: Mutations of the mitochondrial polymerase gamma gene (POLG1) lead to reduced replication and repair of mtDNA. This results in an age-dependent spectrum of different clinical presentations.

Lymphocyte Mitochondrial Depolarization and Apoptosis in HIV-1-Infected HAART Patients

Efavirenz (EFV) and nevirapine (NVP), unlike nucleoside reverse transcriptase inhibitor drugs, do not inhibit mitochondrial (mt) polymerase gamma (Pol-gamma), although EFV has been shown to induce mt depolarization (Deltapsim) in vitro at supratherapeutic concentrations. However, the capacity of nonnucleoside reverse transcriptase inhibitor drugs to induce mt toxicity in vivo remains undetermined. To determine the influence of EFV and NVP on peripheral lymphocyte mt transmembrane potential (Deltapsim) and apoptosis in HIV-1-infected patients treated with these nonnucleoside reverse transcriptase inhibitors. Thirty-two HIV-1-infected patients on highly active antiretroviral therapy (HAART) between 4 and 24 months (12 on EFV, 20 on NVP) and 16 HAART-naive HIV-1-infected patients were enrolled into this study. All participants were black South African patients. Spontaneous peripheral lymphocyte apoptosis and Deltapsim were measured ex vivo by flow cytometry for all patients. : CD4 T-helper apoptosis for the EFV and NVP cohorts was 19.38% +/- 2.62% and 23.35% +/- 1.51% (mean +/- SEM), respectively, whereas total lymphocyte Deltapsim was 27.25% +/- 5.05% and 17.04% +/- 2.98%, respectively. Both parameters for each cohort were significantly lower (P < 0.05) than that of the HAART-naive patients. The NVP cohort exhibited both a significant time-dependent increase in peripheral lymphocyte Deltapsim (P = 0.038) and correlation between T-helper apoptosis and Deltapsim (P = 0.0005). These trends were not observed in the EFV cohort. This study provides evidence that both EFV and NVP induce peripheral lymphocyte Deltapsim in HIV-1-infected patients on nonnucleoside reverse transcriptase inhibitor-based HAART, which in the case of NVP is sufficient to induce the apoptosis cascade.

S12.18 A genetic system to study mitochondrial DNA mutations and their propagation in mice

The number of mutations in mitochondrial DNA (mtDNA) thought to causemetabolic disorders in patients is growing. At present, we lack the technology to introduce these mutations into the mtDNA of model organisms for detailed study. In this study, random mtDNA mutations were generated using the transgenic mtDNA mutator mouse, which expresses an engineered mitochondrial polymerase defective in its proofreading function. Female lines were then established to transmit and segregate the resulting mutations. We observed the selective loss of nonsense mutations in the protein coding genes, consistent with strong purifying selection by the female germline. The selection was evident two generations after the founder mtDNA mutator mouse, eliminating these nonsense mutations before they were detectable by Sanger sequencing methods. Curiously, the tRNA and rRNA genes do not show the same rapid selection against mutations despite strong evolutionary sequence conservation. Despite this purifying selection, putative deleterious mutations were identified and propagated in the mouse lines. Also, mutations analogous to human pathogenic mtDNA mutations were identified. These results will aid in the generation of more accurate models of inheritance of disease-causing mtDNA mutations and allow for the generation of laboratory mouse models of mtDNA mutations known in human disease.

Base Pair Hydrogen Bonds Are Essential for Proofreading Selectivity by the Human Mitochondrial DNA Polymerase

We have characterized the role of Watson-Crick hydrogen bonding in the 3'-terminal base pair on the 3'-5' exonuclease activity of the human mitochondrial DNA polymerase. Nonpolar nucleoside analogs of thymidine (dF) and deoxyadenosine (dQ) were used to eliminate hydrogen bonds while maintaining base pair size and shape. Exonuclease reactions were examined using pre-steady state kinetic methods. The time dependence of removal of natural nucleotides from the primer terminus paired opposite the nonpolar analogs dF and dQ were best fit to a double exponential function. The double exponential kinetics as well as the rates of excision (3-6 s(-1) fast phase, 0.16-0.3 s(-1) slow phase) are comparable with those observed during mismatch removal of natural nucleotides even when the analog was involved in a sterically correct base pair. Additionally, incorporation of the next correct base beyond a nonpolar analog was slow (0.04-0.22 s(-1)), so that more than 95% of terminal base pairs were removed rather than extended. The polymerase responds to all 3'-terminal base pairs containing a nonpolar analog as if it were a mismatch regardless of the identity of the paired base, and kinetic partitioning between polymerase and exonuclease sites failed to discriminate between correct and incorrect base pairs. Thus, sterics alone are insufficient, whereas hydrogen bond formation is essential for proper proofreading selectivity by the mitochondrial polymerase. The enzyme may use the alignment and prevention of fraying provided by proper hydrogen bonding and minor groove hydrogen bonding interactions as critical criteria for correct base pair recognition.

Reversible Valproate Hepatotoxicity and Associated Mitochondrial Disease

A 2-year-old boy with seizures complicated by reversible valproate-induced hepatic failure was subsequently found to have mitochondrial polymerase g gene (POLG1) mutations typical of Alpers-Huttenlocher disease, in a study at Newcastle University, UK.

Reversible valproate hepatotoxicity due to mutations in mitochondrial DNA polymerase γ (POLG1)

We report the case of a 2-year-old boy with seizures who developed hepatic failure shortly after commencing sodium valproate. Unexpectedly, liver function returned to normal on stopping the drug. Sequencing...

Nature of Mitochondrial DNA Deletions in Substantia Nigra Neurons

Mitochondrial DNA (mtDNA) deletions have been investigated in a number of neurodegenerative diseases. This study aimed to investigate the characteristics of mtDNA deletions found in single substantia nigra neurons from three patient groups: controls, Parkinson disease patients, and a patient with Parkinsonism due to multiple mtDNA deletions. We have identified 89 deletions from these neurons and examined the breakpoint characteristics of them. There was no difference in the types of mtDNA deletions detected in these neurons. These results suggest that the mechanism leading to the formation of these deletions in these three distinct groups could be the same.

Stable PNPase RNAi silencing: Its effect on the processing and adenylation of human mitochondrial RNA

Polynucleotide phosphorylase (PNPase) is a diverse enzyme, involved in RNA polyadenylation, degradation, and processing in prokaryotes and organelles. However, in human mitochondria, PNPase is located in the intermembrane space (IMS), where no mitochondrial RNA (mtRNA) is known to be present. In order to determine the nature and degree of its involvement in mtRNA metabolism, we stably silenced PNPase by establishing HeLa cell lines expressing PNPase short-hairpin RNA (shRNA). Processing and polyadenylation of mt-mRNAs were significantly affected, but, to different degrees in different genes. For instance, the stable poly(A) tails at the 3' ends of COX1 transcripts were abolished, while COX3 poly(A) tails remained unaffected and ND5 and ND3 poly(A) extensions increased in length. Despite the lack of polyadenylation at the 3' end, COX1 mRNA and protein accumulated to normal levels, as was the case for all 13 mt-encoded proteins. Interestingly, ATP depletion also altered poly(A) tail length, demonstrating that adenylation of mtRNA can be manipulated by indirect, environmental means and not solely by direct enzymatic activity. When both PNPase and the mitochondrial poly(A)-polymerase (mtPAP) were concurrently silenced, the mature 3' end of ND3 mRNA lacked poly(A) tails but retained oligo(A) extensions. Furthermore, in mtPAP-silenced cells, truncated adenylated COX1 molecules, considered to be degradation intermediates, were present but harbored significantly shorter tails. Together, these results suggest that an additional mitochondrial polymerase, yet to be identified, is responsible for the oligoadenylation of mtRNA and that PNPase, although located in the IMS, is involved, most likely by indirect means, in the processing and polyadenylation of mtRNA.