Dimerization of a pathogenic human mitochondrial tRNA

Mutations of human mitochondrial transfer RNA (tRNA) are implicated in a variety of multisystemic diseases. The most prevalent pathogenic mitochondrial mutation is the A3243G substitution within the gene for tRNA(Leu(UUR)). Here we describe the pronounced structural change promoted by this mutation. The A3243G mutation induces the formation of a tRNA dimer that strongly self-associates under physiological conditions. The dimerization interface in the mutant tRNA is a self-complementary hexanucleotide in the D-stem, a particularly weak structural element within tRNA(Leu(UUR)). Aminoacylation of the A3243G mutant is significantly attenuated, and mutational studies indicate that dimerization is partially responsible for the observed loss of function. The disruption of a conserved tertiary structural contact also contributes to the functional defect. The pathogenic mutation is proposed to interfere with the cellular function of human mitochondrial tRNA(Leu(UUR)) by destabilizing the native structure and facilitating the formation of a dimeric complex with low biological activity.     

Core flexibility of a truncated metazoan mitochondrial tRNA

Secondary and tertiary structures of tRNAs are remarkably preserved from bacteria to humans, the notable exception being the mitochondrial (m) tRNAs of metazoans, which often deviate substantially from the canonical cloverleaf (secondary) or ‘L’-shaped (tertiary) structure. Many metazoan mtRNAs lack either the TψC (T) or dihydrouridine (D) loops of the canonical cloverleaf, which are known to confer structural rigidity to the folded structure. Thus, the absence of canonical TψC–D interactions likely results in greater dispersion of anticodon-acceptor interstem angle than for canonical tRNAs. To test this hypothesis, we have assessed the dispersion of the anticodon-acceptor angle for bovine mtRNA^Ser(AGY), which lacks the canonical D arm and is thus incapable of forming stabilizing interarm interactions. Using the method of transient electric birefringence (TEB), and by changing the helical torsion angle between a core mtRNA bend and a second bend of known angle/rigidity, we have demonstrated that the core of mtRNA^Ser(AGY) has substantially greater flexibility than its well-characterized canonical counterpart, yeast cytoplasmic tRNA^Phe. These results suggest that increased flexibility, in addition to a more open interstem angle, would allow both noncanonical and canonical mtRNAs to utilize the same protein synthetic apparatus.     

PhosphoScan: a probability-based method for phosphorylation site prediction using MS2/MS3 pair information

Phosphopeptide identification and phosphorylation site localization are crucial aspects of many biological studies. Furthermore, multiple phosphorylations of peptides make site localization even more difficult. We developed a probability-based method to unambiguously determine phosphorylation sites within phosphopeptides using MS2/3 pair information. A comparison test was performed with SEQUEST and MASCOT predictions using a spectral data set from a synthetic doubly phosphorylated peptide, and the results showed that PhosphoScan analysis yielded a 63% phosphopeptide localization improvement compared with SEQUEST and a 57% improvement compared with MASCOT.     

Nucleotide sequence of a wheat mitochondrial lysine tRNA gene

We present the sequence of a wheat mitochondrial (mt) lysine tRNA gene (trnK-UUU). This gene more closely resembles its E. coli counterpart than it does the corresponding gene in fungal or mammalian mtDNA. Hybridization experiments with a trnK-specific probe suggest that at least two copies of this tRNALys gene are present in the wheat mitochondrial genome.     

Preeclampsia: multiple approaches for a multifactorial disease

Preeclampsia is a pregnancy-specific disorder characterized by hypertension and excess protein excretion in the urine. It is an important cause of maternal and fetal morbidity and mortality worldwide. The disease is almost exclusive to humans and delivery of the pregnancy continues to be the only effective treatment. The disorder is probably multifactorial, although most cases of preeclampsia are characterized by abnormal maternal uterine vascular remodeling by fetally derived placental trophoblast cells. Numerous in vitro and animal models have been used to study aspects of preeclampsia, the most common being models of placental oxygen dysregulation, abnormal trophoblast invasion, inappropriate maternal vascular damage and anomalous maternal-fetal immune interactions. Investigations into the pathophysiology and treatment of preeclampsia continue to move the field forward, albeit at a frustratingly slow pace. There remains a pressing need for novel approaches, new disease models and innovative investigators to effectively tackle this complex and devastating disorder.     

A whole mitochondrial genome screening in a MELAS patient: a novel mitochondrial tRNA(Val) mutation

Mitochondrial encephalopathy, lactic acidosis and strokelike episodes (MELAS) syndrome is a mitochondrial disorder characterized by a wide variety of clinical presentations and a multisystemic organ involvement. In this study, we report a Tunisian girl with clinical features of MELAS syndrome who was negative for the common m.3243A>G mutation, but also for the reported mitochondrial DNA (mtDNA) mutations and deletions. Screening of the entire mtDNA genome showed several known mitochondrial variants besides to a novel transition m.1640A>G affecting a wobble adenine in the anticodon stem region of the tRNA(Val). This nucleotide was conserved and it was absent in 150 controls suggesting its pathogenicity. In addition, no mutations were found in the nuclear polymerase gamma-1 gene (POLG1). These results suggest further investigation nuclear genes encoding proteins responsible for stability and structural components of the mtDNA or to the oxidative phosphorylation machinery to explain the phenotypic variability in the studied family.     

Proteomic consequences of a human mitochondrial tRNA mutation beyond the frame of mitochondrial translation

Numerous severe neurodegenerative and neuromuscular disorders, characterized biochemically by strong perturbations in energy metabolism, are correlated with single point mutations in mitochondrial genes coding for transfer RNAs. Initial comparative proteomics performed on wild-type and Myoclonic Epilepsy and Ragged Red Fibers (MERRF) mitochondria from sibling human cybrid cell lines revealed the potential of this approach. Here a quantitative analysis of several hundred silver-stained spots separated by two-dimensional gel electrophoresis was performed in the specific case of a couple of mitochondria, containing or not mutation A8344G in the gene for mitochondrial tRNALys, correlated with MERRF syndrome. Computer-assisted analysis allowed us to detect 38 spots with significant quantitative variations, of which 20 could be assigned by mass spectrometry. These include nuclear encoded proteins located in mitochondria such as respiratory chain subunits, metabolic enzymes, a protein of the mitochondrial translation machinery, and cytosolic contaminants. Furthermore, Western blotting combined with mass spectrometry revealed the occurrence of numerous isoforms of pyruvate dehydrogenase subunits, with subtle changes in post-translational modifications. This comparative proteomic approach gives the first insight for nuclear encoded proteins that undergo the largest quantitative changes, and pinpoints new potential molecular partners involved in the cascade of events that connect genotype to phenotype.     

Mutations in mitochondrial tRNA genes: a frequent cause of neuromuscular diseases.

We have sequenced the tRNA genes of mtDNA from patients with chronic progressive external ophthalmoplegia (CPEO) without detectable mtDNA deletions. Four point mutations were identified, located within highly conserved regions of mitochondrial tRNA genes, namely tRNA(Leu)(UAG), tRNA(Ser)(GCU), tRNA(Gly) and tRNA(Lys). One of these mutations (tRNA(Leu)(UAG)) was found in four patients with different forms of mitochondrial myopathy. An accumulation of three different tRNA point mutations (tRNA(Leu)(UAG)), tRNA(Ser)(GCU) and tRNA(Gly) was observed in a single patient, suggesting that mitochondrial tRNA genes represent hotspots for point mutations causing neuromuscular diseases.     

Comparative proteomics as a new tool for exploring human mitochondrial tRNA disorders.

More than 70 different point mutations in human mitochondrial tRNA genes are correlated with severe disorders, including fatal cardiopathies, encephalopathies, myopathies, and others. So far, investigation of the molecular impact(s) of mutations has focused on the affected tRNA itself by seeking structural and/or functional perturbations capable of interfering with synthesis of the 13 mitochondrion-encoded subunits of respiratory chain complexes. Here, a proteomic approach was used to investigate whether such mutations would affect the pattern of mitochondrial proteins at a broader level. Analysis of several hundred mitochondrial proteins from sibling cybrid cell lines by two-dimensional electrophoresis, an approach that takes into account all regulatory steps of mitochondrial and nuclear gene expression, indeed reveals a number of up- and downregulated proteins when healthy and single-point-mutation-carrying mitochondria representative of either MELAS or MERRF syndrome were compared. Assignment by mass spectrometry of the two proteins which exhibit obvious large quantitative decreases in the levels of both pathologic mitochondria identified nuclear-encoded subunits of cytochrome c oxidase, a respiratory chain complex. This clearly shows a linkage between the effects of mutations in mitochondrial tRNA genes and the steady-state level of nuclear-encoded proteins in mitochondria. It opens new routes toward a large-scale exploration of potential proteic partners involved in the genotype-phenotype correlation of mitochondrial disorders.     

ARWEN: a program to detect tRNA genes in metazoan mitochondrial nucleotide sequences

MOTIVATION: Mitochondrial genomes encode their own transfer RNAs (tRNAs). These are often degenerate in sequence and structure compared to tRNAs in their bacterial ancestors. This is one of the reasons why current tRNA gene predictor programs perform poorly identifying mitochondrial tRNA genes. As a consequence there is a need for a new program with the specific aim of predicting these tRNAs. RESULTS: In this study, we present the software ARWEN that identifies tRNA genes in metazoan mitochondrial nucleotide sequences. ARWEN detects close to 100% of previously annotated genes. AVAILABILITY: An online version, software for download and test results are available at www.acgt.se/online.html     

Mitochondrial tRNA valine as a recurrent target for mutations involved in mitochondrial cardiomyopathies

The aim of this study was to identify the genetic defect in two patients having cardiac dysfunction accompanied by neurological symptoms, and in one case MRI evidence of cortical and cerebellar atrophy with hyperintensities in the basal ganglia. Muscle biopsies from each patient revealed single and combined mitochondrial respiratory chain deficiency. The complete mtDNA sequencing of both patients revealed two transitions in the mitochondrial tRNA(Val) gene (MT-TV) (m.1628C>T in Patient 1, and m.1644G>A in Patient 2). The functional and molecular analyses reported here suggest that the MT-TV gene should be routinely considered in the diagnosis of mitochondrial cardiomyopathies.     

MITOPRED: a genome-scale method for prediction of nucleus-encoded mitochondrial proteins

MOTIVATION: Currently available methods for the prediction of subcellular location of mitochondrial proteins rely largely on the presence of mitochondrial targeting signals in the protein sequences. However, a large fraction of mitochondrial proteins lack such signals, making those tools ineffective for genome-scale prediction of mitochondria-targeted proteins. Here, we propose a method for genome-scale prediction of nucleus-encoded mitochondrial proteins. The new method, MITOPRED, is based on the Pfam domain occurrence patterns and the amino acid compositional differences between mitochondrial and non-mitochondrial proteins. RESULTS: MITOPRED could predict mitochondrial proteins with 100% specificity at a 44% sensitivity rate and with 67% specificity at 99% sensitivity. Additionally, it was sufficiently robust to predict mitochondrial proteins across different eukaryotic species with similar accuracy. Based on Matthews correlation coefficient measure, the prediction performance of MITOPRED is clearly superior (0.73) to those of the two popular methods TargetP (0.51) and PSORT (0.53). Using this method, we predicted the nucleus-encoded mitochondrial proteins from six complete genomes (three invertebrate, two vertebrate and one plant species) and estimated the total number in each genome. In human, our method estimated the existence of 1362 mitochondrial proteins corresponding to 4.8% of the total proteome. AVAILABILITY: MITOPRED program is freely accessible at http://mitopred.sdsc.edu. Source code is available on request from the authors. SUPPLEMENTARY INFORMATION: Training data sets are also available at http://mitopred.sdsc.edu     

PLMItRNA, a database for mitochondrial tRNA genes and tRNAs in photosynthetic eukaryotes

The PLMItRNA database for mitochondrial tRNA molecules and genes in VIRIDIPLANTAE: (green plants) [Volpetti,V., Gallerani,R., DeBenedetto,C., Liuni,S., Licciulli,F. and Ceci,L.R. (2000) Nucleic Acids Res., 28, 159-162] has been enlarged to include algae. The database now contains 436 genes and 16 tRNA entries relative to 25 higher plants, eight green algae, four red algae (RHODOPHYTAE:) and two STRAMENOPILES: The PLMItRNA database is accessible via the WWW at http://bio-www.ba.cnr.it:8000/PLMItRNA.