the melas mutation m 3243a g promotes reactivation of fetal cardiac genes and an epithelial mesenchymal transition like program via dysregulation of mirnas

Abstract The pathomechanisms underlying oxidative phosphorylation (OXPHOS) diseases are not well-understood, but they involve maladaptive changes in mitochondria-nucleus communication. Many studies on the mitochondria-nucleus cross-talk triggered by mitochondrial dysfunction have focused on the role played by regulatory proteins, while the participation of miRNAs remains poorly explored. MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) is mostly caused by mutation m.3243A>G in mitochondrial tRNALeu(UUR) gene. Adverse cardiac and neurological events are the commonest causes of early death in m.3243A>G patients. Notably, the incidence of major clinical features associated with this mutation has been correlated to the level of m.3243A>G mutant mitochondrial DNA (heteroplasmy) in skeletal muscle. In this work, we used a transmitochondrial cybrid model of MELAS (100% m.3243A>G mutant mitochondrial DNA) to investigate the participation of miRNAs in the mitochondria-nucleus cross-talk associated with OXPHOS dysfunction. High-throughput analysis of small-RNA-Seq data indicated that expression of 246 miRNAs was significantly altered in MELAS cybrids. Validation of selected miRNAs, including miR-4775 and miR-218-5p, in patient muscle samples revealed miRNAs whose expression declined with high levels of mutant heteroplasmy. We show that miR-218-5p and miR-4775 are direct regulators of fetal cardiac genes such as NODAL, RHOA, ISL1 and RXRB, which are up-regulated in MELAS cybrids and in patient muscle samples with heteroplasmy above 60%. Our data clearly indicate that TGF-β superfamily signaling and an epithelial-mesenchymal transition-like program are activated in MELAS cybrids, and suggest that down-regulation of miRNAs regulating fetal cardiac genes is a risk marker of heart failure in patients with OXPHOS diseases

Further insights into the tRNA modification process controlled by proteins MnmE and GidA of Escherichia coli

In Escherichia coli, proteins GidA and MnmE are involved in the addition of the carboxymethylaminomethyl (cmnm) group onto uridine 34 (U34) of tRNAs decoding two-family box triplets. However, their precise role in the modification reaction remains undetermined. Here, we show that GidA is an FAD-binding protein and that mutagenesis of the N-terminal dinucleotide-binding motif of GidA, impairs capability of this protein to bind FAD and modify tRNA, resulting in defective cell growth. Thus, GidA may catalyse an FAD-dependent reaction that is required for production of cmnmU34. We also show that GidA and MnmE have identical cell location and that both proteins physically interact. Gel filtration and native PAGE experiments indicate that GidA, like MnmE, dimerizes and that GidA and MnmE directly assemble in an α2β2 heterotetrameric complex. Interestingly, high-performance liquid chromatography (HPLC) analysis shows that identical levels of the same undermodified form of U34 are present in tRNA hydrolysates from loss-of-function gidA and mnmE mutants. Moreover, these mutants exhibit similar phenotypic traits. Altogether, these results do not support previous proposals that activity of MnmE precedes that of GidA; rather, our data suggest that MnmE and GidA form a functional complex in which both proteins are interdependent

Structural Basis for Fe–S Cluster Assembly and tRNA Thiolation Mediated by IscS Protein–Protein Interactions

Crystal structures reveal how distinct sites on the cysteine desulfurase IscS bind two different sulfur-acceptor proteins, IscU and TusA, to transfer sulfur atoms for iron-sulfur cluster biosynthesis and tRNA thiolation

YibK is the 2′-O-methyltransferase TrmL that modifies the wobble nucleotide in Escherichia coli tRNALeu isoacceptors

Transfer RNAs are the most densely modified nucleic acid molecules in living cells. In Escherichia coli, more than 30 nucleoside modifications have been characterized, ranging from methylations and pseudouridylations to more complex additions that require multiple enzymatic steps. Most of the modifying enzymes have been identified, although a few notable exceptions include the 2′-O-methyltransferase(s) that methylate the ribose at the nucleotide 34 wobble position in the two leucyl isoacceptors tRNALeuCmAA and tRNALeucmnm5UmAA. Here, we have used a comparative genomics approach to uncover candidate E. coli genes for the missing enzyme(s). Transfer RNAs from null mutants for candidate genes were analyzed by mass spectrometry and revealed that inactivation of yibK leads to loss of 2′-O-methylation at position 34 in both tRNALeuCmAA and tRNALeucmnm5UmAA. Loss of YibK methylation reduces the efficiency of codon–wobble base interaction, as demonstrated in an amber suppressor supP system. Inactivation of yibK had no detectable effect on steady-state growth rate, although a distinct disadvantage was noted in multiple-round, mixed-population growth experiments, suggesting that the ability to recover from the stationary phase was impaired. Methylation is restored in vivo by complementing with a recombinant copy of yibK. Despite being one of the smallest characterized α/β knot proteins, YibK independently catalyzes the methyl transfer from S-adenosyl-L-methionine to the 2′-OH of the wobble nucleotide; YibK recognition of this target requires a pyridine at position 34 and N6-(isopentenyl)-2-methylthioadenosine at position 37. YibK is one of the last remaining E. coli tRNA modification enzymes to be identified and is now renamed TrmL

Defective Expression of the Mitochondrial-tRNA Modifying Enzyme GTPBP3 Triggers AMPK-Mediated Adaptive Responses Involving Complex I Assembly Factors, Uncoupling Protein 2, and the Mitochondrial Pyruvate Carrier

GTPBP3 is an evolutionary conserved protein presumably involved in mitochondrial tRNA (mt-tRNA) modification. In humans, GTPBP3 mutations cause hypertrophic cardiomyopathy with lactic acidosis, and have been associated with a defect in mitochondrial translation, yet the pathomechanism remains unclear. Here we use a GTPBP3 stable-silencing model (shGTPBP3 cells) for a further characterization of the phenotype conferred by the GTPBP3 defect. We experimentally show for the first time that GTPBP3 depletion is associated with an mt-tRNA hypomodification status, as mt-tRNAs from shGTPBP3 cells were more sensitive to digestion by angiogenin than tRNAs from control cells. Despite the effect of stable silencing of GTPBP3 on global mitochondrial translation being rather mild, the steady-state levels and activity of Complex I, and cellular ATP levels were 50\% of those found in the controls. Notably, the ATPase activity of Complex V increased by about 40\% in GTPBP3 depleted cells suggesting that mitochondria consume ATP to maintain the membrane potential. Moreover, shGTPBP3 cells exhibited enhanced antioxidant capacity and a nearly 2-fold increase in the uncoupling protein UCP2 levels. Our data indicate that stable silencing of GTPBP3 triggers an AMPK-dependent retrograde signaling pathway that down-regulates the expression of the NDUFAF3 and NDUFAF4 Complex I assembly factors and the mitochondrial pyruvate carrier (MPC), while up-regulating the expression of UCP2. We also found that genes involved in glycolysis and oxidation of fatty acids are up-regulated. These data are compatible with a model in which high UCP2 levels, together with a reduction in pyruvate transport due to the down-regulation of MPC, promote a shift from pyruvate to fatty acid oxidation, and to an uncoupling of glycolysis and oxidative phosphorylation. These metabolic alterations, and the low ATP levels, may negatively affect heart function.This work has been supported by grants from the Spanish Ministry of Economy and Competitiveness (http://www.mineco.gob.es/portal/site/mineco/) (grant numbers BFU2010-19737 and BFU2014-58673-P to M.-E. A., BFU2011-22630 and SAF2014-54604-C3-2-R to E.K.) and the Generalitat Valenciana (http://www.gva.es/va/inicio/presentacion) (grant numbers ACOMP/2012/065 to M.-E. A., and PROMETEO/2012/061 to M.-E. A. and E.K.), and a PhD fellowship from the Instituto de Salud Carlos III (http://www.isciii.es/) to A.M.-Z. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Mutations in the Caenorhabditis elegans orthologs of human genes required for mitochondrial tRNA modification cause similar electron transport chain defects but different nuclear responses

Several oxidative phosphorylation (OXPHOS) diseases are caused by defects in the post-transcriptional modification of mitochondrial tRNAs (mt-tRNAs). Mutations in MTO1 or GTPBP3 impair the modification of the wobble uridine at position 5 of the pyrimidine ring and cause heart failure. Mutations in TRMU affect modification at position 2 and cause liver disease. Presently, the molecular basis of the diseases and why mutations in the different genes lead to such different clinical symptoms is poorly understood. Here we use Caenorhabditis elegans as a model organism to investigate how defects in the TRMU, GTPBP3 and MTO1 orthologues (designated as mttu-1, mtcu-1, and mtcu-2, respectively) exert their effects. We found that whereas the inactivation of each C. elegans gene is associated with a mild OXPHOS dysfunction, mutations in mtcu-1 or mtcu-2 cause changes in the expression of metabolic and mitochondrial stress response genes that are quite different from those caused by mttu-1 mutations. Our data suggest that retrograde signaling promotes defect-specific metabolic reprogramming, which is able to rescue the OXPHOS dysfunction in the single mutants by stimulating the oxidative tricarboxylic acid cycle flux through complex II. This adaptive response, however, appears to be associated with a biological cost since the single mutant worms exhibit thermosensitivity and decreased fertility and, in the case of mttu-1, longer reproductive cycle. Notably, mttu-1 worms also exhibit increased lifespan. We further show that mtcu-1; mttu-1 and mtcu-2; mttu-1 double mutants display severe growth defects and sterility. The animal models presented here support the idea that the pathological states in humans may initially develop not as a direct consequence of a bioenergetic defect, but from the cell's maladaptive response to the hypomodification status of mt-tRNAs. Our work highlights the important association of the defect-specific metabolic rewiring with the pathological phenotype, which must be taken into consideration in exploring specific therapeutic interventions

An Alternative Homodimerization Interface of MnmG Reveals a Conformational Dynamics that Is Essential for Its tRNA Modification Function

21 páginas, 11 figuras, 4 tablas. Material suplementario en http://dx.doi.org/10.1016/j.jmb.2018.05.035The Escherichia coli homodimeric proteins MnmE and MnmG form a functional complex, MnmEG, that modifies tRNAs using GTP, methylene-tetrahydrofolate, FAD, and glycine or ammonium. MnmE is a tetrahydrofolate- and GTP-binding protein, whereas MnmG is a FAD-binding protein with each protomer composed of the FAD-binding domain, two insertion domains, and the helical C-terminal domain. The detailed mechanism of the MnmEG-mediated reaction remains unclear partially due to incomplete structural information on the free- and substrate-bound forms of the complex. In this study, we show that MnmG can adopt in solution a dimer arrangement (form I) different from that currently considered as the only biologically active (form II). Normal mode analysis indicates that form I can oscillate in a range of open and closed conformations. Using isothermal titration calorimetry and native red electrophoresis, we show that a form-I open conformation, which can be stabilized in vitro by the formation of an interprotomer disulfide bond between the catalytic C277 residues, appears to be involved in the assembly of the MnmEG catalytic center. We also show that residues R196, D253, R436, R554 and E585 are important for the stabilization of form I and the tRNA modification function. We propose that the form I dynamics regulates the alternative access of MnmE and tRNA to the MnmG FAD active site. Finally, we show that the C-terminal region of MnmG contains a sterile alpha motif domain responsible for tRNA-protein and protein-protein interactions.This work was supported by the Spanish Ministry of Economy and Competitiveness (BFU2010-19737 and BFU2014-58673-P to M.-E.A.; BFU2016-78232-P to A.V.-C.), and Generalitat Valenciana(ACOMP/2012/065 to M.-E.A; PROMETEO/2012/061to J.B.). Funding for open access charge: [BFU2014-58673-P to M.-E.A.]Peer reviewe

Defects in the mitochondrial-tRNA modification enzymes MTO1 and GTPBP3 promote different metabolic reprogramming through a HIF-PPARγ-UCP2-AMPK axis

18 páginas 8 figuras. Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-19587-5.Human proteins MTO1 and GTPBP3 are thought to jointly catalyze the modification of the wobble uridine in mitochondrial tRNAs. Defects in each protein cause infantile hypertrophic cardiomyopathy with lactic acidosis. However, the underlying mechanisms are mostly unknown. Using fibroblasts from an MTO1 patient and MTO1 silenced cells, we found that the MTO1 deficiency is associated with a metabolic reprogramming mediated by inactivation of AMPK, down regulation of the uncoupling protein 2 (UCP2) and transcription factor PPARγ, and activation of the hypoxia inducible factor 1 (HIF-1). As a result, glycolysis and oxidative phosphorylation are uncoupled, while fatty acid metabolism is altered, leading to accumulation of lipid droplets in MTO1 fibroblasts. Unexpectedly, this response is different from that triggered by the GTPBP3 defect, as GTPBP3-depleted cells exhibit AMPK activation, increased levels of UCP2 and PPARγ, and inactivation of HIF-1. In addition, fatty acid oxidation and respiration are stimulated in these cells. Therefore, the HIF-PPARγ-UCP2-AMPK axis is operating differently in MTO1- and GTPBP3-defective cells, which strongly suggests that one of these proteins has an additional role, besides mitochondrial-tRNA modification. This work provides new and useful information on the molecular basis of the MTO1 and GTPBP3 defects and on putative targets for therapeutic intervention.Tis work has been supported by grants from the Spanish Ministry of Economy and Competitiveness (grants BFU2010-19737 and BFU2014-58673-P to M.-E.A.; and SAF2016-75004-R to M.C.), Instituto de Salud Carlos III (FIS-ISCIII PI 14/0431 to M.A.M) and Generalitat Valenciana (ACOMP/2012/065 and PROMETEO/2012/061 to M.-E.A. Contribution to COST Action CA15203 MITOEAGLE. R.B. is a recipient of a fellowship from the Generalitat Valenciana (grant GRISOLIA/2014/038). Te authors thank Dr. E. Knecht and Dr. C. Aguado (CIPF, Valencia, Spain) for their valuable advice and for providing facilities in our research work, and Dr. M. Morán (Hospital 12 de Octubre, Madrid, Spain) for growth and maintenance of patient fbroblasts.Peer reviewe

Modification of the wobble uridine (U₃₄) in mitochondrial and bacterial tRNAs.

<p>Schema of the U₃₄ modification pathways in human and yeast mt-tRNAs and <i>Escherichia coli</i> tRNAs <b>(A)</b> and <i>A</i>. <i>suum</i> mt-tRNAs <b>(B)</b>. In A, proteins GTPBP3, MTO1, and TRMU (also named MTU1) from humans, and MSS1, MTO1, and MTU1, from yeast, are orthologous of the bacterial MnmE, MnmG and MnmA proteins, respectively. Taurine (humans) and glycine (<i>E</i>. <i>coli</i> and yeast) are used to introduce the τm and cmnm groups into position 5 of U₃₄. In B, MTCU-1, MTCU-2 and MTTU-1 are the nematode orthologues of GTPBP3, MTO1, and TRMU, respectively, and their roles are inferred from studies of the <i>E</i>. <i>coli</i> and yeast proteins. The fractions of <i>A</i>. <i>suum</i> modified mt-tRNAs, according to the study by Sakurai <i>et al</i>. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006921#pgen.1006921.ref028" target="_blank">28</a>], are indicated below the schema. Note that mt-tRNA^Glu, mt-tRNA^Lys and mt-tRNA^Gln are fully modified at position 5 but lack thiolation at position 2, whereas most (~90%) is modified at both positions (2 and 5), and about 50% of mt-tRNA^Trp molecules do not contain any modification at the U₃₄.</p