Publications by authors named "M-Eugenia Armengod"

27 Publications

  • Page 1 of 1

The MELAS mutation m.3243A>G alters the expression of mitochondrial tRNA fragments.

Biochim Biophys Acta Mol Cell Res 2019 09 11;1866(9):1433-1449. Epub 2019 Jun 11.

RNA Modification and Mitochondrial Diseases Laboratory, Centro de Investigación Príncipe Felipe (CIPF), Carrer d'Eduardo Primo Yúfera 3, Valencia 46012, Spain; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER) node 721, Madrid 28029, Spain. Electronic address:

Recent evidences highlight the importance of mitochondria-nucleus communication for the clinical phenotype of oxidative phosphorylation (OXPHOS) diseases. However, the participation of small non-coding RNAs (sncRNAs) in this communication has been poorly explored. We asked whether OXPHOS dysfunction alters the production of a new class of sncRNAs, mitochondrial tRNA fragments (mt tRFs), and, if so, whether mt tRFs play a physiological role and their accumulation is controlled by the action of mt tRNA modification enzymes. To address these questions, we used a cybrid model of MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes), an OXPHOS disease mostly caused by mutation m.3243A>G in the mitochondrial tRNA gene. High-throughput analysis of small-RNA-Seq data indicated that m.3243A>G significantly changed the expression pattern of mt tRFs. A functional analysis of potential mt tRFs targets (performed under the assumption that these tRFs act as miRNAs) indicated an association with processes that involve the most common affected tissues in MELAS. We present evidences that mt tRFs may be biologically relevant, as one of them (mt i-tRF GluUUC), likely produced by the action of the nuclease Dicer and whose levels are Ago2 dependent, down-regulates the expression of mitochondrial pyruvate carrier 1 (MPC1), promoting the build-up of extracellular lactate. Therefore, our study underpins the idea that retrograde signaling from mitochondria is also mediated by mt tRFs. Finally, we show that accumulation of mt i-tRF GluUUC depends on the modification status of mt tRNAs, which is regulated by the action of stress-responsive miRNAs on mt tRNA modification enzymes.
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http://dx.doi.org/10.1016/j.bbamcr.2019.06.004DOI Listing
September 2019

Bacillus subtilis exhibits MnmC-like tRNA modification activities.

RNA Biol 2018 24;15(9):1167-1173. Epub 2018 Sep 24.

a Laboratory of RNA Modification and Mitochondrial Diseases , Centro de Investigación Príncipe Felipe , Valencia , Spain.

The MnmE-MnmG complex of Escherichia coli uses either ammonium or glycine as a substrate to incorporate the 5-aminomethyl or 5-carboxymethylaminomethyl group into the wobble uridine of certain tRNAs. Both modifications can be converted into a 5-methylaminomethyl group by the independent oxidoreductase and methyltransferase activities of MnmC, which respectively reside in the MnmC(o) and MnmC(m) domains of this bifunctional enzyme. MnmE and MnmG, but not MnmC, are evolutionarily conserved. Bacillus subtilis lacks genes encoding MnmC(o) and/or MnmC(m) homologs. The glycine pathway has been considered predominant in this typical gram-positive species because only the 5-carboxymethylaminomethyl group has been detected in tRNA and bulk tRNA to date. Here, we show that the 5-methylaminomethyl modification is prevalent in B. subtilis tRNA and tRNA. Our data indicate that B. subtilis has evolved MnmC(o)- and MnmC(m)-like activities that reside in non MnmC homologous protein(s), which suggests that both activities provide some sort of biological advantage.
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http://dx.doi.org/10.1080/15476286.2018.1517012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6284559PMC
December 2018

The MELAS mutation m.3243A>G promotes reactivation of fetal cardiac genes and an epithelial-mesenchymal transition-like program via dysregulation of miRNAs.

Biochim Biophys Acta Mol Basis Dis 2018 09 19;1864(9 Pt B):3022-3037. Epub 2018 Jun 19.

RNA Modification and Mitochondrial Diseases Laboratory, Centro de Investigación Príncipe Felipe (CIPF), Carrer d'Eduardo Primo Yúfera 3, Valencia 46012, Spain; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER) node 721, Madrid 28029, Spain. Electronic address:

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 tRNA 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.
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http://dx.doi.org/10.1016/j.bbadis.2018.06.014DOI Listing
September 2018

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

J Mol Biol 2018 08 2;430(17):2822-2842. Epub 2018 Jun 2.

Centro de Investigación Príncipe Felipe, Valencia 46012, Spain; Biomedical Research Networking Centre for Rare Diseases (CIBERER, Node 721), Valencia, Spain. Electronic address:

The 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.
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http://dx.doi.org/10.1016/j.jmb.2018.05.035DOI Listing
August 2018

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

Sci Rep 2018 01 18;8(1):1163. Epub 2018 Jan 18.

RNA Modification and Mitochondrial Diseases Laboratory, Centro de Investigación Príncipe Felipe (CIPF), Valencia, 46012, Spain.

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.
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http://dx.doi.org/10.1038/s41598-018-19587-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5773609PMC
January 2018

microRNA-mediated differential expression of TRMU, GTPBP3 and MTO1 in cell models of mitochondrial-DNA diseases.

Sci Rep 2017 07 24;7(1):6209. Epub 2017 Jul 24.

Laboratory of RNA Modification and Mitochondrial Diseases, Centro de Investigación Príncipe Felipe, Valencia, Spain.

Mitochondrial diseases due to mutations in the mitochondrial (mt) DNA are heterogeneous in clinical manifestations but usually include OXPHOS dysfunction. Mechanisms by which OXPHOS dysfunction contributes to the disease phenotype invoke, apart from cell energy deficit, maladaptive responses to mitochondria-to-nucleus retrograde signaling. Here we used five different cybrid models of mtDNA diseases to demonstrate that the expression of the nuclear-encoded mt-tRNA modification enzymes TRMU, GTPBP3 and MTO1 varies in response to specific pathological mtDNA mutations, thus altering the modification status of mt-tRNAs. Importantly, we demonstrated that the expression of TRMU, GTPBP3 and MTO1 is regulated by different miRNAs, which are induced by retrograde signals like ROS and Ca via different pathways. Our data suggest that the up- or down-regulation of the mt-tRNA modification enzymes is part of a cellular response to cope with a stoichiometric imbalance between mtDNA- and nuclear-encoded OXPHOS subunits. However, this miRNA-mediated response fails to provide full protection from the OXPHOS dysfunction; rather, it appears to aggravate the phenotype since transfection of the mutant cybrids with miRNA antagonists improves the energetic state of the cells, which opens up options for new therapeutic approaches.
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http://dx.doi.org/10.1038/s41598-017-06553-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5524753PMC
July 2017

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

PLoS Genet 2017 Jul 21;13(7):e1006921. Epub 2017 Jul 21.

Modificación del RNA y Enfermedades Mitocondriales, Centro de Investigación Príncipe Felipe, Valencia, Spain.

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.
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http://dx.doi.org/10.1371/journal.pgen.1006921DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5544249PMC
July 2017

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.

PLoS One 2015 7;10(12):e0144273. Epub 2015 Dec 7.

Laboratory of RNA Modification and Mitochondrial Diseases, Centro de Investigación Príncipe Felipe, Valencia, Spain.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0144273PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4671719PMC
June 2016

Modification of the wobble uridine in bacterial and mitochondrial tRNAs reading NNA/NNG triplets of 2-codon boxes.

RNA Biol 2014 ;11(12):1495-507

a Laboratory of RNA Modification and Mitochondrial Diseases ; Centro de Investigación Príncipe Felipe ; Valencia , Spain.

Posttranscriptional modification of the uridine located at the wobble position (U34) of tRNAs is crucial for optimization of translation. Defects in the U34 modification of mitochondrial-tRNAs are associated with a group of rare diseases collectively characterized by the impairment of the oxidative phosphorylation system. Retrograde signaling pathways from mitochondria to nucleus are involved in the pathophysiology of these diseases. These pathways may be triggered by not only the disturbance of the mitochondrial (mt) translation caused by hypomodification of tRNAs, but also as a result of nonconventional roles of mt-tRNAs and mt-tRNA-modifying enzymes. The evolutionary conservation of these enzymes supports their importance for cell and organismal functions. Interestingly, bacterial and eukaryotic cells respond to stress by altering the expression or activity of these tRNA-modifying enzymes, which leads to changes in the modification status of tRNAs. This review summarizes recent findings about these enzymes and sets them within the previous data context.
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http://dx.doi.org/10.4161/15476286.2014.992269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4615368PMC
October 2015

The ROS-sensitive microRNA-9/9* controls the expression of mitochondrial tRNA-modifying enzymes and is involved in the molecular mechanism of MELAS syndrome.

Hum Mol Genet 2015 Jan 22;24(1):167-84. Epub 2014 Aug 22.

Laboratory of RNA Modification and Mitochondrial Diseases, Centro de Investigación Príncipe Felipe, Valencia 46012, Spain CIBERER (node U721), Valencia, Spain

Mitochondrial dysfunction activates mitochondria-to-nucleus signaling pathways whose components are mostly unknown. Identification of these components is important to understand the molecular mechanisms underlying mitochondrial diseases and to discover putative therapeutic targets. MELAS syndrome is a rare neurodegenerative disease caused by mutations in mitochondrial (mt) DNA affecting mt-tRNA(Leu(UUR)). Patient and cybrid cells exhibit elevated oxidative stress. Moreover, mutant mt-tRNAs(Leu(UUR)) lack the taurine-containing modification normally present at the wobble uridine (U34) of wild-type mt-tRNA(Leu(UUR)), which is considered an etiology of MELAS. However, the molecular mechanism is still unclear. We found that MELAS cybrids exhibit a significant decrease in the steady-state levels of several mt-tRNA-modification enzymes, which is not due to transcriptional regulation. We demonstrated that oxidative stress mediates an NFkB-dependent induction of microRNA-9/9*, which acts as a post-transcriptional negative regulator of the mt-tRNA-modification enzymes GTPBP3, MTO1 and TRMU. Down-regulation of these enzymes by microRNA-9/9* affects the U34 modification status of non-mutant tRNAs and contributes to the MELAS phenotype. Anti-microRNA-9 treatments of MELAS cybrids reverse the phenotype, whereas miR-9 transfection of wild-type cells mimics the effects of siRNA-mediated down-regulation of GTPBP3, MTO1 and TRMU. Our data represent the first evidence that an mt-DNA disease can directly affect microRNA expression. Moreover, we demonstrate that the modification status of mt-tRNAs is dynamic and that cells respond to stress by modulating the expression of mt-tRNA-modifying enzymes. microRNA-9/9* is a crucial player in mitochondria-to-nucleus signaling as it regulates expression of nuclear genes in response to changes in the functional state of mitochondria.
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http://dx.doi.org/10.1093/hmg/ddu427DOI Listing
January 2015

The output of the tRNA modification pathways controlled by the Escherichia coli MnmEG and MnmC enzymes depends on the growth conditions and the tRNA species.

Nucleic Acids Res 2014 Feb 30;42(4):2602-23. Epub 2013 Nov 30.

Laboratory of RNA Modification and Mitochondrial Diseases, Príncipe Felipe Research Center, 46012-Valencia, Spain, Department of Molecular Biology, Umeå University, S90187, Sweden and Biomedical Research Networking Centre for Rare Diseases (CIBERER) (node U721), Spain.

In Escherichia coli, the MnmEG complex modifies transfer RNAs (tRNAs) decoding NNA/NNG codons. MnmEG catalyzes two different modification reactions, which add an aminomethyl (nm) or carboxymethylaminomethyl (cmnm) group to position 5 of the anticodon wobble uridine using ammonium or glycine, respectively. In tRNA(cmnm5s2UUG)(Gln) and tRNA(cmnm5UmAA)(Leu), however, cmnm(5) appears as the final modification, whereas in the remaining tRNAs, the MnmEG products are converted into 5-methylaminomethyl (mnm(5)) through the two-domain, bi-functional enzyme MnmC. MnmC(o) transforms cmnm(5) into nm(5), whereas MnmC(m) converts nm(5) into mnm(5), thus producing an atypical network of modification pathways. We investigate the activities and tRNA specificity of MnmEG and the MnmC domains, the ability of tRNAs to follow the ammonium or glycine pathway and the effect of mnmC mutations on growth. We demonstrate that the two MnmC domains function independently of each other and that tRNA(cmnm5s2UUG)(Gln) and tRNA(cmnm5UmAA)(Leu), are substrates for MnmC(m), but not MnmC(o). Synthesis of mnm(5)s(2)U by MnmEG-MnmC in vivo avoids build-up of intermediates in tRNA(mnm5s2UUU)(Lys). We also show that MnmEG can modify all the tRNAs via the ammonium pathway. Strikingly, the net output of the MnmEG pathways in vivo depends on growth conditions and tRNA species. Loss of any MnmC activity has a biological cost under specific conditions.
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http://dx.doi.org/10.1093/nar/gkt1228DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3936742PMC
February 2014

The tRNA-modifying function of MnmE is controlled by post-hydrolysis steps of its GTPase cycle.

Nucleic Acids Res 2013 Jul 28;41(12):6190-208. Epub 2013 Apr 28.

RNA Modification and Mitochondrial Diseases Laboratory, Centro de Investigación Príncipe Felipe, 46012-Valencia, Spain.

MnmE is a homodimeric multi-domain GTPase involved in tRNA modification. This protein differs from Ras-like GTPases in its low affinity for guanine nucleotides and mechanism of activation, which occurs by a cis, nucleotide- and potassium-dependent dimerization of its G-domains. Moreover, MnmE requires GTP hydrolysis to be functionally active. However, how GTP hydrolysis drives tRNA modification and how the MnmE GTPase cycle is regulated remains unresolved. Here, the kinetics of the MnmE GTPase cycle was studied under single-turnover conditions using stopped- and quench-flow techniques. We found that the G-domain dissociation is the rate-limiting step of the overall reaction. Mutational analysis and fast kinetics assays revealed that GTP hydrolysis, G-domain dissociation and Pi release can be uncoupled and that G-domain dissociation is directly responsible for the 'ON' state of MnmE. Thus, MnmE provides a new paradigm of how the ON/OFF cycling of GTPases may regulate a cellular process. We also demonstrate that the MnmE GTPase cycle is negatively controlled by the reaction products GDP and Pi. This feedback mechanism may prevent inefficacious GTP hydrolysis in vivo. We propose a biological model whereby a conformational change triggered by tRNA binding is required to remove product inhibition and initiate a new GTPase/tRNA-modification cycle.
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http://dx.doi.org/10.1093/nar/gkt320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3695501PMC
July 2013

The Escherichia coli RlmN methyltransferase is a dual-specificity enzyme that modifies both rRNA and tRNA and controls translational accuracy.

RNA 2012 Oct 13;18(10):1783-95. Epub 2012 Aug 13.

Laboratorio de Genética Molecular, Centro de Investigación Príncipe Felipe, 46012 Valencia, Spain.

Modifying RNA enzymes are highly specific for substrate-rRNA or tRNA-and the target position. In Escherichia coli, there are very few multisite acting enzymes, and only one rRNA/tRNA dual-specificity enzyme, pseudouridine synthase RluA, has been identified to date. Among the tRNA-modifying enzymes, the methyltransferase responsible for the m(2)A synthesis at purine 37 in a tRNA set still remains unknown. m(2)A is also present at position 2503 in the peptidyl transferase center of 23S RNA, where it is introduced by RlmN, a radical S-adenosyl-L-methionine (SAM) enzyme. Here, we show that E. coli RlmN is a dual-specificity enzyme that catalyzes methylation of both rRNA and tRNA. The ΔrlmN mutant lacks m(2)A in both RNA types, whereas the expression of recombinant RlmN from a plasmid introduced into this mutant restores tRNA modification. Moreover, RlmN performs m(2)A(37) synthesis in vitro using a tRNA chimera as a substrate. This chimera has also proved useful to characterize some tRNA identity determinants for RlmN and other tRNA modification enzymes. Our data suggest that RlmN works in a late step during tRNA maturation by recognizing a precise 3D structure of tRNA. RlmN inactivation increases the misreading of a UAG stop codon. Since loss of m(2)A(37) from tRNA is expected to produce a hyperaccurate phenotype, we believe that the error-prone phenotype exhibited by the ΔrlmN mutant is due to loss of m(2)A from 23S rRNA and, accordingly, that the m(2)A2503 modification plays a crucial role in the proofreading step occurring at the peptidyl transferase center.
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http://dx.doi.org/10.1261/rna.033266.112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3446703PMC
October 2012

Enzymology of tRNA modification in the bacterial MnmEG pathway.

Biochimie 2012 Jul 28;94(7):1510-20. Epub 2012 Feb 28.

Laboratorio de Genética Molecular, Centro de Investigación Príncipe Felipe, Molecular Genetics, Avenida Autopista del Saler, 16-3, 46012-Valencia, Spain.

Among all RNAs, tRNA exhibits the largest number and the widest variety of post-transcriptional modifications. Modifications within the anticodon stem loop, mainly at the wobble position and purine-37, collectively contribute to stabilize the codon-anticodon pairing, maintain the translational reading frame, facilitate the engagement of the ribosomal decoding site and enable translocation of tRNA from the A-site to the P-site of the ribosome. Modifications at the wobble uridine (U34) of tRNAs reading two degenerate codons ending in purine are complex and result from the activity of two multi-enzyme pathways, the IscS-MnmA and MnmEG pathways, which independently work on positions 2 and 5 of the U34 pyrimidine ring, respectively, and from a third pathway, controlled by TrmL (YibK), that modifies the 2'-hydroxyl group of the ribose. MnmEG is the only common pathway to all the mentioned tRNAs, and involves the GTP- and FAD-dependent activity of the MnmEG complex and, in some cases, the activity of the bifunctional enzyme MnmC. The Escherichia coli MnmEG complex catalyzes the incorporation of an aminomethyl group into the C5 atom of U34 using methylene-tetrahydrofolate and glycine or ammonium as donors. The reaction requires GTP hydrolysis, probably to assemble the active site of the enzyme or to carry out substrate recognition. Inactivation of the evolutionarily conserved MnmEG pathway produces a pleiotropic phenotype in bacteria and mitochondrial dysfunction in human cell lines. While the IscS-MnmA pathway and the MnmA-mediated thiouridylation reaction are relatively well understood, we have limited information on the reactions mediated by the MnmEG, MnmC and TrmL enzymes and on the precise role of proteins MnmE and MnmG in the MnmEG complex activity. This review summarizes the present state of knowledge on these pathways and what we still need to know, with special emphasis on the MnmEG pathway.
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http://dx.doi.org/10.1016/j.biochi.2012.02.019DOI Listing
July 2012

Regulation of expression and catalytic activity of Escherichia coli RsmG methyltransferase.

RNA 2012 Apr 15;18(4):795-806. Epub 2012 Feb 15.

Laboratorio de Genética Molecular, Centro de Investigación Príncipe Felipe, 46012 Valencia, Spain.

RsmG is an AdoMet-dependent methyltransferase responsible for the synthesis of m(7)G527 in the 530 loop of bacterial 16S rRNA. This loop is universally conserved, plays a key role in ribosomal accuracy, and is a target for streptomycin binding. Loss of the m(7)G527 modification confers low-level streptomycin resistance and may affect ribosomal functioning. Here, we explore the mechanisms controlling RsmG expression and activity, which may somehow respond to the demand set by the amount of rRNA. We confirm that rsmG is the second member in a bicistronic operon and demonstrate that rsmG also has its own promoter, which appears, in actively growing cells, as a control device to offset both the relatively low stability of RsmG and inhibition of the operon promoter. RsmG levels decrease under conditions that down-regulate rRNA synthesis. However, coordination between rRNA and RsmG expression does not seem to occur at the level of transcription initiation. Instead, it might depend on the activity of an inverted repeated region, located between the rsmG promoter and ribosome binding site, which we show to work as a weak transcriptional terminator. To gain insights into the enzymatic mechanism of RsmG, highly conserved residues were mutated and the abilities of the resulting proteins to confer streptomycin resistance, to modify rRNA, and to bind AdoMet were explored. Our data demonstrate for the first time the critical importance of some residues located in the active site of Escherichia coli RsmG for the m(7)G modification process and suggest a role for them in rRNA binding and catalysis.
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http://dx.doi.org/10.1261/rna.029868.111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3312566PMC
April 2012

YibK is the 2'-O-methyltransferase TrmL that modifies the wobble nucleotide in Escherichia coli tRNA(Leu) isoacceptors.

RNA 2010 Nov 20;16(11):2131-43. Epub 2010 Sep 20.

Laboratorio de Genética Molecular, Centro de Investigación Príncipe Felipe, 46012 Valencia, Spain.

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 tRNA(Leu)(CmAA) and tRNA(Leu)(cmnm5UmAA). 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 tRNA(Leu)(CmAA) and tRNA(Leu)(cmnm5UmAA). 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.
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http://dx.doi.org/10.1261/rna.2245910DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2957053PMC
November 2010

Structural basis for Fe-S cluster assembly and tRNA thiolation mediated by IscS protein-protein interactions.

PLoS Biol 2010 Apr 13;8(4):e1000354. Epub 2010 Apr 13.

Department of Biochemistry, McGill University, Montréal, Québec, Canada.

The cysteine desulfurase IscS is a highly conserved master enzyme initiating sulfur transfer via persulfide to a range of acceptor proteins involved in Fe-S cluster assembly, tRNA modifications, and sulfur-containing cofactor biosynthesis. Several IscS-interacting partners including IscU, a scaffold for Fe-S cluster assembly; TusA, the first member of a sulfur relay leading to sulfur incorporation into the wobble uridine of several tRNAs; ThiI, involved in tRNA modification and thiamine biosynthesis; and rhodanese RhdA are sulfur acceptors. Other proteins, such as CyaY/frataxin and IscX, also bind to IscS, but their functional roles are not directly related to sulfur transfer. We have determined the crystal structures of IscS-IscU and IscS-TusA complexes providing the first insight into their different modes of binding and the mechanism of sulfur transfer. Exhaustive mutational analysis of the IscS surface allowed us to map the binding sites of various partner proteins and to determine the functional and biochemical role of selected IscS and TusA residues. IscS interacts with its partners through an extensive surface area centered on the active site Cys328. The structures indicate that the acceptor proteins approach Cys328 from different directions and suggest that the conformational plasticity of a long loop containing this cysteine is essential for the ability of IscS to transfer sulfur to multiple acceptor proteins. The sulfur acceptors can only bind to IscS one at a time, while frataxin and IscX can form a ternary complex with IscU and IscS. Our data support the role of frataxin as an iron donor for IscU to form the Fe-S clusters.
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http://dx.doi.org/10.1371/journal.pbio.1000354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2854127PMC
April 2010

Structure-function analysis of Escherichia coli MnmG (GidA), a highly conserved tRNA-modifying enzyme.

J Bacteriol 2009 Dec 2;191(24):7614-9. Epub 2009 Oct 2.

Department of Biochemistry, McGill University, Montreal, Quebec, Canada.

The MnmE-MnmG complex is involved in tRNA modification. We have determined the crystal structure of Escherichia coli MnmG at 2.4-A resolution, mutated highly conserved residues with putative roles in flavin adenine dinucleotide (FAD) or tRNA binding and MnmE interaction, and analyzed the effects of these mutations in vivo and in vitro. Limited trypsinolysis of MnmG suggests significant conformational changes upon FAD binding.
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http://dx.doi.org/10.1128/JB.00650-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2786596PMC
December 2009

Evolutionarily conserved proteins MnmE and GidA catalyze the formation of two methyluridine derivatives at tRNA wobble positions.

Nucleic Acids Res 2009 Nov;37(21):7177-93

Laboratorio de Genética Molecular, Centro de Investigación Príncipe Felipe, 46012-Valencia, Spain.

The wobble uridine of certain bacterial and mitochondrial tRNAs is modified, at position 5, through an unknown reaction pathway that utilizes the evolutionarily conserved MnmE and GidA proteins. The resulting modification (a methyluridine derivative) plays a critical role in decoding NNG/A codons and reading frame maintenance during mRNA translation. The lack of this tRNA modification produces a pleiotropic phenotype in bacteria and has been associated with mitochondrial encephalomyopathies in humans. In this work, we use in vitro and in vivo approaches to characterize the enzymatic pathway controlled by the Escherichia coli MnmE*GidA complex. Surprisingly, this complex catalyzes two different GTP- and FAD-dependent reactions, which produce 5-aminomethyluridine and 5-carboxymethylamino-methyluridine using ammonium and glycine, respectively, as substrates. In both reactions, methylene-tetrahydrofolate is the most probable source to form the C5-methylene moiety, whereas NADH is dispensable in vitro unless FAD levels are limiting. Our results allow us to reformulate the bacterial MnmE*GidA dependent pathway and propose a novel mechanism for the modification reactions performed by the MnmE and GidA family proteins.
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http://dx.doi.org/10.1093/nar/gkp762DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2790889PMC
November 2009

Characterization of human GTPBP3, a GTP-binding protein involved in mitochondrial tRNA modification.

Mol Cell Biol 2008 Dec 13;28(24):7514-31. Epub 2008 Oct 13.

Laboratorio de Genética Molecular, Centro de Investigación Príncipe Felipe, Avenida Autopista del Saler, 16-3, 46012 Valencia, Spain.

Human GTPBP3 is an evolutionarily conserved, multidomain protein involved in mitochondrial tRNA modification. Characterization of its biochemical properties and the phenotype conferred by GTPBP3 inactivation is crucial to understanding the role of this protein in tRNA maturation and its effects on mitochondrial respiration. We show that the two most abundant GTPBP3 isoforms exhibit moderate affinity for guanine nucleotides like their bacterial homologue, MnmE, although they hydrolyze GTP at a 100-fold lower rate. This suggests that regulation of the GTPase activity, essential for the tRNA modification function of MnmE, is different in GTPBP3. In fact, potassium-induced dimerization of the G domain leads to stimulation of the GTPase activity in MnmE but not in GTPBP3. The GTPBP3 N-terminal domain mediates a potassium-independent dimerization, which appears as an evolutionarily conserved property of the protein family, probably related to the construction of the binding site for the one-carbon-unit donor in the modification reaction. Partial inactivation of GTPBP3 by small interfering RNA reduces oxygen consumption, ATP production, and mitochondrial protein synthesis, while the degradation of these proteins slightly increases. It also results in mitochondria with defective membrane potential and increased superoxide levels. These phenotypic traits suggest that GTPBP3 defects contribute to the pathogenesis of some oxidative phosphorylation diseases.
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http://dx.doi.org/10.1128/MCB.00946-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2593442PMC
December 2008

Polymorphisms in TRAIL receptor genes and risk of breast cancer in Spanish women.

Cancer Biomark 2007 ;3(2):89-93

Laboratorio de Genética Molecular, Centro de Investigación Príncipe Felipe, Valencia, Spain.

TRAIL is a potent inducer of apoptosis in malignant but not in normal cells. TRAIL binds to the proapoptotic death receptor DR4 and DR5 as well as to the decoy receptors DcR1 and DcR2. To evaluate the involvement of TRAIL receptor genes in breast cancer, we carried out a case-control study of eight selected polymorphisms in a large sample of Spanish women. Three of the eight selected SNPs (626G/C and 1322G/A in DR4 and 2699A/G in DcR2) showed some evidence of different genotype distributions in a random selection of 535 cases and 480 controls and were therefore studied in our entire sample (1008 cases and 768 controls). For the two DR4 polymorphisms, no differences in genotype or haplotype distribution were found between cases and controls. Interestingly, allele 2699G in the decoy receptor DcR2 appears associated with reduced breast cancer risk (P=0.05). Given that it is located in the 3' UTR, its effect might be related to DcR2 mRNA instability, or linkage disequilibrium with a functional variant residing in either DcR2 or neighbouring genes. A decreased efficiency of DcR2 to work as decoy receptor for TRAIL, would facilitate the apoptotic pathway in cells at risk.
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http://dx.doi.org/10.3233/cbm-2007-3203DOI Listing
July 2007

Efficient selection of silenced primary cells by flow cytometry.

Cytometry A 2007 Aug;71(8):599-604

Laboratorio de Genética Molecular, Centro de Investigación Príncipe Felipe, Avda. Autopista del Saler 16-3, 46013-Valencia, Spain.

Background: RNA interference has emerged as a new and potent tool to knockdown the expression of target genes and to investigate their functions. For short time experiments with mammalian cell lines, RNA interference is typically induced by transfecting small interfering RNAs (siRNAs). Primary cells constitute important experimental systems in many studies because of their similarity to their in vivo counterparts; however, transfection of these cells has been found to be difficult. As a consequence, RNA interference of primary cells may result in mixed phenotypes because of the simultaneous presence in the same preparation of transfected and nontransfected cells. This may be particularly inconvenient when certain experiments (for example, biochemical analysis) should be performed.

Methods: We use fluorescently labeled siRNAs to induce RNA interference in fibroblasts, and flow-cytometry associated cell sorting to separate subpopulations of transfected cells according to fluorescence intensity.

Results: Flow cytometry allows one to discriminate between strongly- and weakly- or nonsilenced fibroblasts, since the fluorescence intensity of transfected cells is related to the number of internalized siRNA copies and to the mRNA knockdown efficiency.

Conclusions: The use of fluorescently labeled siRNAs may allow one to isolate by flow-cytometry associated cell sorting the most efficiently silenced primary cells for subsequent analysis.
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http://dx.doi.org/10.1002/cyto.a.20413DOI Listing
August 2007

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

Nucleic Acids Res 2006 24;34(20):5892-905. Epub 2006 Oct 24.

Laboratorio de Genética Molecular, Centro de Investigación Príncipe Felipe, Avda. Autopista del Saler 16-3, 46013 Valencia, Spain.

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 alpha2beta2 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.
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http://dx.doi.org/10.1093/nar/gkl752DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1635325PMC
December 2006

Effects of mutagenesis in the switch I region and conserved arginines of Escherichia coli MnmE protein, a GTPase involved in tRNA modification.

J Biol Chem 2005 Sep 27;280(35):30660-70. Epub 2005 Jun 27.

Laboratorio de Genética Molecular, Centro de Investigación Príncipe Felipe, Valencia, Spain.

MnmE is an evolutionarily conserved, three domain GTPase involved in tRNA modification. In contrast to Ras proteins, MnmE exhibits a high intrinsic GTPase activity and requires GTP hydrolysis to be functionally active. Its G domain conserves the GTPase activity of the full protein, and thus, it should contain the catalytic residues responsible for this activity. In this work, mutational analysis of all conserved arginine residues of the MnmE G-domain indicates that MnmE, unlike other GTPases, does not use an arginine finger to drive catalysis. In addition, we show that residues in the G2 motif (249GTTRD253), which resides in the switch I region, are not important for GTP binding but play some role in stabilizing the transition state, specially Gly249 and Thr251. On the other hand, G2 mutations leading to a minor loss of the GTPase activity result in a non-functional MnmE protein. This indicates that GTP hydrolysis is a required but non-sufficient condition so that MnmE can mediate modification of tRNA. The conformational change of the switch I region associated with GTP hydrolysis seems to be crucial for the function of MnmE, and the invariant threonine (Thr251) of the G2 motif would be essential for such a change, because it cannot be substituted by serine. MnmE defects result in impaired growth, a condition that is exacerbated when defects in other genes involved in the decoding process are simultaneously present. This behavior is reminiscent to that found in yeast and stresses the importance of tRNA modification for gene expression.
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http://dx.doi.org/10.1074/jbc.M503223200DOI Listing
September 2005

The structure of the TrmE GTP-binding protein and its implications for tRNA modification.

EMBO J 2005 Jan 16;24(1):23-33. Epub 2004 Dec 16.

Max-Planck Institut für Molekulare Physiologie, Dortmund, Germany.

TrmE is a 50 kDa guanine nucleotide-binding protein conserved between bacteria and man. It is involved in the modification of uridine bases (U34) at the first anticodon (wobble) position of tRNAs decoding two-family box triplets. The precise role of TrmE in the modification reaction is hitherto unknown. Here, we report the X-ray structure of TrmE from Thermotoga maritima. The structure reveals a three-domain protein comprising the N-terminal alpha/beta domain, the central helical domain and the G domain, responsible for GTP binding and hydrolysis. The N-terminal domain induces dimerization and is homologous to the tetrahydrofolate-binding domain of N,N-dimethylglycine oxidase. Biochemical and structural studies show that TrmE indeed binds formyl-tetrahydrofolate. A cysteine residue, necessary for modification of U34, is located close to the C1-group donor 5-formyl-tetrahydrofolate, suggesting a direct role of TrmE in the modification analogous to DNA modification enzymes. We propose a reaction mechanism whereby TrmE actively participates in the formylation reaction of uridine and regulates the ensuing hydrogenation reaction of a Schiff's base intermediate.
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http://dx.doi.org/10.1038/sj.emboj.7600507DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC544919PMC
January 2005

The variant E233G of the RAD51D gene could be a low-penetrance allele in high-risk breast cancer families without BRCA1/2 mutations.

Int J Cancer 2004 Jul;110(6):845-9

Department of Human Genetics, Spanish National Cancer Centre, C/Melchior Fernández Almagro 3, 28029 Madrid, Spain.

Six SNPs have been detected in the DNA repair genes RAD51C and RAD51D, not previously characterized. The novel variant E233G in RAD51D is more highly represented in high-risk, site-specific, familial breast cancer cases that are not associated with the BRCA1/2 genes, with a frequency of 5.74% (n = 174) compared to a control population (n = 567) and another subset of breast cancer patients (n = 765) with a prevalence of around 2% only (comparison to controls, OR = 2.6, 95% CI 1.12-6.03; p < 0.021). We found that the immunohistochemical profile detected in available tumors from these patients differs slightly from those described in non-BRCA1/2 tumors. Finally, the structural prediction of the putative functional consequence of this change indicates that it can diminish protein stability and structure. This suggests a role for E233G as a low-penetrance susceptibility gene in the specific subgroup of high-risk familial breast cancer cases that are not related to BRCA1/2.
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http://dx.doi.org/10.1002/ijc.20169DOI Listing
July 2004
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