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Abstract: 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. [Copyright &y& Elsevier]

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-terminala/ßdomain, 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. [ABSTRACT FROM AUTHOR]

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 tRNALeu(UUR) 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. Unlabelled Image • Main MELAS mutation changes the mt tRF expression pattern in relation to controls. • Expression of selected mt tRFs correlates with heteroplasmy in MELAS cells. • At least one mt tRF (mt i-tRF GluUUC) seems to be involved in nuclear gene regulation. • Stress-responsive miRNAs control mt i-tRF GluUUC yield via mt tRNA modification enzymes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The evolutionarily conserved 50K protein of Escherichia coli, encoded by o454, contains a consensus GTP-binding motif. Here we show that 50K is a GTPase that differs extensively from regulatory GTPase such as p21. Thus, 50K exhibits a very high intrinsic GTPase hydrolysis rate, rather low affinity for GTP, and extremely low affinity for GDP. Moreover, it can form self-assemblies. Strikingly, the 17 kDa GTPase domain of 50K conserves the guanine nucleotide-binding and GTPase activities of the intact 50K molecule. Therefore, the structural requirements for GTP binding and GTP hydrolysis by 50K are without precedent and justify a separate classification in the GTPase superfamily. Immunoelectron microscopy reveals that 50K is a cytoplasmic protein partially associated with the inner membrane. We prove that o454 is allelic with trmE, a gene involved in the biosynthesis of the hypermodified nucleoside 5-methylaminomethyl-2-thiouridine, which is found in the wobble position of some tRNAs. Our results demonstrate that 50K is essential for variability depending on the genetic background. We propose that combination of mutations affecting the decoding process, which separately do not reveal an obvious defect in growth, can give rise to lethal phenotypes, most likely due to synergism. [ABSTRACT FROM AUTHOR]

The β subunit of DNA polymerase III holoenzyme, the Escherichia coli chromosomal replicase, is a sliding DNA clamp responsible for tethering the polymerase to DNA and endowing it with high processivity. The gene encoding , dnaN, maps between dnaA and recF, which are involved in initiation of DNA replication at oriC and resumption of DNA replication at disrupted replication forks, respectively. In exponentially growing cells, dnaN and recF are expressed predominantly from the dnaA promoters. However, we have found that stationary phase induction of the dnaN promoters drastically changes the expression pattern of the dnaA operon genes. As a striking consequence, synthesis of the β subunit and RecF protein increases when cell metabolism is slowing down. Such an induction is dependent on the stationary phase · factor, RpoS, although the accumulation of this factor alone is not sufficient to activate the dnaN promoters. These promoters are located in DNA regions without static bending, and the ­35 hexamer element is essential for their RpoS-dependent induction. Our results suggest that stationary phase-dependent mechanisms have evolved in order to coordinate expression of dnaN and recF independently of the dnaA regulatory region. These mechanisms might be part of a developmental programme aimed at maintaining DNA integrity under stress conditions. [ABSTRACT FROM AUTHOR]

The MnmE-MnmG complex is involved in tRNA modification. We have determined the crystal structure of Escherichia coli MnmG at 2.4-Å 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]