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

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 Ca2+ 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

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

Stationary phase induction of dnaN and recF, two genes of Escherichia coli involved in DNA replication and repair.

The beta 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 beta, 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 beta subunit and RecF protein increases when cell metabolism is slowing down. Such an induction is dependent on the stationary phase sigma 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

The Escherichia coli trmE (mnmE) gene, involved in tRNA modification, codes for an evolutionarily conserved GTPase with unusual biochemical properties.

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 GTPases 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 viability 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