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

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

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

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

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.This work was supported by grant GSP-48370 from the Canadian Institutes of Health Research (to M.C. and A.M.) and grant BFU2007-66509 from the Ministerio de Ciencia e Innovación (to M.-E.A.).Peer reviewe

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

Regulation of expression and catalytic activity of Escherichia coli RsmG methyltransferase

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

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

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