RESUMEN
BACKGROUND: Mitochondrial tRNA (mt-tRNA) variants have been found to cause disease. Post-transcriptional queuosine (Q) modifications of mt-tRNA can promote efficient mitochondrial mRNA translation. Q modifications of mt-tRNAAsn have recently been identified. Here, the therapeutic effectiveness of queuine was investigated in cells from patients with mt-tRNAAsn variants. METHODS: Six patients (from four families) carrying mt-tRNAAsn variants were included in the study. Queuine levels were quantified by mass spectrometry. Clinical, genetic, histochemical, biochemical, and molecular analysis was performed on muscle tissues and lymphoblastoid cell lines (LCLs) from patients to investigate the pathogenicity of the novel m.5708 C > T variant. The use of queuine in mitigating mitochondrial dysfunction resulting from the mt-tRNAAsn variants was evaluated. RESULTS: The variants included the m.5701 delA, m.5708 C > T, m.5709 C > T, and m.5698 G > A variants in mt-tRNAAsn. The pathogenicity of the novel m.5708 C > T variant was confirmed, as demonstrated by a decreased steady-state level of mt-tRNAAsn, mtDNA-encoded protein levels, oxygen consumption rate (OCR), and the respiratory complex activity. Notably, the serum queuine level was significantly reduced in these patients and in vitro queuine supplementation was found to restore the reductions in mitochondrial protein activities, mitochondrial membrane potential, OCR, and increases in reactive oxygen species. CONCLUSIONS: The study not only confirmed the pathogenicity of the m.5708 C > T variant but also explored the therapeutic potential of queuine in individuals with mt-tRNAAsn variants. The recognition of the novel m.5708 C > T variant's pathogenic nature contributes to our comprehension of mitochondrial disorders. Furthermore, the results emphasize queuine supplementation as a promising approach to enhance the stability of mt-tRNAAsn and rescue mitochondrial dysfunction caused by mt-tRNAAsn variants, indicating potential implications for the development of targeted therapies for patients with mt-tRNAAsn variants.
Asunto(s)
Mitocondrias , Humanos , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Masculino , Femenino , Adulto , Persona de Mediana Edad , ADN Mitocondrial/genética , Variación Genética , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Nucleósido Q/metabolismo , ARN Mitocondrial/genética , ARN Mitocondrial/metabolismo , ARN de Transferencia de Alanina/genética , ARN de Transferencia de Alanina/metabolismoRESUMEN
Alanyl-tRNA synthetase (AlaRS) retains a conserved prototype structure throughout its biology, consisting of catalytic, tRNA-recognition, editing, and C-Ala domains. The catalytic and tRNA-recognition domains catalyze aminoacylation, the editing domain hydrolyzes mischarged tRNAAla, and C-Ala-the major tRNA-binding module-targets the elbow of the L-shaped tRNAAla. Interestingly, a mini-AlaRS lacking the editing and C-Ala domains is recovered from the Tupanvirus of the amoeba Acanthamoeba castellanii. Here we show that Tupanvirus AlaRS (TuAlaRS) is phylogenetically related to its host's AlaRS. Despite lacking the conserved amino acid residues responsible for recognition of the identity element of tRNAAla (G3:U70), TuAlaRS still specifically recognized G3:U70-containing tRNAAla. In addition, despite lacking C-Ala, TuAlaRS robustly binds and charges microAla (an RNA substrate corresponding to the acceptor stem of tRNAAla) as well as tRNAAla, indicating that TuAlaRS exclusively targets the acceptor stem. Moreover, this mini-AlaRS could functionally substitute for yeast AlaRS in vivo. This study suggests that TuAlaRS has developed a new tRNA-binding mode to compensate for the loss of C-Ala.
Asunto(s)
Alanina-ARNt Ligasa , Alanina-ARNt Ligasa/genética , Alanina-ARNt Ligasa/química , Alanina-ARNt Ligasa/metabolismo , ARN de Transferencia de Alanina/química , ARN de Transferencia de Alanina/genética , ARN de Transferencia de Alanina/metabolismo , Escherichia coli/genética , ARN de Transferencia/metabolismoRESUMEN
By linking amino acids to their codon assignments, transfer RNAs (tRNAs) are essential for protein synthesis and translation fidelity. Some human tRNA variants cause amino acid mis-incorporation at a codon or set of codons. We recently found that a naturally occurring tRNASer variant decodes phenylalanine codons with serine and inhibits protein synthesis. Here, we hypothesized that human tRNA variants that misread glycine (Gly) codons with alanine (Ala) will also disrupt protein homeostasis. The A3G mutation occurs naturally in tRNAGly variants (tRNAGlyCCC, tRNAGlyGCC) and creates an alanyl-tRNA synthetase (AlaRS) identity element (G3 : U70). Because AlaRS does not recognize the anticodon, the human tRNAAlaAGC G35C (tRNAAlaACC) variant may function similarly to mis-incorporate Ala at Gly codons. The tRNAGly and tRNAAla variants had no effect on protein synthesis in mammalian cells under normal growth conditions; however, tRNAGlyGCC A3G depressed protein synthesis in the context of proteasome inhibition. Mass spectrometry confirmed Ala mistranslation at multiple Gly codons caused by the tRNAGlyGCC A3G and tRNAAlaAGC G35C mutants, and in some cases, we observed multiple mistranslation events in the same peptide. The data reveal mistranslation of Ala at Gly codons and defects in protein homeostasis generated by natural human tRNA variants that are tolerated under normal conditions. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.
Asunto(s)
Alanina-ARNt Ligasa , Biosíntesis de Proteínas , Humanos , Alanina/genética , Alanina/química , Alanina/metabolismo , Alanina-ARNt Ligasa/química , Alanina-ARNt Ligasa/genética , Alanina-ARNt Ligasa/metabolismo , Codón/genética , Glicina/genética , Glicina/metabolismo , Proteostasis , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , ARN de Transferencia de Alanina/química , ARN de Transferencia de Alanina/genética , ARN de Transferencia de Alanina/metabolismo , ARN de Transferencia de Glicerina/metabolismoRESUMEN
Aminoacyl-tRNA synthetases (aaRSs) are enzymes that synthesize aminoacyl-tRNAs to facilitate translation of the genetic code. Quality control by aaRS proofreading and other mechanisms maintains translational accuracy, which promotes cellular viability. Systematic disruption of proofreading, as recently demonstrated for alanyl-tRNA synthetase (AlaRS), leads to dysregulation of the proteome and reduced viability. Recent studies showed that environmental challenges such as exposure to reactive oxygen species can also alter aaRS synthetic and proofreading functions, prompting us to investigate if oxidation might positively or negatively affect AlaRS activity. We found that while oxidation leads to modification of several residues in Escherichia coli AlaRS, unlike in other aaRSs, this does not affect proofreading activity against the noncognate substrates serine and glycine and only results in a 1.6-fold decrease in efficiency of cognate Ala-tRNAAla formation. Mass spectrometry analysis of oxidized AlaRS revealed that the critical proofreading residue in the editing site, Cys666, and three methionine residues (M217 in the active site, M658 in the editing site, and M785 in the C-Ala domain) were modified to cysteine sulfenic acid and methionine sulfoxide, respectively. Alanine scanning mutagenesis showed that none of the identified residues were solely responsible for the change in cognate tRNAAla aminoacylation observed under oxidative stress, suggesting that these residues may act as reactive oxygen species "sinks" to protect catalytically critical sites from oxidative damage. Combined, our results indicate that E. coli AlaRS proofreading is resistant to oxidative damage, providing an important mechanism of stress resistance that helps to maintain proteome integrity and cellular viability.
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Alanina-ARNt Ligasa , Escherichia coli , Alanina-ARNt Ligasa/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/metabolismo , Estrés Oxidativo , Proteoma , ARN de Transferencia de Alanina/genética , ARN de Transferencia de Alanina/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Unlike many other aminoacyl-tRNA synthetases, alanyl-tRNA synthetase (AlaRS) retains a conserved prototype structure throughout biology. While Caenorhabditis elegans cytoplasmic AlaRS (CeAlaRSc) retains the prototype structure, its mitochondrial counterpart (CeAlaRSm) contains only a residual C-terminal domain (C-Ala). We demonstrated herein that the C-Ala domain from CeAlaRSc robustly binds both tRNA and DNA. It bound different tRNAs but preferred tRNAAla. Deletion of this domain from CeAlaRSc sharply reduced its aminoacylation activity, while fusion of this domain to CeAlaRSm selectively and distinctly enhanced its aminoacylation activity toward the elbow-containing (or L-shaped) tRNAAla. Phylogenetic analysis showed that CeAlaRSm once possessed the C-Ala domain but later lost most of it during evolution, perhaps in response to the deletion of the T-arm (part of the elbow) from its cognate tRNA. This study underscores the evolutionary gain of C-Ala for docking AlaRS to the L-shaped tRNAAla.
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Alanina-ARNt Ligasa , Aminoacil-ARNt Sintetasas , Alanina-ARNt Ligasa/genética , Aminoacil-ARNt Sintetasas/genética , Aminoacilación , Filogenia , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , ARN de Transferencia de Alanina/genéticaRESUMEN
Diseases caused by heteroplasmic mitochondrial DNA mutations have no effective treatment or cure. In recent years, DNA editing enzymes were tested as tools to eliminate mutant mtDNA in heteroplasmic cells and tissues. Mitochondrial-targeted restriction endonucleases, ZFNs, and TALENs have been successful in shifting mtDNA heteroplasmy, but they all have drawbacks as gene therapy reagents, including: large size, heterodimeric nature, inability to distinguish single base changes, or low flexibility and effectiveness. Here we report the adaptation of a gene editing platform based on the I-CreI meganuclease known as ARCUS®. These mitochondrial-targeted meganucleases (mitoARCUS) have a relatively small size, are monomeric, and can recognize sequences differing by as little as one base pair. We show the development of a mitoARCUS specific for the mouse m.5024C>T mutation in the mt-tRNAAla gene and its delivery to mice intravenously using AAV9 as a vector. Liver and skeletal muscle show robust elimination of mutant mtDNA with concomitant restoration of mt-tRNAAla levels. We conclude that mitoARCUS is a potential powerful tool for the elimination of mutant mtDNA.
Asunto(s)
Enzimas de Restricción del ADN/metabolismo , ADN Mitocondrial/metabolismo , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Enfermedades Mitocondriales/terapia , Animales , Enzimas de Restricción del ADN/genética , ADN Mitocondrial/genética , Dependovirus/genética , Modelos Animales de Enfermedad , Fibroblastos , Edición Génica/métodos , Vectores Genéticos/genética , Células HeLa , Humanos , Ratones , Ratones Transgénicos , Mitocondrias/genética , Mitocondrias/metabolismo , Enfermedades Mitocondriales/genética , Mutación Puntual , Cultivo Primario de Células , ARN de Transferencia de Alanina/genéticaRESUMEN
Most mammalian cytoplasmic tRNAs contain ribothymidine (T) and pseudouridine (Ψ) at positions 54 and 55, respectively. However, some tRNAs contain Ψ at both positions. Several Ψ54-containing tRNAs function as primers in retroviral DNA synthesis. The Ψ54 of these tRNAs is produced by PUS10, which can also synthesize Ψ55. Two other enzymes, TRUB1 and TRUB2, can also produce Ψ55. By nearest-neighbor analyses of tRNAs treated with recombinant proteins and subcellular extracts of wild-type and specific Ψ55 synthase knockdown cells, we determined that while TRUB1, PUS10, and TRUB2 all have tRNA Ψ55 synthase activities, they have different tRNA structural requirements. Moreover, these activities are primarily present in the nucleus, cytoplasm, and mitochondria, respectively, suggesting a compartmentalization of Ψ55 synthase activity. TRUB1 produces the Ψ55 of most elongator tRNAs, but cytoplasmic PUS10 produces both Ψs of the tRNAs with Ψ54Ψ55. The nuclear isoform of PUS10 is catalytically inactive and specifically binds the unmodified U54U55 versions of Ψ54Ψ55-containing tRNAs, as well as the A54U55-containing tRNAiMet This binding inhibits TRUB1-mediated U55 to Ψ55 conversion in the nucleus. Consequently, the U54U55 of Ψ54Ψ55-containing tRNAs are modified by the cytoplasmic PUS10. Nuclear PUS10 does not bind the U55 versions of T54Ψ55- and A54Ψ55-containing elongator tRNAs. Therefore, TRUB1 is able to produce Ψ55 in these tRNAs. In summary, the tRNA Ψ55 synthase activities of TRUB1 and PUS10 are not redundant but rather are compartmentalized and act on different sets of tRNAs. The significance of this compartmentalization needs further study.
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Núcleo Celular/genética , Citoplasma/genética , Hidroliasas/genética , Mitocondrias/genética , Seudouridina/metabolismo , ARN de Transferencia de Alanina/genética , ARN de Transferencia de Metionina/genética , ARN de Transferencia de Triptófano/genética , Animales , Sitios de Unión , Compartimento Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Expresión Génica , Células HEK293 , Humanos , Hidroliasas/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Mitocondrias/metabolismo , Células PC-3 , Unión Proteica , ARN de Transferencia de Alanina/metabolismo , ARN de Transferencia de Metionina/metabolismo , ARN de Transferencia de Triptófano/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , SpodopteraRESUMEN
Aborted translation produces large ribosomal subunits obstructed with tRNA-linked nascent chains, which are substrates of ribosome-associated quality control (RQC). Bacterial RqcH, a widely conserved RQC factor, senses the obstruction and recruits tRNAAla(UGC) to modify nascent-chain C termini with a polyalanine degron. However, how RqcH and its eukaryotic homologs (Rqc2 and NEMF), despite their relatively simple architecture, synthesize such C-terminal tails in the absence of a small ribosomal subunit and mRNA has remained unknown. Here, we present cryoelectron microscopy (cryo-EM) structures of Bacillus subtilis RQC complexes representing different Ala tail synthesis steps. The structures explain how tRNAAla is selected via anticodon reading during recruitment to the A-site and uncover striking hinge-like movements in RqcH leading tRNAAla into a hybrid A/P-state associated with peptidyl-transfer. Finally, we provide structural, biochemical, and molecular genetic evidence identifying the Hsp15 homolog (encoded by rqcP) as a novel RQC component that completes the cycle by stabilizing the P-site tRNA conformation. Ala tailing thus follows mechanistic principles surprisingly similar to canonical translation elongation.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Extensión de la Cadena Peptídica de Translación , ARN Bacteriano/metabolismo , ARN de Transferencia de Alanina/metabolismo , Bacillus subtilis/ultraestructura , Proteínas Bacterianas/genética , Microscopía por Crioelectrón , ARN Bacteriano/genética , ARN de Transferencia de Alanina/genéticaRESUMEN
BACKGROUND Although mutations and dysfunction of mitochondrial DNA (mtDNA) are related to a variety of diseases, few studies have focused on the relationship between mtDNA and coronary artery disease (CAD), especially the relationship between rare variants and CAD. MATERIAL AND METHODS Two-stage high-throughput sequencing was performed to detect mtDNA variants or heteroplasmy and the relationship between them and CAD phenotypes. In the discovery stage, mtDNA was analyzed by high-throughput sequencing of long-range PCR products generated from the peripheral blood of 85 CAD patients and 80 demographically matched controls. In the validation stage, high-throughput sequencing for mtDNA target regions captured by GenCap Kit was performed on 100 CAD samples and 100 controls. Finally, tRNA fine mapping was performed between our study and the reported Chinese CAD study. RESULTS Among the tRNA genes, we confirmed a highly conserved rare variant, A5592G, previously reported in the Chinese CAD study, and 2 novel rare mutations that reached Bonferroni's correction significance in the combined analysis were found (P=7.39×10-4 for T5628C in tRNAAla and P=1.01×10-5 for T681C in 12S rRNA) in the CAD study. Both of them were predicted to be pathological, with T5628C disrupting an extremely conservative base-pairing at the AC stem of tRNAAla. Furthermore, we confirmed the controversial issue that the number of non-synonymous heteroplasmic sites per sample was significantly higher in CAD patients. CONCLUSIONS In conclusion, our study confirmed the contribution of rare variants in CAD and showed that CAD patients had more non-synonymous heterogeneity mutations, which may be helpful in identifying the genetic and molecular basis of CAD.
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Enfermedad de la Arteria Coronaria/genética , ADN Mitocondrial/análisis , ARN de Transferencia de Alanina/genética , Anciano , China , Femenino , Heteroplasmia , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Mitocondrias/genética , MutaciónRESUMEN
The unique G3:U70 base pair in the acceptor stem of tRNAAla has been shown to be a critical recognition site by alanyl-tRNA synthetase (AlaRS). The base pair resides on one of the arms of the L-shaped structure of tRNA (minihelix) and the genetic code has likely evolved from a primordial tRNA-aaRS (aminoacyl-tRNA synthetase) system. In terms of the evolution of tRNA, incorporation of a G:U base pair in the structure would be important. Here, we found that two independent short hairpin RNAs change their conformation through kissing-loop interactions, finally forming a minihelix-like structure, in which the G3:U70 base pair is incorporated. The RNA system can be properly aminoacylated by the minimal Escherichia coli AlaRS variant with alanylation activity (AlaRS442N). Thus, characteristic structural features produced via kissing-loop interactions may provide important clues into the evolution of RNA.
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Aminoacilación/genética , Evolución Molecular , Conformación de Ácido Nucleico , ARN Interferente Pequeño/genética , ARN de Transferencia de Alanina/genética , Alanina-ARNt Ligasa , Aminoacil-ARNt Sintetasas , Emparejamiento Base , Escherichia coli/genética , Transferencia Resonante de Energía de Fluorescencia , Modelos Moleculares , Pliegue del ARN , ARN Interferente Pequeño/metabolismo , ARN de Transferencia de Alanina/metabolismoRESUMEN
Certain mutations in mitochondrial DNA (mtDNA) are associated with Leber's hereditary optic neuropathy (LHON). In particular, the wellknown NADH dehydrogenase 4 (ND4) m.11778G>A mutation is one of the most common LHONassociated primary mutations worldwide. However, how specific mtDNA mutations, or variants, affect LHON penetrance is not fully understood. The aim of the current study was to explore the relationship between mtDNA mutations and LHON, and to provide useful information for early detection and prevention of this disease. Following the molecular characterization of a Han Chinese family with maternally inherited LHON, four out of eight matrilineal relatives demonstrated varying degrees of both visual impairment and age of onset. Through PCR amplification of mitochondrial genomes and direct Sanger sequencing analysis, a homoplasmic mitochondrialencoded ND4 m.11778G>A mutation, alongside a set of genetic variations belonging to human mtDNA haplogroup B5b1 were identified. Among these sequence variants, alanine transfer RNA (tRNA)Ala m.5601C>T was of particular interest. This variant occurred at position 59 in the TψC loop and altered the base pairing, which led to mitochondrial RNA (mtRNA) metabolism failure and defects in mitochondrial protein synthesis. Bioinformatics analysis suggested that the m.5601C>T variant altered tRNAAla structure. Therefore, impaired mitochondrial functions caused by the ND4 m.11778G>A mutation may be enhanced by the mttRNAAla m.5601C>T variant. These findings suggested that the tRNAAla m.5601C>T variant might modulate the clinical manifestation of the LHONassociated primary mutation.
Asunto(s)
ADN Mitocondrial/genética , Mitocondrias/genética , NADH Deshidrogenasa/genética , Atrofia Óptica Hereditaria de Leber/genética , ARN de Transferencia de Alanina/genética , Adolescente , Adulto , Pueblo Asiatico/genética , Niño , Biología Computacional , Familia , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación , Atrofia Óptica Hereditaria de Leber/sangre , Atrofia Óptica Hereditaria de Leber/metabolismo , Atrofia Óptica Hereditaria de Leber/patología , Linaje , Penetrancia , Filogenia , Polimorfismo Genético , ARN de Transferencia de Alanina/químicaRESUMEN
Alanyl-tRNA synthetases (AlaRSs) from three domains of life predominantly rely on a single wobble base pair, G3-U70, of tRNAAla as a major determinant. However, this base pair is divergent in human mitochondrial tRNAAla, but instead with a translocated G5-U68. How human mitochondrial AlaRS (hmtAlaRS) recognizes tRNAAla, in particular, in the acceptor stem region, remains unknown. In the present study, we found that hmtAlaRS is a monomer and recognizes mitochondrial tRNAAla in a G3-U70-independent manner, requiring several elements in the acceptor stem. In addition, we found that hmtAlaRS misactivates noncognate Gly and catalyzes strong transfer RNA (tRNA)-independent pre-transfer editing for Gly. A completely conserved residue outside of the editing active site, Arg663, likely functions as a tRNA translocation determinant to facilitate tRNA entry into the editing domain during editing. Finally, we investigated the effects of the severe infantile-onset cardiomyopathy-associated R592W mutation of hmtAlaRS on the canonical enzymatic activities of hmtAlaRS. Overall, our results provide fundamental information about tRNA recognition and deepen our understanding of translational quality control mechanisms by hmtAlaRS.
Asunto(s)
Conformación de Ácido Nucleico , ARN Mitocondrial/genética , ARN de Transferencia de Alanina/genética , ARN de Transferencia/genética , Alanina-ARNt Ligasa/genética , Emparejamiento Base/genética , Dominio Catalítico , Escherichia coli/genética , Humanos , Cinética , Modelos Moleculares , Especificidad por SustratoRESUMEN
Adenosine deaminase acting on transfer RNA (ADAT) is an essential eukaryotic enzyme that catalyzes the deamination of adenosine to inosine at the first position of tRNA anticodons. Mammalian ADATs modify eight different tRNAs, having increased their substrate range from a bacterial ancestor that likely deaminated exclusively tRNAArg Here we investigate the recognition mechanisms of tRNAArg and tRNAAla by human ADAT to shed light on the process of substrate expansion that took place during the evolution of the enzyme. We show that tRNA recognition by human ADAT does not depend on conserved identity elements, but on the overall structural features of tRNA. We find that ancestral-like interactions are conserved for tRNAArg, while eukaryote-specific substrates use alternative mechanisms. These recognition studies show that human ADAT can be inhibited by tRNA fragments in vitro, including naturally occurring fragments involved in important regulatory pathways.
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Adenosina Desaminasa/metabolismo , Anticodón/química , ARN de Transferencia de Alanina/química , ARN de Transferencia de Arginina/química , Adenosina/metabolismo , Adenosina Desaminasa/genética , Anticodón/genética , Anticodón/metabolismo , Secuencia de Bases , Desaminación , Evolución Molecular , Expresión Génica , Humanos , Inosina/metabolismo , Conformación de Ácido Nucleico , ARN de Transferencia de Alanina/genética , ARN de Transferencia de Alanina/metabolismo , ARN de Transferencia de Arginina/genética , ARN de Transferencia de Arginina/metabolismo , Alineación de Secuencia , Especificidad por SustratoRESUMEN
Sarcoptes scabiei (Acari: Sarcoptidae) causes a common contagious skin disease that affects many mammals. Here, the complete mitochondrial genome of a mite, S. scabiei var. nyctereutis, from Japanese wild raccoon dogs was analyzed. The 13,837bp circular genome contained 13 protein-coding genes, two rRNA genes, and 22 tRNA genes. For the first time, two tRNAs (alanine and tyrosine), that were thought to be absent in scabies mites from other animals, were predicted to have short, non-cloverleaf structures by in silico annotation and detected by RT-PCR, sequencing, and northern analysis. The mitochondrial genome structure of S. scabiei is similar to that of Psoroptes cuniculi and Dermatophagoides farinae. While small and unusual tRNA genes seem to be common among acariform mites, further experimental evidence for their presence is needed. Furthermore, through an analysis of the cox1 gene, we have provided new evidence to confirm the transmission of this mite between different animal hosts.
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Genoma Mitocondrial , ARN de Transferencia de Alanina/genética , ARN de Transferencia de Tirosina/genética , Sarcoptes scabiei/genética , Animales , Filogenia , ARN de Transferencia de Alanina/química , ARN de Transferencia de Tirosina/química , Perros Mapache/parasitología , Sarcoptes scabiei/clasificaciónRESUMEN
The genetic code used in nuclear genes is almost universal, but here we report that it changed three times in parallel during the evolution of budding yeasts. All three changes were reassignments of the codon CUG, which is translated as serine (in 2 yeast clades), alanine (1 clade), or the 'universal' leucine (2 clades). The newly discovered Ser2 clade is in the final stages of a genetic code transition. Most species in this clade have genes for both a novel tRNASer(CAG) and an ancestral tRNALeu(CAG) to read CUG, but only tRNASer(CAG) is used in standard growth conditions. The coexistence of these alloacceptor tRNA genes indicates that the genetic code transition occurred via an ambiguous translation phase. We propose that the three parallel reassignments of CUG were not driven by natural selection in favor of their effects on the proteome, but by selection to eliminate the ancestral tRNALeu(CAG).
Asunto(s)
Codón , Código Genético , Genoma Fúngico , ARN de Transferencia de Alanina/genética , ARN de Transferencia de Leucina/genética , ARN de Transferencia de Serina/genética , Saccharomycetales/genética , Alanina/genética , Alanina/metabolismo , Evolución Molecular , Leucina/genética , Leucina/metabolismo , Conformación de Ácido Nucleico , Filogenia , Biosíntesis de Proteínas , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN de Transferencia de Alanina/metabolismo , ARN de Transferencia de Leucina/metabolismo , ARN de Transferencia de Serina/metabolismo , Saccharomycetales/clasificación , Saccharomycetales/metabolismo , Selección Genética , Serina/genética , Serina/metabolismoRESUMEN
Defects in the respiratory chain, interfering with energy production in the cell, are major underlying causes of mitochondrial diseases. In spite of this, the surprising variety of clinical symptoms, disparity between ages of onset, as well as the involvement of mitochondrial impairment in ageing and age-related diseases continue to challenge our understanding of the pathogenic processes. This complexity can be in part attributed to the unique metabolic needs of organs or of various cell types. In this view, it remains essential to investigate mitochondrial dysfunction at the cellular level. For this purpose, we developed a novel enzyme histochemical method that enables precise quantification in fresh-frozen tissues using competing redox reactions which ultimately lead to the reduction of tetrazolium salts and formazan deposition in cytochrome c oxidase-deficient mitochondria. We demonstrate that the loss of oxidative activity is detected at very low levels - this achievement is unequalled by previous techniques and opens up new opportunities for the study of early disease processes or comparative investigations. Moreover, human biopsy samples of mitochondrial disease patients of diverse genotypic origins were used and the successful detection of COX-deficient cells suggests a broad application for this new method. Lastly, the assay can be adapted to a wide range of tissues in the mouse and extends to other animal models, which we show here with the fruit fly, Drosophila melanogaster. Overall, the new assay provides the means to quantify and map, on a cell-by-cell basis, the full extent of COX deficiency in tissues, thereby expending new possibilities for future investigation. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
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Deficiencia de Citocromo-c Oxidasa/diagnóstico , Complejo IV de Transporte de Electrones/metabolismo , Análisis de la Célula Individual/métodos , Coloración y Etiquetado/métodos , Animales , Deficiencia de Citocromo-c Oxidasa/enzimología , Deficiencia de Citocromo-c Oxidasa/genética , Modelos Animales de Enfermedad , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Metabolismo Energético , Humanos , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Metosulfato de Metilfenazonio/química , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Mitocondriales/deficiencia , Proteínas Mitocondriales/genética , Mutación , Proteínas de Neoplasias/deficiencia , Proteínas de Neoplasias/genética , Nitroazul de Tetrazolio/química , Oxidación-Reducción , Valor Predictivo de las Pruebas , ARN de Transferencia de Alanina/genéticaRESUMEN
Mutations in mitochondrial DNA, especially in mitochondrial tRNA (mt-tRNA) genes, are the important causes for maternally inherited hypertension. In this study, we reported the clinical, genetic, and molecular characterization of a Han Chinese family with hypertension. Most strikingly, this family exhibited a high penetrance and expressivity of hypertension. Sequence analysis of the complete mt-tRNA genes showed the presence of tRNAMet A4435G and tRNAAla C5601T mutations. The A4435G had already been reported as a pathogenic mutation associated with hypertension; in addition, the C5601T mutation, which was located at the highly conserved nucleotide of T arm of tRNAAla, created a novel Watson-Crick base pairing and may result in failure of tRNA metabolism. Moreover, bioinformatics analysis indicated that the C5601T mutation altered the secondary structure of tRNAAla. Thus, the mitochondrial dysfunction, caused by the A4435G mutation, may be worsened by the C5601T mutation. Taken together, our data indicated that the co-occurrence of the A4435G and C5601T mutations may account for the high penetrance and expressivity of hypertension in this family. Therefore, our study provided novel insight into the pathophysiology of maternally inherited hypertension.
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Pueblo Asiatico/genética , ADN Mitocondrial/genética , Regulación de la Expresión Génica/genética , Hipertensión/genética , Mutación/genética , ARN de Transferencia de Alanina/genética , ARN de Transferencia de Metionina/genética , Adulto , Anciano , Familia , Femenino , Humanos , Masculino , Persona de Mediana Edad , LinajeRESUMEN
Horizontal gene transfer is crucial for the adaptation of microorganisms to environmental cues. The acidophilic, bioleaching bacterium Acidithiobacillus ferrooxidans encodes an integrative-conjugative genetic element (ICEAfe1) inserted in the gene encoding a tRNAAla. This genetic element is actively excised from the chromosome upon induction of DNA damage. A similar genetic element (ICEAcaTY.2) is also found in an equivalent position in the genome of Acidithiobacillus caldus. The local genomic context of both mobile genetic elements is highly syntenous and the cognate integrases are well conserved. By means of site directed mutagenesis, target site deletions and in vivo integrations assays in the heterologous model Escherichia coli, we assessed the target sequence requirements for site-specific recombination to be catalyzed by these integrases. We determined that each enzyme recognizes a specific small DNA segment encoding the anticodon stem/loop of the tRNA as target site and that specific positions in these regions are well conserved in the target attB sites of orthologous integrases. Also, we demonstrate that the local genetic context of the target sequence is not relevant for the integration to take place. These findings shed new light on the mechanism of site-specific integration of integrative-conjugative elements in members of Acidithiobacillus genus.
Asunto(s)
Acidithiobacillus/genética , Elementos Transponibles de ADN , ADN Bacteriano/genética , Transferencia de Gen Horizontal , ARN de Transferencia de Alanina/genética , Acidithiobacillus/metabolismo , Anticodón/química , Anticodón/metabolismo , Sitios de Ligazón Microbiológica , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Bacterianos/química , Cromosomas Bacterianos/metabolismo , Daño del ADN , ADN Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Integrasas/genética , Integrasas/metabolismo , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , ARN de Transferencia de Alanina/metabolismo , Recombinación Genética , SinteníaRESUMEN
Aminoacyl-tRNAs containing a deoxy substitution in the penultimate nucleotide (C75 2'OH â 2'H) have been widely used in translation for incorporation of unnatural amino acids (AAs). However, this supposedly innocuous modification surprisingly increased peptidyl-tRNAAlaugc drop off in biochemical assays of successive incorporations. Here we predict the function of this tRNA 2'OH in the ribosomal A, P and E sites using recent co-crystal structures of ribosomes and tRNA substrates and test these structure-function models by systematic kinetics analyses. Unexpectedly, the C75 2'H did not affect A- to P-site translocation nor peptidyl donor activity of tRNAAlaugc. Rather, the peptidyl acceptor activity of the A-site Ala-tRNAAlaugc and the translocation of the P-site deacylated tRNAAlaugc to the E site were impeded. Delivery by EF-Tu was not significantly affected. This broadens our view of the roles of 2'OH groups in tRNAs in translation.
Asunto(s)
Sitios Internos de Entrada al Ribosoma , Biosíntesis de Proteínas , ARN de Transferencia de Alanina/química , Ribosomas/genética , Cristalografía por Rayos X , Cinética , Modelos Moleculares , Conformación de Ácido Nucleico , Factor Tu de Elongación Peptídica/genética , Factor Tu de Elongación Peptídica/metabolismo , ARN de Transferencia de Alanina/genética , ARN de Transferencia de Alanina/metabolismo , Ribosomas/metabolismo , Ribosomas/ultraestructuraRESUMEN
Mutations in mitochondrial DNA are associated with the pathogenesis of essential hypertension. We report here the clinical, genetic, and molecular characterization of a three-generation Han Chinese family with essential hypertension. Most strikingly, this family exhibited a high penetrance of essential hypertension. Sequence analysis of the mitochondrial genome showed the presence of a homoplasmic T5655C mutation in tRNAAla, together with the A4401G mutation in the adjacent region between tRNAMet and tRNAGln. Notably, the T5655C mutation was localized at the acceptor arm of tRNAAla, disrupted the high conserved base-pairing (1A-72T), and may impair the tRNA function. Moreover, the A4401G mutation was reported to decrease the steady-state level of tRNAMet and tRNAGln, and consequently caused the mitochondrial dysfunction responsible for hypertension. Taken together, the combination of T5655C and A4401G mutations in mitochondrial tRNA genes may account for the high penetrance and expressivity of hypertension in this Chinese family. Thus, our findings may provide new insight into the pathogenesis of this disorder.