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1.
Neurology ; 92(11): e1225-e1237, 2019 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-30737337

RESUMO

OBJECTIVE: To describe the leukodystrophy caused by pathogenic variants in LARS2 and KARS, encoding mitochondrial leucyl transfer RNA (tRNA) synthase and mitochondrial and cytoplasmic lysyl tRNA synthase, respectively. METHODS: We composed a group of 5 patients with leukodystrophy, in whom whole-genome or whole-exome sequencing revealed pathogenic variants in LARS2 or KARS. Clinical information, brain MRIs, and postmortem brain autopsy data were collected. We assessed aminoacylation activities of purified mutant recombinant mitochondrial leucyl tRNA synthase and performed aminoacylation assays on patients' lymphoblasts and fibroblasts. RESULTS: Patients had a combination of early-onset deafness and later-onset neurologic deterioration caused by progressive brain white matter abnormalities on MRI. Female patients with LARS2 pathogenic variants had premature ovarian failure. In 2 patients, MRI showed additional signs of early-onset vascular abnormalities. In 2 other patients with LARS2 and KARS pathogenic variants, magnetic resonance spectroscopy revealed elevated white matter lactate, suggesting mitochondrial disease. Pathology in one patient with LARS2 pathogenic variants displayed evidence of primary disease of oligodendrocytes and astrocytes with lack of myelin and deficient astrogliosis. Aminoacylation activities of purified recombinant mutant leucyl tRNA synthase showed a 3-fold loss of catalytic efficiency. Aminoacylation assays on patients' lymphoblasts and fibroblasts showed about 50% reduction of enzyme activity. CONCLUSION: This study adds LARS2 and KARS pathogenic variants as gene defects that may underlie deafness, ovarian failure, and leukodystrophy with mitochondrial signature. We discuss the specific MRI characteristics shared by leukodystrophies caused by mitochondrial tRNA synthase defects. We propose to add aminoacylation assays as biochemical diagnostic tools for leukodystrophies.

2.
PLoS One ; 13(12): e0209805, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30592748

RESUMO

The life cycle of Plasmodium falciparum, the agent responsible for malaria, depends on both cytosolic and apicoplast translation fidelity. Apicoplast aminoacyl-tRNA synthetases (aaRS) are bacterial-like enzymes devoted to organellar tRNA aminoacylation. They are all encoded by the nuclear genome and are translocated into the apicoplast only after cytosolic biosynthesis. Apicoplast aaRSs contain numerous idiosyncratic sequence insertions: An understanding of the roles of these insertions has remained elusive and they hinder efforts to heterologously overexpress these proteins. Moreover, the A/T rich content of the Plasmodium genome leads to A/U rich apicoplast tRNA substrates that display structural plasticity. Here, we focus on the P. falciparum apicoplast tyrosyl-tRNA synthetase (Pf-apiTyrRS) and its cognate tRNATyr substrate (Pf-apitRNATyr). Cloning and expression strategies used to obtain an active and functional recombinant Pf-apiTyrRS are reported. Functional analyses established that only three weak identity elements in the apitRNATyr promote specific recognition by the cognate Pf-apiTyrRS and that positive identity elements usually found in the tRNATyr acceptor stem are excluded from this set. This finding brings to light an unusual behavior for a tRNATyr aminoacylation system and suggests that Pf-apiTyrRS uses primarily negative recognition elements to direct tyrosylation specificity.


Assuntos
Apicoplastos/enzimologia , Apicoplastos/metabolismo , Plasmodium falciparum/metabolismo , RNA de Transferência de Tirosina/metabolismo , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Humanos , Malária Falciparum/fisiopatologia , Plasmodium falciparum/enzimologia , Plasmodium falciparum/patogenicidade , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA de Transferência de Tirosina/genética , Tirosina-tRNA Ligase/genética , Tirosina-tRNA Ligase/metabolismo
3.
Haematologica ; 2018 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-30026338

RESUMO

YARS2 variants have previously been described in patients with myopathy, lactic acidosis and sideroblastic anemia 2 (MLASA2). YARS2 encodes the mitochondrial tyrosyl-tRNA synthetase, which is responsible for conjugating tyrosine to its cognate mt-tRNA for mitochondrial protein synthesis. Here we describe 14 individuals from 11 families presenting with sideroblastic anemia and with YARS2 variants that we identified using a sideroblastic anemia gene panel or exome sequencing. The phenotype of these patients ranged from MLASA to isolated congenital sideroblastic anemia. As in previous cases, inter- and intra-familial phenotypic variability was observed, however this report includes the first cases with isolated sideroblastic anemia and patients with biallelic YARS2 variants that have no clinically ascertainable phenotype. We identified ten novel YARS2 variants and three previously reported variants. In vitro amino-acylation assays of three five novel missense variants showed they that three had less effect on the catalytic activity of YARS2 than the most commonly reported variant, p.(Phe52Leu), associated with MLASA2, which may explain the milder phenotypes in patients with these variants. However, the other two missense variants had a more severe effect on YARS2 catalytic efficiency. Several patients carried the common YARS2 c.572 G>T, p.(Gly191Val) variant (minor allele frequency = 0.1259) in trans with a rare deleterious YARS2 variant. We have previously shown that the p.(Gly191Val) variant reduces YARS2 catalytic activity. Consequently, we suggest that biallelic YARS2 variants, including severe loss-of-function alleles in trans of the common p.(Gly191Val) variant, should be considered as a cause of isolated congenital sideroblastic anemia, as well as the MLASA syndromic phenotype.

4.
JIMD Rep ; 28: 49-57, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26537577

RESUMO

Pathogenic variants in mitochondrial aminoacyl-tRNA synthetases result in a broad range of mitochondrial respiratory chain disorders despite their shared role in mitochondrial protein synthesis. LARS2 encodes the mitochondrial leucyl-tRNA synthetase, which attaches leucine to its cognate tRNA. Sequence variants in LARS2 have previously been associated with Perrault syndrome, characterized by premature ovarian failure and hearing loss (OMIM #615300). In this study, we report variants in LARS2 that are associated with a severe multisystem metabolic disorder. The proband was born prematurely with severe lactic acidosis, hydrops, and sideroblastic anemia. She had multisystem complications with hyaline membrane disease, impaired cardiac function, a coagulopathy, pulmonary hypertension, and progressive renal disease and succumbed at 5 days of age. Whole exome sequencing of patient DNA revealed compound heterozygous variants in LARS2 (c.1289C>T; p.Ala430Val and c.1565C>A; p.Thr522Asn). The c.1565C>A (p.Thr522Asn) LARS2 variant has previously been associated with Perrault syndrome and both identified variants are predicted to be damaging (SIFT, PolyPhen). Muscle and liver samples from the proband did not display marked mitochondrial respiratory chain enzyme deficiency. Immunoblotting of patient muscle and liver showed LARS2 levels were reduced in liver and complex I protein levels were reduced in patient muscle and liver. Aminoacylation assays revealed p.Ala430Val LARS2 had an 18-fold loss of catalytic efficiency and p.Thr522Asn a 9-fold loss compared to wild-type LARS2. We suggest that the identified LARS2 variants are responsible for the severe multisystem clinical phenotype seen in this baby and that mutations in LARS2 can result in variable phenotypes.

5.
RNA Biol ; 12(12): 1301-13, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26327585

RESUMO

The canonical activity of glycyl-tRNA synthetase (GARS) is to charge glycine onto its cognate tRNAs. However, outside translation, GARS also participates in many other functions. A single gene encodes both the cytosolic and mitochondrial forms of GARS but 2 mRNA isoforms were identified. Using immunolocalization assays, in vitro translation assays and bicistronic constructs we provide experimental evidence that one of these mRNAs tightly controls expression and localization of human GARS. An intricate regulatory domain was found in its 5'-UTR which displays a functional Internal Ribosome Entry Site and an upstream Open Reading Frame. Together, these elements hinder the synthesis of the mitochondrial GARS and target the translation of the cytosolic enzyme to ER-bound ribosomes. This finding reveals a complex picture of GARS translation and localization in mammals. In this context, we discuss how human GARS expression could influence its moonlighting activities and its involvement in diseases.


Assuntos
Regulação Enzimológica da Expressão Gênica , Glicina-tRNA Ligase/genética , Glicina-tRNA Ligase/metabolismo , Sítios Internos de Entrada Ribossomal/genética , Fases de Leitura Aberta/genética , Animais , Sequência de Bases , Células COS , Cercopithecus aethiops , Códon de Terminação/genética , Retículo Endoplasmático/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Iniciação Traducional da Cadeia Peptídica , Transporte Proteico , Capuzes de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Homologia de Sequência de Aminoácidos
6.
Orphanet J Rare Dis ; 8: 193, 2013 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-24344687

RESUMO

BACKGROUND: Mutations in the mitochondrial tyrosyl-tRNA synthetase (YARS2) gene have previously been identified as a cause of the tissue specific mitochondrial respiratory chain (RC) disorder, Myopathy, Lactic Acidosis, Sideroblastic Anaemia (MLASA). In this study, a cohort of patients with a mitochondrial RC disorder for who anaemia was a feature, were screened for mutations in YARS2. METHODS: Twelve patients were screened for YARS2 mutations by Sanger sequencing. Clinical data were compared. Functional assays were performed to confirm the pathogenicity of the novel mutations and to investigate tissue specific effects. RESULTS: PathogenicYARS2 mutations were identified in three of twelve patients screened. Two patients were found to be homozygous for the previously reported p.Phe52Leu mutation, one severely and one mildly affected. These patients had different mtDNA haplogroups which may contribute to the observed phenotypic variability. A mildly affected patient was a compound heterozygote for two novel YARS2 mutations, p.Gly191Asp and p.Arg360X. The p.Gly191Asp mutation resulted in a 38-fold loss in YARS2 catalytic efficiency and the p.Arg360X mutation did not produce a stable protein. The p.Phe52Leu and p.Gly191Asp/p.Arg360X mutations resulted in more severe RC deficiency of complexes I, III and IV in muscle cells compared to fibroblasts, but had relatively normal YARS2 protein levels. The muscle-specific RC deficiency can be related to the increased requirement for RC complexes in muscle. There was also a failure of mtDNA proliferation upon myogenesis in patient cells which may compound the RC defect. Patient muscle had increased levels of PGC1-α and TFAM suggesting mitochondrial biogenesis was activated as a potential compensatory mechanism. CONCLUSION: In this study we have identified novel YARS2 mutations and noted marked phenotypic variability among YARS2 MLASA patients, with phenotypes ranging from mild to lethal, and we suggest that the background mtDNA haplotype may be contributing to the phenotypic variability. These findings have implications for diagnosis and prognostication of the MLASA and related phenotypes.


Assuntos
Acidose Láctica/genética , Anemia Sideroblástica/genética , Miopatias Mitocondriais/genética , Tirosina-tRNA Ligase/genética , Acidose Láctica/metabolismo , Adolescente , Adulto , Anemia Sideroblástica/metabolismo , Criança , Pré-Escolar , DNA Mitocondrial/genética , Feminino , Humanos , Hidroliases/genética , Hidroliases/metabolismo , Lactente , Recém-Nascido , Miopatias Mitocondriais/metabolismo , Tirosina-tRNA Ligase/metabolismo , Adulto Jovem
7.
J Biol Chem ; 288(51): 36361-71, 2013 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-24196969

RESUMO

Genome sequencing revealed an extreme AT-rich genome and a profusion of asparagine repeats associated with low complexity regions (LCRs) in proteins of the malarial parasite Plasmodium falciparum. Despite their abundance, the function of these LCRs remains unclear. Because they occur in almost all families of plasmodial proteins, the occurrence of LCRs cannot be associated with any specific metabolic pathway; yet their accumulation must have given selective advantages to the parasite. Translation of these asparagine-rich LCRs demands extraordinarily high amounts of asparaginylated tRNA(Asn). However, unlike other organisms, Plasmodium codon bias is not correlated to tRNA gene copy number. Here, we studied tRNA(Asn) accumulation as well as the catalytic capacities of the asparaginyl-tRNA synthetase of the parasite in vitro. We observed that asparaginylation in this parasite can be considered standard, which is expected to limit the availability of asparaginylated tRNA(Asn) in the cell and, in turn, slow down the ribosomal translation rate when decoding asparagine repeats. This observation strengthens our earlier hypothesis considering that asparagine rich sequences act as "tRNA sponges" and help cotranslational folding of parasite proteins. However, it also raises many questions about the mechanistic aspects of the synthesis of asparagine repeats and about their implications in the global control of protein expression throughout Plasmodium life cycle.


Assuntos
Plasmodium falciparum/metabolismo , RNA de Transferência de Asparagina/metabolismo , Aminoacilação de RNA de Transferência , Sequência de Aminoácidos , Aminoacil-tRNA Sintetases/metabolismo , Asparagina/química , Asparagina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Humanos , Cinética , Dados de Sequência Molecular , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Pyrococcus abyssi/enzimologia , RNA de Transferência de Asparagina/biossíntese , Sequências Repetitivas de Aminoácidos
8.
Proc Natl Acad Sci U S A ; 108(40): E794-802, 2011 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-21896722

RESUMO

Several classes of small noncoding RNAs are key players in cellular metabolism including mRNA decoding, RNA processing, and mRNA stability. Here we show that a tRNA(Asp) isodecoder, corresponding to a human tRNA-derived sequence, binds to an embedded Alu RNA element contained in the 3' UTR of the human aspartyl-tRNA synthetase mRNA. This interaction between two well-known classes of RNA molecules, tRNA and Alu RNA, is driven by an unexpected structural motif and induces a global rearrangement of the 3' UTR. Besides, this 3' UTR contains two functional polyadenylation signals. We propose a model where the tRNA/Alu interaction would modulate the accessibility of the two alternative polyadenylation sites and regulate the stability of the mRNA. This unique regulation mechanism would link gene expression to RNA polymerase III transcription and may have implications in a primate-specific signal pathway.


Assuntos
Regiões 3' não Traduzidas/genética , Elementos Alu/fisiologia , Aspartato-tRNA Ligase/metabolismo , Modelos Biológicos , Conformação Proteica , Dobramento de RNA/fisiologia , Processamento Pós-Transcricional do RNA/fisiologia , RNA de Transferência de Ácido Aspártico/metabolismo , Elementos Alu/genética , Aspartato-tRNA Ligase/genética , Sequência de Bases , Northern Blotting , Primers do DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Células HeLa , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Processamento Pós-Transcricional do RNA/genética , RNA de Transferência de Ácido Aspártico/genética , Transfecção
9.
Am J Hum Genet ; 87(1): 52-9, 2010 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-20598274

RESUMO

Mitochondrial respiratory chain disorders are a heterogeneous group of disorders in which the underlying genetic defect is often unknown. We have identified a pathogenic mutation (c.156C>G [p.F52L]) in YARS2, located at chromosome 12p11.21, by using genome-wide SNP-based homozygosity analysis of a family with affected members displaying myopathy, lactic acidosis, and sideroblastic anemia (MLASA). We subsequently identified the same mutation in another unrelated MLASA patient. The YARS2 gene product, mitochondrial tyrosyl-tRNA synthetase (YARS2), was present at lower levels in skeletal muscle whereas fibroblasts were relatively normal. Complex I, III, and IV were dysfunctional as indicated by enzyme analysis, immunoblotting, and immunohistochemistry. A mitochondrial protein-synthesis assay showed reduced levels of respiratory chain subunits in myotubes generated from patient cell lines. A tRNA aminoacylation assay revealed that mutant YARS2 was still active; however, enzyme kinetics were abnormal compared to the wild-type protein. We propose that the reduced aminoacylation activity of mutant YARS2 enzyme leads to decreased mitochondrial protein synthesis, resulting in mitochondrial respiratory chain dysfunction. MLASA has previously been associated with PUS1 mutations; hence, the YARS2 mutation reported here is an alternative cause of MLASA.


Assuntos
Acidose Láctica/genética , Anemia Sideroblástica/genética , Mitocôndrias/enzimologia , Doenças Musculares/genética , Tirosina-tRNA Ligase/genética , Adolescente , Adulto , Sequência de Aminoácidos , Criança , Pré-Escolar , Cromossomos Humanos Par 12/genética , Consanguinidade , Complexo I de Transporte de Elétrons/fisiologia , Complexo III da Cadeia de Transporte de Elétrons/fisiologia , Complexo IV da Cadeia de Transporte de Elétrons/fisiologia , Feminino , Estudos de Associação Genética , Ligação Genética , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Lactente , Masculino , Dados de Sequência Molecular , Mutação , Linhagem , Síndrome , Adulto Jovem
10.
FEBS Lett ; 584(2): 448-54, 2010 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-19900443

RESUMO

In most organisms, the information necessary to specify the native 3D-structures of proteins is encoded in the corresponding mRNA sequences. Translational accuracy and efficiency are coupled and sequences that are slowly translated play an essential role in the concomitant folding of protein domains. Here, we suggest that the well-known mechanisms for the regulation of translational efficiency, which involves mRNA structure and/or asymmetric tRNA abundance, do not apply to all organisms. We propose that Plasmodium, the parasite responsible for malaria, uses an alternative strategy to slow down ribosomal speed and avoid multidomain protein misfolding during translation. In our model, the abundant Low Complexity Regions present in Plasmodium proteins replace the codon preferences, which influence the assembly of protein secondary structures.


Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Plasmodium falciparum/metabolismo , Biossíntese de Proteínas , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Sequência de Aminoácidos , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/genética , Dados de Sequência Molecular , Mutagênese Insercional , Dobramento de Proteína , Estrutura Secundária de Proteína , RNA Mensageiro/metabolismo
11.
RNA ; 14(4): 641-8, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18268021

RESUMO

A growing number of human pathologies are ascribed to mutations in mitochondrial tRNA genes. Here, we report biochemical investigations on three mt-tRNA(Tyr) molecules with point substitutions associated with diseases. The mutations occur in the atypical T- and D-loops at positions homologous to those involved in the tertiary interaction network of canonical tRNAs. They do not correspond to tyrosine identity positions and likely do not contact the mitochondrial tyrosyl-tRNA synthetase during the aminoacylation process. The impact of these substitutions on mt-tRNA(Tyr) tyrosylation and structure was investigated using the corresponding tRNA transcripts. In vitro tyrosylation efficiency is decreased 600-fold for mutant A22G (mitochondrial gene mutation T5874C), 40-fold for G15A (C5877T), and is without significant effect on U54C (A5843G). Comparative solution probings with lead and nucleases on mutant and wild-type tRNA(Tyr) molecules reveal a greater sensitivity to single-strand specific probes for mutants G15A and A22G. For both transcripts, the mutation triggers a structural destabilization in the D-loop that propagates toward the anticodon arm and thus hinders efficient tyrosylation. Further probing analysis combined with phylogenetic data support the participation of G15 and A22 in the tertiary network of human mt-tRNA(Tyr) via nonclassical Watson-Crick G15-C48 and G13-A22 pairings. In contrast, the pathogenic effect of the tyrosylable mutant U54C, where structure is only marginally affected, has to be sought at another level of the tRNA(Tyr) life cycle.


Assuntos
Mutação Puntual , RNA de Transferência de Tirosina/química , RNA de Transferência de Tirosina/genética , RNA/química , RNA/genética , Aminoacilação de RNA de Transferência , Sequência de Bases , Humanos , Técnicas In Vitro , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA/metabolismo , Estabilidade de RNA , RNA Mitocondrial , RNA de Transferência de Tirosina/metabolismo
12.
Structure ; 15(11): 1505-16, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17997975

RESUMO

We report the structure of a strictly mitochondrial human synthetase, namely tyrosyl-tRNA synthetase (mt-TyrRS), in complex with an adenylate analog at 2.2 A resolution. The structure is that of an active enzyme deprived of the C-terminal S4-like domain and resembles eubacterial TyrRSs with a canonical tyrosine-binding pocket and adenylate-binding residues typical of class I synthetases. Two bulges at the enzyme surface, not seen in eubacterial TyrRSs, correspond to conserved sequences in mt-TyrRSs. The synthetase electrostatic surface potential differs from that of other TyrRSs, including the human cytoplasmic homolog and the mitochondrial one from Neurospora crassa. The homodimeric human mt-TyrRS shows an asymmetry propagating from the dimer interface toward the two catalytic sites and extremities of each subunit. Mutagenesis of the catalytic domain reveals functional importance of Ser200 in line with an involvement of A73 rather than N1-N72 in tyrosine identity.


Assuntos
Mitocôndrias/enzimologia , Tirosina-tRNA Ligase/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , RNA de Transferência/química , RNA de Transferência/metabolismo , Alinhamento de Sequência , Tirosina-tRNA Ligase/genética , Tirosina-tRNA Ligase/metabolismo
13.
J Virol ; 81(22): 12406-17, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17855524

RESUMO

Aminoacyl-tRNA synthetases are pivotal in determining how the genetic code is translated in amino acids and in providing the substrate for protein synthesis. As such, they fulfill a key role in a process universally conserved in all cellular organisms from their most complex to their most reduced parasitic forms. In contrast, even complex viruses were not found to encode much translation machinery, with the exception of isolated components such as tRNAs. In this context, the discovery of four aminoacyl-tRNA synthetases encoded in the genome of mimivirus together with a full set of translation initiation, elongation, and termination factors appeared to blur what was once a clear frontier between the cellular and viral world. Functional studies of two mimivirus tRNA synthetases confirmed the MetRS specificity for methionine and the TyrRS specificity for tyrosine and conformity with the identity rules for tRNA(Tyr) for archea/eukarya. The atomic structure of the mimivirus tyrosyl-tRNA synthetase in complex with tyrosinol exhibits the typical fold and active-site organization of archaeal-type TyrRS. However, the viral enzyme presents a unique dimeric conformation and significant differences in its anticodon binding site. The present work suggests that mimivirus aminoacyl-tRNA synthetases function as regular translation enzymes in infected amoebas. Their phylogenetic classification does not suggest that they have been acquired recently by horizontal gene transfer from a cellular host but rather militates in favor of an intricate evolutionary relationship between large DNA viruses and ancestral eukaryotes.


Assuntos
Acanthamoeba/virologia , Vírus de DNA/enzimologia , Metionina tRNA Ligase/química , Tirosina-tRNA Ligase/química , Proteínas Virais/química , Animais , Anticódon/química , Anticódon/metabolismo , Cristalografia por Raios X , Metionina tRNA Ligase/classificação , Metionina tRNA Ligase/genética , Filogenia , Estrutura Secundária de Proteína , RNA de Transferência de Metionina/química , RNA de Transferência de Metionina/metabolismo , RNA de Transferência de Tirosina/química , RNA de Transferência de Tirosina/metabolismo , Tirosina-tRNA Ligase/classificação , Tirosina-tRNA Ligase/genética , Proteínas Virais/classificação , Proteínas Virais/genética
14.
Artigo em Inglês | MEDLINE | ID: mdl-17401211

RESUMO

Human mitochondrial tyrosyl-tRNA synthetase and a truncated version with its C-terminal S4-like domain deleted were purified and crystallized. Only the truncated version, which is active in tyrosine activation and Escherichia coli tRNA(Tyr) charging, yielded crystals suitable for structure determination. These tetragonal crystals, belonging to space group P4(3)2(1)2, were obtained in the presence of PEG 4000 as a crystallizing agent and diffracted X-rays to 2.7 A resolution. Complete data sets could be collected and led to structure solution by molecular replacement.


Assuntos
Mitocôndrias/enzimologia , Tirosina-tRNA Ligase/química , Sequência de Bases , Cristalização , Cristalografia por Raios X , Primers do DNA , Humanos , Conformação Proteica
15.
Nucleic Acids Res ; 34(17): 4987-95, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16982637

RESUMO

In metazoans, cell-cycle-dependent histones are produced from poly(A)-lacking mRNAs. The 3' end of histone mRNAs is formed by an endonucleolytic cleavage of longer precursors between a conserved stem-loop structure and a purine-rich histone downstream element (HDE). The cleavage requires at least two trans-acting factors: the stem-loop binding protein (SLBP), which binds to the stem-loop and the U7 snRNP, which anchors to histone pre-mRNAs by annealing to the HDE. Using RNA structure-probing techniques, we determined the secondary structure of the 3'-untranslated region (3'-UTR) of mouse histone pre-mRNAs H4-12, H1t and H2a-614. Surprisingly, the HDE is embedded in hairpin structures and is therefore not easily accessible for U7 snRNP anchoring. Probing of the 3'-UTR in complex with SLBP revealed structural rearrangements leading to an overall opening of the structure especially at the level of the HDE. Electrophoretic mobility shift assays demonstrated that the SLBP-induced opening of HDE actually facilitates U7 snRNA anchoring on the histone H4-12 pre-mRNAs 3' end. These results suggest that initial binding of the SLBP functions in making the HDE more accessible for U7 snRNA anchoring.


Assuntos
Regiões 3' não Traduzidas/química , Histonas/genética , Proteínas Nucleares/metabolismo , Processamento de Terminações 3' de RNA , Precursores de RNA/química , RNA Nuclear Pequeno/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Regiões 3' não Traduzidas/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Humanos , Camundongos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Pegadas de Proteínas , Precursores de RNA/metabolismo
16.
J Mol Biol ; 355(5): 873-8, 2006 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-16337653

RESUMO

Tobacco mosaic virus (TMV) and Nemesia ring necrosis virus (NeRNV) belong to the Tobamoviridae and Tymoviridae families, respectively. Although their RNAs present different 5'-untranslated regions and different family-specific genomic organizations, they share common 3'-ends organized into three consecutive pseudoknot structures followed by a histidylatable tRNA-like structure (TLS). We investigate here whether the histidine residue becomes incorporated into viral proteins and if the TLSs of TMV and NeRNV play a role in viral translation. Our results indicate that, regardless of the genomic context, the histidine moiety does not become incorporated in proteins via ribosomal translation, and that disruption of the TLS in either viral RNA does not perturb the viral translation patterns. In the light of the present data and of previous results on tymoviral TLSVal and bromoviral TLSTyr showing differential effects on translation, we suggest that the key role for the TLS in promoting translation initiation appears to be dictated by the TLS architecture and identity.


Assuntos
Conformação de Ácido Nucleico , Biossíntese de Proteínas , RNA de Transferência , RNA Viral , Vírus do Mosaico do Tabaco/genética , Tymoviridae/metabolismo , Genoma Viral , Histidina/química , Plantas/virologia , RNA de Transferência/química , RNA de Transferência/metabolismo
17.
Biochimie ; 87(9-10): 813-7, 2005 Sep-Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16164991

RESUMO

In the methanogenic archae Methanosarcina barkeri, insertion of pyrrolysine, the 22nd amino acid, results from the decoding of an amber UAG codon in the mRNA of monomethylamine methyltransferases (MtmB). Sequence comparisons combined with structural enzymatic and chemical probing on M. barkeri MtmB1 mRNA demonstrate the presence of a hairpin motif located immediately after the redefined UAG codon. This structure of 86 nucleotides differs slightly from a proposal given in the literature and comprises four successive stems separated by three internal loops and closed by a large apical loop. Sequence alignments of MtmB mRNAs of different Methanosarcinacae reveal a conservation of the motif in both sequence and folding levels. The functional role of this motif as a signal leading to pyrrolysine insertion is discussed.


Assuntos
Proteínas Arqueais/genética , Códon/genética , Lisina/análogos & derivados , Methanosarcina barkeri/genética , Metiltransferases/genética , Biossíntese de Proteínas , RNA Mensageiro/química , Autorradiografia , Sequência de Bases , Sequência Conservada , Elementos de DNA Transponíveis , Lisina/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Mensageiro/metabolismo , RNA de Transferência , Ribossomos , Alinhamento de Sequência
18.
Biochimie ; 87(9-10): 873-83, 2005 Sep-Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16164994

RESUMO

The tRNA identity rules ensuring fidelity of translation are globally conserved throughout evolution except for tyrosyl-tRNA synthetases (TyrRSs) that display species-specific tRNA recognition. This discrimination originates from the presence of a conserved identity pair, G1-C72, located at the top of the acceptor stem of tRNA(Tyr) from eubacteria that is invariably replaced by an unusual C1-G72 pair in archaeal and eubacterial tRNA(Tyr). In addition to the key role of pair 1-72 in tyrosylation, discriminator base A73, the anticodon triplet and the large variable region (present in eubacterial tRNA(Tyr) but not found in eukaryal tRNA(Tyr)) contribute to tyrosylation with variable strengths. Crystallographic structures of two tRNA(Tyr)/TyrRS complexes revealed different interaction modes in accordance with the phylum-specificity. Recent functional studies on the human mitochondrial tRNA(Tyr)/TyrRS system indicates strong deviations from the canonical tyrosylation rules. These differences are discussed in the light of the present knowledge on TyrRSs.


Assuntos
Aminoacilação/genética , Evolução Molecular , RNA de Transferência/química , Tirosina-tRNA Ligase/química , Alanina-tRNA Ligase , Anticódon , Sequência de Bases , Sequência Conservada , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Conformação Proteica , RNA de Transferência/metabolismo , Especificidade da Espécie , Relação Estrutura-Atividade , Especificidade por Substrato , Tirosina-tRNA Ligase/genética , Tirosina-tRNA Ligase/metabolismo
19.
RNA ; 11(5): 558-62, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15840810

RESUMO

Human tyrosyl-tRNA synthetase from mitochondria (mt-TyrRS) presents dual sequence features characteristic of eubacterial and archaeal TyrRSs, especially in the region containing amino acids recognizing the N1-N72 tyrosine identity pair. This would imply that human mt-TyrRS has lost the capacity to discriminate between the G1-C72 pair typical of eubacterial and mitochondrial tRNATyr and the reverse pair C1-G72 present in archaeal and eukaryal tRNATyr. This expectation was verified by a functional analysis of wild-type or mutated tRNATyr molecules, showing that mt-TyrRS aminoacylates with similar catalytic efficiency its cognate tRNATyr with G1-C72 and its mutated version with C1-G72. This provides the first example of a TyrRS lacking specificity toward N1-N72 and thus of a TyrRS disobeying the identity rules. Sequence comparisons of mt-TyrRSs across phylogeny suggest that the functional behavior of the human mt-TyrRS is conserved among all vertebrate mt-TyrRSs.


Assuntos
Mitocôndrias/enzimologia , RNA de Transferência de Tirosina/metabolismo , Tirosina-tRNA Ligase/metabolismo , Tirosina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Domínio Catalítico , Humanos , Dados de Sequência Molecular , RNA de Transferência de Tirosina/genética , Especificidade por Substrato , Tirosina/genética , Tirosina-tRNA Ligase/química
20.
Biochemistry ; 44(12): 4805-16, 2005 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-15779907

RESUMO

The human mitochondrion possesses a translational machinery devoted to the synthesis of 13 proteins. While the required tRNAs and rRNAs are produced by transcription of the mitochondrial genome, all other factors needed for protein synthesis are synthesized in the cytosol and imported. This is the case for aminoacyl-tRNA synthetases, the enzymes which esterify their cognate tRNA with the specific amino acid. The genes for the full set of cytosolic aaRSs are well defined, but only nine genes for mitochondrial synthetases are known. Here we describe the genes for human mitochondrial aspartyl- and tyrosyl-tRNA synthetases and the initial characterization of the enzymes. Both belong to the expected class of synthetases, have a dimeric organization, and aminoacylate Escherichia coli tRNAs as well as in vitro transcribed human mitochondrial tRNAs. Genes for the remaining missing synthetases were also found with the exception of glutaminyl-tRNA synthetase. Their sequence analysis confirms and further extends the view that, except for lysyl- and glycyl-tRNA synthetases, human mitochondrial and cytosolic enzymes are coded by two different sets of genes.


Assuntos
Aspartato-tRNA Ligase/química , Mitocôndrias/enzimologia , Tirosina-tRNA Ligase/química , Sequência de Aminoácidos , Aspartato-tRNA Ligase/genética , Aspartato-tRNA Ligase/isolamento & purificação , Sequência de Bases , Clonagem Molecular , Biologia Computacional/métodos , Bases de Dados de Ácidos Nucleicos , Humanos , Mitocôndrias/genética , Dados de Sequência Molecular , RNA de Transferência de Ácido Aspártico/metabolismo , RNA de Transferência de Tirosina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Aminoacilação de RNA de Transferência , Tirosina-tRNA Ligase/genética , Tirosina-tRNA Ligase/isolamento & purificação
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