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1.
J Mol Neurosci ; 73(11-12): 912-920, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37845428

RESUMO

Parkinson's disease (PD) is speculated with genetic and environmental factors. At molecular level, the mitochondrial impact is stated to be one of the causative reasons for PD. In this study, we investigated the mitochondrial membrane potential (MMP), reactive oxygen species (ROS) and adenosine triphosphate (ATP) levels along with mitochondrial tRNA alterations among three age categories of PD. By determining the genetic and organellar functionality using molecular techniques, the ROS levels were reported to be high with decreased MMP and ATP in the late-onset age group than in other two age categories. Likewise, the tRNA significancy in tRNAThr and tRNAGln was noticed with C4335T and G15927A mutations in late-onset and early-onset PD groups respectively. Therefore, from the findings, ageing has shown a disruption in tRNA metabolism leading to critical functioning of ATP synthesis and MMP, causing oxidative stress in PD patients. These physiological outcomes show that ageing has a keen role in the divergence of mitochondrial function, thereby proving a correlation with ageing and maintenance of mitochondrial homeostasis in PD.


Assuntos
Doença de Parkinson , RNA de Transferência de Treonina , Humanos , RNA de Transferência de Treonina/genética , RNA de Transferência de Treonina/metabolismo , RNA de Transferência de Glutamina/genética , RNA de Transferência de Glutamina/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Índia , Mitocôndrias/genética , Mitocôndrias/metabolismo , Trifosfato de Adenosina/metabolismo
2.
Nucleic Acids Res ; 50(7): 4012-4028, 2022 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-35357504

RESUMO

METTL8 has recently been identified as the methyltransferase catalyzing 3-methylcytidine biogenesis at position 32 (m3C32) of mitochondrial tRNAs. METTL8 also potentially participates in mRNA methylation and R-loop biogenesis. How METTL8 plays multiple roles in distinct cell compartments and catalyzes mitochondrial tRNA m3C formation remain unclear. Here, we discovered that alternative mRNA splicing generated several isoforms of METTL8. One isoform (METTL8-Iso1) was targeted to mitochondria via an N-terminal pre-sequence, while another one (METTL8-Iso4) mainly localized to the nucleolus. METTL8-Iso1-mediated m3C32 modification of human mitochondrial tRNAThr (hmtRNAThr) was not reliant on t6A modification at A37 (t6A37), while that of hmtRNASer(UCN) critically depended on i6A modification at A37 (i6A37). We clarified the hmtRNAThr substrate recognition mechanism, which was obviously different from that of hmtRNASer(UCN), in terms of requiring a G35 determinant. Moreover, SARS2 (mitochondrial seryl-tRNA synthetase) interacted with METTL8-Iso1 in an RNA-independent manner and modestly accelerated m3C modification activity. We further elucidated how nonsubstrate tRNAs in human mitochondria were efficiently discriminated by METTL8-Iso1. In summary, our results established the expression pattern of METTL8, clarified the molecular basis for m3C32 modification by METTL8-Iso1 and provided the rationale for the involvement of METTL8 in tRNA modification, mRNA methylation or R-loop biogenesis.


Assuntos
Metiltransferases/metabolismo , Mitocôndrias/metabolismo , RNA de Transferência , Processamento Alternativo , Humanos , Metiltransferases/genética , Mitocôndrias/genética , RNA Mensageiro , RNA de Transferência/genética , RNA de Transferência/metabolismo , RNA de Transferência de Treonina/genética
3.
Nat Commun ; 13(1): 209, 2022 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-35017528

RESUMO

Modified nucleotides in tRNAs are important determinants of folding, structure and function. Here we identify METTL8 as a mitochondrial matrix protein and active RNA methyltransferase responsible for installing m3C32 in the human mitochondrial (mt-)tRNAThr and mt-tRNASer(UCN). METTL8 crosslinks to the anticodon stem loop (ASL) of many mt-tRNAs in cells, raising the question of how methylation target specificity is achieved. Dissection of mt-tRNA recognition elements revealed U34G35 and t6A37/(ms2)i6A37, present concomitantly only in the ASLs of the two substrate mt-tRNAs, as key determinants for METTL8-mediated methylation of C32. Several lines of evidence demonstrate the influence of U34, G35, and the m3C32 and t6A37/(ms2)i6A37 modifications in mt-tRNAThr/Ser(UCN) on the structure of these mt-tRNAs. Although mt-tRNAThr/Ser(UCN) lacking METTL8-mediated m3C32 are efficiently aminoacylated and associate with mitochondrial ribosomes, mitochondrial translation is mildly impaired by lack of METTL8. Together these results define the cellular targets of METTL8 and shed new light on the role of m3C32 within mt-tRNAs.


Assuntos
Anticódon/química , Metiltransferases/genética , Mitocôndrias/genética , RNA Mitocondrial/química , RNA de Transferência de Serina/química , RNA de Transferência de Treonina/química , Anticódon/metabolismo , Pareamento de Bases , Citosina/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Metilação , Metiltransferases/metabolismo , Mitocôndrias/metabolismo , Conformação de Ácido Nucleico , Ligação Proteica , Biossíntese de Proteínas , RNA Mitocondrial/genética , RNA Mitocondrial/metabolismo , RNA de Transferência de Serina/genética , RNA de Transferência de Serina/metabolismo , RNA de Transferência de Treonina/genética , RNA de Transferência de Treonina/metabolismo , Transdução de Sinais
4.
Hum Mol Genet ; 31(4): 523-534, 2022 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-34508595

RESUMO

TARS2 encodes human mitochondrial threonyl tRNA-synthetase that is responsible for generating mitochondrial Thr-tRNAThr and clearing mischarged Ser-tRNAThr during mitochondrial translation. Pathogenic variants in TARS2 have hitherto been reported in a pair of siblings and an unrelated patient with an early onset mitochondrial encephalomyopathy and a combined respiratory chain enzyme deficiency in muscle. We here report five additional unrelated patients with TARS2-related mitochondrial diseases, expanding the clinical phenotype to also include epilepsy, dystonia, hyperhidrosis and severe hearing impairment. In addition, we document seven novel TARS2 variants-one nonsense variant and six missense variants-that we demonstrate are pathogenic and causal of the disease presentation based on population frequency, homology modeling and functional studies that show the effects of the pathogenic variants on TARS2 stability and/or function.


Assuntos
Doenças Mitocondriais , Encefalomiopatias Mitocondriais , Treonina-tRNA Ligase , Humanos , Doenças Mitocondriais/genética , Doenças Mitocondriais/patologia , Encefalomiopatias Mitocondriais/genética , Mutação , Fenótipo , RNA de Transferência de Treonina/genética , Treonina-tRNA Ligase/genética
5.
J Diabetes Res ; 2020: 2057187, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32083134

RESUMO

We report here the clinical, genetic, and molecular characteristics of type 2 diabetes in a Chinese family. There are differences in the severity and age of onset in diabetes among these families. By molecular analysis of the complete mitochondrial genome in this family, we identified the homoplasmic m.15897G>A mutation underwent sequence analysis of whole mitochondrial DNA genome, which localized at conventional position ten of tRNAThr, and distinct sets of mtDNA polymorphisms belonging to haplogroup D4b1. This mutation has been implicated to be important for tRNA identity and stability. Using cybrid cell models, the decreased efficiency of mitochondrial tRNAThr levels caused by the m.15897G>A mutation results in respiratory deficiency, protein synthesis and assembly, mitochondrial ATP synthesis, and mitochondrial membrane potential. These mitochondrial dysfunctions caused an increase in the production of reactive oxygen species in the mutant cell lines. These data provide a direct evidence that a novel tRNA mutation was associated with T2DM. Thus, our findings provide a new insight into the understanding of pathophysiology of maternally inherited diabetes.


Assuntos
Diabetes Mellitus Tipo 2/genética , RNA de Transferência de Treonina/genética , Adulto , Idoso , Povo Asiático/genética , Northern Blotting , Western Blotting , China , Diabetes Mellitus Tipo 2/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Eletroforese em Gel de Poliacrilamida , Feminino , Genoma Mitocondrial , Humanos , Masculino , Herança Materna , Potencial da Membrana Mitocondrial/genética , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Linhagem , Filogenia , Mutação Puntual , Espécies Reativas de Oxigênio/metabolismo
6.
Nucleic Acids Res ; 48(6): 3181-3194, 2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-32047918

RESUMO

N 6-Threonylcarbamoyladenosine (t6A) is a universal tRNA modification essential for translational accuracy and fidelity. In human mitochondria, YrdC synthesises an l-threonylcarbamoyl adenylate (TC-AMP) intermediate, and OSGEPL1 transfers the TC-moiety to five tRNAs, including human mitochondrial tRNAThr (hmtRNAThr). Mutation of hmtRNAs, YrdC and OSGEPL1, affecting efficient t6A modification, has been implicated in various human diseases. However, little is known about the tRNA recognition mechanism in t6A formation in human mitochondria. Herein, we showed that OSGEPL1 is a monomer and is unique in utilising C34 as an anti-determinant by studying the contributions of individual bases in the anticodon loop of hmtRNAThr to t6A modification. OSGEPL1 activity was greatly enhanced by introducing G38A in hmtRNAIle or the A28:U42 base pair in a chimeric tRNA containing the anticodon stem of hmtRNASer(AGY), suggesting that sequences of specific hmtRNAs are fine-tuned for different modification levels. Moreover, using purified OSGEPL1, we identified multiple acetylation sites, and OSGEPL1 activity was readily affected by acetylation via multiple mechanisms in vitro and in vivo. Collectively, we systematically elucidated the nucleotide requirement in the anticodon loop of hmtRNAs, and revealed mechanisms involving tRNA sequence optimisation and post-translational protein modification that determine t6A modification levels.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Mitocôndrias/genética , Biossíntese de Proteínas , RNA de Transferência de Treonina/genética , Acetilação , Adenosina/análogos & derivados , Adenosina/genética , Anticódon/genética , Escherichia coli/genética , Proteínas de Ligação ao GTP/genética , Humanos , Mutação , Conformação de Ácido Nucleico , Processamento Pós-Transcricional do RNA/genética , RNA de Transferência/genética , Proteínas de Ligação a RNA/genética
7.
BMC Cardiovasc Disord ; 19(1): 293, 2019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31842766

RESUMO

BACKGROUND: Coronary heart disease (CHD) is the most common cause of mortality globally, yet mitochondrial genetic mutations associated with CHD development remain incompletely understood. METHODS: The subjects from three Chinese families with LHON underwent clinical, genetic, molecular, and biochemical evaluations. Biochemical characterizations included measuring the effects of the15910C > T mutation on tRNAThr levels, enzymatic activity of electron transport chain complexes, membrane permeability, and the mitochondria-mediated generation of both reactive oxygen species (ROS) and adenosine triphosphate (ATP). RESULTS: We characterize mitochondrial genetic mutations in a three-generation Chinese family exhibiting signs of maternally inherited CHD. Of the 24 different family members in this pedigree we assessed, CHD was detected in 6, with variable severity and age of first appearance. When we sequenced the mitochondrial genomes of these individuals, we found a tRNAThr 15910C > T mutation of the Eastern Asian haplogroup M7b'c. This mutation is predicted to destabilize the strongly conserved (24C-10G) base-pairing, thereby disrupting tRNAThr functionality. When we performed Northern blotting, we detected we observed a 37.5% reduction in tRNAThr levels at baseline in cybrid cell lines bearing the 15910C > T mutation. When we conducted western blot analysis, we detected a ~ 24.96% decrease in mitochondrial translation rates in these same cells. CONCLUSIONS: In the present report, Together these findings suggest a possible link between this 15910C > T tRNAThr mutation and CHD, potentially offering new avenues for future disease intervention.


Assuntos
Doença das Coronárias/genética , Herança Materna , Mitocôndrias Cardíacas/genética , Mutação , RNA Mitocondrial/genética , RNA de Transferência de Treonina/genética , Trifosfato de Adenosina/metabolismo , Adulto , Idade de Início , Idoso , Povo Asiático/genética , Estudos de Casos e Controles , Linhagem Celular , China/epidemiologia , Doença das Coronárias/diagnóstico , Doença das Coronárias/etnologia , Metabolismo Energético , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Mitocôndrias Cardíacas/metabolismo , Linhagem , Fenótipo , Biossíntese de Proteínas , RNA Mitocondrial/metabolismo , RNA de Transferência de Treonina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fatores de Risco , Índice de Gravidade de Doença
8.
Curr Mol Med ; 19(2): 136-146, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30854964

RESUMO

BACKGROUND: Mutations in mitochondrial tRNA (mt-tRNA) genes have been found to be associated with both syndromic and non-syndromic hearing impairment. However, the pathophysiology underlying mt-tRNA mutations in clinical expression of hearing loss remains poorly understood. OBJECTIVE: The aim of this study was to explore the potential association between mttRNA mutations and hearing loss. METHODS AND RESULTS: We reported here the molecular features of a pedigree with maternally transmitted non-syndromic hearing loss. Among 12 matrilineal relatives, five of them suffered variable degree of hearing impairment, but none of them had any medical history of using aminoglycosides antibiotics (AmAn). Genetic screening of the complete mitochondrial genomes from the matrilineal relatives identified the coexistence of mt-tRNAHis G12192A and mt-tRNAThr G15927A mutations, together with a set of polymorphisms belonging to human mitochondrial haplogroup B5b1b. Interestingly, the G12192A mutation occurred 2-bp from the 3' end of the TψC loop of mt-tRNAHis, which was evolutionarily conserved from various species. In addition, the well-known G15927A mutation, which disrupted the highly conserved C-G base-pairing at the anticodon stem of mt-tRNAThr, may lead to the failure in mt-tRNA metabolism. Furthermore, a significant decreased in ATP production and an increased ROS generation were observed in polymononuclear leukocytes (PMNs) which were isolated from the deaf patients carrying these mt-tRNA mutations, suggested that the G12192A and G15927A mutations may cause mitochondrial dysfunction that was responsible for deafness. However, the absence of any functional mutations/variants in GJB2, GJB3, GJB6 and TRMU genes suggested that the nuclear genes may not play important roles in the clinical expression of non-syndromic hearing loss in this family. CONCLUSION: Our data indicated that mt-tRNAHis G12192A mutation may increase the penetrance and expressivity of deafness-associated m-tRNAThr G15927A mutation in this family.


Assuntos
Povo Asiático/genética , Surdez/genética , Surdez/fisiopatologia , Mitocôndrias/genética , Mutação , RNA de Transferência de Histidina/genética , RNA de Transferência de Treonina/genética , Adulto , Sequência de Bases , DNA Mitocondrial/análise , Feminino , Genes Mitocondriais , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Penetrância , Fenótipo
9.
Nat Commun ; 10(1): 118, 2019 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-30631057

RESUMO

In the absence of extensive transcription control mechanisms the pathogenic parasite Trypanosoma brucei crucially depends on translation regulation to orchestrate gene expression. However, molecular insight into regulating protein biosynthesis is sparse. Here we analyze the small non-coding RNA (ncRNA) interactome of ribosomes in T. brucei during different growth conditions and life stages. Ribosome-associated ncRNAs have recently been recognized as unprecedented regulators of ribosome functions. Our data show that the tRNAThr 3´half is produced during nutrient deprivation and becomes one of the most abundant tRNA-derived RNA fragments (tdRs). tRNAThr halves associate with ribosomes and polysomes and stimulate translation by facilitating mRNA loading during stress recovery once starvation conditions ceased. Blocking or depleting the endogenous tRNAThr halves mitigates this stimulatory effect both in vivo and in vitro. T. brucei and its close relatives lack the well-described mammalian enzymes for tRNA half processing, thus hinting at a unique tdR biogenesis in these parasites.


Assuntos
Biossíntese de Proteínas/genética , RNA Mensageiro/genética , RNA de Transferência/genética , Ribossomos/genética , Trypanosoma brucei brucei/genética , Polirribossomos/genética , Polirribossomos/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Mensageiro/metabolismo , RNA de Protozoário/genética , RNA de Protozoário/metabolismo , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , RNA de Transferência/metabolismo , RNA de Transferência de Treonina/genética , RNA de Transferência de Treonina/metabolismo , Ribossomos/metabolismo , Estresse Fisiológico , Trypanosoma brucei brucei/metabolismo
10.
Nucleic Acids Res ; 47(4): 2056-2074, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30541130

RESUMO

The tissue specificity of mitochondrial tRNA mutations remains largely elusive. In this study, we demonstrated the deleterious effects of tRNAThr 15927G>A mutation that contributed to pathogenesis of coronary artery disease. The m.15927G>A mutation abolished the highly conserved base-pairing (28C-42G) of anticodon stem of tRNAThr. Using molecular dynamics simulations, we showed that the m.15927G>A mutation caused unstable tRNAThr structure, supported by decreased melting temperature and slower electrophoretic mobility of mutated tRNA. Using cybrids constructed by transferring mitochondria from a Chinese family carrying the m.15927G>A mutation and a control into mitochondrial DNA (mtDNA)-less human umbilical vein endothelial cells, we demonstrated that the m.15927G>A mutation caused significantly decreased efficiency in aminoacylation and steady-state levels of tRNAThr. The aberrant tRNAThr metabolism yielded variable decreases in mtDNA-encoded polypeptides, respiratory deficiency, diminished membrane potential and increased the production of reactive oxygen species. The m.15927G>A mutation promoted the apoptosis, evidenced by elevated release of cytochrome c into cytosol and increased levels of apoptosis-activated proteins: caspases 3, 7, 9 and PARP. Moreover, the lower wound healing cells and perturbed tube formation were observed in mutant cybrids, indicating altered angiogenesis. Our findings provide new insights into the pathophysiology of coronary artery disease, which is manifested by tRNAThr mutation-induced alterations.


Assuntos
Mitocôndrias/genética , Neovascularização Patológica/genética , RNA de Transferência de Treonina/química , RNA de Transferência/genética , Apoptose/genética , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/patologia , DNA Mitocondrial/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Mitocôndrias/patologia , Mutação , Conformação de Ácido Nucleico , RNA de Transferência de Treonina/genética , Espécies Reativas de Oxigênio/metabolismo
11.
Nucleic Acids Res ; 46(9): 4662-4676, 2018 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-29648639

RESUMO

Six pathogenic mutations have been reported in human mitochondrial tRNAThr (hmtRNAThr); however, the pathogenic molecular mechanism remains unclear. Previously, we established an activity assay system for human mitochondrial threonyl-tRNA synthetase (hmThrRS). In the present study, we surveyed the structural and enzymatic effects of pathogenic mutations in hmtRNAThr and then focused on m.15915 G > A (G30A) and m.15923A > G (A38G). The harmful evolutionary gain of non-Watson-Crick base pair A29/C41 caused hmtRNAThr to be highly susceptible to mutations disrupting the G30-C40 base pair in various ways; for example, structural integrity maintenance, modification and aminoacylation of tRNAThr, and editing mischarged tRNAThr. A similar phenomenon was observed for hmtRNATrp with an A29/C41 non-Watson-Crick base pair, but not in bovine mtRNAThr with a natural G29-C41 base pair. The A38G mutation caused a severe reduction in Thr-acceptance and editing of hmThrRS. Importantly, A38 is a nucleotide determinant for the t6A modification at A37, which is essential for the coding properties of hmtRNAThr. In summary, our results revealed the crucial role of the G30-C40 base pair in maintaining the proper structure and function of hmtRNAThr because of A29/C41 non-Watson-Crick base pair and explained the molecular outcome of pathogenic G30A and A38G mutations.


Assuntos
Mutação , RNA Mitocondrial/química , RNA de Transferência de Treonina/química , Anticódon , Pareamento de Bases , Humanos , Mitocôndrias/enzimologia , Edição de RNA , RNA Mitocondrial/genética , RNA Mitocondrial/metabolismo , RNA de Transferência de Treonina/genética , RNA de Transferência de Treonina/metabolismo , Treonina-tRNA Ligase/metabolismo , Aminoacilação de RNA de Transferência
12.
Mitochondrion ; 42: 84-91, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29225014

RESUMO

PURPOSE: The purpose of this study was to investigate the pathophysiology underlying Leber's hereditary optic neuropathy (LHON)-associated mitochondrial tRNA mutation. METHODS: Severn hundred ninety-seven Han Chinese subjects underwent clinical and genetic evaluation and analysis of mitochondrial DNA (mtDNA). The cybrid cell lines were constructed by transferring mitochondria from lymphoblastoid cell lines derived from a Chinese family into mtDNA-less (ρo) cells. These cell lines were assayed by tRNA Northern blot and Western blot analyses, respiratory enzymatic activities, the rate of ATP production and the generation of reactive oxygen species. RESULTS: The tRNAThr 15927G>A mutation was identified in eight probands with suggestively maternal inheritance among 352 Han Chinese probands lacking these known LHON-associated mtDNA mutations. The m.15927G>A mutation affected a highly conserved guanine at position 42 at the anticodon-stem of tRNAThr, destabilizing the conservative base pairing (28C-42G). We therefore hypothesized that the m.15927G>A mutation, and altered the structure and function of tRNAThr. Northern blot analysis revealed 60% decrease in the steady-state level of tRNAThr in the mutant cell lines. Western blot analysis showed the variable reductions of 4 mtDNA encoding proteins, especially for marked decrease of ND1 and CYTB observed in mutant cell lines. Furthermore, we demonstrated that the m.15927G>A mutation decreased the activities of mitochondrial complexes I and III, markedly diminished mitochondrial ATP levels, and increased the production of reactive oxygen species in the mutant cells. CONCLUSIONS: Our data demonstrated the first mitochondrial tRNA mutation leading to LHON. Our findings may provide new insights into the understanding of pathophysiology of LHON.


Assuntos
Mitocôndrias/genética , Mitocôndrias/metabolismo , Atrofia Óptica Hereditária de Leber/genética , Mutação Puntual , RNA de Transferência de Treonina/genética , Adolescente , Adulto , Idoso , Povo Asiático , Northern Blotting , Western Blotting , Criança , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Espécies Reativas de Oxigênio/metabolismo , Adulto Jovem
13.
Nature ; 542(7642): 494-497, 2017 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-28230119

RESUMO

Nucleic acids undergo naturally occurring chemical modifications. Over 100 different modifications have been described and every position in the purine and pyrimidine bases can be modified; often the sugar is also modified. Despite recent progress, the mechanism for the biosynthesis of most modifications is not fully understood, owing, in part, to the difficulty associated with reconstituting enzyme activity in vitro. Whereas some modifications can be efficiently formed with purified components, others may require more intricate pathways. A model for modification interdependence, in which one modification is a prerequisite for another, potentially explains a major hindrance in reconstituting enzymatic activity in vitro. This model was prompted by the earlier discovery of tRNA cytosine-to-uridine editing in eukaryotes, a reaction that has not been recapitulated in vitro and the mechanism of which remains unknown. Here we show that cytosine 32 in the anticodon loop of Trypanosoma brucei tRNAThr is methylated to 3-methylcytosine (m3C) as a pre-requisite for C-to-U deamination. Formation of m3C in vitro requires the presence of both the T. brucei m3C methyltransferase TRM140 and the deaminase ADAT2/3. Once formed, m3C is deaminated to 3-methyluridine (m3U) by the same set of enzymes. ADAT2/3 is a highly mutagenic enzyme, but we also show that when co-expressed with the methyltransferase its mutagenicity is kept in check. This helps to explain how T. brucei escapes 'wholesale deamination' of its genome while harbouring both enzymes in the nucleus. This observation has implications for the control of another mutagenic deaminase, human AID, and provides a rationale for its regulation.


Assuntos
Metiltransferases/metabolismo , Nucleosídeo Desaminases/metabolismo , Edição de RNA , RNA de Transferência de Treonina/química , RNA de Transferência de Treonina/metabolismo , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/genética , Anticódon/metabolismo , Sequência de Bases , Citosina/análogos & derivados , Citosina/metabolismo , Desaminação , Metilação , RNA de Transferência de Treonina/genética , Uridina/metabolismo
14.
RNA ; 23(3): 406-419, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28003514

RESUMO

The 3-methylcytidine (m3C) modification is ubiquitous in eukaryotic tRNA, widely found at C32 in the anticodon loop of tRNAThr, tRNASer, and some tRNAArg species, as well as in the variable loop (V-loop) of certain tRNASer species. In the yeast Saccharomyces cerevisiae, formation of m3C32 requires Trm140 for six tRNA substrates, including three tRNAThr species and three tRNASer species, whereas in Schizosaccharomyces pombe, two Trm140 homologs are used, one for tRNAThr and one for tRNASer The occurrence of a single Trm140 homolog is conserved broadly among Ascomycota, whereas multiple Trm140-related homologs are found in metazoans and other fungi. We investigate here how S. cerevisiae Trm140 protein recognizes its six tRNA substrates. We show that Trm140 has two modes of tRNA substrate recognition. Trm140 recognizes G35-U36-t6A37 of the anticodon loop of tRNAThr substrates, and this sequence is an identity element because it can be used to direct m3C modification of tRNAPhe However, Trm140 recognition of tRNASer substrates is different, since their anticodons do not share G35-U36 and do not have any nucleotides in common. Rather, specificity of Trm140 for tRNASer is achieved by seryl-tRNA synthetase and the distinctive tRNASer V-loop, as well as by t6A37 and i6A37 We provide evidence that all of these components are important in vivo and that seryl-tRNA synthetase greatly stimulates m3C modification of tRNASer(CGA) and tRNASer(UGA) in vitro. In addition, our results show that Trm140 binding is a significant driving force for tRNA modification and suggest separate contributions from each recognition element for the modification.


Assuntos
Anticódon/química , Citidina/análogos & derivados , Proteínas dos Microfilamentos/metabolismo , RNA de Transferência de Serina/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , tRNA Metiltransferases/metabolismo , Anticódon/metabolismo , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Citidina/genética , Citidina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Proteínas dos Microfilamentos/genética , Conformação de Ácido Nucleico , Ligação Proteica , Biossíntese de Proteínas , Domínios Proteicos , RNA de Transferência de Fenilalanina/química , RNA de Transferência de Fenilalanina/genética , RNA de Transferência de Fenilalanina/metabolismo , RNA de Transferência de Serina/genética , RNA de Transferência de Serina/metabolismo , RNA de Transferência de Treonina/química , RNA de Transferência de Treonina/genética , RNA de Transferência de Treonina/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por Substrato , tRNA Metiltransferases/genética
15.
Methods ; 113: 132-138, 2017 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-27847344

RESUMO

Several recent reports have found a connection between specific aminoacyl-tRNA synthetases and the regulation of angiogenesis. As this new area of research is explored, it is important to have reliable assays to assess the specific angiogenesis functions of these enzymes. This review provides information about specific in vitro and in vivo methods that were used to assess the angiogenic functions of threonyl-tRNA synthetase including endothelial cell migration and tube assays as well as chorioallantoic membrane and tumor vascularization assays. The theory and discussion include best methods of analysis and quantification along with the advantages and limitations of each type of assay.


Assuntos
Bioensaio , Membrana Corioalantoide/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Neovascularização Patológica/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Treonina-tRNA Ligase/antagonistas & inibidores , Aminoacilação de RNA de Transferência , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Movimento Celular/efeitos dos fármacos , Embrião de Galinha , Membrana Corioalantoide/irrigação sanguínea , Membrana Corioalantoide/enzimologia , Colágeno/química , Combinação de Medicamentos , Feminino , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Laminina/química , Camundongos , Neovascularização Patológica/enzimologia , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/genética , Neoplasias Ovarianas/irrigação sanguínea , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Proteoglicanas/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA de Transferência de Treonina/genética , RNA de Transferência de Treonina/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Treonina-tRNA Ligase/genética , Treonina-tRNA Ligase/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
16.
J Biol Chem ; 291(46): 24293-24303, 2016 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-27703015

RESUMO

Human NSun6 is an RNA methyltransferase that catalyzes the transfer of the methyl group from S-adenosyl-l-methionine (SAM) to C72 of tRNAThr and tRNACys In the current study, we used mass spectrometry to demonstrate that human NSun6 indeed introduces 5-methylcytosine (m5C) into tRNA, as expected. To further reveal the tRNA recognition mechanism of human NSun6, we measured the methylation activity of human NSun6 and its kinetic parameters for different tRNA substrates and their mutants. We showed that human NSun6 requires a well folded, full-length tRNA as its substrate. In the acceptor region, the CCA terminus, the target site C72, the discriminator base U73, and the second and third base pairs (2:71 and 3:70) of the acceptor stem are all important RNA recognition elements for human NSun6. In addition, two specific base pairs (11:24 and 12:23) in the D-stem of the tRNA substrate are involved in interacting with human NSun6. Together, our findings suggest that human NSun6 relies on a delicate network for RNA recognition, which involves both the primary sequence and tertiary structure of tRNA substrates.


Assuntos
Dobramento de Proteína , RNA de Transferência de Treonina/química , tRNA Metiltransferases/química , Humanos , Metilação , Domínios Proteicos , RNA de Transferência de Treonina/genética , RNA de Transferência de Treonina/metabolismo , tRNA Metiltransferases/genética , tRNA Metiltransferases/metabolismo
17.
PLoS One ; 11(2): e0148367, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26841043

RESUMO

Plasmids play critical roles in bacterial fitness and evolution of Pseudomonas aeruginosa. Here two plasmids found in a drug-resistant P. aeruginosa clinical isolate HS87 were completely sequenced. The pHS87b plasmid (11.2 kb) carries phage-related genes and function-unknown genes. Notably, pHS87b encodes an integrase and has an adjacent tRNAThr-associated attachment site. A corresponding integrated form of pHS87b at the tRNAThr locus was identified on the chromosome of P. aeruginosa, showing that pHS87b is able to site-specifically integrate into the 3'-end of the tRNAThr gene. The pHS87a plasmid (26.8 kb) displays a plastic structure containing a putative replication module, stability factors and a variable region. The RepA of pHS87a shows significant similarity to the replication proteins of pPT23A-family plasmids. pHS87a carries a transposon Tn6049, a truncated insertion sequence ΔIS1071 and a Tn402-like class 1 integron which contains an aacA4 cassette that may confer aminoglycoside resistance. Thus, pHS87b is a site-specific integrative plasmid whereas pHS87a is a plastic antibiotic resistance plasmid. The two native plasmids may promote the fitness and evolution of P. aeruginosa.


Assuntos
Aminoglicosídeos , Cromossomos Bacterianos/genética , Farmacorresistência Bacteriana/genética , Evolução Molecular , Plasmídeos/genética , Pseudomonas aeruginosa/genética , Elementos de DNA Transponíveis/genética , RNA de Transferência de Treonina/genética , Origem de Replicação/genética
18.
Nucleic Acids Res ; 44(3): 1342-53, 2016 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-26657638

RESUMO

Human polypyrimidine tract-binding protein PTB is a multifunctional RNA-binding protein with four RNA recognition motifs (RRM1 to RRM4). PTB is a nucleocytoplasmic shuttle protein that functions as a key regulator of alternative pre-mRNA splicing in the nucleoplasm and promotes internal ribosome entry site-mediated translation initiation of viral and cellular mRNAs in the cytoplasm. Here, we demonstrate that PTB and its paralogs, nPTB and ROD1, specifically interact with mitochondrial (mt) tRNA(Thr) both in human and mouse cells. In vivo and in vitro RNA-binding experiments demonstrate that PTB forms a direct interaction with the T-loop and the D-stem-loop of mt tRNA(Thr) using its N-terminal RRM1 and RRM2 motifs. RNA sequencing and cell fractionation experiments show that PTB associates with correctly processed and internally modified, mature mt tRNA(Thr) in the cytoplasm outside of mitochondria. Consistent with this, PTB activity is not required for mt tRNA(Thr) biogenesis or for correct mitochondrial protein synthesis. PTB association with mt tRNA(Thr) is largely increased upon induction of apoptosis, arguing for a potential role of the mt tRNA(Thr)/PTB complex in apoptosis. Our results lend strong support to the recently emerging conception that human mt tRNAs can participate in novel cytoplasmic processes independent from mitochondrial protein synthesis.


Assuntos
Citoplasma/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , RNA de Transferência de Treonina/metabolismo , Motivos de Aminoácidos/genética , Animais , Apoptose/genética , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Células HEK293 , Células HeLa , Humanos , Camundongos , Mitocôndrias/genética , Dados de Sequência Molecular , Células NIH 3T3 , Proteínas do Tecido Nervoso/genética , Conformação de Ácido Nucleico , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Ligação Proteica , Interferência de RNA , RNA de Transferência de Treonina/química , RNA de Transferência de Treonina/genética
19.
Nucleic Acids Res ; 44(3): 1428-39, 2016 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-26704982

RESUMO

In mitochondria of Saccharomyces cerevisiae, a single aminoacyl-tRNA synthetase (aaRS), MST1, aminoacylates two isoacceptor tRNAs, tRNA1(Thr) and tRNA2(Thr), that harbor anticodon loops of different size and sequence. As a result of this promiscuity, reassignment of the CUN codon box from leucine to threonine is facilitated. However, the mechanism by which a single aaRS binds distinct anticodon loops with high specificity is not well understood. Herein, we present the crystal structure of MST1 in complex with the canonical tRNA2(Thr) and non-hydrolyzable analog of threonyl adenylate. Our structure reveals that the dimeric arrangement of MST1 is essential for binding the 5'-phosphate, the second base pair of the acceptor stem, the first two base pairs of the anticodon stem and the first nucleotide of the variable arm. Further, in contrast to the bacterial ortholog that 'reads' the entire anticodon sequence, MST1 recognizes bases in the second and third position and the nucleotide upstream of the anticodon sequence. We speculate that a flexible loop linking strands ß4 and ß5 may be allosteric regulator that establishes cross-subunit communication between the aminoacylation and tRNA-binding sites. We also propose that structural features of the anticodon-binding domain in MST1 permit binding of the enlarged anticodon loop of tRNA1(Thr).


Assuntos
Proteínas de Escherichia coli/metabolismo , RNA de Transferência de Treonina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Treonina-tRNA Ligase/metabolismo , Anticódon/química , Anticódon/genética , Anticódon/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Cristalografia por Raios X , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Cinética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Terciária de Proteína , RNA Fúngico/química , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA de Transferência de Treonina/química , RNA de Transferência de Treonina/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Treonina-tRNA Ligase/química , Treonina-tRNA Ligase/genética
20.
Artigo em Inglês | MEDLINE | ID: mdl-25186221

RESUMO

Mutations in mitochondrial DNA (mtDNA) were the most important causes of Leber's hereditary optic neuropathy (LHON). To date, approximately 25 LHON-associated mtDNA mutations have been identified in various ethnic populations. Three primary mutations, the 3460G > A, 11778G > A and 14484T > C, in genes encoding the subunits of respiratory chain complex I, were the most common LHON-associated mtDNA mutations. Moreover, secondary mutations in mt-tRNA genes have been reported increasingly to be associated with LHON, simply due to the high mutation rates of mt-tRNAs. There is a lack of functional analysis and a poor genetic evaluation of a certain mt-tRNA mutation, which failed to meet the classic pathogenicity scoring system. As a result, how to classify a pathogenic mutation in mt-tRNA gene became important for both geneticist and clinician to diagnosis the LHON or the suspicious of LHON. In this study, we reassessed the role of a point mutation in mt-tRNA(Thr) gene which had been reported to be a mutation associated with LHON, the pathogenicity of this mutation has been discussed in this context.


Assuntos
DNA Mitocondrial/química , Mutação , Atrofia Óptica Hereditária de Leber/genética , RNA de Transferência de Treonina/química , Sequência de Bases , China , Sequência Conservada , Humanos , Conformação de Ácido Nucleico , RNA de Transferência de Treonina/genética , Análise de Sequência de DNA
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