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1.
PLoS Genet ; 17(7): e1009675, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34324497

RESUMO

Emerging evidence indicates that tRNA-derived small RNAs (tsRNAs) are involved in fine-tuning gene expression and become dysregulated in various cancers. We recently showed that the 22nt LeuCAG3´tsRNA from the 3´ end of tRNALeu is required for efficient translation of a ribosomal protein mRNA and ribosome biogenesis. Inactivation of this 3´tsRNA induced apoptosis in rapidly dividing cells and suppressed the growth of a patient-derived orthotopic hepatocellular carcinoma in mice. The mechanism involved in the generation of the 3´tsRNAs remains elusive and it is unclear if the 3´-ends of 3´tsRNAs are aminoacylated. Here we report an enzymatic method utilizing exonuclease T to determine the 3´charging status of tRNAs and tsRNAs. Our results showed that the LeuCAG3´tsRNA, and two other 3´tsRNAs are fully aminoacylated. When the leucyl-tRNA synthetase (LARS1) was inhibited, there was no change in the total tRNALeu concentration but a reduction in both the charged tRNALeu and LeuCAG3´tsRNA, suggesting the 3´tsRNAs are fully charged and originated solely from the charged mature tRNA. Altering LARS1 expression or the expression of various tRNALeu mutants were also shown to affect the generation of the LeuCAG3´tsRNA further suggesting they are created in a highly regulated process. The fact that the 3´tsRNAs are aminoacylated and their production is regulated provides additional insights into their importance in post-transcriptional gene regulation that includes coordinating the production of the protein synthetic machinery.


Assuntos
RNA de Transferência/biossíntese , RNA de Transferência/genética , Aminoacilação de RNA de Transferência/genética , Aminoácidos/genética , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Células HeLa , Humanos , Leucina/genética , Leucina/metabolismo , Processamento Pós-Transcricional do RNA , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , RNA de Transferência/metabolismo , Proteínas Ribossômicas , Aminoacilação de RNA de Transferência/fisiologia
2.
Nucleic Acids Res ; 49(8): 4689-4704, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33836087

RESUMO

Deficient maturations of mitochondrial transcripts are linked to clinical abnormalities but their pathophysiology remains elusive. Previous investigations showed that pathogenic variants in MTO1 for the biosynthesis of τm5U of tRNAGlu, tRNAGln, tRNALys, tRNATrp and tRNALeu(UUR) were associated with hypertrophic cardiomyopathy (HCM). Using mto1 knock-out(KO) zebrafish generated by CRISPR/Cas9 system, we demonstrated the pleiotropic effects of Mto1 deficiency on mitochondrial RNA maturations. The perturbed structure and stability of tRNAs caused by mto1 deletion were evidenced by conformation changes and sensitivity to S1-mediated digestion of tRNAGln, tRNALys, tRNATrp and tRNALeu(UUR). Notably, mto1KO zebrafish exhibited the global decreases in the aminoacylation of mitochondrial tRNAs with the taurine modification. Strikingly, ablated mto1 mediated the expression of MTPAP and caused the altered polyadenylation of cox1, cox3, and nd1 mRNAs. Immunoprecipitation assay indicated the interaction of MTO1 with MTPAP related to mRNA polyadenylation. These alterations impaired mitochondrial translation and reduced activities of oxidative phosphorylation complexes. These mitochondria dysfunctions caused heart development defects and hypertrophy of cardiomyocytes and myocardial fiber disarray in ventricles. These cardiac defects in the mto1KO zebrafish recapitulated the clinical phenotypes in HCM patients carrying the MTO1 mutation(s). Our findings highlighted the critical role of MTO1 in mitochondrial transcript maturation and their pathological consequences in hypertrophic cardiomyopathy.


Assuntos
Cardiomiopatia Hipertrófica/genética , Coração/embriologia , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , RNA Mitocondrial/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Cardiomiopatia Hipertrófica/fisiopatologia , Perfilação da Expressão Gênica , Coração/fisiopatologia , Hibridização In Situ , Microscopia Eletrônica de Transmissão , Mitocôndrias/enzimologia , Mitocôndrias/genética , Mitocôndrias/patologia , Proteínas Mitocondriais/genética , Mutação , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fosforilação Oxidativa , Poliadenilação/genética , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas de Ligação a RNA/genética , Aminoacilação de RNA de Transferência/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
3.
Biochemistry ; 60(6): 477-488, 2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33523633

RESUMO

Aminoacylated tRNAs are the substrates for ribosomal protein synthesis in all branches of life, implying an ancient origin for aminoacylation chemistry. In the 1970s, Orgel and colleagues reported potentially prebiotic routes to aminoacylated nucleotides and their RNA-templated condensation to form amino acid-bridged dinucleotides. However, it is unclear whether such reactions would have aided or impeded non-enzymatic RNA replication. Determining whether aminoacylated RNAs could have been advantageous in evolution prior to the emergence of protein synthesis remains a key challenge. We therefore tested the ability of aminoacylated RNA to participate in both templated primer extension and ligation reactions. We find that at low magnesium concentrations that favor fatty acid-based protocells, these reactions proceed orders of magnitude more rapidly than when initiated from the cis-diol of unmodified RNA. We further demonstrate that amino acid-bridged RNAs can act as templates in a subsequent round of copying. Our results suggest that aminoacylation facilitated non-enzymatic RNA replication, thus outlining a potentially primordial functional link between aminoacylation chemistry and RNA replication.


Assuntos
Aminoacilação de RNA de Transferência/fisiologia , Aminoacilação/fisiologia , Fosfatos de Dinucleosídeos/metabolismo , Conformação de Ácido Nucleico , Nucleotídeos/metabolismo , RNA/metabolismo , Moldes Genéticos , Aminoacilação de RNA de Transferência/genética
4.
Int J Mol Sci ; 22(2)2021 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-33419045

RESUMO

The global rise in type 2 diabetes results from a combination of genetic predisposition with environmental assaults that negatively affect insulin action in peripheral tissues and impair pancreatic ß-cell function and survival. Nongenetic heritability of metabolic traits may be an important contributor to the diabetes epidemic. Transfer RNAs (tRNAs) are noncoding RNA molecules that play a crucial role in protein synthesis. tRNAs also have noncanonical functions through which they control a variety of biological processes. Genetic and environmental effects on tRNAs have emerged as novel contributors to the pathogenesis of diabetes. Indeed, altered tRNA aminoacylation, modification, and fragmentation are associated with ß-cell failure, obesity, and insulin resistance. Moreover, diet-induced tRNA fragments have been linked with intergenerational inheritance of metabolic traits. Here, we provide a comprehensive review of how perturbations in tRNA biology play a role in the pathogenesis of monogenic and type 2 diabetes.


Assuntos
Diabetes Mellitus Tipo 2/genética , Predisposição Genética para Doença/genética , Biossíntese de Proteínas/genética , RNA de Transferência/genética , Aminoacilação de RNA de Transferência/genética , Animais , Diabetes Mellitus Tipo 2/metabolismo , Interação Gene-Ambiente , Humanos , Células Secretoras de Insulina/metabolismo , Processamento Pós-Transcricional do RNA/genética , RNA de Transferência/metabolismo
5.
J Biosci ; 452020.
Artigo em Inglês | MEDLINE | ID: mdl-32385222

RESUMO

Leucyl-tRNA synthetases (LRS) catalyze the linkage of leucine with tRNALeu. A large insertion CP1 domain (Connective Polypeptide 1) in LRS is responsible for post-transfer editing of mis-charged aminoacyl-tRNAs. Here, we characterized the CP1 domain of Leishmania donovani, a protozoan parasite, and its role in editing activity and interaction with broad spectrum anti-fungal, AN2690. The deletion mutant of LRS, devoid of CP1 domain (LRS-CP1Δ) was constructed, followed by determination of its role in editing and aminoacylation. Binding of AN2690 and different amino acids with CP1 deletion mutant and full length LRS was evaluated using isothermal titration calorimetry (ITC) and molecular dynamics simulations. The recombinant LRS-CP1Δ protein did not catalyze the aminoacylation and the editing reaction when compared to full-length LRS. Thus, indicating that CP1 domain was imperative for both aminoacylation and editing activities of LRS. Binding studies with different amino acids indicated selectivity of isoleucine by CP1 domain over other amino acids. These studies also indicated high affinity of AN2690 with the editing domain. Molecular docking studies indicated that AN2690-CP1 domain complex was stabilized by hydrogen bonding and hydrophobic interactions resulting in high binding affinity between the two. Our data suggests CP1 is crucial for the function of L.donovani LRS.


Assuntos
Antiprotozoários/farmacologia , Compostos de Boro/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Leishmania donovani/química , Leucina-tRNA Ligase/antagonistas & inibidores , Peptídeos/química , Processamento de Proteína Pós-Traducional , Proteínas de Protozoários/antagonistas & inibidores , Sequência de Aminoácidos , Antifúngicos/química , Antifúngicos/farmacologia , Antiprotozoários/química , Sítios de Ligação , Compostos de Boro/química , Compostos Bicíclicos Heterocíclicos com Pontes/química , Reposicionamento de Medicamentos , Expressão Gênica , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Leishmania donovani/enzimologia , Leishmania donovani/genética , Leucina-tRNA Ligase/química , Leucina-tRNA Ligase/genética , Leucina-tRNA Ligase/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA de Transferência de Leucina/química , RNA de Transferência de Leucina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Aminoacilação de RNA de Transferência/genética
6.
RNA ; 26(9): 1291-1298, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32439717

RESUMO

Queuosine (Q) is a conserved tRNA modification in bacteria and eukaryotes. Eukaryotic Q-tRNA modification occurs through replacing the guanine base with the scavenged metabolite queuine at the wobble position of tRNAs with G34U35N36 anticodon (Tyr, His, Asn, Asp) by the QTRT1/QTRT2 heterodimeric enzyme encoded in the genome. In humans, Q-modification in tRNATyr and tRNAAsp are further glycosylated with galactose and mannose, respectively. Although galactosyl-Q (galQ) and mannosyl-Q (manQ) can be measured by LC/MS approaches, the difficulty of detecting and quantifying these modifications with low sample inputs has hindered their biological investigations. Here we describe a simple acid denaturing gel and nonradioactive northern blot method to detect and quantify the fraction of galQ/manQ-modified tRNA using just microgram amounts of total RNA. Our method relies on the secondary amine group of galQ/manQ becoming positively charged to slow their migration in acid denaturing gels commonly used for tRNA charging studies. We apply this method to determine the Q and galQ/manQ modification kinetics in three human cells lines. For Q-modification, tRNAAsp is modified the fastest, followed by tRNAHis, tRNATyr, and tRNAAsn Compared to Q-modification, glycosylation occurs at a much slower rate for tRNAAsp, but at a similar rate for tRNATyr Our method enables easy access to study the function of these enigmatic tRNA modifications.


Assuntos
Géis/química , Nucleosídeo Q/química , RNA de Transferência/química , RNA de Transferência/genética , Anticódon/química , Anticódon/genética , Linhagem Celular Tumoral , Glicosilação , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Nucleosídeo Q/genética , Aminoacilação de RNA de Transferência/genética
7.
Biochim Biophys Acta Proteins Proteom ; 1868(8): 140438, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32330624

RESUMO

tRNA synthetases are responsible for decoding the molecular information, from codons to amino acids. Seryl-tRNA synthetase (SerRS), besides the five isoacceptors of tRNASer, recognizes tRNA[Ser]Sec for the incorporation of selenocysteine (Sec, U) into selenoproteins. The selenocysteine synthesis pathway is known and is dependent on several protein-protein and protein-RNA interactions. Those interactions are not fully described, in particular, involving tRNA[Ser]Sec and SerRS. Here we describe the molecular interactions between the Escherichia coli Seryl-tRNA synthetase (EcSerRS) and tRNA[Ser]Sec in order to determine their specificity, selectivity and binding order, leading to tRNA aminoacylation. The dissociation constant of EcSerRS and tRNA[Ser]Sec was determined as (126 ± 20) nM. We also demonstrate that EcSerRS binds initially to tRNA[Ser]Sec in the presence of ATP for further recognition by E. coli selenocysteine synthetase (EcSelA) for Ser to Sec conversion. The proposed studies clarify the mechanism of tRNA[Ser]Sec incorporation in Bacteria as well as of other domains of life.


Assuntos
Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , RNA de Transferência Aminoácido-Específico/metabolismo , RNA de Transferência de Cisteína/metabolismo , Serina-tRNA Ligase/metabolismo , Transferases/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Escherichia coli/genética , Cinética , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA de Transferência Aminoácido-Específico/genética , RNA de Transferência de Cisteína/genética , Serina-tRNA Ligase/genética , Termodinâmica , Aminoacilação de RNA de Transferência/genética , Transferases/genética
8.
RNA ; 26(8): 910-936, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32303649

RESUMO

The aminoacyl-tRNA synthetases are an essential and universally distributed family of enzymes that plays a critical role in protein synthesis, pairing tRNAs with their cognate amino acids for decoding mRNAs according to the genetic code. Synthetases help to ensure accurate translation of the genetic code by using both highly accurate cognate substrate recognition and stringent proofreading of noncognate products. While alterations in the quality control mechanisms of synthetases are generally detrimental to cellular viability, recent studies suggest that in some instances such changes facilitate adaption to stress conditions. Beyond their central role in translation, synthetases are also emerging as key players in an increasing number of other cellular processes, with far-reaching consequences in health and disease. The biochemical versatility of the synthetases has also proven pivotal in efforts to expand the genetic code, further emphasizing the wide-ranging roles of the aminoacyl-tRNA synthetase family in synthetic and natural biology.


Assuntos
Aminoacil-tRNA Sintetases/genética , RNA de Transferência/genética , Animais , Código Genético , Humanos , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , Aminoacilação de RNA de Transferência/genética
9.
Trends Genet ; 36(2): 105-117, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31839378

RESUMO

Aminoacyl-tRNA synthetases (ARS) are ubiquitously expressed, essential enzymes that charge tRNA with cognate amino acids. Variants in genes encoding ARS enzymes lead to myriad human inherited diseases. First, missense alleles cause dominant peripheral neuropathy. Second, missense, nonsense, and frameshift alleles cause recessive multisystem disorders that differentially affect tissues depending on which ARS is mutated. A preponderance of evidence has shown that both phenotypic classes are associated with loss-of-function alleles, suggesting that tRNA charging plays a central role in disease pathogenesis. However, it is currently unclear how perturbation in the function of these ubiquitously expressed enzymes leads to tissue-specific or tissue-predominant phenotypes. Here, we review our current understanding of ARS-associated disease phenotypes and discuss potential explanations for the observed tissue specificity.


Assuntos
Aminoacil-tRNA Sintetases/genética , Predisposição Genética para Doença , Doenças do Sistema Nervoso Periférico/genética , RNA de Transferência/genética , Alelos , Aminoácidos/genética , Humanos , Mutação/genética , Doenças do Sistema Nervoso Periférico/patologia , Fenótipo , Aminoacilação de RNA de Transferência/genética
10.
Elife ; 82019 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-30724162

RESUMO

In eukaryotes, ribosome profiling provides insight into the mechanism of protein synthesis at the codon level. In bacteria, however, the method has been more problematic and no consensus has emerged for how to best prepare profiling samples. Here, we identify the sources of these problems and describe new solutions for arresting translation and harvesting cells in order to overcome them. These improvements remove confounding artifacts and improve the resolution to allow analyses of ribosome behavior at the codon level. With a clearer view of the translational landscape in vivo, we observe that filtering cultures leads to translational pauses at serine and glycine codons through the reduction of tRNA aminoacylation levels. This observation illustrates how bacterial ribosome profiling studies can yield insight into the mechanism of protein synthesis at the codon level and how these mechanisms are regulated in response to changes in the physiology of the cell.


Assuntos
Escherichia coli/genética , Biossíntese de Proteínas/genética , Ribossomos/genética , Aminoacilação de RNA de Transferência/genética , Códon/genética , Glicina/genética , RNA Mensageiro/genética , Serina/genética
11.
Hum Genet ; 137(4): 293-303, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29691655

RESUMO

Progressive microcephaly and neurodegeneration are genetically heterogenous conditions, largely associated with genes that are essential for the survival of neurons. In this study, we interrogate the genetic etiology of two siblings from a non-consanguineous family with severe early onset of neurological manifestations. Whole exome sequencing identified novel compound heterozygous mutations in VARS that segregated with the proband: a missense (c.3192G>A; p.Met1064Ile) and a splice site mutation (c.1577-2A>G). The VARS gene encodes cytoplasmic valyl-tRNA synthetase (ValRS), an enzyme that is essential during eukaryotic translation. cDNA analysis on patient derived fibroblasts revealed that the splice site acceptor variant allele led to nonsense mediated decay, thus resulting in a null allele. Three-dimensional modeling of ValRS predicts that the missense mutation lies in a highly conserved region and could alter side chain packing, thus affecting tRNA binding or destabilizing the interface between the catalytic and tRNA binding domains. Further quantitation of the expression of VARS showed remarkably reduced levels of mRNA and protein in skin derived fibroblasts. Aminoacylation experiments on patient derived cells showed markedly reduced enzyme activity of ValRS suggesting the mutations to be loss of function. Bi-allelic mutations in cytoplasmic amino acyl tRNA synthetases are well-known for their role in neurodegenerative disorders, yet human disorders associated with VARS mutations have not yet been clinically well characterized. Our study describes the phenotype associated with recessive VARS mutations and further functional delineation of the pathogenicity of novel variants identified, which widens the clinical and genetic spectrum of patients with progressive microcephaly.


Assuntos
Atrofia/genética , Microcefalia/genética , Convulsões/genética , Valina-tRNA Ligase/genética , Alelos , Aminoacil-tRNA Sintetases/genética , Atrofia/fisiopatologia , Pré-Escolar , Regulação Enzimológica da Expressão Gênica , Humanos , Lactente , Mutação com Perda de Função/genética , Masculino , Microcefalia/fisiopatologia , Linhagem , RNA de Transferência/genética , Proteínas de Ligação a RNA/genética , Convulsões/fisiopatologia , Aminoacilação de RNA de Transferência/genética , Sequenciamento do Exoma
12.
Nucleic Acids Res ; 46(7): 3643-3656, 2018 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-29579307

RESUMO

TARS and TARS2 encode cytoplasmic and mitochondrial threonyl-tRNA synthetases (ThrRSs) in mammals, respectively. Interestingly, in higher eukaryotes, a third gene, TARSL2, encodes a ThrRS-like protein (ThrRS-L), which is highly homologous to cytoplasmic ThrRS but with a different N-terminal extension (N-extension). Whether ThrRS-L has canonical functions is unknown. In this work, we studied the organ expression pattern, cellular localization, canonical aminoacylation and editing activities of mouse ThrRS-L (mThrRS-L). Tarsl2 is ubiquitously but unevenly expressed in mouse tissues. Different from mouse cytoplasmic ThrRS (mThrRS), mThrRS-L is located in both the cytoplasm and nucleus; the nuclear distribution is mediated via a nuclear localization sequence at its C-terminus. Native mThrRS-L enriched from HEK293T cells was active in aminoacylation and editing. To investigate the in vitro catalytic properties of mThrRS-L accurately, we replaced the N-extension of mThrRS-L with that of mThrRS. The chimeric protein (mThrRS-L-NT) has amino acid activation, aminoacylation and editing activities. We compared the activities and cross-species tRNA recognition between mThrRS-L-NT and mThrRS. Despite having a similar aminoacylation activity, mThrRS-L-NT and mThrRS exhibit differences in tRNA recognition and editing capacity. Our results provided the first analysis of the aminoacylation and editing activities of ThrRS-L, and improved our understanding of Tarsl2.


Assuntos
RNA de Transferência/genética , Treonina-tRNA Ligase/genética , Sequência de Aminoácidos/genética , Aminoacilação/genética , Animais , Células HEK293 , Humanos , Camundongos , Especificidade da Espécie , Treonina/genética , Aminoacilação de RNA de Transferência/genética
13.
Nat Commun ; 9(1): 511, 2018 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-29410408

RESUMO

D-aminoacyl-tRNA deacylase (DTD), a bacterial/eukaryotic trans-editing factor, removes D-amino acids mischarged on tRNAs and achiral glycine mischarged on tRNAAla. An invariant cross-subunit Gly-cisPro motif forms the mechanistic basis of L-amino acid rejection from the catalytic site. Here, we present the identification of a DTD variant, named ATD (Animalia-specific tRNA deacylase), that harbors a Gly-transPro motif. The cis-to-trans switch causes a "gain of function" through L-chiral selectivity in ATD resulting in the clearing of L-alanine mischarged on tRNAThr(G4•U69) by eukaryotic AlaRS. The proofreading activity of ATD is conserved across diverse classes of phylum Chordata. Animalia genomes enriched in tRNAThr(G4•U69) genes are in strict association with the presence of ATD, underlining the mandatory requirement of a dedicated factor to proofread tRNA misaminoacylation. The study highlights the emergence of ATD during genome expansion as a key event associated with the evolution of Animalia.


Assuntos
Alanina/química , Aminoaciltransferases/química , Aminoacil-RNA de Transferência/química , Treonina/química , Aminoacilação de RNA de Transferência/genética , Alanina/genética , Alanina/metabolismo , Sequência de Aminoácidos , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismo , Animais , Apicomplexa/genética , Apicomplexa/metabolismo , Bactérias/genética , Bactérias/metabolismo , Sítios de Ligação , Evolução Biológica , Clonagem Molecular , Cristalografia por Raios X , Expressão Gênica , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Aminoacil-RNA de Transferência/genética , Aminoacil-RNA de Transferência/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Treonina/genética , Treonina/metabolismo
14.
RNA Biol ; 15(4-5): 667-677, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29345185

RESUMO

Inhibition of tRNA aminoacylation has proven to be an effective antimicrobial strategy, impeding an essential step of protein synthesis. Mupirocin, the well-known selective inhibitor of bacterial isoleucyl-tRNA synthetase, is one of three aminoacylation inhibitors now approved for human or animal use. However, design of novel aminoacylation inhibitors is complicated by the steadfast requirement to avoid off-target inhibition of protein synthesis in human cells. Here we review available data regarding known aminoacylation inhibitors as well as key amino-acid residues in aminoacyl-tRNA synthetases (aaRSs) and nucleotides in tRNA that determine the specificity and strength of the aaRS-tRNA interaction. Unlike most ligand-protein interactions, the aaRS-tRNA recognition interaction represents coevolution of both the tRNA and aaRS structures to conserve the specificity of aminoacylation. This property means that many determinants of tRNA recognition in pathogens have diverged from those of humans-a phenomenon that provides a valuable source of data for antimicrobial drug development.


Assuntos
Antibacterianos/farmacologia , Isoleucina-tRNA Ligase/genética , Inibidores da Síntese de Proteínas/farmacologia , RNA de Transferência de Leucina/genética , Aminoacilação de RNA de Transferência/efeitos dos fármacos , Antibacterianos/química , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Álcoois Graxos/química , Álcoois Graxos/farmacologia , Humanos , Isoleucina-tRNA Ligase/antagonistas & inibidores , Isoleucina-tRNA Ligase/metabolismo , Mupirocina/química , Mupirocina/farmacologia , Piperidinas/química , Piperidinas/farmacologia , Inibidores da Síntese de Proteínas/química , Quinazolinonas/química , Quinazolinonas/farmacologia , RNA de Transferência de Leucina/antagonistas & inibidores , RNA de Transferência de Leucina/metabolismo , Especificidade da Espécie , Relação Estrutura-Atividade , Thermus thermophilus/efeitos dos fármacos , Thermus thermophilus/enzimologia , Thermus thermophilus/genética , Aminoacilação de RNA de Transferência/genética
15.
Trends Genet ; 34(3): 218-231, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29352613

RESUMO

Errors during mRNA translation can lead to a reduction in the levels of functional proteins and an increase in deleterious molecules. Advances in next-generation sequencing have led to the discovery of rare genetic disorders, many caused by mutations in genes encoding the mRNA translation machinery, as well as to a better understanding of translational dynamics through ribosome profiling. We discuss here multiple neurological disorders that are linked to errors in tRNA aminoacylation and ribosome decoding. We draw on studies from genetic models, including yeast and mice, to enhance our understanding of the translational defects observed in these diseases. Finally, we emphasize the importance of tRNA, their associated enzymes, and the inextricable link between accuracy and efficiency in the maintenance of translational fidelity.


Assuntos
Mutação , Doenças do Sistema Nervoso/genética , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , Animais , Humanos , Modelos Genéticos , Saccharomyces cerevisiae/genética , Aminoacilação de RNA de Transferência/genética
16.
Nucleic Acids Res ; 46(2): 849-860, 2018 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-29228266

RESUMO

Accuracy of protein synthesis is enabled by the selection of amino acids for tRNA charging by aminoacyl-tRNA synthetases (ARSs), and further enhanced by the proofreading functions of some of these enzymes for eliminating tRNAs mischarged with noncognate amino acids. Mouse models of editing-defective cytoplasmic alanyl-tRNA synthetase (AlaRS) have previously demonstrated the importance of proofreading for cytoplasmic protein synthesis, with embryonic lethal and progressive neurodegeneration phenotypes. Mammalian mitochondria import their own set of nuclear-encoded ARSs for translating critical polypeptides of the oxidative phosphorylation system, but the importance of editing by the mitochondrial ARSs for mitochondrial proteostasis has not been known. We demonstrate here that the human mitochondrial AlaRS is capable of editing mischarged tRNAs in vitro, and that loss of the proofreading activity causes embryonic lethality in mice. These results indicate that tRNA proofreading is essential in mammalian mitochondria, and cannot be overcome by other quality control mechanisms.


Assuntos
Alanina-tRNA Ligase/genética , Mitocôndrias/genética , Edição de RNA , RNA de Transferência/genética , Aminoacilação de RNA de Transferência/genética , Alanina-tRNA Ligase/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Mamíferos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Mutação , Biossíntese de Proteínas/genética , RNA de Transferência/metabolismo , Homologia de Sequência de Aminoácidos
17.
RNA Biol ; 15(4-5): 659-666, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29168435

RESUMO

Aminoacyl-tRNA synthetases (aaRSs) catalyze the aminoacylation of tRNAs to produce the aminoacyl-tRNAs (aa-tRNAs) required by ribosomes for translation of the genetic message into proteins. To ensure the accuracy of tRNA aminoacylation, and consequently the fidelity of protein synthesis, some aaRSs exhibit a proofreading (editing) site, distinct from the aa-tRNA synthetic site. The aaRS editing site hydrolyzes misacylated products formed when a non-cognate amino acid is used during tRNA charging. Because aaRSs play a central role in protein biosynthesis and cellular life, these proteins represent longstanding targets for therapeutic drug development to combat infectious diseases. Most existing aaRS inhibitors target the synthetic site, and it is only recently that drugs targeting the proofreading site have been considered. In the present study, we developed a robust assay for the high-throughput screening of libraries of inhibitors targeting both the synthetic and the proofreading sites of up to four aaRSs simultaneously. Thus, this assay allows for screening of eight distinct enzyme active sites in a single experiment. aaRSs from several prominent human pathogens (i.e., Mycobacterium tuberculosis, Plasmodium falciparum, and Escherichia coli) were used for development of this assay.


Assuntos
Aminoacil-tRNA Sintetases/genética , Ensaios de Triagem em Larga Escala , Processamento Pós-Transcricional do RNA , RNA de Transferência/genética , Aminoacilação de RNA de Transferência/efeitos dos fármacos , Aminoacil-tRNA Sintetases/metabolismo , Clonagem Molecular , Descoberta de Drogas , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Mupirocina/farmacologia , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Plasmodium falciparum/enzimologia , Plasmodium falciparum/genética , Inibidores da Síntese de Proteínas/farmacologia , RNA de Transferência/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Aminoacilação de RNA de Transferência/genética
18.
Nucleic Acids Res ; 45(14): e133, 2017 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-28586482

RESUMO

Transfer RNA (tRNA) decodes mRNA codons when aminoacylated (charged) with an amino acid at its 3' end. Charged tRNAs turn over rapidly in cells, and variations in charged tRNA fractions are known to be a useful parameter in cellular responses to stress. tRNA charging fractions can be measured for individual tRNA species using acid denaturing gels, or comparatively at the genome level using microarrays. These hybridization-based approaches cannot be used for high resolution analysis of mammalian tRNAs due to their large sequence diversity. Here we develop a high-throughput sequencing method that enables accurate determination of charged tRNA fractions at single-base resolution (Charged DM-tRNA-seq). Our method takes advantage of the recently developed DM-tRNA-seq method, but includes additional chemical steps that specifically remove the 3'A residue in uncharged tRNA. Charging fraction is obtained by counting the fraction of A-ending reads versus A+C-ending reads for each tRNA species in the same sequencing reaction. In HEK293T cells, most cytosolic tRNAs are charged at >80% levels, whereas tRNASer and tRNAThr are charged at lower levels. These low charging levels were validated using acid denaturing gels. Our method should be widely applicable for investigations of tRNA charging as a parameter in biological regulation.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Aminoacil-RNA de Transferência/genética , RNA de Transferência/genética , Aminoacilação de RNA de Transferência/genética , Aminoacilação , Northern Blotting , Células HEK293 , Humanos , Modelos Genéticos , RNA de Transferência/metabolismo , Aminoacil-RNA de Transferência/metabolismo
19.
Adv Nutr ; 7(4): 773S-9S, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27422514

RESUMO

Translation of genetic information into functional proteins is critical for all cellular life. Accurate protein synthesis relies on proper aminoacylation of transfer RNAs (tRNAs) and decoding of mRNAs by the ribosome with the use of aminoacyl-tRNAs. Mistranslation can lead to pathologic consequences. All cells contain elaborate quality control mechanisms in translation, although translational fidelity may be regulated by various factors such as nutrient limitation or reactive oxygen species. Translation fidelity is maintained via the accuracy of tRNA aminoacylation by the aminoacyl-tRNA synthetases and matching of the mRNA codon with the tRNA anticodon by the ribosome. Stringent substrate discrimination and proofreading are critical in aminoacylating tRNAs with their cognate amino acid to maintain high accuracy of translation. Although the composition of the cellular proteome generally adheres to the genetic code, accumulating evidence indicates that cells can also deliberately mistranslate; they synthesize mutant proteins that deviate from the genetic code in response to stress or environmental changes. Mistranslation with tRNA charged with noncognate amino acids can expand the proteome to enhance stress response and help adaptation. Here, we review current knowledge on mistranslation through tRNA misacylation and describe advances in our understanding of translational control in the regulation of stress response and human diseases.


Assuntos
Adaptação Biológica/genética , Aminoácidos/metabolismo , Biossíntese de Proteínas/genética , Estresse Fisiológico/genética , Animais , Código Genético , Humanos , Mutação/genética , Proteínas , RNA de Transferência/genética , Aminoacilação de RNA de Transferência/genética
20.
Methods ; 106: 105-11, 2016 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-27163863

RESUMO

Detection of rare small RNA species whose sizes are overlapping with tRNAs often suffers from insufficient sensitivity due to the overwhelming abundance of tRNAs. We here report a method, named tRid (tRNA rid), for removing abundant tRNAs from small RNA fractions regardless of tRNA sequence species. By means of tRid, we are able to selectively enrich small RNAs which have been previously difficult to access due to mass existence of tRNAs in such fractions. A flexible tRNA-acylation ribozyme, known as flexizyme, is a key tool where the total tRNAs are aminoacylated with N-biotinylated phenylalanine regardless of tRNA sequences, and therefore the biotin-tagged tRNAs could be readily removed from the small RNA fractions by the use of streptavidin-immobilized magnetic beads. Next generation sequencing of the isolated small RNA fraction revealed that small RNAs with less than 200nt were effectively enriched, allowing us to identify previously unknown small RNAs in HeLa and E. coli.


Assuntos
RNA Catalítico/genética , Pequeno RNA não Traduzido/isolamento & purificação , RNA de Transferência/genética , Aminoacil-tRNA Sintetases , Sequenciamento de Nucleotídeos em Larga Escala , Conformação de Ácido Nucleico , RNA Catalítico/química , Pequeno RNA não Traduzido/genética , RNA de Transferência/química , Aminoacilação de RNA de Transferência/genética
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