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1.
Elife ; 102021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34382933

RESUMO

N1-methylation of G37 is required for a subset of tRNAs to maintain the translational reading-frame. While loss of m1G37 increases ribosomal +1 frameshifting, whether it incurs additional translational defects is unknown. Here, we address this question by applying ribosome profiling to gain a genome-wide view of the effects of m1G37 deficiency on protein synthesis. Using E coli as a model, we show that m1G37 deficiency induces ribosome stalling at codons that are normally translated by m1G37-containing tRNAs. Stalling occurs during decoding of affected codons at the ribosomal A site, indicating a distinct mechanism than that of +1 frameshifting, which occurs after the affected codons leave the A site. Enzyme- and cell-based assays show that m1G37 deficiency reduces tRNA aminoacylation and in some cases peptide-bond formation. We observe changes of gene expression in m1G37 deficiency similar to those in the stringent response that is typically induced by deficiency of amino acids. This work demonstrates a previously unrecognized function of m1G37 that emphasizes its role throughout the entire elongation cycle of protein synthesis, providing new insight into its essentiality for bacterial growth and survival.


Assuntos
Escherichia coli/genética , Mudança da Fase de Leitura do Gene Ribossômico , Expressão Gênica , Biossíntese de Proteínas/fisiologia , RNA de Transferência/genética , RNA de Transferência/metabolismo , Aminoácidos/genética , Aminoácidos/metabolismo , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Metilação , Biossíntese de Proteínas/genética , Especificidade por Substrato
2.
Nucleic Acids Res ; 49(17): 10046-10060, 2021 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-34417618

RESUMO

Inducing tRNA +1 frameshifting to read a quadruplet codon has the potential to incorporate a non-natural amino acid into the polypeptide chain. While this strategy is being considered for genome expansion in biotechnology and bioengineering endeavors, a major limitation is a lack of understanding of where the shift occurs in an elongation cycle of protein synthesis. Here, we use the high-efficiency +1-frameshifting SufB2 tRNA, containing an extra nucleotide in the anticodon loop, to address this question. Physical and kinetic measurements of the ribosome reading frame of SufB2 identify twice exploration of +1 frameshifting in one elongation cycle, with the major fraction making the shift during translocation from the aminoacyl-tRNA binding (A) site to the peptidyl-tRNA binding (P) site and the remaining fraction making the shift within the P site upon occupancy of the A site in the +1-frame. We demonstrate that the twice exploration of +1 frameshifting occurs during active protein synthesis and that each exploration is consistent with ribosomal conformational dynamics that permits changes of the reading frame. This work indicates that the ribosome itself is a determinant of changes of the reading frame and reveals a mechanistic parallel of +1 frameshifting with -1 frameshifting.

3.
Nat Commun ; 12(1): 4644, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34330903

RESUMO

Frameshifting of mRNA during translation provides a strategy to expand the coding repertoire of cells and viruses. How and where in the elongation cycle +1-frameshifting occurs remains poorly understood. We describe seven ~3.5-Å-resolution cryo-EM structures of 70S ribosome complexes, allowing visualization of elongation and translocation by the GTPase elongation factor G (EF-G). Four structures with a + 1-frameshifting-prone mRNA reveal that frameshifting takes place during translocation of tRNA and mRNA. Prior to EF-G binding, the pre-translocation complex features an in-frame tRNA-mRNA pairing in the A site. In the partially translocated structure with EF-G•GDPCP, the tRNA shifts to the +1-frame near the P site, rendering the freed mRNA base to bulge between the P and E sites and to stack on the 16S rRNA nucleotide G926. The ribosome remains frameshifted in the nearly post-translocation state. Our findings demonstrate that the ribosome and EF-G cooperate to induce +1 frameshifting during tRNA-mRNA translocation.


Assuntos
Mudança da Fase de Leitura do Gene Ribossômico/genética , Elongação Traducional da Cadeia Peptídica/genética , Fator G para Elongação de Peptídeos/genética , RNA Mensageiro/genética , RNA de Transferência/genética , Ribossomos/genética , Biocatálise , Microscopia Crioeletrônica , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Fator G para Elongação de Peptídeos/química , Fator G para Elongação de Peptídeos/metabolismo , Conformação Proteica , RNA Mensageiro/química , RNA Mensageiro/metabolismo , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Ribossomos/ultraestrutura , tRNA Metiltransferases/genética , tRNA Metiltransferases/metabolismo
4.
Stem Cell Res ; 53: 102332, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33857832

RESUMO

When studying patient specific induced pluripotent stem cells (iPS cells) as a disease model, the ideal control is an isogenic line that has corrected the point mutation, instead of iPS cells from siblings or other healthy subjects. However, repairing a point mutation in iPS cells even with the newly developed CRISPR-Cas9 technique remains difficult and time-consuming. Here we report a strategy that makes the Cas9 "knock-in" methodology both hassle-free and error-free. Instead of selecting a Cas9 recognition site close to the point mutation, we chose a site located in the nearest intron. We constructed a donor template with the fragment containing the corrected point mutation as one of the homologous recombination arms flanking a PGK-PuroR cassette. After selection with puromycin, positive clones were identified and further transfected with a CRE vector to remove the PGK-PuroR cassette. Using this methodology, we successfully repaired the point mutation G2019S of the LRRK2 gene in a Parkinson Disease (PD) patient iPS line and the point mutation R329H of the AARS1 gene in a Charcot-Marie-Tooth disease (CMT) patient iPS line. These isogenic iPS lines are ideal as a control in future studies.


Assuntos
Células-Tronco Pluripotentes Induzidas , Sistemas CRISPR-Cas/genética , Células Clonais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Recombinação Homóloga , Humanos , Mutação , Mutação Puntual
5.
Nat Commun ; 12(1): 328, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436566

RESUMO

While genome recoding using quadruplet codons to incorporate non-proteinogenic amino acids is attractive for biotechnology and bioengineering purposes, the mechanism through which such codons are translated is poorly understood. Here we investigate translation of quadruplet codons by a +1-frameshifting tRNA, SufB2, that contains an extra nucleotide in its anticodon loop. Natural post-transcriptional modification of SufB2 in cells prevents it from frameshifting using a quadruplet-pairing mechanism such that it preferentially employs a triplet-slippage mechanism. We show that SufB2 uses triplet anticodon-codon pairing in the 0-frame to initially decode the quadruplet codon, but subsequently shifts to the +1-frame during tRNA-mRNA translocation. SufB2 frameshifting involves perturbation of an essential ribosome conformational change that facilitates tRNA-mRNA movements at a late stage of the translocation reaction. Our results provide a molecular mechanism for SufB2-induced +1 frameshifting and suggest that engineering of a specific ribosome conformational change can improve the efficiency of genome recoding.


Assuntos
Mudança da Fase de Leitura do Gene Ribossômico/genética , Genoma Bacteriano , RNA de Transferência/genética , Salmonella typhimurium/genética , Aminoácidos/metabolismo , Aminoacilação , Anticódon/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Códon/genética , Escherichia coli/metabolismo , Transferência Ressonante de Energia de Fluorescência , Guanosina Trifosfato/metabolismo , Hidrólise , Metilação , Modelos Moleculares , Conformação de Ácido Nucleico , Motivos de Nucleotídeos/genética , RNA de Transferência/química , RNA de Transferência/metabolismo , Ribossomos/metabolismo
6.
Genes (Basel) ; 11(10)2020 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-33036365

RESUMO

Aminoacylation of tRNA generates an aminoacyl-tRNA (aa-tRNA) that is active for protein synthesis on the ribosome. Quantification of aminoacylation of tRNA is critical to understand the mechanism of specificity and the flux of the aa-tRNA into the protein synthesis machinery, which determines the rate of cell growth. Traditional assays for the quantification of tRNA aminoacylation involve radioactivity, either with a radioactive amino acid or with a [3'-32P]-labeled tRNA. We describe here a label-free assay that monitors aminoacylation by biotinylation-streptavidin (SA) conjugation to the α-amine or the α-imine of the aminoacyl group on the aa-tRNA. The conjugated aa-tRNA product is readily separated from the unreacted tRNA by a denaturing polyacrylamide gel, allowing for quantitative measurement of aminoacylation. This label-free assay is applicable to a wide range of amino acids and tRNA sequences and to both classes of aminoacylation. It is more sensitive and robust than the assay with a radioactive amino acid and has the potential to explore a wider range of tRNA than the assay with a [3'-32P]-labeled tRNA. This label-free assay reports kinetic parameters of aminoacylation quantitatively similar to those reported by using a radioactive amino acid, suggesting its broad applicability to research relevant to human health and disease.

7.
ACS Catal ; 10(15): 8058-8068, 2020 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-32904895

RESUMO

Mg2+ is required for the catalytic activity of TrmD, a bacteria-specific methyltransferase that is made up of a protein topological knot-fold, to synthesize methylated m1G37-tRNA to support life. However, neither the location of Mg2+ in the structure of TrmD nor its role in the catalytic mechanism is known. Using molecular dynamics (MD) simulations, we identify a plausible Mg2+ binding pocket within the active site of the enzyme, wherein the ion is coordinated by two aspartates and a glutamate. In this position, Mg2+ additionally interacts with the carboxylate of a methyl donor cofactor S-adenosylmethionine (SAM). The computational results are validated by experimental mutation studies, which demonstrate the importance of the Mg2+-binding residues for the catalytic activity. The presence of Mg2+ in the binding pocket induces SAM to adopt a unique bent shape required for the methyl transfer activity and causes a structural reorganization of the active site. Quantum mechanical calculations show that the methyl transfer is energetically feasible only when Mg2+ is bound in the position revealed by the MD simulations, demonstrating that its function is to align the active site residues within the topological knot-fold in a geometry optimal for catalysis. The obtained insights provide the opportunity for developing a strategy of antibacterial drug discovery based on targeting of Mg2+-binding to TrmD.

8.
Cell Death Dis ; 11(7): 563, 2020 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-32703935

RESUMO

17-beta-hydroxysteroid dehydrogenase 10 (HSD17B10) plays an important role in mitochondrial fatty acid metabolism and is also involved in mitochondrial tRNA maturation. HSD17B10 missense mutations cause HSD10 mitochondrial disease (HSD10MD). HSD17B10 with mutations identified from cases of HSD10MD show loss of function in dehydrogenase activity and mitochondrial tRNA maturation, resulting in mitochondrial dysfunction. It has also been implicated to play roles in the development of Alzheimer disease (AD) and tumorigenesis. Here, we found that HSD17B10 is a new substrate of NAD-dependent deacetylase Sirtuin 3 (SIRT3). HSD17B10 is acetylated at lysine residues K79, K99 and K105 by the acetyltransferase CBP, and the acetylation is reversed by SIRT3. HSD17B10 acetylation regulates its enzymatic activity and the formation of mitochondrial RNase P. Furthermore, HSD17B10 acetylation regulates the intracellular functions, affecting cell growth and cell resistance in response to stresses. Our results demonstrated that acetylation is an important regulation mechanism for HSD17B10 and may provide insight into interrupting the development of AD.


Assuntos
3-Hidroxiacil-CoA Desidrogenases/metabolismo , Estresse Oxidativo , Sirtuína 3/metabolismo , Estresse Fisiológico , 3-Hidroxiacil-CoA Desidrogenases/química , Acetilação , Processamento Alternativo/genética , Sequência de Aminoácidos , Proliferação de Células , Células HCT116 , Células HEK293 , Humanos , Mitocôndrias/metabolismo , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , RNA de Transferência/genética , Sialoglicoproteínas/metabolismo
9.
Ann Neurol ; 88(4): 830-842, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32715519

RESUMO

OBJECTIVE: The objective of this study was to identify the genetic cause for progressive peripheral nerve disease in a Venezuelan family. Despite the growing list of genes associated with Charcot-Marie-Tooth disease, many patients with axonal forms lack a genetic diagnosis. METHODS: A pedigree was constructed, based on family clinical data. Next-generation sequencing of mitochondrial DNA (mtDNA) was performed for 6 affected family members. Muscle biopsies from 4 family members were used for analysis of muscle histology and ultrastructure, mtDNA sequencing, and RNA quantification. Ultrastructural studies were performed on sensory nerve biopsies from 2 affected family members. RESULTS: Electrodiagnostic testing showed a motor and sensory axonal polyneuropathy. Pedigree analysis revealed inheritance only through the maternal line, consistent with mitochondrial transmission. Sequencing of mtDNA identified a mutation in the mitochondrial tRNAVal (mt-tRNAVal ) gene, m.1661A>G, present at nearly 100% heteroplasmy, which disrupts a Watson-Crick base pair in the T-stem-loop. Muscle biopsies showed chronic denervation/reinnervation changes, whereas biochemical analysis of electron transport chain (ETC) enzyme activities showed reduction in multiple ETC complexes. Northern blots from skeletal muscle total RNA showed severe reduction in abundance of mt-tRNAVal , and mildly increased mt-tRNAPhe , in subjects compared with unrelated age- and sex-matched controls. Nerve biopsies from 2 affected family members demonstrated ultrastructural mitochondrial abnormalities (hyperplasia, hypertrophy, and crystalline arrays) consistent with a mitochondrial neuropathy. CONCLUSION: We identify a previously unreported cause of Charcot-Marie-Tooth (CMT) disease, a mutation in the mt-tRNAVal , in a Venezuelan family. This work expands the list of CMT-associated genes from protein-coding genes to a mitochondrial tRNA gene. ANN NEUROL 2020;88:830-842.


Assuntos
Doença de Charcot-Marie-Tooth/genética , RNA Mitocondrial/genética , RNA de Transferência/genética , Adolescente , Adulto , Idoso de 80 Anos ou mais , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Linhagem , Venezuela , Adulto Jovem
10.
Wiley Interdiscip Rev RNA ; 11(6): e1609, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32533808

RESUMO

A major threat to public health is the resistance and persistence of Gram-negative bacteria to multiple drugs during antibiotic treatment. The resistance is due to the ability of these bacteria to block antibiotics from permeating into and accumulating inside the cell, while the persistence is due to the ability of these bacteria to enter into a nonreplicating state that shuts down major metabolic pathways but remains active in drug efflux. Resistance and persistence are permitted by the unique cell envelope structure of Gram-negative bacteria, which consists of both an outer and an inner membrane (OM and IM, respectively) that lay above and below the cell wall. Unexpectedly, recent work reveals that m1 G37 methylation of tRNA, at the N1 of guanosine at position 37 on the 3'-side of the tRNA anticodon, controls biosynthesis of both membranes and determines the integrity of cell envelope structure, thus providing a novel link to the development of bacterial resistance and persistence to antibiotics. The impact of m1 G37-tRNA methylation on Gram-negative bacteria can reach further, by determining the ability of these bacteria to exit from the persistence state when the antibiotic treatment is removed. These conceptual advances raise the possibility that successful targeting of m1 G37-tRNA methylation can provide new approaches for treating acute and chronic infections caused by Gram-negative bacteria. This article is categorized under: Translation > Translation Regulation RNA Processing > RNA Editing and Modification RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems.

11.
Cell Chem Biol ; 27(7): 839-849.e4, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32553119

RESUMO

Arginyltransferase ATE1 mediates posttranslational arginylation and plays key roles in multiple physiological processes. ATE1 utilizes arginyl (Arg)-tRNAArg as the donor of Arg, putting this reaction into a direct competition with the protein synthesis machinery. Here, we address the question of ATE1- Arg-tRNAArg specificity as a potential mechanism enabling this competition in vivo. Using in vitro arginylation assays and Ate1 knockout models, we find that, in addition to full-length tRNA, ATE1 is also able to utilize short tRNAArg fragments that bear structural resemblance to tRNA-derived fragments (tRF), a recently discovered class of small regulatory non-coding RNAs with global emerging biological role. Ate1 knockout cells show a decrease in tRFArg generation and a significant increase in the ratio of tRNAArg:tRFArg compared with wild type, suggesting a functional link between tRFArg and arginylation. We propose that generation of physiologically important tRFs can serve as a switch between translation and protein arginylation.

12.
J Clin Invest ; 129(12): 5568-5583, 2019 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-31557132

RESUMO

Gene therapy approaches are being deployed to treat recessive genetic disorders by restoring the expression of mutated genes. However, the feasibility of these approaches for dominantly inherited diseases - where treatment may require reduction in the expression of a toxic mutant protein resulting from a gain-of-function allele - is unclear. Here we show the efficacy of allele-specific RNAi as a potential therapy for Charcot-Marie-Tooth disease type 2D (CMT2D), caused by dominant mutations in glycyl-tRNA synthetase (GARS). A de novo mutation in GARS was identified in a patient with a severe peripheral neuropathy, and a mouse model precisely recreating the mutation was produced. These mice developed a neuropathy by 3-4 weeks of age, validating the pathogenicity of the mutation. RNAi sequences targeting mutant GARS mRNA, but not wild-type, were optimized and then packaged into AAV9 for in vivo delivery. This almost completely prevented the neuropathy in mice treated at birth. Delaying treatment until after disease onset showed modest benefit, though this effect decreased the longer treatment was delayed. These outcomes were reproduced in a second mouse model of CMT2D using a vector specifically targeting that allele. The effects were dose dependent, and persisted for at least 1 year. Our findings demonstrate the feasibility of AAV9-mediated allele-specific knockdown and provide proof of concept for gene therapy approaches for dominant neuromuscular diseases.


Assuntos
Doença de Charcot-Marie-Tooth/terapia , Terapia Genética , Glicina-tRNA Ligase/genética , Interferência de RNA , Alelos , Animais , Modelos Animais de Doenças , Células HEK293 , Humanos , Camundongos , Mutação
13.
Structure ; 27(8): 1190-1191, 2019 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-31390544

RESUMO

The origin of protein backbone threading through a topological knot remains elusive. To understand the evolutionary origin of protein knots, in this issue of StructureKo et al. (2019) used circular permutation to untie a knotted protein. They showed that a domain-swapped dimer releases the knot and the associated high-energy state for substrate binding.


Assuntos
Dobramento de Proteína , RNA , Metiltransferases , Proteínas
14.
Neurol Genet ; 5(2): e565, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31192300

RESUMO

Objective: To expand the clinical spectrum of lysyl-tRNA synthetase (KARS) gene-related diseases, which so far includes Charcot-Marie-Tooth disease, congenital visual impairment and microcephaly, and nonsyndromic hearing impairment. Methods: Whole-exome sequencing was performed on index patients from 4 unrelated families with leukoencephalopathy. Candidate pathogenic variants and their cosegregation were confirmed by Sanger sequencing. Effects of mutations on KARS protein function were examined by aminoacylation assays and yeast complementation assays. Results: Common clinical features of the patients in this study included impaired cognitive ability, seizure, hypotonia, ataxia, and abnormal brain imaging, suggesting that the CNS involvement is the main clinical presentation. Six previously unreported and 1 known KARS mutations were identified and cosegregated in these families. Two patients are compound heterozygous for missense mutations, 1 patient is homozygous for a missense mutation, and 1 patient harbored an insertion mutation and a missense mutation. Functional and structural analyses revealed that these mutations impair aminoacylation activity of lysyl-tRNA synthetase, indicating that defective KARS function is responsible for the phenotypes in these individuals. Conclusions: Our results demonstrate that patients with loss-of-function KARS mutations can manifest CNS disorders, thus broadening the phenotypic spectrum associated with KARS-related disease.

16.
Cell Syst ; 8(4): 302-314.e8, 2019 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-30981730

RESUMO

Gram-negative bacteria are intrinsically resistant to drugs because of their double-membrane envelope structure that acts as a permeability barrier and as an anchor for efflux pumps. Antibiotics are blocked and expelled from cells and cannot reach high-enough intracellular concentrations to exert a therapeutic effect. Efforts to target one membrane protein at a time have been ineffective. Here, we show that m1G37-tRNA methylation determines the synthesis of a multitude of membrane proteins via its control of translation at proline codons near the start of open reading frames. Decreases in m1G37 levels in Escherichia coli and Salmonella impair membrane structure and sensitize these bacteria to multiple classes of antibiotics, rendering them incapable of developing resistance or persistence. Codon engineering of membrane-associated genes reduces their translational dependence on m1G37 and confers resistance. These findings highlight the potential of tRNA methylation in codon-specific translation to control the development of multi-drug resistance in Gram-negative bacteria.


Assuntos
Farmacorresistência Bacteriana , Proteínas de Escherichia coli/genética , Processamento Pós-Transcricional do RNA , RNA de Transferência/metabolismo , tRNA Metiltransferases/genética , Antibacterianos/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Escherichia coli , Proteínas de Escherichia coli/metabolismo , Metilação , RNA de Transferência/genética , Salmonella , Transcriptoma , tRNA Metiltransferases/metabolismo
17.
Am J Hum Genet ; 104(3): 520-529, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30824121

RESUMO

Aminoacyl-tRNA synthetases (ARSs) are essential enzymes responsible for charging tRNA molecules with cognate amino acids. Consistent with the essential function and ubiquitous expression of ARSs, mutations in 32 of the 37 ARS-encoding loci cause severe, early-onset recessive phenotypes. Previous genetic and functional data suggest a loss-of-function mechanism; however, our understanding of the allelic and locus heterogeneity of ARS-related disease is incomplete. Cysteinyl-tRNA synthetase (CARS) encodes the enzyme that charges tRNACys with cysteine in the cytoplasm. To date, CARS variants have not been implicated in any human disease phenotype. Here, we report on four subjects from three families with complex syndromes that include microcephaly, developmental delay, and brittle hair and nails. Each affected person carries bi-allelic CARS variants: one individual is compound heterozygous for c.1138C>T (p.Gln380∗) and c.1022G>A (p.Arg341His), two related individuals are compound heterozygous for c.1076C>T (p.Ser359Leu) and c.1199T>A (p.Leu400Gln), and one individual is homozygous for c.2061dup (p.Ser688Glnfs∗2). Measurement of protein abundance, yeast complementation assays, and assessments of tRNA charging indicate that each CARS variant causes a loss-of-function effect. Compared to subjects with previously reported ARS-related diseases, individuals with bi-allelic CARS variants are unique in presenting with a brittle-hair-and-nail phenotype, which most likely reflects the high cysteine content in human keratins. In sum, our efforts implicate CARS variants in human inherited disease, expand the locus and clinical heterogeneity of ARS-related clinical phenotypes, and further support impaired tRNA charging as the primary mechanism of recessive ARS-related disease.


Assuntos
Aminoacil-tRNA Sintetases/genética , Doença de Charcot-Marie-Tooth/etiologia , Deficiências do Desenvolvimento/etiologia , Doenças do Cabelo/etiologia , Microcefalia/etiologia , Mutação , Doenças da Unha/etiologia , Adulto , Sequência de Aminoácidos , Doença de Charcot-Marie-Tooth/enzimologia , Doença de Charcot-Marie-Tooth/patologia , Deficiências do Desenvolvimento/enzimologia , Deficiências do Desenvolvimento/patologia , Feminino , Genes Recessivos , Predisposição Genética para Doença , Doenças do Cabelo/enzimologia , Doenças do Cabelo/patologia , Humanos , Masculino , Microcefalia/enzimologia , Microcefalia/patologia , Doenças da Unha/enzimologia , Doenças da Unha/patologia , Linhagem , Fenótipo , Prognóstico , Homologia de Sequência , Adulto Jovem
18.
Nature ; 564(7736): E37, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30459470

RESUMO

In Fig. 1b of this Article, a U was inadvertently inserted after G15 in the D loop. The original Article has not been corrected.

19.
RNA ; 24(12): 1878-1885, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30217865

RESUMO

Amino acids are attached to the tRNA 3'-end as a prerequisite for entering the ribosome for protein synthesis. Amino acid attachment also gives tRNA access to nonribosomal cellular activities. However, the normal attachment is via an ester linkage between the carboxylic group of the amino acid and the 3'-hydroxyl of the terminal A76 ribose in tRNA. The instability of this ester linkage has severely hampered studies of aminoacyl-tRNAs. Although the use of 3'-amino-3'-deoxy A76 in a 3'-amino-tailed tRNA provides stable aminoacyl attachment via an amide linkage, there are multiple tailing protocols and the efficiency of each relative to the others is unknown. Here we compare five different tailing protocols in parallel, all dependent on the CCA-adding enzyme [CTP(ATP): tRNA nucleotidyl transferase; abbreviated as the CCA enzyme] to exchange the natural ribose with the modified one. We show that the most efficient protocol is achieved by the CCA-catalyzed pyrophosphorolysis removal of the natural A76 in equilibrium with the addition of the appropriate ATP analog to synthesize the modified 3'-end. This protocol for 3'-amino-tailing affords quantitative and stable attachment of a broad range of amino acids to tRNA, indicating its general utility for studies of aminoacyl-tRNAs in both canonical and noncanonical activities.


Assuntos
Aminoácidos/genética , Aminoacil-tRNA Sintetases/genética , RNA de Transferência/genética , Trifosfato de Adenosina/química , Aminoácidos/química , Aminoacil-tRNA Sintetases/química , Sítios de Ligação/genética , Catálise , Escherichia coli/química , Escherichia coli/genética , Biossíntese de Proteínas/genética , RNA de Transferência/química , Aminoacil-RNA de Transferência/química , Aminoacil-RNA de Transferência/genética
20.
Hum Mol Genet ; 27(23): 4036-4050, 2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30124830

RESUMO

Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed enzymes implicated in several dominant and recessive disease phenotypes. The canonical function of ARSs is to couple an amino acid to a cognate transfer RNA (tRNA). We identified three novel disease-associated missense mutations in the alanyl-tRNA synthetase (AARS) gene in three families with dominant axonal Charcot-Marie-Tooth (CMT) disease. Two mutations (p.Arg326Trp and p.Glu337Lys) are located near a recurrent pathologic change in AARS, p.Arg329His. The third (p.Ser627Leu) is in the editing domain of the protein in which hitherto only mutations associated with recessive encephalopathies have been described. Yeast complementation assays demonstrated that two mutations (p.Ser627Leu and p.Arg326Trp) represent loss-of-function alleles, while the third (p.Glu337Lys) represents a hypermorphic allele. Further, aminoacylation assays confirmed that the third mutation (p.Glu337Lys) increases tRNA charging velocity. To test the effect of each mutation in the context of a vertebrate nervous system, we developed a zebrafish assay. Remarkably, all three mutations caused a pathological phenotype of neural abnormalities when expressed in zebrafish, while expression of the human wild-type messenger RNA (mRNA) did not. Our data indicate that not only functional null or hypomorphic alleles, but also hypermorphic AARS alleles can cause dominantly inherited axonal CMT disease.


Assuntos
Alanina-tRNA Ligase/genética , Aminoacil-tRNA Sintetases/genética , Doença de Charcot-Marie-Tooth/genética , RNA de Transferência/genética , Adulto , Alelos , Aminoácidos/genética , Animais , Doença de Charcot-Marie-Tooth/patologia , Feminino , Regulação Enzimológica da Expressão Gênica/genética , Heterogeneidade Genética , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/genética , Linhagem , Leveduras/genética , Peixe-Zebra/genética
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