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  1 / 466 MEDLINE  
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[PMID]:28854731
[Au] Autor:Credle JJ; Itoh CY; Yuan T; Sharma R; Scott ER; Workman RE; Fan Y; Housseau F; Llosa NJ; Bell WR; Miller H; Zhang SX; Timp W; Larman HB
[Ad] Endereço:Division of Immunology, Department of Pathology, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA.
[Ti] Título:Multiplexed analysis of fixed tissue RNA using Ligation in situ Hybridization.
[So] Source:Nucleic Acids Res;45(14):e128, 2017 Aug 21.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Clinical tissues are prepared for histological analysis and long-term storage via formalin fixation and paraffin embedding (FFPE). The FFPE process results in fragmentation and chemical modification of RNA, rendering it less suitable for analysis by techniques that rely on reverse transcription (RT) such as RT-qPCR and RNA-Seq. Here we describe a broadly applicable technique called 'Ligation in situ Hybridization' ('LISH'), which is an alternative methodology for the analysis of FFPE RNA. LISH utilizes the T4 RNA Ligase 2 to efficiently join adjacent chimeric RNA-DNA probe pairs hybridized in situ on fixed RNA target sequences. Subsequent treatment with RNase H releases RNA-templated ligation products into solution for downstream analysis. We demonstrate several unique advantages of LISH-based assays using patient-derived FFPE tissue. These include >100-plex capability, compatibility with common histochemical stains and suitability for analysis of decade-old materials and exceedingly small microdissected tissue fragments. High-throughput DNA sequencing modalities, including single molecule sequencing, can be used to analyze ligation products from complex panels of LISH probes ('LISH-seq'), which can be amplified efficiently and with negligible bias. LISH analysis of FFPE RNA is a novel methodology with broad applications that range from multiplexed gene expression analysis to the sensitive detection of infectious organisms.
[Mh] Termos MeSH primário: Hibridização In Situ/métodos
Inclusão em Parafina/métodos
RNA/genética
Fixação de Tecidos/métodos
[Mh] Termos MeSH secundário: Perfilação da Expressão Gênica/métodos
Sequenciamento de Nucleotídeos em Larga Escala/métodos
Seres Humanos
Microscopia de Fluorescência
RNA/análise
RNA/metabolismo
RNA Ligase (ATP)/metabolismo
Reação em Cadeia da Polimerase em Tempo Real
Reprodutibilidade dos Testes
Ribonuclease H/metabolismo
Proteínas Virais/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Viral Proteins); 63231-63-0 (RNA); EC 3.1.26.4 (Ribonuclease H); EC 6.5.1.3 (RNA Ligase (ATP)); EC 6.5.1.3 (bacteriophage T4 RNA ligase 2)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171023
[Lr] Data última revisão:
171023
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170901
[St] Status:MEDLINE
[do] DOI:10.1093/nar/gkx471


  2 / 466 MEDLINE  
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[PMID]:28707320
[Au] Autor:Nandy A; Saenz-Méndez P; Gorman AM; Samali A; Eriksson LA
[Ad] Endereço:Apoptosis Research Center, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland.
[Ti] Título:Homology model of the human tRNA splicing ligase RtcB.
[So] Source:Proteins;85(11):1983-1993, 2017 Nov.
[Is] ISSN:1097-0134
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:RtcB is an essential human tRNA ligase required for ligating the 2',3'-cyclic phosphate and 5'-hydroxyl termini of cleaved tRNA halves during tRNA splicing and XBP1 fragments during endoplasmic reticulum stress. Activation of XBP1 has been implicated in various human tumors including breast cancer. Here we present, for the first time, a homology model of human RtcB (hRtcB) in complex with manganese and covalently bound GMP built from the Pyrococcus horikoshii RtcB (bRtcB) crystal structure, PDB ID 4DWQA. The structure is analyzed in terms of stereochemical quality, folding reliability, secondary structure similarity with bRtcB, druggability of the active site binding pocket and its metal-binding microenvironment. In comparison with bRtcB, loss of a manganese-coordinating water and movement of Asn226 (Asn202 in 4DWQA) to form metal-ligand coordination, demonstrates the uniqueness of the hRtcB model. Rotation of GMP leads to the formation of an additional metal-ligand coordination (Mn-O). Umbrella sampling simulations of Mn binding in wild type and the catalytically inactive C122A mutant reveal a clear reduction of Mn binding ability in the mutant, thus explaining the loss of activity therein. Our results furthermore clearly show that the GTP binding site of the enzyme is a well-defined pocket that can be utilized as target site for in silico drug discovery.
[Mh] Termos MeSH primário: Simulação de Dinâmica Molecular
RNA Ligase (ATP)/química
Homologia de Sequência de Aminoácidos
[Mh] Termos MeSH secundário: Animais
Proteínas de Bactérias/química
Domínio Catalítico
Seres Humanos
Manganês/química
Manganês/metabolismo
RNA Ligase (ATP)/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Bacterial Proteins); 42Z2K6ZL8P (Manganese); EC 6.5.1.- (tRNA splicing ligase); EC 6.5.1.3 (RNA Ligase (ATP))
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171030
[Lr] Data última revisão:
171030
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170715
[St] Status:MEDLINE
[do] DOI:10.1002/prot.25352


  3 / 466 MEDLINE  
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[PMID]:28334744
[Au] Autor:Kiliszek A; Blaszczyk L; Kierzek R; Rypniewski W
[Ad] Endereço:Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland.
[Ti] Título:Stabilization of RNA hairpins using non-nucleotide linkers and circularization.
[So] Source:Nucleic Acids Res;45(10):e92, 2017 Jun 02.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:An RNA hairpin is an essential structural element of RNA. Hairpins play crucial roles in gene expression and intermolecular recognition but are also involved in the pathogenesis of some congenital diseases. Structural studies of the hairpin motifs are impeded by their thermodynamic instability, as they tend to unfold to form duplexes, especially at high concentrations required for crystallography or nuclear magnetic resonance spectroscopy. We have elaborated techniques to stabilize the RNA hairpins by linking the free ends of the RNA strand at the base of the hairpin stem. One method involves stilbene diether or hexaethylene glycol linkers and circularization by T4 RNA ligase. Another method uses click chemistry to stitch the RNA ends with a triazole linker. Both techniques are efficient and easy to perform. They should be useful in making stable, biologically relevant RNA constructs for structural studies.
[Mh] Termos MeSH primário: Etilenoglicóis/química
Sequências Repetidas Invertidas
RNA Ligase (ATP)/química
RNA/química
Triazóis/química
Proteínas Virais/química
[Mh] Termos MeSH secundário: Bacteriófago T4/química
Pareamento de Bases
Sequência de Bases
Química Click
Ciclização
Éteres/química
Conformação de Ácido Nucleico
RNA/genética
RNA Ligase (ATP)/genética
Estabilidade de RNA
Termodinâmica
Proteínas Virais/genética
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Ethers); 0 (Ethylene Glycols); 0 (Triazoles); 0 (Viral Proteins); 2615-15-8 (hexaethylene glycol); 63231-63-0 (RNA); EC 6.5.1.3 (RNA Ligase (ATP))
[Em] Mês de entrada:1709
[Cu] Atualização por classe:170912
[Lr] Data última revisão:
170912
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170324
[St] Status:MEDLINE
[do] DOI:10.1093/nar/gkx122


  4 / 466 MEDLINE  
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[PMID]:28130425
[Au] Autor:Morelli A; Cabezas Y; Mills LJ; Seelig B
[Ad] Endereço:Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.
[Ti] Título:Extensive libraries of gene truncation variants generated by in vitro transposition.
[So] Source:Nucleic Acids Res;45(10):e78, 2017 Jun 02.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:The detailed analysis of the impact of deletions on proteins or nucleic acids can reveal important functional regions and lead to variants with improved macromolecular properties. We present a method to generate large libraries of mutants with deletions of varying length that are randomly distributed throughout a given gene. This technique facilitates the identification of crucial sequence regions in nucleic acids or proteins. The approach utilizes in vitro transposition to generate 5΄ and 3΄ fragment sub-libraries of a given gene, which are then randomly recombined to yield a final library comprising both terminal and internal deletions. The method is easy to implement and can generate libraries in three to four days. We used this approach to produce a library of >9000 random deletion mutants of an artificial RNA ligase enzyme representing 32% of all possible deletions. The quality of the library was assessed by next-generation sequencing and detailed bioinformatics analysis. Finally, we subjected this library to in vitro selection and obtained fully functional variants with deletions of up to 18 amino acids of the parental enzyme that had been 95 amino acids in length.
[Mh] Termos MeSH primário: Sequência de Aminoácidos
DNA/genética
Biblioteca Gênica
Deleção de Sequência
Transposases/genética
[Mh] Termos MeSH secundário: Região 3'-Flanqueadora
Região 5'-Flanqueadora
Biologia Computacional
DNA/metabolismo
Primers do DNA/genética
Primers do DNA/metabolismo
Escherichia coli/genética
Escherichia coli/metabolismo
Diester Fosfórico Hidrolases/genética
Diester Fosfórico Hidrolases/metabolismo
Plasmídeos/química
Plasmídeos/metabolismo
Reação em Cadeia da Polimerase
RNA Ligase (ATP)/genética
RNA Ligase (ATP)/metabolismo
Proteínas Recombinantes/genética
Proteínas Recombinantes/metabolismo
Transposases/metabolismo
beta-Lactamases/genética
beta-Lactamases/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (DNA Primers); 0 (Recombinant Proteins); 9007-49-2 (DNA); EC 2.7.7.- (Transposases); EC 3.1.4.- (Phosphoric Diester Hydrolases); EC 3.1.4.46 (glycerophosphodiester phosphodiesterase); EC 3.5.2.6 (beta-Lactamases); EC 6.5.1.3 (RNA Ligase (ATP))
[Em] Mês de entrada:1709
[Cu] Atualização por classe:170912
[Lr] Data última revisão:
170912
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170129
[St] Status:MEDLINE
[do] DOI:10.1093/nar/gkx030


  5 / 466 MEDLINE  
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[PMID]:28126918
[Au] Autor:Schmier BJ; Chen X; Wolin S; Shuman S
[Ad] Endereço:Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.
[Ti] Título:Deletion of the rnl gene encoding a nick-sealing RNA ligase sensitizes Deinococcus radiodurans to ionizing radiation.
[So] Source:Nucleic Acids Res;45(7):3812-3821, 2017 Apr 20.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Deinococcus radiodurans RNA ligase (DraRnl) seals 3΄-OH/5΄-PO4 nicks in duplex nucleic acids in which the 3΄-OH nick terminus consists of two or more ribonucleotides. DraRnl exemplifies a widely distributed Rnl5 family of nick-sealing RNA ligases, the physiological functions of which are uncharted. Here we show via gene knockout that whereas DraRnl is inessential for growth of D. radiodurans, its absence sensitizes the bacterium to killing by ionizing radiation (IR). DraRnl protein is present in exponentially growing and stationary phase cells, but is depleted during the early stages of recovery from 10 kGy of IR and subsequently replenished during the late phase of post-IR genome reassembly. Absence of DraRnl elicts a delay in reconstitution of the 10 kGy IR-shattered D. radiodurans replicons that correlates with the timing of DraRnl replenishment in wild-type cells. Complementation with a catalytically dead mutant highlights that nick sealing activity is important for the radioprotective function of DraRnl. Our findings suggest a scenario in which DraRnl acts at genomic nicks resulting from gap-filling by a ribonucleotide-incorporating repair polymerase.
[Mh] Termos MeSH primário: Proteínas de Bactérias/metabolismo
Deinococcus/enzimologia
RNA Ligase (ATP)/metabolismo
[Mh] Termos MeSH secundário: Deinococcus/genética
Deinococcus/crescimento & desenvolvimento
Deinococcus/efeitos da radiação
Raios gama
Deleção de Genes
Genoma Bacteriano
Óperon
RNA Ribossômico/efeitos da radiação
Tolerância a Radiação
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Bacterial Proteins); 0 (RNA, Ribosomal); EC 6.5.1.3 (RNA Ligase (ATP))
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171005
[Lr] Data última revisão:
171005
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170128
[St] Status:MEDLINE
[do] DOI:10.1093/nar/gkx038


  6 / 466 MEDLINE  
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[PMID]:27715457
[Au] Autor:Zhang L; Tripathi A
[Ad] Endereço:a Center for Biomedical Engineering, School of Engineering, Brown University , Providence , RI , USA.
[Ti] Título:Archaeal RNA ligase from thermoccocus kodakarensis for template dependent ligation.
[So] Source:RNA Biol;14(1):36-44, 2017 Jan 02.
[Is] ISSN:1555-8584
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Nicking-sealing RNA ligases play a significant biological role in host defense and cellular repair, and have become an important molecular tool in biomedical engineering. Due to the propensity for RNA to form secondary structures, RNA modifying enzymes with elevated optimum temperatures are highly desired. Current characterized double stranded RNA ligases, such as the bacteriophage T4 RNA ligase 2, while possessing good template dependency, are not active at elevated temperatures. The few characterized RNA ligases from thermophiles exhibit high template independency. We synthesize and characterize here, KOD RNA ligase (KOD1Rnl), a thermostable and template dependent RNA ligase from the archaeon, Thermoccocus Kodakarensis. We disclose that a 13 time reduction in template independent ligation can be achieved with the addition of a single stranded DNase, such as RecJ. We also elucidate the effects of the presence of blood proteins on the activity of KOD1Rnl. Template dependent and thermostable RNA ligases, such as KOD RNA ligase, can be utilized in RNA detection, modification and sequencing.
[Mh] Termos MeSH primário: RNA Ligase (ATP)/metabolismo
Moldes Genéticos
Thermococcus/enzimologia
[Mh] Termos MeSH secundário: Sequência de Aminoácidos
Sequência de Bases
Ebolavirus/genética
Modelos Moleculares
Conformação Proteica
RNA Ligase (ATP)/química
RNA Viral/genética
Especificidade por Substrato
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (RNA, Viral); EC 6.5.1.3 (RNA Ligase (ATP))
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171019
[Lr] Data última revisão:
171019
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:161008
[St] Status:MEDLINE
[do] DOI:10.1080/15476286.2016.1239688


  7 / 466 MEDLINE  
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[PMID]:27492257
[Au] Autor:Remus BS; Schwer B; Shuman S
[Ad] Endereço:Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10065, USA.
[Ti] Título:Characterization of the tRNA ligases of pathogenic fungi Aspergillus fumigatus and Coccidioides immitis.
[So] Source:RNA;22(10):1500-9, 2016 Oct.
[Is] ISSN:1469-9001
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Yeast tRNA ligase (Trl1) is an essential trifunctional enzyme that repairs RNA breaks with 2',3'-cyclic-PO4 and 5'-OH ends. Trl1 is composed of C-terminal cyclic phosphodiesterase and central polynucleotide kinase domains that heal the broken ends to generate the 3'-OH, 2'-PO4, and 5'-PO4 termini required for sealing by an N-terminal ligase domain. Trl1 enzymes are found in all human fungal pathogens and they are promising targets for antifungal drug discovery because: (i) their domain structures and biochemical mechanisms are unique compared to the mammalian RtcB-type tRNA splicing enzyme; and (ii) there are no obvious homologs of the Trl1 ligase domain in mammalian proteomes. Here we characterize the tRNA ligases of two human fungal pathogens: Coccidioides immitis and Aspergillus fumigatus The biological activity of CimTrl1 and AfuTrl1 was verified by showing that their expression complements a Saccharomyces cerevisiae trl1Δ mutant. Purified recombinant AfuTrl1 and CimTrl1 proteins were catalytically active in joining 2',3'-cyclic-PO4 and 5'-OH ends in vitro, either as full-length proteins or as a mixture of separately produced healing and sealing domains. The biochemical properties of CimTrl1 and AfuTrl1 are similar to those of budding yeast Trl1, particularly with respect to their preferential use of GTP as the phosphate donor for the polynucleotide kinase reaction. Our findings provide genetic and biochemical tools to screen for inhibitors of tRNA ligases from pathogenic fungi.
[Mh] Termos MeSH primário: Aspergillus fumigatus/enzimologia
Coccidioides/enzimologia
Proteínas Fúngicas/metabolismo
RNA Ligase (ATP)/metabolismo
[Mh] Termos MeSH secundário: Aspergillus fumigatus/genética
Coccidioides/genética
Proteínas Fúngicas/genética
Guanosina Trifosfato/metabolismo
RNA Ligase (ATP)/genética
Processamento de RNA
RNA de Transferência/genética
RNA de Transferência/metabolismo
Saccharomyces cerevisiae/genética
Saccharomyces cerevisiae/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Fungal Proteins); 86-01-1 (Guanosine Triphosphate); 9014-25-9 (RNA, Transfer); EC 6.5.1.3 (RNA Ligase (ATP))
[Em] Mês de entrada:1707
[Cu] Atualização por classe:171001
[Lr] Data última revisão:
171001
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:160806
[St] Status:MEDLINE
[do] DOI:10.1261/rna.057455.116


  8 / 466 MEDLINE  
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[PMID]:26896806
[Au] Autor:Gu H; Yoshinari S; Ghosh R; Ignatochkina AV; Gollnick PD; Murakami KS; Ho CK
[Ad] Endereço:Department of Biological Sciences, State University of New York, Buffalo, NY 14260, USA.
[Ti] Título:Structural and mutational analysis of archaeal ATP-dependent RNA ligase identifies amino acids required for RNA binding and catalysis.
[So] Source:Nucleic Acids Res;44(5):2337-47, 2016 Mar 18.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:An ATP-dependent RNA ligase from Methanobacterium thermoautotrophicum (MthRnl) catalyzes intramolecular ligation of single-stranded RNA to form a closed circular RNA via covalent ligase-AMP and RNA-adenylylate intermediate. Here, we report the X-ray crystal structures of an MthRnl•ATP complex as well as the covalent MthRnl-AMP intermediate. We also performed structure-guided mutational analysis to survey the functions of 36 residues in three component steps of the ligation pathway including ligase-adenylylation (step 1), RNA adenylylation (step 2) and phosphodiester bond synthesis (step 3). Kinetic analysis underscored the importance of motif 1a loop structure in promoting phosphodiester bond synthesis. Alanine substitutions of Thr117 or Arg118 favor the reverse step 2 reaction to deadenylate the 5'-AMP from the RNA-adenylate, thereby inhibiting step 3 reaction. Tyr159, Phe281 and Glu285, which are conserved among archaeal ATP-dependent RNA ligases and are situated on the surface of the enzyme, are required for RNA binding. We propose an RNA binding interface of the MthRnl based on the mutational studies and two sulfate ions that co-crystallized at the active site cleft in the MthRnl-AMP complex.
[Mh] Termos MeSH primário: Proteínas Arqueais/química
Methanobacterium/enzimologia
RNA Ligase (ATP)/química
RNA Arqueal/química
RNA/química
[Mh] Termos MeSH secundário: Motivos de Aminoácidos
Substituição de Aminoácidos
Proteínas Arqueais/genética
Proteínas Arqueais/metabolismo
Biocatálise
Clonagem Molecular
Cristalografia por Raios X
Análise Mutacional de DNA
Escherichia coli/genética
Escherichia coli/metabolismo
Expressão Gênica
Cinética
Methanobacterium/química
Modelos Moleculares
Dados de Sequência Molecular
Mutação
Estrutura Secundária de Proteína
Estrutura Terciária de Proteína
RNA/metabolismo
RNA Ligase (ATP)/genética
RNA Ligase (ATP)/metabolismo
RNA Arqueal/metabolismo
Proteínas Recombinantes/química
Proteínas Recombinantes/genética
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, N.I.H., EXTRAMURAL; RESEARCH SUPPORT, U.S. GOV'T, NON-P.H.S.
[Nm] Nome de substância:
0 (Archaeal Proteins); 0 (RNA, Archaeal); 0 (RNA, circular); 0 (Recombinant Proteins); 63231-63-0 (RNA); EC 6.5.1.3 (RNA Ligase (ATP))
[Em] Mês de entrada:1608
[Cu] Atualização por classe:161019
[Lr] Data última revisão:
161019
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:160221
[St] Status:MEDLINE
[do] DOI:10.1093/nar/gkw094


  9 / 466 MEDLINE  
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[PMID]:26820526
[Au] Autor:Nagashima Y; Iwata Y; Mishiba K; Koizumi N
[Ad] Endereço:Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1, Gakuen-cho, 599-8531, Sakai, Japan.
[Ti] Título:Arabidopsis tRNA ligase completes the cytoplasmic splicing of bZIP60 mRNA in the unfolded protein response.
[So] Source:Biochem Biophys Res Commun;470(4):941-6, 2016 Feb 19.
[Is] ISSN:1090-2104
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Arabidopsis bZIP60 is a major transcription factor that activates the unfolded protein response and is regulated by cytoplasmic splicing. Two Arabidopsis inositol-requiring 1s (IRE1A and IRE1B) cleave bZIP60 mRNA; however, the ligase that connects the two half-molecules of the split bZIP60 mRNA has not yet been identified. We aimed to determine whether the Arabidopsis tRNA ligase RLG1 catalyzes the ligation of cleaved bZIP60 mRNA. Recombinant IRE1B containing the ribonuclease domain correctly cleaved synthetic RNA covering the cleaved site of bZIP60 in vitro. Recombinant RLG1 then ligated the two cleaved fragments. The cytoplasmic form of RLG1 was expressed in a T-DNA insertion mutant whose homozygote exhibited a lethal phenotype and when the transgene was substituted with endogenous RLG1, the plants grew normally. RLG1 proteins derived from transgene were mainly found in the cytoplasm; however, some were in the microsomal fraction, possibly on the ER membrane. This intracellular distribution of RLG1 is discussed.
[Mh] Termos MeSH primário: Proteínas de Arabidopsis/metabolismo
Arabidopsis/fisiologia
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo
Retículo Endoplasmático/metabolismo
RNA Ligase (ATP)/metabolismo
Processamento de RNA/fisiologia
Resposta a Proteínas não Dobradas/fisiologia
[Mh] Termos MeSH secundário: Proteínas de Arabidopsis/genética
Fatores de Transcrição de Zíper de Leucina Básica/genética
Citoplasma/metabolismo
Retículo Endoplasmático/genética
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Arabidopsis Proteins); 0 (Basic-Leucine Zipper Transcription Factors); 0 (bZIP60 protein, Arabidopsis); EC 6.5.1.3 (RNA Ligase (ATP))
[Em] Mês de entrada:1606
[Cu] Atualização por classe:160213
[Lr] Data última revisão:
160213
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:160129
[St] Status:MEDLINE


  10 / 466 MEDLINE  
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[PMID]:26812183
[Au] Autor:Naimuddin M; Kubo T
[Ad] Endereço:Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology , Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
[Ti] Título:A High Performance Platform Based on cDNA Display for Efficient Synthesis of Protein Fusions and Accelerated Directed Evolution.
[So] Source:ACS Comb Sci;18(2):117-29, 2016 Feb 08.
[Is] ISSN:2156-8944
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:We describe a high performance platform based on cDNA display technology by developing a new modified puromycin linker-oligonucleotide. The linker consists of four major characteristics: a "ligation site" for hybridization and ligation of mRNA by T4 RNA ligase, a "puromycin arm" for covalent linkage of the protein, a "polyadenosine site" for a longer puromycin arm and purification of protein fusions (optional) using oligo-dT matrices, and a "reverse transcription site" for the formation of stable cDNA protein fusions whose cDNA is covalently linked to its encoded protein. The linker was synthesized by a novel branching strategy and provided >8-fold higher yield than previous linkers. This linker enables rapid and highly efficient ligation of mRNA (>90%) and synthesis of protein fusions (∼ 50-95%) in various cell-free expression systems. Overall, this new cDNA display method provides 10-200 fold higher end-usage fusions than previous methods and benefits higher diversity libraries crucial for directed protein/peptide evolution. With the increased efficiency, this system was able to reduce the time for one selection cycle to <8 h and is potentially amenable to high-throughput systems. We demonstrate the efficiency of this system for higher throughput selections of various biomolecular interactions and achieved 30-40-fold enrichment per selection cycle. Furthermore, a 4-fold higher enrichment of Flag-tag was obtained from a doped mixture compared with that of the previous cDNA display method. A three-finger protein library was evolved to isolate superior nanomolar range binding candidates for vascular endothelial growth factor. This method is expected to provide a beneficial impact to accelerated drug discovery and proteome analysis.
[Mh] Termos MeSH primário: DNA Complementar/química
Evolução Molecular Direcionada/métodos
Ensaios de Triagem em Larga Escala/métodos
Proteínas Recombinantes/metabolismo
[Mh] Termos MeSH secundário: Oligonucleotídeos/química
Biblioteca de Peptídeos
Peptídeos
Conformação Proteica
Puromicina/química
RNA Ligase (ATP)
Proteínas Recombinantes/química
Proteínas Virais
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (DNA, Complementary); 0 (Oligonucleotides); 0 (Peptide Library); 0 (Peptides); 0 (Recombinant Proteins); 0 (Viral Proteins); 4A6ZS6Q2CL (Puromycin); EC 6.5.1.3 (RNA Ligase (ATP)); EC 6.5.1.3 (bacteriophage T4 RNA ligase 2)
[Em] Mês de entrada:1610
[Cu] Atualização por classe:161230
[Lr] Data última revisão:
161230
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:160127
[St] Status:MEDLINE
[do] DOI:10.1021/acscombsci.5b00139



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