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1.
Nat Commun ; 10(1): 2544, 2019 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-31186424

RESUMO

Cas13d, the type VI-D CRISPR-Cas effector, is an RNA-guided ribonuclease that has been repurposed to edit RNA in a programmable manner. Here we report the detailed structural and functional analysis of the uncultured Ruminococcus sp. Cas13d (UrCas13d)-crRNA complex. Two hydrated Mg2+ ions aid in stabilizing the conformation of the crRNA repeat region. Sequestration of divalent metal ions does not alter pre-crRNA processing, but abolishes target cleavage by UrCas13d. Notably, the pre-crRNA processing is executed by the HEPN-2 domain. Furthermore, both the structure and sequence of the nucleotides U(-8)-C(-1) within the repeat region are indispensable for target cleavage, and are specifically recognized by UrCas13d. Moreover, correct base pairings within two separate spacer regions (an internal and a 3'-end region) are essential for target cleavage. These findings provide a framework for the development of Cas13d into a tool for a wide range of applications.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Ribonucleases/metabolismo , Ruminococcus/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Conformação de Ácido Nucleico , Domínios Proteicos , Processamento Pós-Transcricional do RNA , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Guia/genética , Ribonucleases/química , Ribonucleases/genética , Ruminococcus/enzimologia
3.
Analyst ; 144(10): 3216-3220, 2019 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-30984925

RESUMO

A DSN-RNAse-TdT-T7 exo probing system allows the detection of miRNA 21 with very high sensitivity (LOD = 2.57 fM) and selectivity-the result of (i) avoiding the false-positive signal from miRNA reacting with TdT polymerase and (ii) signal amplification occurring through a FRET-breaking mechanism involving T7 exo.


Assuntos
DNA Nucleotidilexotransferase/química , Exodesoxirribonucleases/química , MicroRNAs/sangue , Ribonucleases/química , Bacteriófago T7/enzimologia , Sondas de DNA/síntese química , Sondas de DNA/genética , DNA de Cadeia Simples/síntese química , DNA de Cadeia Simples/genética , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/química , Humanos , Limite de Detecção , MicroRNAs/genética , Técnicas de Amplificação de Ácido Nucleico , Hibridização de Ácido Nucleico , RNA Mensageiro/genética
4.
Plant Mol Biol ; 100(4-5): 367-378, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30937702

RESUMO

KEY MESSAGE: S-RNase was demonstrated to be predominantly recognized by an S locus F-box-like protein and an S haplotype-specific F-box-like protein in compatible pollen tubes of sweet cherry. Self-incompatibility (SI) is a reproductive barrier that rejects self-pollen and inhibits self-fertilization to promote outcrossing. In Solanaceae and Rosaceae, S-RNase-based gametophytic SI (GSI) comprises S-RNase and F-box protein(s) as the pistil and pollen S determinants, respectively. Compatible pollen tubes are assumed to detoxify the internalized cytotoxic S-RNases to maintain growth. S-RNase detoxification is conducted by the Skp1-cullin1-F-box protein complex (SCF) formed by pollen S determinants, S locus F-box proteins (SLFs), in Solanaceae. In Prunus, the general inhibitor (GI), but not pollen S determinant S haplotype-specific F-box protein (SFB), is hypothesized to detoxify S-RNases. Recently, SLF-like proteins 1-3 (SLFL1-3) were suggested as GI candidates, although it is still possible that other proteins function predominantly in GI. To identify the other GI candidates, we isolated four other pollen-expressed SLFL and SFB-like (SFBL) proteins PavSLFL6, PavSLFL7A, PavSFBL1, and PavSFBL2 in sweet cherry. Binding assays with four PavS-RNases indicated that PavSFBL2 bound to PavS1, 6-RNase while the others bound to nothing. PavSFBL2 was confirmed to form an SCF complex in vitro. A co-immunoprecipitation assay using the recombinant PavS6-RNase as bait against pollen extracts and a mass spectrometry analysis identified the SCF complex components of PavSLFLs and PavSFBL2, M-locus-encoded glutathione S-transferase (MGST), DnaJ-like protein, and other minor proteins. These results suggest that SLFLs and SFBLs could act as predominant GIs in Prunus-specific S-RNase-based GSI.


Assuntos
Proteínas F-Box/fisiologia , Proteínas de Plantas/fisiologia , Prunus/metabolismo , Autoincompatibilidade em Angiospermas , Clonagem Molecular , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Espectrometria de Massas , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tubo Polínico/metabolismo , Tubo Polínico/fisiologia , Prunus/enzimologia , Reprodução , Ribonucleases/química , Ribonucleases/metabolismo
5.
Molecules ; 24(4)2019 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-30769777

RESUMO

Peptide nucleic acid (PNA)-neocuproine conjugates have been shown to efficiently catalyse the cleavage of RNA target sequences in the presence of Cu2+ ions in a site-specific manner. These artificial enzymes are designed to force the formation of a bulge in the RNA target, the sequence of which has been shown to be key to the catalytic activity. Here, we present a further investigation into the action of Cu2+-dependent PNAzymes with respect to the dependence on bulge composition in 3- and 4-nucleotide bulge systems. Cu2+-dependent PNAzymes were shown to have a clear preference for 4-nucleotide bulges, as the cleavage of 3-nucleotide bulge-forming RNA sequences was significantly slower, which is illustrated by a shift in the half-lives from approximately 30 min to 24 h. Nonetheless, the nucleotide preferences at different positions in the bulge displayed similar trends in both systems. Moreover, the cleavage site was probed by introducing critical chemical modifications to one of the cleavage site nucleotides of the fastest cleaved 4-nucleotide RNA bulge. Namely, the exclusion of the exocyclic amine of the central adenine and the replacement of the 2'-hydroxyl nucleophile with 2'-H or 2'-OMe substituents in the RNA severely diminished the rate of RNA cleavage by the Cu2+-dependent PNAzyme, giving insight into the mechanism of cleavage. Moreover, the shorter recognition arm of the RNA/PNAzyme complex was modified by extending the PNAzyme by two additional nucleobases. The new PNAzyme was able to efficiently promote the cleavage of RNA when fully hybridised to a longer RNA target and even outperform the previous fastest PNAzyme. The improvement was demonstrated in cleavage studies with stoichiometric amounts of either PNAzyme present, and the extended PNAzyme was also shown to give turnover with a 10-fold excess of the RNA target.


Assuntos
Enzimas de Restrição do DNA/química , Engenharia de Proteínas , RNA/química , Ribonucleases/química , Sequência de Bases , Catálise , Cobre/química , Enzimas de Restrição do DNA/genética , Cinética , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Nucleotídeos/química , Nucleotídeos/genética , RNA/genética , Ribonucleases/genética
6.
Biosens Bioelectron ; 130: 81-87, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30731349

RESUMO

A novel electrochemical biosensor was developed based on multiwall carbon nanotubes/graphene oxide nanoribbons (MWCNTs/GONRs) for sensitive analysis of microRNA-21. Signal-amplified strategy was achieved by duplex-specific nuclease assisted target recycling and alkaline phosphatase-induced redox reactions. At the fabrication process of the sensor, ssDNA capture probes were immobilized on the surface of the MWCNTs@GONRs/AuNPs modified electrode through the Au-S bond, and the streptavidin-conjugated alkaline phosphatase (SA-ALP) was attached to the end of the probe. In the absence of miRNA-21, SA-ALP catalysed the conversion of ascorbic acid 2-phosphate (AAP) into ascorbic acid (AA), triggered a redox reaction under iodine, producing a marked electrochemical response. When miRNA-21 was hybridized to the capture probe, the duplex would be cleaved by the duplex-specific nuclease (DSN), causing the electrochemical signals being significantly decreased as a result of SA-ALP detached from the electrode surface. Under the optimized conditions, our biosensor showed satisfactory sensitivity (detection limit, 0.034 fM), excellent selectivity and good accuracy (recoveries, 77.4-120.2%; RSD, 5.2-7.3%) after systematic evaluations. The proposed approach was applied to detect miRNA-21 from human serum samples, which indicated that it was reliable and could be widely used as an effective tool for rapid detection of the target in serums.


Assuntos
Técnicas Biossensoriais , Técnicas Eletroquímicas , MicroRNAs/isolamento & purificação , Ribonucleases/química , Ácido Ascórbico/química , DNA de Cadeia Simples/química , Grafite/química , Humanos , Iodo/química , Limite de Detecção , Nanopartículas Metálicas/química , MicroRNAs/química , Nanotubos de Carbono/química , Oxirredução
7.
Artigo em Inglês | MEDLINE | ID: mdl-30663497

RESUMO

Properties of cationic peptides bearing amino or guanidino groups with various side chain lengths that bind to double stranded RNAs (dsRNAs) were investigated. Peptides with shorter side chain lengths effectively bound to dsRNAs (12mers) increasing their thermal stability. NMR measurements suggested that the cationic peptide binds to the inner side of the major groove of dsRNA. These peptides also increased the thermal stability of siRNA and effectively protected from RNase A digestion. On the other hand, both peptides containing amino groups and guanidine groups did not disturb RNAi activity.


Assuntos
Peptídeos/química , Interferência de RNA , RNA Interferente Pequeno/química , Ribonucleases/química , Aminas/química , Apolipoproteínas B/genética , Apolipoproteínas B/metabolismo , Cátions , Linhagem Celular Tumoral , Guanidinas/química , Humanos , Peptídeos/metabolismo , Transição de Fase , Estabilidade de RNA , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/metabolismo , Ribonucleases/metabolismo , Termodinâmica
8.
Mutat Res ; 813: 1-12, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30544007

RESUMO

Missense mutations in certain genes of the Ribonuclease (RNASE) superfamily cause amyotrophic lateral sclerosis (ALS) through loss of either ribonucleolytic or nuclear translocation or both of these activities. While rare ANG/RNASE5 variants have been previously shown to be ALS causative, it is not yet known if any of the reported rare RNASE4 variants can also trigger ALS. The study aims to understand whether rare variants of RNASE4 can manifest ALS through similar loss-of-function mechanisms. Molecular dynamics (MD) simulations were performed on wild-type and all reported rare RNASE4 variants to study the structural and dynamic changes in the catalytic triad and nuclear localization signal residues responsible for ribonucleolytic and nuclear translocation activities respectively. Our systematic study comprising a total of 2.1 µs MD simulations reveal that three rare variants M29I, H72P and R95W would lose their ribonucleolytic activity as a result of conformational alteration of catalytic residue His116, and the R31T and R32W variants would lose their nuclear translocation ability due to local folding and reduced solvent accessibility of 30QRR32 residues. Our results show that five among the 20 known rare variants in RNASE4 (M29I, H72P, R95W, R31T and R32W) are possibly deleterious and may manifest ALS due to loss-of-function mechanisms. Overall, this is the first study to demonstrate that although rare and not yet clinically correlated, certain rare RNASE4 variants could cause ALS due to their loss-of-function characteristics and highlights the need to discover novel RNASE variants for a comprehensive understanding of structure-function-disease relationships and design effective therapeutics.


Assuntos
Esclerose Amiotrófica Lateral/genética , Predisposição Genética para Doença , Mutação de Sentido Incorreto , Ribonucleases/genética , Sequência de Aminoácidos , Esclerose Amiotrófica Lateral/enzimologia , Catálise , Núcleo Celular/metabolismo , Cristalografia por Raios X , Histidina/química , Humanos , Simulação de Dinâmica Molecular , Conformação Proteica , Transporte Proteico , Ribonucleases/química , Ribonucleases/metabolismo , Homologia de Sequência de Aminoácidos
9.
Biochim Biophys Acta Gen Subj ; 1863(1): 105-117, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30287244

RESUMO

BACKGROUND: Human RNase6 is a small cationic antimicrobial protein that belongs to the vertebrate RNaseA superfamily. All members share a common catalytic mechanism, which involves a conserved catalytic triad, constituted by two histidines and a lysine (His15/His122/Lys38 in RNase6 corresponding to His12/His119/Lys41 in RNaseA). Recently, our first crystal structure of human RNase6 identified an additional His pair (His36/His39) and suggested the presence of a secondary active site. METHODS: In this work we have explored RNase6 and RNaseA subsite architecture by X-ray crystallography, site-directed mutagenesis and kinetic characterization. RESULTS: The analysis of two novel crystal structures of RNase6 in complex with phosphate anions at atomic resolution locates a total of nine binding sites and reveals the contribution of Lys87 to phosphate-binding at the secondary active center. Contribution of the second catalytic triad residues to the enzyme activity is confirmed by mutagenesis. RNase6 catalytic site architecture has been compared with an RNaseA engineered variant where a phosphate-binding subsite is converted into a secondary catalytic center (RNaseA-K7H/R10H). CONCLUSIONS: We have identified the residues that participate in RNase6 second catalytic triad (His36/His39/Lys87) and secondary phosphate-binding sites. To note, residues His39 and Lys87 are unique within higher primates. The RNaseA/RNase6 side-by-side comparison correlates the presence of a dual active site in RNase6 with a favored endonuclease-type cleavage pattern. GENERAL SIGNIFICANCE: An RNase dual catalytic and extended binding site arrangement facilitates the cleavage of polymeric substrates. This is the first report of the presence of two catalytic centers in a single monomer within the RNaseA superfamily.


Assuntos
Endonucleases/química , Exonucleases/química , Fosfatos/química , Polímeros/química , Ribonucleases/química , Catálise , Domínio Catalítico , Cristalografia por Raios X , Histidina/química , Humanos , Cinética , Lisina/química , Mutagênese Sítio-Dirigida , Mutação , Estrutura Secundária de Proteína , Ribonuclease Pancreático/química
10.
J Biochem ; 164(5): 329-339, 2018 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-29905832

RESUMO

Colicins are protein toxins produced by and toxic to Escherichia coli strains. Colicin D consists of an N-terminal domain (NTD), central domain (CD) and C-terminal RNase domain (CRD). The cognate immunity protein, ImmD, is co-synthesized in producer cells to block the toxic tRNase activity of the CRD. Previous studies have reported the crystal structure of CRD/ImmD complex. Colicin D hijacks the surface receptor FepA and the energy transducer TonB system using the NTD for translocation across the outer membrane of the target cells. The CD is required for endoproteolytic processing and the translocation of CRD across the inner membrane, and the membrane-associated protease FtsH and the signal peptidase LepB are exploited in this process. Although several regions of the CD have been identified in interactions with the hijacked inner membrane system or immunity protein, the structural basis of the CD is unknown. In this study, we determined the crystal structure of colicin D, containing both the CD and CRD. The full-length colicin D/ImmD heterodimer structure was built by superimposing the CD-CRD structure with the previously determined partial structures. The overall translocation process of colicin D, including the interaction between CD and LepB, is discussed.


Assuntos
Proteínas de Escherichia coli/química , Ribonucleases/química , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Estrutura Terciária de Proteína , Transporte Proteico , Ribonucleases/metabolismo
11.
J Chromatogr A ; 1560: 91-96, 2018 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-29778448

RESUMO

The labeling by amino acids and peptides was investigated for sensitive and fast analyses of oligosaccharides and N-linked glycans by capillary electrophoresis-mass spectrometry (CE-MS). Peptide tags with a various number of histidine residues were tested for maltooligosaccharide labeling in order to investigate the effect of the size of labels and a number of charges on CE-MS analysis. Nevertheless, the reductive amination labeling of N-linked glycans by a hexahistidine tag resulted in a multiple products formation, therefore a peptide tag was modified by hydrazine functionality in order to perform labeling by hydrazone formation chemistry. This labeling approach significantly improved sensitivity with LOD of labeled maltopentaose determined to be 40 nmol/L and also significantly reduced separation time of neutral maltooligosaccharides and N-linked glycans released from bovine ribonuclease B. Furthermore, the labeling by this multi-cationic peptide hydrazine tag also allowed performing analysis of acidic glycans by CE-MS in a positive ion mode as demonstrated by separation of sialylated N-linked glycans released from bovine fetuin.


Assuntos
Eletroforese Capilar/métodos , Histidina/química , Espectrometria de Massas/métodos , Oligopeptídeos/química , Oligossacarídeos/análise , Polissacarídeos/análise , Ribonucleases/química , alfa-2-Glicoproteína-HS/química , Animais , Bovinos , Oligossacarídeos/química , Oligossacarídeos/isolamento & purificação , Polissacarídeos/química , Polissacarídeos/isolamento & purificação
12.
Plant Physiol Biochem ; 128: 66-71, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29758474

RESUMO

In this study, two pathogenesis-related (PR) class 10 protein isoforms, ASPR-1 and ASPR-2, were purified from fresh roots of the Chinese medicinal plant Angelica sinensis (A. sinensis) using 80% ammonium sulfate precipitation, Sephadex G50 gel filtration chromatography, and DEAE-Sepharose ion-exchange chromatography. The molecular masses of ASPR-1 and ASPR-2 were estimated to be 16.66 kDa and 16.46 kDa, respectively, using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The isoforms are both glycoproteins containing glycosyl contents of 1.8% (ASPR-1) and 3.4% (ASPR-2). The two isoforms were predominantly present as monomers, but they partially dimerized in solution. The 15 N-terminal amino acids of ASPR-1 were determined to be GIQKTEVEAPSTVSA, with significant sequence homology to certain PR-10 proteins. ASPR-2 was also identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) analysis to be a PR-10 protein. The isoforms both exhibited ribonuclease (RNase) activity, with ASPR-2 having higher specific activity (128.85 U mg-1) than ASPR-1 (68.67 U mg-1). The isoforms had the same optimal temperature of 50 °C but different optimal pH values of 5.0 (ASPR-1) and 6.0 (ASPR-2). The RNase activities of the isoforms were both stable for 30 min at 50 °C, rapidly decreasing at higher or lower processing temperatures. However, ASPR-1 retained higher residual activity (89.4%-80.9%) than ASPR-2 (74.3%-67.9%) at temperatures from 40 °C to 60 °C. These results provide additional information to enrich the current knowledge of poorly annotated A. sinensis proteins.


Assuntos
Angelica sinensis/química , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Raízes de Plantas/química , Ribonucleases/química , Ribonucleases/isolamento & purificação , Angelica sinensis/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/metabolismo , Ribonucleases/metabolismo
13.
Biochemistry ; 57(20): 2971-2983, 2018 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-29683663

RESUMO

Understanding how oxidatively damaged RNA interacts with ribonucleases is important because of its proposed role in the development and progression of disease. Thus, understanding structural aspects of RNA containing lesions generated under oxidative stress, as well as its interactions with other biopolymers, is fundamental. We explored the reactivity of RNase A, RNase T1, and RNase H toward oligonucleotides of RNA containing 8-oxo-7,8-dihydroguanosine (8oxoG). This is the first example that addresses this relationship and will be useful for understanding (1) how these RNases can be used to characterize the structural impact that this lesion has on RNA and (2) how oxidatively modified RNA may be handled intracellularly. 8-OxoG was incorporated into 10-16-mers of RNA, and its reactivity with each ribonuclease was assessed via electrophoretic analyses, circular dichroism, and the use of other C8-purine-modified analogues (8-bromoguanosine, 8-methoxyguanosine, and 8-oxoadenosine). RNase T1 does not recognize sites containing 8-oxoG, while RNase A recognizes and cleaves RNA at positions containing this lesion while differentiating if it is involved in H-bonding. The selectivity of RNase A followed the order C > 8-oxoG ≈ U. In addition, isothermal titration calorimetry showed that an 8-oxoG-C3'-methylphosphate derivative can inhibit RNase A activity. Cleavage patterns obtained from RNase H displayed changes in reactivity in a sequence- and concentration-dependent manner and displayed recognition at sites containing the modification in some cases. These data will aid in understanding how this modification affects reactivity with ribonucleases and will enable the characterization of global and local structural changes in oxidatively damaged RNA.


Assuntos
Oligonucleotídeos/genética , Ribonuclease H/genética , Ribonuclease T1/genética , Ribonuclease Pancreático/genética , Dicroísmo Circular , Guanosina/análogos & derivados , Guanosina/química , Guanosina/genética , Humanos , Oligonucleotídeos/química , Estresse Oxidativo/genética , RNA/química , RNA/genética , Ribonuclease H/química , Ribonuclease T1/química , Ribonuclease Pancreático/química , Ribonucleases/química , Ribonucleases/genética , Especificidade por Substrato
14.
Acta Biochim Biophys Sin (Shanghai) ; 50(4): 391-398, 2018 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-29566107

RESUMO

Utilizing cytotoxic proteins linked to tumor targeting molecules as anti-tumor drugs is a promising approach. However, most cytotoxins derived from bacteria or plants have inherent problems such as large molecular weights and they trigger a strong immune system reaction, which leads to drug failure and serious side effects. Amphinase (Amph) is a ribonuclease with a low molecular weight that is found in northern leopard frog oocytes. It has strong cytotoxicity against tumor cell lines in vitro and weak immunogenicity in vivo, and is a promising candidate in the development of targeted drugs. Transforming growth factor-α (TGF-α) that binds to the epidermal growth factor receptor (EGFR) is being used as a targeting molecule for the treatment of EGFR high-expressing tumors. In this study, we expressed and purified a recombinant amphinase and its TGF-α fusion protein (AGT) separately from Escherichia coli. AGT exhibited more significant cytotoxicity in vitro on EGFR high-expressing tumor cell lines, and stronger anti-tumor effects in vivo. This fusion protein also exhibited unusual thermostability, low in vivo immunogenicity, and side effects. Our results provide a new entry point for the development of novel, highly efficient anti-tumor targeting biological agents with low immunogenicity.


Assuntos
Antineoplásicos/química , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/química , Ribonucleases/química , Células A549 , Animais , Apoptose , Linhagem Celular Tumoral , Escherichia coli/metabolismo , Feminino , Células HL-60 , Humanos , Concentração Inibidora 50 , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Camundongos Nus , Oócitos/metabolismo , Plasmídeos/metabolismo , Ranidae , Proteínas Recombinantes de Fusão/química , Fator de Crescimento Transformador alfa/metabolismo
15.
RNA ; 24(5): 739-748, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29440319

RESUMO

Tudor staphylococcal nuclease (TSN) is an evolutionarily conserved ribonuclease in eukaryotes that is composed of five staphylococcal nuclease-like domains (SN1-SN5) and a Tudor domain. TSN degrades hyper-edited double-stranded RNA, including primary miRNA precursors containing multiple I•U and U•I pairs, and mature miRNA during miRNA decay. However, how TSN binds and degrades its RNA substrates remains unclear. Here, we show that the C. elegans TSN (cTSN) is a monomeric Ca2+-dependent ribonuclease, cleaving RNA chains at the 5'-side of the phosphodiester linkage to produce degraded fragments with 5'-hydroxyl and 3'-phosphate ends. cTSN degrades single-stranded RNA and double-stranded RNA containing mismatched base pairs, but is not restricted to those containing multiple I•U and U•I pairs. cTSN has at least two catalytic active sites located in the SN1 and SN3 domains, since mutations of the putative Ca2+-binding residues in these two domains strongly impaired its ribonuclease activity. We further show by small-angle X-ray scattering that rice osTSN has a flexible two-lobed structure with open to closed conformations, indicating that TSN may change its conformation upon RNA binding. We conclude that TSN is a structure-specific ribonuclease targeting not only single-stranded RNA, but also unstructured regions of double-stranded RNA. This study provides the molecular basis for how TSN cooperates with RNA editing to eliminate duplex RNA in cell defense, and how TSN selects and degrades RNA during microRNA decay.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , MicroRNAs/metabolismo , Estabilidade de RNA , Ribonucleases/metabolismo , Proteínas de Caenorhabditis elegans/química , Cálcio/metabolismo , Domínio Catalítico , Inosina/análise , RNA/metabolismo , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/metabolismo , Ribonucleases/química
16.
J Appl Microbiol ; 124(4): 1008-1016, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29464825

RESUMO

AIMS: Detection/Quantification of RNA viruses is mostly done by reverse-transcriptase (RT)-(q)PCR, but it does not distinguish between infectious and noninfectious viruses. Our aim was to test, how different pretreatments before RT-qPCR could eliminate positivity originated from external nucleic acids or genomes of damaged particles. METHODS AND RESULTS: Heat-inactivated (80°C for 10 min) rotavirus Wa strain and faecal samples containing rotavirus or norovirus were treated with PMA/PMAxx, benzonase or crude extract RNase prior to RT-qPCR. PMA/PMAxx pretreatments were not consistently efficient for RV, although they seemed to work to some extent for heat-inactivated norovirus. Benzonase and RNase provided consistently 2·2-2·8 log10 reductions in the titre of faecal rotavirus. CONCLUSIONS: All pretreatments need to be further validated for each virus separately, taking into account sample matrix and inactivation conditions. Although none of the pretreatments could completely render inactivated viruses undetectable, RNase worked most consistently for both rota- and norovirus. SIGNIFICANCE AND IMPACT OF THE STUDY: This study sheds light on capacity of the most common pre-RT-qPCR treatments to eliminate damaged, noninfectious rotaviruses and noroviruses after thermal treatment. To our knowledge, this is the first time, when benzonase has been used in this context.


Assuntos
Infecções por Caliciviridae/virologia , Norovirus/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Infecções por Rotavirus/virologia , Rotavirus/isolamento & purificação , Análise Discriminante , Endodesoxirribonucleases/química , Endorribonucleases/química , Temperatura Alta , Humanos , Norovirus/química , Norovirus/genética , Norovirus/fisiologia , Ribonucleases/química , Rotavirus/química , Rotavirus/genética , Rotavirus/fisiologia , Inativação de Vírus
17.
Artigo em Inglês | MEDLINE | ID: mdl-29314657

RESUMO

Correct balance between bacterial RNA degradation and synthesis is essential for controlling expression level of all RNAs. The RNA polymerase, which performs the RNA synthesis, is highly conserved across the bacterial domain. However, this is surprisingly not the case for the RNA degradation machinery, which is composed of different subunits and performs different enzymatic reactions, depending on the organism. In Escherichia coli, the RNA decay is performed by the degradosome complex, which forms around the membrane-associated endoribonuclease RNase E, and is stable enough to be purified without falling apart. In contrast, many Firmicutes, for example, Bacillus subtilis, Staphylococcus aureus, and Streptococcus pneumoniae, do not encode an RNase E homolog, but instead have the endoribonuclease RNase Y and the exo- and endo-ribonuclease RNase J complex. A wide range of experiments have been performed, mainly with B. subtilis and S. aureus, to determine which interactions exist between the various RNA decay enzymes in the Firmicutes, with the goal of understanding how RNA degradation (and thus gene expression homeostasis and regulation) is organized in these organisms. The in vivo and in vitro data is diverse, and does not always concur. This overview gathers the data on interactions between Firmicute RNA degradation factors, to highlight the similarities and differences between experimental data from different experiments and from different organisms. WIREs RNA 2018, 9:e1460. doi: 10.1002/wrna.1460 This article is categorized under: RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms RNA Turnover and Surveillance > Regulation of RNA Stability.


Assuntos
Firmicutes/genética , Estabilidade de RNA/genética , Firmicutes/metabolismo , Ribonucleases/química , Ribonucleases/genética , Ribonucleases/metabolismo , Ribossomos
18.
Tree Physiol ; 38(3): 485-501, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29329457

RESUMO

Mountain pine beetle (Dendroctonus ponderosae Hopkins; MPB) is an economically and ecologically important pest of pine species in western North America. Mountain pine beetles form complex multipartite relationships with microbial partners, including the ophiostomoid fungi Grosmannia clavigera (Robinson-Jeffrey and Davidson) Zipfel, de Beer and Wingfield, Ophiostoma montium (Rumbold) von Arx, Grosmannia aurea (Robinson-Jeffrey and Davidson) Zipfel, de Beer and Wingfield, Leptographium longiclavatum (Lee, Kim, and Breuil) and Leptographium terebrantis (Barras and Perry). These fungi are vectored by MPB to new pine hosts, where the fungi overcome host defenses to grow into the sapwood. A tree's relative susceptibility to these fungi is conventionally assessed by measuring lesions that develop in response to fungal inoculation. However, these lesions represent a symptom of infection, representing both fungal growth and tree defense capacity. In order to more objectively assess fungal virulence and host tree susceptibility in studies of host-pathogen interactions, a reliable, consistent, sensitive method is required to accurately identify and quantify MPB-associated fungal symbionts in planta. We have adapted RNase H2-dependent PCR, a technique originally designed for rare allele discrimination, to develop a novel RNase H2-dependent quantitative PCR (rh-qPCR) assay that shows greater specificity and sensitivity than previously published PCR-based methods to quantify MPB fungal symbionts in pine xylem and MPB whole beetles. Two sets of assay probes were designed: one that amplifies a broad range of ophiostomoid species, and a second that amplifies G. clavigera but not other MPB-associated ophiostomoid species. Using these primers to quantify G. clavigera in pine stems, we provide evidence that lesion length does not accurately reflect the extent of fungal colonization along the stem nor the quantity of fungal growth within this colonized portion of stem. The sensitivity, specificity, reproducibility, cost effectiveness and high-throughput potential of the rh-qPCR assay makes the technology suitable for identification and quantification of a wide array of pathogenic and beneficial microbes that form associations with plants and other organisms, even when the microbial partner is present in low abundance.


Assuntos
Micologia/métodos , Ophiostomatales/isolamento & purificação , Pinus/microbiologia , Doenças das Plantas/microbiologia , Reação em Cadeia da Polimerase em Tempo Real , Gorgulhos/microbiologia , Animais , Ophiostomatales/fisiologia , Ribonucleases/química , Simbiose
19.
J Pharm Biomed Anal ; 149: 365-373, 2018 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-29145098

RESUMO

The lack of a highly sensitive and simple method for the quantitative analysis of glycan has impeded the exploration of protein glycosylation patterns (glycomics), evaluation of antibody drug stability, and screening of disease glycan biomarkers. In this study, we describe a novel and simplified quantitative glycomics strategy. Quantitation by mutant enzyme reaction stable isotope labeling (QMERSIL) to label the N-glycans with either a nondeuterated (d0-) or deuterated (d8-) 4-(2,4-Dinitro-5-piperazin-1-yl-phenyl)-1,1-dimethyl-piperazin-1-ium (MPDPZ)-Boc-asparaginyl-N-acetyl-d-glucosamine (Boc-Asn-GlcNAc) acceptor of a positive charge structure through the glycosynthase (Endo-M-N175Q) transglycosylation reaction with mass spectrometry facilitates comparative glycomics. The sialylglycopeptide (SGP) of the complex type was used to demonstrate that QMERSIL facilitates the relative quantitation over a linear dynamic range (up to d0/d8=0.02:20) of 3 orders of magnitude. The area ratios of the N-glycan peaks from the QMERSIL method showed a good linearity (d0/d8, R2=0.9999; d8/d0, R2=0.9978). The reproducibility and accuracy assay precisions were all less than 6.12%, and the mean recoveries (%) of SGP spiked in the human plasma were 97.34%. Moreover, the QMERSIL using LC-MS/MS was evaluated with various molar ratios (1:1, 1:5, 5:1) of d0(d8)- MPDPZ-Boc-Asn-GlcNAc-labeled glycans from ribonuclease B, bovine fetuin, and ovalbumin. The ratios of the relative intensity between the isotopically MPDPZ-Boc-Asn-GlcNAc labeled N-glycans were almost equal a close to the theoretical values (1:1, 1:5, 5:1). Finally, this method was used for the relative quantitative comparison of the N-Linked oligosaccharides in human plasma.


Assuntos
Glicômica/métodos , Marcação por Isótopo/métodos , Polissacarídeos/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Adulto , Calibragem , Cromatografia Líquida de Alta Pressão/métodos , Deutério/química , Fetuínas/química , Fetuínas/metabolismo , Glicopeptídeos/química , Glicopeptídeos/metabolismo , Glicosídeo Hidrolases/metabolismo , Glicosilação , Voluntários Saudáveis , Humanos , Masculino , Ovalbumina/química , Ovalbumina/metabolismo , Polissacarídeos/química , Reprodutibilidade dos Testes , Ribonucleases/química , Ribonucleases/metabolismo , Espectrometria de Massas em Tandem/métodos
20.
Plant Cell Physiol ; 59(2): 234-247, 2018 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-29149301

RESUMO

Petunia inflata possesses a self-incompatibility (SI) mechanism, which involves S-RNase and multiple S-locus F-box (SLF) genes at the polymorphic S-locus. For a given S-haplotype, each SLF is thought to interact with some of its non-self S-RNases, but not with its self S-RNase. In this work, we studied an allelic pair of SLF1, S2-SLF1 and S3-SLF1, which differ in 44 amino acids and show differential interactions with S3-RNase. We first used an in vivo transgenic assay to determine whether four chimeric proteins of S2-SLF1 and S3-SLF1, each with one of the three functional domains swapped, interact with S3-RNase. The results narrowed the candidate amino acids for specific interaction of S2-SLF1 with S3-RNase to the 16 in domain FD3. We then examined seven additional chimeric proteins by dividing FD3 into two subdomains and four mini-domains (A, B, C and D). The results further narrowed the candidate amino acids to four in mini-domain A and four in mini-domain D. Molecular modeling of interactions between S3-RNase and S2-SLF1 revealed that three of these eight are at the interaction surface, and all three are conserved in S1-SLF1 and S6a-SLF1, both of which interact with S3-RNase based on the in vivo transgenic assay. Three of the chimeric proteins were used for the in vivo transgenic assay to determine whether FD3 alone contains the amino acids required for S2-SLF1 to interact with S7-RNase and S13-RNase. The results revealed the diversity and complexity of interactions between SLF proteins and S-RNases.


Assuntos
Alelos , Aminoácidos/genética , Loci Gênicos , Petunia/genética , Ribonucleases/química , Ribonucleases/genética , Sequência de Aminoácidos , Regulação da Expressão Gênica de Plantas , Genótipo , Simulação de Acoplamento Molecular , Fenótipo , Plantas Geneticamente Modificadas , Pólen/genética , Domínios Proteicos , Relação Estrutura-Atividade , Transgenes
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