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1.
Nat Commun ; 12(1): 5201, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34465779

RESUMO

N6-methyladenosine (m6A) is a post-transcriptional modification that controls gene expression by recruiting proteins to RNA sites. The modification also slows biochemical processes through mechanisms that are not understood. Using temperature-dependent (20°C-65°C) NMR relaxation dispersion, we show that m6A pairs with uridine with the methylamino group in the anti conformation to form a Watson-Crick base pair that transiently exchanges on the millisecond timescale with a singly hydrogen-bonded low-populated (1%) mismatch-like conformation in which the methylamino group is syn. This ability to rapidly interchange between Watson-Crick or mismatch-like forms, combined with different syn:anti isomer preferences when paired (~1:100) versus unpaired (~10:1), explains how m6A robustly slows duplex annealing without affecting melting at elevated temperatures via two pathways in which isomerization occurs before or after duplex annealing. Our model quantitatively predicts how m6A reshapes the kinetic landscape of nucleic acid hybridization and conformational transitions, and provides an explanation for why the modification robustly slows diverse cellular processes.


Assuntos
Adenosina/análogos & derivados , DNA/química , DNA/metabolismo , Adenosina/química , Adenosina/genética , Adenosina/metabolismo , Pareamento de Bases , DNA/genética , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Processamento Pós-Transcricional do RNA , Uridina/química , Uridina/genética , Uridina/metabolismo
2.
Microbiol Res ; 249: 126773, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33940365

RESUMO

Purpureocillium lilacinum (formerly Paecilomyces lilacinus) is widely commercialized for controlling plant-parasitic nematodes and represents a potential cell factory for enzyme production. This nematicidal fungus is intrinsically resistant to common antifungal agents used for genetic transformation. Therefore, molecular investigations in P. lilacinum are still limited so far. In the present study, we have established a new Agrobacterium tumefaciens-mediated transformation (ATMT) system in P. lilacinum based on the uridine/uracil auxotrophic mechanism. Here, uridine/uracil auxotrophic mutants were simply generated via UV irradiation instead of a complicated genetic approach for the pyrG gene deletion. A stable uridine/uracil auxotrophic mutant was then selected as a recipient for fungal transformation. We further indicated that the pyrG gene from Aspergillus niger can be used as a selectable marker for genetic transformation of P. lilacinum. Under optimized conditions for ATMT, the transformation efficiency reached 2873 ± 224 transformants per 106 spores. Using the constructed ATMT system, we succeeded in expressing the DsRed reporter gene in P. lilacinum. Additionally, we have identified a very promising mutant for chitinase production from a collection of T-DNA insertion transformants. This mutant possesses a special phenotype of hyper-branching mycelium and produces more conidia in comparison to the wild strain. Conclusively, our ATMT system can be exploited for overexpression of target genes or for T-DNA insertion mutagenesis in the agriculturally important fungus P. lilacinum. The genetic approach in the present work may also be applied for developing similar ATMT systems in other fungi, especially for fungi that their genome databases are currently not available.


Assuntos
Agrobacterium tumefaciens/genética , Hypocreales/genética , Transformação Genética , Antifúngicos/farmacologia , Quitinases/genética , Quitinases/metabolismo , DNA Bacteriano/genética , Genes Fúngicos , Genes Reporter , Hypocreales/efeitos dos fármacos , Hypocreales/metabolismo , Mutagênese Insercional , Mutação , Uracila/metabolismo , Uridina/metabolismo
3.
Nat Commun ; 12(1): 2170, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33859181

RESUMO

Regulation of mRNA translation elongation impacts nascent protein synthesis and integrity and plays a critical role in disease establishment. Here, we investigate features linking regulation of codon-dependent translation elongation to protein expression and homeostasis. Using knockdown models of enzymes that catalyze the mcm5s2 wobble uridine tRNA modification (U34-enzymes), we show that gene codon content is necessary but not sufficient to predict protein fate. While translation defects upon perturbation of U34-enzymes are strictly dependent on codon content, the consequences on protein output are determined by other features. Specific hydrophilic motifs cause protein aggregation and degradation upon codon-dependent translation elongation defects. Accordingly, the combination of codon content and the presence of hydrophilic motifs define the proteome whose maintenance relies on U34-tRNA modification. Together, these results uncover the mechanism linking wobble tRNA modification to mRNA translation and aggregation to maintain proteome homeostasis.


Assuntos
Aminoácidos/química , Complexos Multienzimáticos/metabolismo , Elongação Traducional da Cadeia Peptídica , Processamento Pós-Transcricional do RNA , RNA de Transferência/metabolismo , Aminoácidos/genética , Aminoácidos/metabolismo , Linhagem Celular Tumoral , Uso do Códon , Técnicas de Silenciamento de Genes , Humanos , Interações Hidrofóbicas e Hidrofílicas , Complexos Multienzimáticos/genética , Agregados Proteicos/genética , Proteólise , Proteômica , RNA Mensageiro/metabolismo , RNA de Transferência/genética , Uridina/metabolismo
4.
Nat Chem Biol ; 17(6): 703-710, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33723432

RESUMO

The protein complexes of the mitochondrial electron transport chain exist in isolation and in higher order assemblies termed supercomplexes (SCs) or respirasomes (SC I+III2+IV). The association of complexes I, III and IV into the respirasome is regulated by unknown mechanisms. Here, we designed a nanoluciferase complementation reporter for complex III and IV proximity to determine in vivo respirasome levels. In a chemical screen, we found that inhibitors of the de novo pyrimidine synthesis enzyme dihydroorotate dehydrogenase (DHODH) potently increased respirasome assembly and activity. By-passing DHODH inhibition via uridine supplementation decreases SC assembly by altering mitochondrial phospholipid composition, specifically elevated peroxisomal-derived ether phospholipids. Cell growth rates upon DHODH inhibition depend on ether lipid synthesis and SC assembly. These data reveal that nucleotide pools signal to peroxisomes to modulate synthesis and transport of ether phospholipids to mitochondria for SC assembly, which are necessary for optimal cell growth in conditions of nucleotide limitation.


Assuntos
Transporte de Elétrons , Nucleotídeos/química , Peroxissomos/química , Fosfolipídeos/química , Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lipídeos/biossíntese , Metabolômica , Mitocôndrias/metabolismo , Estrutura Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Consumo de Oxigênio , Éteres Fosfolipídicos , Uridina/metabolismo
5.
Nucleic Acids Res ; 49(6): 3557-3572, 2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33677542

RESUMO

Uridine insertion/deletion editing of mitochondrial mRNAs is a characteristic feature of kinetoplastids, including Trypanosoma brucei. Editing is directed by trans-acting gRNAs and catalyzed by related RNA Editing Core Complexes (RECCs). The non-catalytic RNA Editing Substrate Binding Complex (RESC) coordinates interactions between RECC, gRNA and mRNA. RESC is a dynamic complex comprising GRBC (Guide RNA Binding Complex) and heterogeneous REMCs (RNA Editing Mediator Complexes). Here, we show that RESC10 is an essential, low abundance, RNA binding protein that exhibits RNase-sensitive and RNase-insensitive interactions with RESC proteins, albeit its minimal in vivo interaction with RESC13. RESC10 RNAi causes extensive RESC disorganization, including disruption of intra-GRBC protein-protein interactions, as well as mRNA depletion from GRBC and accumulation on REMCs. Analysis of mitochondrial RNAs at single nucleotide resolution reveals transcript-specific effects: RESC10 dramatically impacts editing progression in pan-edited RPS12 mRNA, but is critical for editing initiation in mRNAs with internally initiating gRNAs, pointing to distinct initiation mechanisms for these RNA classes. Correlations between sites at which editing pauses in RESC10 depleted cells and those in knockdowns of previously studied RESC proteins suggest that RESC10 acts upstream of these factors and that RESC is particularly important in promoting transitions between uridine insertion and deletion RECCs.


Assuntos
Proteínas de Protozoários/fisiologia , Edição de RNA , RNA Mensageiro/metabolismo , RNA Mitocondrial/metabolismo , Proteínas de Ligação a RNA/fisiologia , Trypanosoma brucei brucei/genética , RNA Guia/metabolismo , RNA Mensageiro/química , RNA Mitocondrial/química , Proteínas de Ligação a RNA/metabolismo , Trypanosoma brucei brucei/crescimento & desenvolvimento , Uridina/metabolismo
6.
Virol J ; 18(1): 37, 2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33602251

RESUMO

BACKGROUND: Hepatitis B virus (HBV) infection is difficult to cure. HBV-specific immune tolerance plays a key role in HBV persistence, and enhancing cellular and humoral immunity will improve the control of HBV infection. The purpose of the study was to explore the anti-HBV and immunostimulatory effects of msiRNAs that introduce unpaired uridine bulges in the passenger strand. METHODS: msiRNAs targeting the HBV S and X genes were designed and named msiHBs and msiHBx, respectively. HepG2 cells were cotransfected with siRNA or msiRNA and the HBV replication-competent plasmid pHY106-wta or pHY106-X15. HepG2.215 cells were transfected with siRNA or msiRNA. The levels of HBsAg, HBeAg, and the cytokines TNF-α, IFN-α, IFN-ß, IL-1α, and IL-6 in the culture supernatant was detected by ELISA. The levels of intracellular HBV RNA, nuclear HBV replication intermediates, and HBV DNA in the supernatant were measured by quantitative RT-PCR and PCR. The levels of HBV replication intermediates were detected by Southern blotting. Peripheral blood mononuclear cells were transfected with siRNA or msiRNA, and the levels of secreted cytokines IFN-α and IFN-ß were detected by ELISA. The bioactivity of type I interferons in the supernatants was detected by the virus protection assay. RESULTS: msiHBx treatment led to a significant decrease in HBsAg (to a negative level) and HBV DNA (95.5%) in the supernatant and intrahepatocellular HBV replication intermediates (89.8%) in HepG2 cells with transient HBV replication and in HepG2.2.15 cells. There was no significant difference between msiHBx and siHBx in terms of the reduction in HBV proteins and HBV replication (P > 0.05). Compared with siHBx, msiHBx treatment of HepG2 cells transfected with the HBV replication-competent plasmid led to a significant increase in the levels of the antiviral cytokines TNF-α (3.3-fold), IFN-α (1.4-fold), and IFN-ß (2.5-fold) (P < 0.01), without upregulation of the proinflammatory cytokines IL-1α and IL-6. The virus protection assay results showed msiHBx-mediated type I interferons effectively protected L929 cells against ECMV infection. CONCLUSIONS: msiHBx could effectively inhibit HBV expression and replication and induce an antiviral innate immune response without proinflammatory activation. The dual RNAi and immunostimulatory activity of msiRNAs may play an important role in the control of HBV infection.


Assuntos
Vírus da Hepatite B/genética , Vírus da Hepatite B/imunologia , Hepatite B/imunologia , Imunidade Inata , RNA Interferente Pequeno/química , RNA Interferente Pequeno/imunologia , Uridina/metabolismo , Genes Virais , Células Hep G2 , Humanos , Imunização , Leucócitos Mononucleares/metabolismo , RNA Interferente Pequeno/síntese química , RNA Interferente Pequeno/genética , Transfecção , Uridina/genética , Replicação Viral
7.
PLoS One ; 16(2): e0247684, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33635895

RESUMO

Superoxide dismutase 1 (SOD1) is known to be involved in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS) and is therefore considered to be an important ALS drug target. Identifying potential drug leads that bind to SOD1 and characterizing their interactions by nuclear magnetic resonance (NMR) spectroscopy is complicated by the fact that SOD1 is a homodimer. Creating a monomeric version of SOD1 could alleviate these issues. A specially designed monomeric form of human superoxide dismutase (T2M4SOD1) was cloned into E. coli and its expression significantly enhanced using a number of novel DNA sequence, leader peptide and growth condition optimizations. Uniformly 15N-labeled T2M4SOD1 was prepared from minimal media using 15NH4Cl as the 15N source. The T2M4SOD1 monomer (both 15N labeled and unlabeled) was correctly folded as confirmed by 1H-NMR spectroscopy and active as confirmed by an in-gel enzymatic assay. To demonstrate the utility of this new SOD1 expression system for NMR-based drug screening, eight pyrimidine compounds were tested for binding to T2M4SOD1 by monitoring changes in their 1H NMR and/or 19F-NMR spectra. Weak binding to 5-fluorouridine (FUrd) was observed via line broadening, but very minimal spectral changes were seen with uridine, 5-bromouridine or trifluridine. On the other hand, 1H-NMR spectra of T2M4SOD1 with uracil or three halogenated derivatives of uracil changed dramatically suggesting that the pyrimidine moiety is the crucial binding component of FUrd. Interestingly, no change in tryptophan 32 (Trp32), the putative receptor for FUrd, was detected in the 15N-NMR spectra of 15N-T2M4SOD1 when mixed with these uracil analogs. Molecular docking and molecular dynamic (MD) studies indicate that interaction with Trp32 of SOD1 is predicted to be weak and that there was hydrogen bonding with the nearby aspartate (Asp96), potentiating the Trp32-uracil interaction. These studies demonstrate that monomeric T2M4SOD1 can be readily used to explore small molecule interactions via NMR.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Bromouracila/análogos & derivados , Clonagem Molecular/métodos , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Trifluridina/metabolismo , Uridina/análogos & derivados , Esclerose Amiotrófica Lateral/genética , Sequência de Bases , Bromouracila/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Ligação de Hidrogênio , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutação , Dobramento de Proteína , Espectroscopia de Prótons por Ressonância Magnética/métodos , Superóxido Dismutase-1/química , Triptofano/metabolismo , Uridina/metabolismo
8.
Nat Commun ; 12(1): 1298, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33637717

RESUMO

Uridylation is a widespread modification destabilizing eukaryotic mRNAs. Yet, molecular mechanisms underlying TUTase-mediated mRNA degradation remain mostly unresolved. Here, we report that the Arabidopsis TUTase URT1 participates in a molecular network connecting several translational repressors/decapping activators. URT1 directly interacts with DECAPPING 5 (DCP5), the Arabidopsis ortholog of human LSM14 and yeast Scd6, and this interaction connects URT1 to additional decay factors like DDX6/Dhh1-like RNA helicases. Nanopore direct RNA sequencing reveals a global role of URT1 in shaping poly(A) tail length, notably by preventing the accumulation of excessively deadenylated mRNAs. Based on in vitro and in planta data, we propose a model that explains how URT1 could reduce the accumulation of oligo(A)-tailed mRNAs both by favoring their degradation and because 3' terminal uridines intrinsically hinder deadenylation. Importantly, preventing the accumulation of excessively deadenylated mRNAs avoids the biogenesis of illegitimate siRNAs that silence endogenous mRNAs and perturb Arabidopsis growth and development.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , RNA Nucleotidiltransferases/metabolismo , RNA Interferente Pequeno/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas Correpressoras/metabolismo , RNA Helicases DEAD-box/metabolismo , Regulação da Expressão Gênica de Plantas , Humanos , Proteínas Proto-Oncogênicas/metabolismo , RNA Nucleotidiltransferases/genética , Estabilidade de RNA/genética , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Tabaco/genética , Transcriptoma , Uridina/metabolismo
9.
Commun Biol ; 4(1): 193, 2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33564093

RESUMO

SARS-CoV-2 Nsp15 is a uridine-specific endoribonuclease with C-terminal catalytic domain belonging to the EndoU family that is highly conserved in coronaviruses. As endoribonuclease activity seems to be responsible for the interference with the innate immune response, Nsp15 emerges as an attractive target for therapeutic intervention. Here we report the first structures with bound nucleotides and show how the enzyme specifically recognizes uridine moiety. In addition to a uridine site we present evidence for a second base binding site that can accommodate any base. The structure with a transition state analog, uridine vanadate, confirms interactions key to catalytic mechanisms. In the presence of manganese ions, the enzyme cleaves unpaired RNAs. This acquired knowledge was instrumental in identifying Tipiracil, an FDA approved drug that is used in the treatment of colorectal cancer, as a potential anti-COVID-19 drug. Using crystallography, biochemical, and whole-cell assays, we demonstrate that Tipiracil inhibits SARS-CoV-2 Nsp15 by interacting with the uridine binding pocket in the enzyme's active site. Our findings provide new insights for the development of uracil scaffold-based drugs.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , COVID-19/virologia , Endorribonucleases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Pirrolidinas/farmacologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , Timina/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Células A549 , Antivirais/química , Antivirais/farmacocinética , Domínio Catalítico , Cristalografia por Raios X , Endorribonucleases/química , Endorribonucleases/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Humanos , Ligantes , Modelos Moleculares , Conformação Proteica , Pirrolidinas/química , Pirrolidinas/farmacocinética , Timina/química , Timina/farmacocinética , Uridina/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo
10.
Circ Heart Fail ; 14(1): e007275, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33464957

RESUMO

BACKGROUND: Heart failure (HF) is a heterogeneous disease characterized by significant metabolic disturbances; however, the breadth of metabolic dysfunction before the onset of overt disease is not well understood. The purpose of this study was to determine the association of circulating metabolites with incident HF to uncover novel metabolic pathways to disease. METHODS: We performed targeted plasma metabolomic profiling in a deeply phenotyped group of Black adults from the JHS (Jackson Heart Study; n=2199). We related metabolites associated with incident HF to established etiological mechanisms, including increased left ventricular mass index and incident coronary heart disease. Furthermore, we evaluated differential associations of metabolites with HF with preserved ejection fraction versus HF with reduced ejection fraction. RESULTS: Metabolites associated with incident HF included products of posttranscriptional modifications of RNA, as well as polyamine and nitric oxide metabolism. A subset of metabolite-HF associations was independent of well-established HF pathways such as increased left ventricular mass index and incident coronary heart disease and included homoarginine (per 1 SD increase in metabolite level, hazard ratio, 0.77; P=1.2×10-3), diacetylspermine (hazard ratio, 1.34; P=3.4×10-3), and uridine (hazard ratio, 0.79; P, 3×10-4). Furthermore, metabolites involved in pyrimidine metabolism (orotic acid) and collagen turnover (N-methylproline) among others were part of a distinct metabolic signature that differentiated individuals with HF with preserved ejection fraction versus HF with reduced ejection fraction. CONCLUSIONS: The integration of clinical phenotyping with plasma metabolomic profiling uncovered novel metabolic processes in nontraditional disease pathways underlying the heterogeneity of HF development in Black adults.


Assuntos
Afro-Americanos , Doença das Coronárias/metabolismo , Insuficiência Cardíaca/metabolismo , Hipertrofia Ventricular Esquerda/metabolismo , Metabolômica , Adulto , Idoso , Estudos de Casos e Controles , Colágeno/metabolismo , Doença das Coronárias/epidemiologia , Modificador do Efeito Epidemiológico , Grupo com Ancestrais do Continente Europeu , Feminino , Fatores de Risco de Doenças Cardíacas , Insuficiência Cardíaca/epidemiologia , Insuficiência Cardíaca/fisiopatologia , Homoarginina/metabolismo , Humanos , Hipertrofia Ventricular Esquerda/epidemiologia , Incidência , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Óxido Nítrico/metabolismo , Ácido Orótico/metabolismo , Poliaminas/metabolismo , Prolina/análogos & derivados , Prolina/metabolismo , Modelos de Riscos Proporcionais , Pirimidinas/metabolismo , Processamento Pós-Transcricional do RNA , Risco , Espermina/análogos & derivados , Espermina/metabolismo , Volume Sistólico/fisiologia , Uridina/metabolismo
11.
Nat Commun ; 12(1): 428, 2021 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-33462199

RESUMO

The human prototypical SR protein SRSF1 is an oncoprotein that contains two RRMs and plays a pivotal role in RNA metabolism. We determined the structure of the RRM1 bound to RNA and found that the domain binds preferentially to a CN motif (N is for any nucleotide). Based on this solution structure, we engineered a protein containing a single glutamate to asparagine mutation (E87N), which gains the ability to bind to uridines and thereby activates SMN exon7 inclusion, a strategy that is used to cure spinal muscular atrophy. Finally, we revealed that the flexible inter-RRM linker of SRSF1 allows RRM1 to bind RNA on both sides of RRM2 binding site. Besides revealing an unexpected bimodal mode of interaction of SRSF1 with RNA, which will be of interest to design new therapeutic strategies, this study brings a new perspective on the mode of action of SRSF1 in cells.


Assuntos
Motivo de Reconhecimento de RNA/genética , Sítios de Splice de RNA/genética , Splicing de RNA , Fatores de Processamento de Serina-Arginina/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Substituição de Aminoácidos , Asparagina/genética , Biologia Computacional , Éxons/genética , Ácido Glutâmico/genética , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/terapia , Ressonância Magnética Nuclear Biomolecular , Engenharia de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/isolamento & purificação , Fatores de Processamento de Serina-Arginina/ultraestrutura , Uridina/metabolismo
12.
Mol Pharmacol ; 99(2): 147-162, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33262250

RESUMO

Equilibrative nucleoside transporters (ENTs) 1 and 2 facilitate nucleoside transport across the blood-testis barrier (BTB). Improving drug entry into the testes with drugs that use endogenous transport pathways may lead to more effective treatments for diseases within the reproductive tract. In this study, CRISPR/CRISPR-associated protein 9 was used to generate HeLa cell lines in which ENT expression was limited to ENT1 or ENT2. We characterized uridine transport in these cell lines and generated Bayesian models to predict interactions with the ENTs. Quantification of [3H]uridine uptake in the presence of the ENT-specific inhibitor S-(4-nitrobenzyl)-6-thioinosine (NBMPR) demonstrated functional loss of each transporter. Nine nucleoside reverse-transcriptase inhibitors and 37 nucleoside/heterocycle analogs were evaluated to identify ENT interactions. Twenty-one compounds inhibited uridine uptake and abacavir, nevirapine, ticagrelor, and uridine triacetate had different IC50 values for ENT1 and ENT2. Total accumulation of four identified inhibitors was measured with and without NBMPR to determine whether there was ENT-mediated transport. Clofarabine and cladribine were ENT1 and ENT2 substrates, whereas nevirapine and lexibulin were ENT1 and ENT2 nontransported inhibitors. Bayesian models generated using Assay Central machine learning software yielded reasonably high internal validation performance (receiver operator characteristic > 0.7). ENT1 IC50-based models were generated from ChEMBL; subvalidations using this training data set correctly predicted 58% of inhibitors when analyzing activity by percent uptake and 63% when using estimated-IC50 values. Determining drug interactions with these transporters can be useful in identifying and predicting compounds that are ENT1 and ENT2 substrates and can thereby circumvent the BTB through this transepithelial transport pathway in Sertoli cells. SIGNIFICANCE STATEMENT: This study is the first to predict drug interactions with equilibrative nucleoside transporter (ENT) 1 and ENT2 using Bayesian modeling. Novel CRISPR/CRISPR-associated protein 9 functional knockouts of ENT1 and ENT2 in HeLa S3 cells were generated and characterized. Determining drug interactions with these transporters can be useful in identifying and predicting compounds that are ENT1 and ENT2 substrates and can circumvent the blood-testis barrier through this transepithelial transport pathway in Sertoli cells.


Assuntos
Acetatos/farmacologia , Didesoxinucleosídeos/farmacologia , Transportador Equilibrativo 1 de Nucleosídeo/genética , Transportador Equilibrativo 2 de Nucleosídeo/genética , Nevirapina/farmacologia , Ticagrelor/farmacologia , Uridina/análogos & derivados , Uridina/metabolismo , Teorema de Bayes , Transporte Biológico , Sistemas CRISPR-Cas , Linhagem Celular , Interações Medicamentosas , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Transportador Equilibrativo 2 de Nucleosídeo/metabolismo , Técnicas de Inativação de Genes , Células HeLa , Humanos , Aprendizado de Máquina , Tioinosina/análogos & derivados , Tioinosina/farmacologia , Uridina/farmacologia
13.
Biochem Biophys Res Commun ; 534: 604-609, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33213836

RESUMO

CmoB utilizes carboxy-S-adenosyl-l-methionine (CxSAM) to carry out unusual carboxymethyl transfer to form 5-carboxymethoxyuridine (cmo5U) of several tRNA species in Gram-negative bacteria. In this report, we present three X-ray crystal structures of CmoB from Vibrio vulnificus representing different states in the course of the reaction pathway; i.e., apo-, substrate-bound, and product-bound forms. Especially, the crystal structure of apo-CmoB unveils a novel open state of the enzyme, capturing unprecedented conformational dynamics around the substrate-binding site. The apo-structure demonstrates that the open conformation favors the release of CxSAM thus representing an inactive form. Our crystal structures of CmoB complexed with CxSAM and S-adenosyl-l-homocysteine (SAH) and combined binding assay results support the proposed mechanism underlying the cofactor selectivity, where CmoB preferentially senses negative charge around amino acid residues Lys-91, Tyr-200, and Arg-315.


Assuntos
Proteínas de Bactérias/metabolismo , Metiltransferases/metabolismo , RNA Bacteriano/metabolismo , RNA de Transferência/metabolismo , Uridina/análogos & derivados , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Domínio Catalítico , Cristalografia por Raios X , Ligantes , Metiltransferases/química , Metiltransferases/genética , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , RNA Bacteriano/química , RNA Bacteriano/genética , RNA de Transferência/química , RNA de Transferência/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/análogos & derivados , S-Adenosilmetionina/metabolismo , Uridina/química , Uridina/metabolismo , Vibrio vulnificus/enzimologia , Vibrio vulnificus/genética
14.
Bioorg Med Chem ; 30: 115932, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33316720

RESUMO

Small interfering RNA (siRNA) exhibits gene-specific RNAi activity by the formation of RISC complex with mRNA of gene. The structural modification of siRNA at appropriate positions affects the structure of RISC complex and then RNAi activity. The modified siRNA are mostly prepared from the incorporation of sugar ring modified, and nucleobase modified RNA nucleotides. It is learned that the introduction of the sterically hindered nucleoside at the specific position of siRNA, severely affects siRNA-RISC complex formation. This report describes the syntheses of bulkier siRNA from 2'-caged-tethered-siRNAs, containing bulkier photolabile protecting group (o-nitrobenzyl) at 2'-position of ribose nucleoside. Importantly, these 2'-caged-siRNAs exhibit the light-dependent RNA interference (RNAi) activity into HEK293T cells for the GFP gene expression. The 2'-caged-siRNAs are synthesized by caging the sense and antisense strand of siRNA. The biochemical evaluations of these caged-siRNAs show that antisense-strand caged-siRNAs decrease RNAi activity temporarily in dark while enhancing RNAi activity, almost like control, after exposure withUV- light. However, 2'-caged sense strand siRNA increase RNAi activity temporarily while decreasing RNAi activity after exposure with light. These caged-siRNAs are also stable in the serum (fetal bovine serum) as like native siRNA. Hence these results strongly support that 2'-caged-tethered-siRNAs are promising analogues to control RNAi activity by UV-light.


Assuntos
Proteínas de Fluorescência Verde/metabolismo , Nucleosídeos/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Raios Ultravioleta , Uridina/metabolismo , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Nucleosídeos/síntese química , Nucleosídeos/química , Uridina/análogos & derivados , Uridina/química
15.
Methods Mol Biol ; 2192: 69-73, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33230766

RESUMO

The incorporation of nucleoside analogs is a useful tool to study the various functions of DNA and RNA. These analogs can be detected directly by fluorescence or by immunolabeling, allowing to visualize, track, or measure the nucleic acid molecules in which they have been incorporated. In this chapter, methodologies to measure human mitochondrial transcription are described. The nascent RNA that is transcribed from mitochondrial DNA (mtDNA) has been shown to assemble into large ribonucleoprotein complexes that form discrete foci. These structures were called mitochondrial RNA granules (MRGs) and can be observed in vitro by the incorporation of a 5-Bromouridine (BrU), which is subsequently visualized by fluorescent immunolabeling. Here, a combined protocol for the MRGs detection is detailed, consisting of BrU labeling and visualization of one of their bona fide protein components, Fas-activated serine-threonine kinase domain 2 (FASTKD2). Based on immunodetection, the half-life and kinetics of the MRGs under various experimental conditions can further be determined by chasing the BrU pulse with an excess of Uridine.


Assuntos
Bromouracila/análogos & derivados , Imuno-Histoquímica/métodos , Complexos Multiproteicos/metabolismo , RNA Mitocondrial/metabolismo , Ribonucleoproteínas/metabolismo , Uridina/análogos & derivados , Bromouracila/metabolismo , DNA Mitocondrial/metabolismo , Meia-Vida , Células HeLa , Humanos , Cinética , Complexos Multiproteicos/química , Proteínas Serina-Treonina Quinases/metabolismo , Ribonucleoproteínas/química , Transcrição Genética , Uridina/metabolismo
16.
Biomolecules ; 10(12)2020 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-33291826

RESUMO

BOK is an evolutionarily conserved BCL-2 family member that resembles the apoptotic effectors BAK and BAX in sequence and structure. Based on these similarities, BOK has traditionally been classified as a BAX-like pro-apoptotic protein. However, the mechanism of action and cellular functions of BOK remains controversial. While some studies propose that BOK could replace BAK and BAX to elicit apoptosis, others attribute to this protein an indirect way of apoptosis regulation. Adding to the debate, BOK has been associated with a plethora of non-apoptotic functions that makes this protein unpredictable when dictating cell fate. Here, we compile the current knowledge and open questions about this paradoxical protein with a special focus on its structural features as the key aspect to understand BOK biological functions.


Assuntos
Apoptose/genética , Células Eucarióticas/metabolismo , Regulação da Expressão Gênica , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Humanos , Membranas Mitocondriais/metabolismo , Modelos Moleculares , Permeabilidade , Conformação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais , Uridina/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
17.
Int J Mol Sci ; 21(21)2020 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-33152999

RESUMO

Elp3, the catalytic subunit of the eukaryotic Elongator complex, is a lysine acetyltransferase that acetylates the C5 position of wobble-base uridines (U34) in transfer RNAs (tRNAs). This Elongator-dependent RNA acetylation of anticodon bases affects the ribosomal translation elongation rates and directly links acetyl-CoA metabolism to both protein synthesis rates and the proteome integrity. Of note, several human diseases, including various cancers and neurodegenerative disorders, correlate with the dysregulation of Elongator's tRNA modification activity. In this review, we focus on recent findings regarding the structure of Elp3 and the role of acetyl-CoA during its unique modification reaction.


Assuntos
Histona Acetiltransferases/metabolismo , Processamento Pós-Transcricional do RNA , RNA de Transferência/metabolismo , Acetilação , Animais , Sequência de Bases , Sítios de Ligação , Histona Acetiltransferases/fisiologia , Humanos , Lisina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Elongação Traducional da Cadeia Peptídica/genética , Uridina/metabolismo
18.
Bioorg Chem ; 104: 104328, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33142406

RESUMO

The catalytic core of an 8-17 DNAzyme directed against STAT 3 was modified using (2'R) and (2'S) 2'-deoxy-2'-C-methyluridine and cytidine. While 2'-deoxy-2'-C-methyluridine significantly diminished the catalytic activity, 2'-deoxy-2'-C-methylcytidine replacement was better accepted, being the kact of modified DNAzymes at 8- and 11-positions comparable to the non-modified one. When 2'-O-methyl and phosphorothioate nucleotides were tested in the binding arms together with core modified DNAzymes the kcat was affected in a non predictable way, emphasizing the fact that both chemical substitutions should be considered globally. Finally, 2'-deoxy-2'-C-methyl modified DNAzymes stability was assayed finding that the double 2'-C-methyl modification in the catalytic core enhanced 70% the stability against a T47D cell lysate compared to a non-modified control.


Assuntos
DNA Catalítico/metabolismo , Biocatálise , DNA Catalítico/química , Desoxicitidina/análogos & derivados , Desoxicitidina/química , Desoxicitidina/metabolismo , Estrutura Molecular , Uridina/análogos & derivados , Uridina/química , Uridina/metabolismo
19.
Future Microbiol ; 15: 1343-1352, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-33085541

RESUMO

Aim: The inference of coronavirus evolution is largely based on mutations in SARS-CoV-2 genome. Misinterpretation of these mutations would mislead people about the evolution of SARS-CoV-2. Materials & methods: With 4521 lines of SARS-CoV-2, we obtained 3169 unique point mutation sites. We counted the numbers and calculated the minor allele frequency (MAF) of each mutation type. Results: Nearly half of the point mutations are C-T mismatches and 20% are A-G mismatches. The MAF of C-T and A-G mismatches is significantly higher than MAF of other mutation types. Conclusion: The excessive C-T mismatches do not resemble the random mutation profile. They are likely to be caused by the cytosine-to-uridine deamination system in hosts.


Assuntos
Betacoronavirus/genética , Mutação , RNA Viral/metabolismo , Pareamento Incorreto de Bases , COVID-19 , Uso do Códon , Infecções por Coronavirus/virologia , Citosina/metabolismo , Bases de Dados Genéticas , Desaminação , Frequência do Gene , Genoma Viral , Humanos , Pandemias , Pneumonia Viral/virologia , Polimorfismo de Nucleotídeo Único , SARS-CoV-2 , Uridina/metabolismo
20.
BMC Biol ; 18(1): 115, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32887607

RESUMO

BACKGROUND: Modification of RNAs, particularly at the terminals, is critical for various essential cell processes; for example, uridylation is implicated in tumorigenesis, proliferation, stem cell maintenance, and immune defense against viruses and retrotransposons. Ribosomal RNAs can be regulated by antisense ribosomal siRNAs (risiRNAs), which downregulate pre-rRNAs through the nuclear RNAi pathway in Caenorhabditis elegans. However, the biogenesis and regulation of risiRNAs remain obscure. Previously, we showed that 26S rRNAs are uridylated at the 3'-ends by an unknown terminal polyuridylation polymerase before the rRNAs are degraded by a 3' to 5' exoribonuclease SUSI-1(ceDIS3L2). RESULTS: Here, we found that CDE-1, one of the three C.elegans polyuridylation polymerases (PUPs), is specifically involved in suppressing risiRNA production. CDE-1 localizes to perinuclear granules in the germline and uridylates Argonaute-associated 22G-RNAs, 26S, and 5.8S rRNAs at the 3'-ends. Immunoprecipitation followed by mass spectrometry (IP-MS) revealed that CDE-1 interacts with SUSI-1(ceDIS3L2). Consistent with these results, both CDE-1 and SUSI-1(ceDIS3L2) are required for the inheritance of RNAi. CONCLUSIONS: This work identified a rRNA surveillance machinery of rRNAs that couples terminal polyuridylation and degradation.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , RNA de Helmintos/metabolismo , RNA Ribossômico/metabolismo , RNA Interferente Pequeno/metabolismo , Uridina/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Inativação Gênica , Células Germinativas/metabolismo , Interferência de RNA
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