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1.
Spectrochim Acta A Mol Biomol Spectrosc ; 224: 117452, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31408792

RESUMO

Nucleoside drugs are known for their remarkable anticancer and antiviral properties. The development of nucleoside drugs has attracted much attention and generated a great deal of research interest. ß-L-cytidine and ß-D-cytidine are a pair of cytosine nucleoside enantiomers. In this work, the interactions between cytosine nucleoside enantiomers and human serum albumin were studied by ultraviolet-visible spectra, fluorescence spectrum and circular dichroism spectrum under simulated human physiological environment. The data of fluorescence spectra were corrected for the inner-filter effect to improve accuracy. Stern-Volmer quenching constants and binding constants in addition to thermodynamic parameters have been analyzed, which established that complexes formation have taken place via static quenching mechanism, and that hydrophobic force involved in these interactions. CD spectrum revealed that on addition of cytosine nucleoside enantiomers, the α-helix% of HSA increased slightly. What's more, molecular modeling method indicated that cytosine nucleoside enantiomers prefer binding at the IIIA site of HSA.


Assuntos
Citidina/química , Citidina/metabolismo , Albumina Sérica Humana/química , Albumina Sérica Humana/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Simulação de Acoplamento Molecular , Ligação Proteica , Espectrometria de Fluorescência , Estereoisomerismo
2.
Nucleic Acids Res ; 47(14): 7676-7689, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31424549

RESUMO

The potent antiretroviral protein APOBEC3G (A3G) specifically targets and deaminates deoxycytidine nucleotides, generating deoxyuridine, in single stranded DNA (ssDNA) intermediates produced during HIV replication. A non-catalytic domain in A3G binds strongly to RNA, an interaction crucial for recruitment of A3G to the virion; yet, A3G displays no deamination activity for cytidines in viral RNA. Here, we report NMR and molecular dynamics (MD) simulation analysis for interactions between A3Gctd and multiple substrate or non-substrate DNA and RNA, in combination with deamination assays. NMR ssDNA-binding experiments revealed that the interaction with residues in helix1 and loop1 (T201-L220) distinguishes the binding mode of substrate ssDNA from non-substrate. Using 2'-deoxy-2'-fluorine substituted cytidines, we show that a 2'-endo sugar conformation of the target deoxycytidine is favored for substrate binding and deamination. Trajectories of the MD simulation indicate that a ribose 2'-hydroxyl group destabilizes the π-π stacking of the target cytosine and H257, resulting in dislocation of the target cytosine base from the catalytic position. Interestingly, APOBEC3A, which can deaminate ribocytidines, retains the ribocytidine in the catalytic position throughout the MD simulation. Our results indicate that A3Gctd catalytic selectivity against RNA is dictated by both the sugar conformation and 2'-hydroxyl group.


Assuntos
Desaminase APOBEC-3G/metabolismo , DNA de Cadeia Simples/metabolismo , DNA/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Simulação de Dinâmica Molecular , RNA/metabolismo , Desaminase APOBEC-3G/química , Desaminase APOBEC-3G/genética , Biocatálise , Citidina/química , Citidina/metabolismo , DNA/química , DNA/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Desaminação , HIV-1/genética , HIV-1/metabolismo , Humanos , Ligação Proteica , RNA/química , RNA/genética , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , Especificidade por Substrato , Vírion/genética , Vírion/metabolismo
3.
Genes Dev ; 33(13-14): 741-746, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31171702

RESUMO

Site-specific 2'-O-ribose methylation of mammalian rRNAs and RNA polymerase II-synthesized spliceosomal small nuclear RNAs (snRNAs) is mediated by small nucleolar and small Cajal body (CB)-specific box C/D ribonucleoprotein particles (RNPs) in the nucleolus and the nucleoplasmic CBs, respectively. Here, we demonstrate that 2'-O-methylation of the C34 wobble cytidine of human elongator tRNAMet(CAT) is achieved by collaboration of a nucleolar and a CB-specific box C/D RNP carrying the SNORD97 and SCARNA97 box C/D 2'-O-methylation guide RNAs. Methylation of C34 prevents site-specific cleavage of tRNAMet(CAT) by the stress-induced endoribonuclease angiogenin, implicating box C/D guide RNPs in controlling stress-responsive production of putative regulatory tRNA fragments.


Assuntos
Nucléolo Celular/metabolismo , Corpos Enovelados/metabolismo , Citidina/metabolismo , RNA de Transferência/metabolismo , Ribonucleoproteínas/metabolismo , Linhagem Celular , Regulação da Expressão Gênica , Células HeLa , Humanos , Metilação , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , RNA de Transferência/genética , Ribonuclease Pancreático/metabolismo , Ribonucleoproteínas/genética , Estresse Fisiológico
4.
ACS Chem Biol ; 14(7): 1418-1425, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31188562

RESUMO

RNA contains diverse modifications that exert important influences in a variety of cellular processes. So far more than 150 modifications have been identified in various RNA species, mainly in rRNA and tRNA. Recent research advances in RNA modifications have been sparked by the discovery of dynamic and reversible modifications in mRNA. Moving beyond the abundant tRNA and rRNA to mRNA is opening new directions in understanding RNA modification-mediated regulation of gene expression. Recently, it was reported that N3-methylcytidine (m3C) existed in mRNA of mammalian cells, and methyltransferase-like 8 (METTL8) was identified to be the writer enzyme of m3C. However, little is known about the eraser enzyme of m3C in mRNA. In the current study, we found that the AlkB homologue 1 (ALKBH1) was capable of demethylating m3C in mRNA of mammalian cells in vitro. Overexpression and knockdown of ALKBH1 in cultured human cells can induce decrease and increase of the level of m3C in mRNA, respectively, revealing the eraser enzyme property of ALKBH1 on m3C in mRNA. In addition, we observed significant decrease of the level of m3C in mRNA in hepatocellular carcinoma (HCC) tissues compared to tumor-adjacent normal tissues, which could be attributed to the increased expression of ALKBH1 as well as the decreased expression of METTL8 in HCC tissues. These results indicated that m3C in mRNA may play certain roles in tumorigenesis. Our study shed light on understanding the demethylation of m3C in mRNA.


Assuntos
Homólogo AlkB 1 da Histona H2a Dioxigenase/metabolismo , Citidina/análogos & derivados , RNA Mensageiro/metabolismo , Animais , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Citidina/metabolismo , Desmetilação , Células HEK293 , Humanos , Neoplasias Hepáticas/metabolismo , Mamíferos
5.
ACS Chem Biol ; 14(7): 1403-1409, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31243956

RESUMO

Post-transcriptional modifications to messenger RNAs (mRNAs) have the potential to alter the biological function of this important class of biomolecules. The study of mRNA modifications is a rapidly emerging field, and the full complement of chemical modifications in mRNAs is not yet established. We sought to identify and quantify the modifications present in yeast mRNAs using an ultra-high performance liquid chromatography tandem mass spectrometry method to detect 40 nucleoside variations in parallel. We observe six modified nucleosides with high confidence in highly purified mRNA samples (N7-methylguanosine, N6-methyladenosine, 2'-O-methylguanosine, 2'-O-methylcytidine, N4-acetylcytidine, and 5-formylcytidine) and identify the yeast protein responsible for N4-acetylcytidine incorporation in mRNAs (Rra1). In addition, we find that mRNA modification levels change in response to heat shock, glucose starvation, and/or oxidative stress. This work expands the repertoire of potential chemical modifications in mRNAs and highlights the value of integrating mass spectrometry tools in the mRNA modification discovery and characterization pipeline.


Assuntos
Nucleosídeos/análise , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina/análogos & derivados , Adenosina/análise , Adenosina/metabolismo , Citidina/análogos & derivados , Citidina/análise , Citidina/metabolismo , Glucose/metabolismo , Guanosina/análogos & derivados , Guanosina/análise , Guanosina/metabolismo , Resposta ao Choque Térmico , Nucleosídeos/metabolismo , Estresse Oxidativo , RNA Fúngico/química , RNA Mensageiro/química , Saccharomyces cerevisiae/química
6.
Methods Enzymol ; 621: 31-51, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31128786

RESUMO

Posttranscriptional modifications of RNA represent an emerging class of regulatory elements in human biology. Improved methods for studying how these elements are controlled and where they occur has the potential to transform our understanding of gene expression in development and disease. Here we describe a chemical method for nucleotide resolution sequencing of N4-acetylcytidine (ac4C), a highly conserved modified nucleobase whose formation is catalyzed by the essential cytidine acetyltransferase enzyme NAT10. This approach enables the sensitive, PCR-amplifiable detection of individual ac4C sites from nanograms of unfractionated cellular RNA. The sensitive and quantitative nature of this assay provides a powerful tool to understand how cytidine acetylation is targeted, profile RNA acetyltransferase dynamics, and validate the sites and stoichiometry of ac4C in novel RNA species.


Assuntos
Citidina/análogos & derivados , RNA/química , Análise de Sequência de RNA/métodos , Acetilação , Animais , Linhagem Celular , Citidina/análise , Citidina/genética , Citidina/metabolismo , Humanos , Acetiltransferases N-Terminal/metabolismo , Reação em Cadeia da Polimerase/métodos , RNA/genética , RNA/metabolismo , Processamento Pós-Transcricional do RNA
7.
Chem Asian J ; 14(13): 2235-2241, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31116511

RESUMO

Human APOBEC3G (A3G) inhibits the replication of human immunodeficiency virus-1 by deaminating cytidine at the 3'-end in the target motif 5'-CCC-3' in viral cDNA during reverse transcription. It in vitro deaminates two consecutive cytidines in a 3'->5' order. Although a crystal structure of the A3G catalytic domain (A3G-CD2) with DNA was reported, it is unknown why residues involved in enzymatic reaction are distributed widely. Here, we introduced an iodine atom into the C-5 position of cytidine (dC6 I ) in DNA 5'-ATTC4 C5 C6 I A7 ATT-3' (TCCC6 I ). It switches the deamination sequence preference from CCC to TCC, although small dC6 I deamination was observed. Solution structures of A3G-CD2 in complexes with products DNA TCUC6 I and TCUU6 I indicate that the substrate DNA binds A3G-CD2 in TCC and CCC modes. The dC6 deamination correlates with the 4th base type. The CCC mode favours dC6 deamination, while the TCC mode results in dC5 deamination. These studies present an extensive basis to design inhibitors to impede viral evolvability.


Assuntos
Desaminase APOBEC-3G/metabolismo , Citidina/metabolismo , DNA/metabolismo , Desaminase APOBEC-3G/química , Sequência de Bases , Domínio Catalítico , Citidina/química , DNA/química , Desaminação , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Espectroscopia de Prótons por Ressonância Magnética
8.
J Med Food ; 22(4): 408-415, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30990754

RESUMO

Salmon milt extract contains high levels of nucleic acids and has antioxidant potential. Although salmon milt extract is known to improve impaired brain function in animal models with brain disease, its effects on learning and memory ability in healthy subjects is unknown. The purpose of the present study was to clarify the effect of hydrolyzed salmon milt extract (HSME) on object recognition and object location memory under normal conditions. A diet containing 2.5% HSME induced normal mice to devote more time to exploring novel and moved objects than in exploring familiar and unmoved objects, as observed during novel object recognition and spatial recognition tests, respectively. A diet containing 2.5% nucleic acid fraction purified from HSME also induced similar effects, as measured by the same behavioral tests. This suggests that the nucleic acids may be a functional component contributing to the effects of HSME on brain function. Quantitative polymerase chain reaction analysis revealed that gene expression of the markers for brain parenchymal cells, including neural stem cells, astrocytes, oligodendrocytes, and microglia, in the hippocampi of mice on an HSME diet was higher than that in mice on a control diet. Oral administration of HSME increased concentrations of cytosine, cytidine, and deoxycytidine in the hippocampus. Overall, ingestion of HSME may enhance object recognition and object location memory under normal conditions in mice, at least, in part, via the activation of brain parenchymal cells. Our results thus indicate that dietary intake of this easily ingestible food might enhance brain function in healthy individuals.


Assuntos
Citidina/metabolismo , Hipocampo/metabolismo , Memória , Salmão/metabolismo , Sêmen/química , Animais , Encéfalo/fisiologia , Aprendizagem , Masculino , Camundongos
9.
Chem Commun (Camb) ; 55(16): 2328-2331, 2019 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-30723849

RESUMO

5-Methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), two of the best-studied DNA modifications, play crucial roles in normal development and disease in mammals. Although 5-methylcytidine (m5C) and 5-hydroxymethylcytidine (hm5C) have also been identified in RNA, their distribution and biological function in RNA remain largely unexplored, due to the lack of suitable sequencing methods. Here, we report a base-resolution sequencing method for hm5C in RNA. We applied the selective oxidation of hm5C to trihydroxylated-thymine (thT) mediated by peroxotungstate. thT was subsequently converted to T during cDNA synthesis using a thermostable group II intron reverse transcriptase (TGIRT). Base-resolution analysis of the hm5C sites in RNA was performed using Sanger sequencing. Furthermore, in combination with the TET enzyme oxidation of m5C to hm5C in RNA, we expand the use of peroxotungstate oxidation to detect m5C in RNA at base-resolution. By using this method, we confirmed three known m5C sites in human tRNA, demonstrating the applicability of our method in analyzing real RNA samples.


Assuntos
Citidina/análogos & derivados , RNA/genética , RNA/metabolismo , Análise de Sequência de RNA/métodos , Compostos de Tungstênio/química , Compostos de Tungstênio/metabolismo , Sequência de Bases , Citidina/metabolismo
10.
Nat Commun ; 10(1): 439, 2019 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-30683865

RESUMO

RNA-guided nucleases of the CRISPR/Cas type can be repurposed as programmable nucleotide deaminases to mediate targeted nucleotide substitutions. Such base editors have enormous potential in genome editing, gene therapy and precision breeding. However, current editors suffer from limited specificity in that they edit different and/or multiple bases within a larger sequence window. Using cytidine deaminase base editors that elicit C-to-T mutations, we show here that high editing precision can be achieved by engineering the connection between the deaminase domain and the Cas domain of the editor. By systematically testing different linker sequences and removing non-essential sequences from the deaminase, we obtain high-precision base editors with narrow activity windows that can selectively edit a single cytidine at a specific position with high accuracy and efficiency. These base editors will enable the use of genome editing in applications where single-nucleotide changes are required and off-target editing of adjacent nucleotides is not tolerable.


Assuntos
Desaminase APOBEC-1/genética , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Edição de Genes/métodos , Saccharomyces cerevisiae/genética , Desaminase APOBEC-1/química , Desaminase APOBEC-1/metabolismo , Sistemas de Transporte de Aminoácidos Básicos/genética , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Sequência de Bases , Proteína 9 Associada à CRISPR/química , Proteína 9 Associada à CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Citidina/genética , Citidina/metabolismo , Engenharia Genética/métodos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Mutagênese Sítio-Dirigida , RNA Guia/genética , RNA Guia/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sensibilidade e Especificidade , Timidina/genética , Timidina/metabolismo
11.
Methods ; 156: 91-101, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30395967

RESUMO

Ribonucleic acids (RNA) are extensively modified. These modifications are quantified by mass spectrometry (LC-MS/MS) to determine the abundance of a modification under certain conditions or in various genetic backgrounds. With LC-MS/MS the steady state of modifications is determined, and thus we only have a static view of the dynamics of RNA modifications. With nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS) we overcome this limitation and get access to the dynamics of RNA modifications. We describe labeling techniques for E. coli, S. cerevisiae and human cell culture and the current instrumental limitations. We present the power of NAIL-MS but we also outline validation experiments, which are necessary for correct data interpretation. As an example, we apply NAIL-MS to study the demethylation of adenine and cytidine, which are methylated by the damaging agent methyl-methanesulfonate in E. coli. With NAIL-MS we exclude the concurrent processes for removal of RNA methylation, namely RNA degradation, turnover and dilution. We use our tool to study the speed and efficiency of 1-methyladenosine and 3-methylcytidine demethylation. We further outline current limitations of NAIL-MS but also potential future uses for e.g. relative quantification of tRNA isoacceptor abundances.


Assuntos
Adenosina/análogos & derivados , Citidina/análogos & derivados , Marcação por Isótopo/métodos , Espectrometria de Massas/métodos , Processamento Pós-Transcricional do RNA , RNA Mensageiro/química , RNA de Transferência/química , Adenosina/química , Adenosina/metabolismo , Isótopos de Carbono , Citidina/química , Citidina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Células HEK293 , Humanos , Hidrólise , Metanossulfonato de Metila/química , Isótopos de Nitrogênio , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcriptoma
12.
Int Immunol ; 31(3): 167-173, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30535046

RESUMO

Toll-like receptor 8 (TLR8), a sensor for pathogen-derived single-stranded RNA (ssRNA), binds to uridine (Uri) and ssRNA to induce defense responses. We here show that cytidine (Cyd) with ssRNA also activated TLR8 in peripheral blood leukocytes (PBLs) and a myeloid cell line U937, but not in an embryonic kidney cell line 293T. Cyd deaminase (CDA), an enzyme highly expressed in leukocytes, deaminates Cyd to Uri. CDA expression enabled TLR8 response to Cyd and ssRNA in 293T cells. CDA deficiency and a CDA inhibitor both reduced TLR8 responses to Cyd and ssRNA in U937. The CDA inhibitor also reduced PBL response to Cyd and ssRNA. A Cyd analogue, azacytidine, is used for the therapy of myelodysplastic syndrome and acute myeloid leukemia. Azacytidine with ssRNA induced tumor necrosis factor-α expression in U937 and PBLs in a manner dependent on CDA and TLR8. These results suggest that CDA enables TLR8 activation by Cyd or its analogues with ssRNA through deaminating activity. Nucleoside metabolism might impact TLR8 responses in a variety of situations such as the treatment with nucleoside analogues.


Assuntos
Citidina Desaminase/metabolismo , Citidina/análogos & derivados , Citidina/metabolismo , Receptor 8 Toll-Like/metabolismo , Citidina/química , Humanos , Monócitos/metabolismo , Monócitos/patologia , Células Mieloides/metabolismo , Células Mieloides/patologia , Células Tumorais Cultivadas , Células U937
13.
IUBMB Life ; 71(1): 105-112, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30304569

RESUMO

Uridine-cytidine kinases (encoded by UCK1, UCKL1, and UCK2) catalyze the phosphorylation of uridine and cytidine to uridine monophosphate (UMP) and cytidine monophosphate (CMP). In this study, using data from the Cancer Genome Atlas (TCGA), we analyzed the expression profile of uridine-cytidine kinase genes in hepatocellular carcinoma (HCC), their prognostic value, and the epigenetic alterations associated with their dysregulation. Results showed that UCKL1 and UCK2, but not UCK1 were significantly upregulated in HCC tissues than in adjacent normal tissues. Only UCK2 was significantly upregulated in the deceased group and the recurrence group, compared to the control groups. Multivariate analysis confirmed that increased UCK2 expression was an independent prognostic indicator of shorter overall survival (OS) (HR: 1.760, 95% CI: 1.398-2.216, P < 0.001) and recurrence-free survival (RFS) (HR: 1.543, 95% CI: 1.232-1.933, P < 0.001). Two CpG sites (cg09277749 and cg21143899) were significantly hypomethylated in HCC tissues than in adjacent normal tissues and were negatively correlated with UCK2 expression. However, survival analysis showed that only high methylation of cg0927774 was associated with better OS and RFS of HCC patients. Based on the findings above, we infer that UCK2 upregulation might be a valuable prognostic marker in HCC. The methylation of status cg0927774 might play a critical role in its expression. © 2018 IUBMB Life, 71(1):105-112, 2019.


Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Prognóstico , Uridina Quinase/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Citidina/metabolismo , Metilação de DNA/genética , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Núcleosídeo-Fosfato Quinase/genética , Fosforilação , Uridina/metabolismo
15.
Nat Commun ; 9(1): 4865, 2018 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-30451861

RESUMO

The precise interplay between the mRNA codon and the tRNA anticodon is crucial for ensuring efficient and accurate translation by the ribosome. The insertion of RNA nucleobase derivatives in the mRNA allowed us to modulate the stability of the codon-anticodon interaction in the decoding site of bacterial and eukaryotic ribosomes, allowing an in-depth analysis of codon recognition. We found the hydrogen bond between the N1 of purines and the N3 of pyrimidines to be sufficient for decoding of the first two codon nucleotides, whereas adequate stacking between the RNA bases is critical at the wobble position. Inosine, found in eukaryotic mRNAs, is an important example of destabilization of the codon-anticodon interaction. Whereas single inosines are efficiently translated, multiple inosines, e.g., in the serotonin receptor 5-HT2C mRNA, inhibit translation. Thus, our results indicate that despite the robustness of the decoding process, its tolerance toward the weakening of codon-anticodon interactions is limited.


Assuntos
2-Aminopurina/análogos & derivados , Anticódon/química , Códon/química , Inosina/metabolismo , Biossíntese de Proteínas , Receptor 5-HT2C de Serotonina/genética , 2-Aminopurina/química , 2-Aminopurina/metabolismo , Anticódon/metabolismo , Bacteriófago T7/genética , Bacteriófago T7/metabolismo , Sequência de Bases , Códon/metabolismo , Citidina/análogos & derivados , Citidina/genética , Citidina/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Células HEK293 , Humanos , Ligação de Hidrogênio , Inosina/genética , Piridonas/química , Piridonas/metabolismo , RNA de Transferência de Glicina/genética , RNA de Transferência de Glicina/metabolismo , Receptor 5-HT2C de Serotonina/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo
16.
Cell ; 175(7): 1872-1886.e24, 2018 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-30449621

RESUMO

Generation of the "epitranscriptome" through post-transcriptional ribonucleoside modification embeds a layer of regulatory complexity into RNA structure and function. Here, we describe N4-acetylcytidine (ac4C) as an mRNA modification that is catalyzed by the acetyltransferase NAT10. Transcriptome-wide mapping of ac4C revealed discretely acetylated regions that were enriched within coding sequences. Ablation of NAT10 reduced ac4C detection at the mapped mRNA sites and was globally associated with target mRNA downregulation. Analysis of mRNA half-lives revealed a NAT10-dependent increase in stability in the cohort of acetylated mRNAs. mRNA acetylation was further demonstrated to enhance substrate translation in vitro and in vivo. Codon content analysis within ac4C peaks uncovered a biased representation of cytidine within wobble sites that was empirically determined to influence mRNA decoding efficiency. These findings expand the repertoire of mRNA modifications to include an acetylated residue and establish a role for ac4C in the regulation of mRNA translation.


Assuntos
Citidina/análogos & derivados , Acetiltransferase N-Terminal E/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Acetilação , Citidina/genética , Citidina/metabolismo , Células HeLa , Humanos , Acetiltransferase N-Terminal E/genética , RNA Mensageiro/genética
17.
PLoS One ; 13(9): e0203532, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30192840

RESUMO

Purine nucleoside phosphorylases (PNPs) play an important role in the blood fluke parasite Schistosoma mansoni as a key enzyme of the purine salvage pathway. Here we present the structural and kinetic characterization of a new PNP isoform from S. mansoni, SmPNP2. Thermofluorescence screening of different ligands suggested cytidine and cytosine are potential ligands. The binding of cytosine and cytidine were confirmed by isothermal titration calorimetry, with a KD of 27 µM for cytosine, and a KM of 76.3 µM for cytidine. SmPNP2 also displays catalytic activity against inosine and adenosine, making it the first described PNP with robust catalytic activity towards both pyrimidines and purines. Crystal structures of SmPNP2 with different ligands were obtained and comparison of these structures with the previously described S. mansoni PNP (SmPNP1) provided clues for the unique capacity of SmPNP2 to bind pyrimidines. When compared with the structure of SmPNP1, substitutions in the vicinity of SmPNP2 active site alter the architecture of the nucleoside base binding site thus permitting an alternative binding mode for nucleosides, with a 180° rotation from the canonical binding mode. The remarkable plasticity of this binding site enhances our understanding of the correlation between structure and nucleotide selectivity, thus suggesting new ways to analyse PNP activity.


Assuntos
Nucleosídeos/metabolismo , Purina-Núcleosídeo Fosforilase/química , Purina-Núcleosídeo Fosforilase/metabolismo , Schistosoma mansoni/enzimologia , Schistosoma mansoni/genética , Adenosina/metabolismo , Animais , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Citidina/metabolismo , Citosina/metabolismo , Proteínas de Helminto/química , Proteínas de Helminto/metabolismo , Inosina/metabolismo , Cinética , Modelos Moleculares , Mutação , Conformação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Purina-Núcleosídeo Fosforilase/genética , Schistosoma mansoni/química , Especificidade por Substrato
18.
Anal Chem ; 90(20): 11735-11740, 2018 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-30256094

RESUMO

Enzymes that catalyze DNA modifying activities including cytidine deamination and cytosine methylation play important biological roles and have been implicated pathologically in diseases such as cancer. Here, we report Direct Resolution of ONE dalton difference (DRONE), an ultra high performance liquid chromatography (UHPLC)-based analytical method to track a single dalton change in the cytosine-to-uracil conversion catalyzed by the human apolipoprotein B m-RNA editing catalytic polypeptide-like 3 (APOBEC3) cytidine deaminases, implicated in cancer and antiviral defense. Additionally, we demonstrate broad applicability by tracking other important DNA modifications and assessing epigenetic enzyme inhibition. We have extended our methodology to obtain data on two distinct deamination events in the same oligonucleotide substrate designed from a putative APOBEC substrate, diversifying the utility of the described method. DRONE provides an important foundation for in-depth analysis of DNA-modifying enzymes and versatile detection of novel DNA modifications of interest.


Assuntos
Citidina/metabolismo , Citosina Desaminase/metabolismo , DNA/metabolismo , Cromatografia Líquida de Alta Pressão , Citidina/química , Citidina Desaminase , DNA/química , Desaminação , Humanos , Cinética , Estrutura Molecular
19.
Methods ; 150: 19-23, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30076894

RESUMO

The use of CRISPR/Cas9 to knockout genes in zebrafish has been well established. However, to better model many human diseases that are caused by point mutations, a robust methodology for generating desirable DNA base changes is still needed. Recently, Cas9-linked cytidine deaminases (base editors) evolved as a strategy to introduce single base mutations in model organisms. They have been used to convert cytidine to thymine at specific genomic loci. Here we describe a protocol for using the base editing system in zebrafish and its application to reproduce a single base mutation observed in human Ablepharon-Macrostomia Syndrome.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Peixe-Zebra/genética , Anormalidades Múltiplas/genética , Animais , Sequência de Bases/genética , Citidina/metabolismo , Citidina Desaminase/genética , Modelos Animais de Doenças , Embrião não Mamífero , Anormalidades do Olho/genética , Feminino , Humanos , Macrostomia/genética , Masculino , Mutagênese Sítio-Dirigida/métodos , Mutação Puntual/genética , RNA Guia , Timina/metabolismo , Proteína 2 Relacionada a Twist/genética , Proteínas de Peixe-Zebra/genética
20.
Nat Commun ; 9(1): 2717, 2018 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-30006570

RESUMO

Cytidine base editors (CBEs) and adenine base editors (ABEs), composed of a cytidine deaminase or an evolved adenine deaminase fused to Cas9 nickase, enable the conversion of C·G to T·A or A·T to G·C base pair in organisms, respectively. Here, we show that BE3 and ABE7.10 systems can achieve a targeted mutation efficiency of 53-88% and 44-100%, respectively, in both blastocysts and Founder (F0) rabbits. Meanwhile, this strategy can be used to precisely mimic human pathologies by efficiently inducing nonsense or missense mutations as well as RNA mis-splicing in rabbit. In addition, the reduced frequencies of indels with higher product purity are also determined in rabbit blastocysts by BE4-Gam, which is an updated version of the BE3 system. Collectively, this work provides a simple and efficient method for targeted point mutations and generation of disease models in rabbit.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Modelos Animais de Doenças , Edição de Genes/métodos , RNA Guia/genética , Coelhos/genética , Adenina/metabolismo , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Animais , Pareamento de Bases , Sequência de Bases , Blastocisto/citologia , Blastocisto/metabolismo , Proteína 9 Associada à CRISPR/metabolismo , Códon sem Sentido , Citidina/metabolismo , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Efeito Fundador , Humanos , Mutação INDEL , Mutação de Sentido Incorreto , RNA Guia/metabolismo , Alinhamento de Sequência
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