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1.
Biochim Biophys Acta Mol Cell Res ; 1871(7): 119807, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39106920

RESUMO

Post-transcriptional modification of nucleosides in transfer RNAs (tRNAs) is an important process for accurate and efficient translation of the genetic information during protein synthesis in all domains of life. In particular, specific enzymes catalyze the biosynthesis of sulfur-containing nucleosides, such as the derivatives of 2-thiouridine (s2U), 4-thiouridine (s4U), 2-thiocytidine (s2C), and 2-methylthioadenosine (ms2A), within tRNAs. Whereas the mechanism that has prevailed for decades involved persulfide chemistry, more and more tRNA thiolation enzymes have now been shown to contain a [4Fe-4S] cluster. This review summarizes the information over the last ten years concerning the biochemical, spectroscopic and structural characterization of [4Fe-4S]-dependent non-redox tRNA thiolation enzymes.


Assuntos
Proteínas Ferro-Enxofre , RNA de Transferência , RNA de Transferência/metabolismo , RNA de Transferência/química , RNA de Transferência/genética , Proteínas Ferro-Enxofre/metabolismo , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/genética , Compostos de Sulfidrila/metabolismo , Compostos de Sulfidrila/química , Oxirredução , Processamento Pós-Transcricional do RNA , Humanos , Tiouridina/análogos & derivados , Tiouridina/metabolismo , Tiouridina/química
2.
Nucleic Acids Res ; 52(17): 10543-10562, 2024 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-39166491

RESUMO

In this study, we present an extensive analysis of a widespread group of bacterial tRNA de-modifying enzymes, dubbed RudS, which consist of a TudS desulfidase fused to a Domain of Unknown Function 1722 (DUF1722). RudS enzymes exhibit specific de-modification activity towards the 4-thiouridine modification (s4U) in tRNA molecules, as indicated by our experimental findings. The heterologous overexpression of RudS genes in Escherichia coli significantly reduces the tRNA 4-thiouridine content and diminishes UVA-induced growth delay, indicating the enzyme's role in regulating photosensitive tRNA s4U modification. Through a combination of protein modeling, docking studies, and molecular dynamics simulations, we have identified amino acid residues involved in catalysis and tRNA binding. Experimental validation through targeted mutagenesis confirms the TudS domain as the catalytic core of RudS, with the DUF1722 domain facilitating tRNA binding in the anticodon region. Our results suggest that RudS tRNA modification eraser proteins may play a role in regulating tRNA during prokaryotic stress responses.


Assuntos
Escherichia coli , RNA de Transferência , Tiouridina , Tiouridina/metabolismo , Tiouridina/análogos & derivados , Tiouridina/química , RNA de Transferência/metabolismo , RNA de Transferência/genética , Escherichia coli/genética , Escherichia coli/enzimologia , Simulação de Dinâmica Molecular , Simulação de Acoplamento Molecular , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Domínio Catalítico , Liases de Carbono-Enxofre/metabolismo , Liases de Carbono-Enxofre/genética , Liases de Carbono-Enxofre/química , Anticódon/genética
3.
Angew Chem Int Ed Engl ; 63(33): e202405040, 2024 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-38785103

RESUMO

Nucleoside and nucleotide analogues have proven to be transformative in the treatment of viral infections and cancer. One branch of structural modification to deliver new nucleoside analogue classes explores replacement of canonical ribose oxygen with a sulfur atom. Whilst biological activity of such analogues has been shown in some cases, widespread exploration of this compound class is hitherto hampered by the lack of a straightforward and universal nucleobase diversification strategy. Herein, we present a synergistic platform enabling both biocatalytic nucleobase diversification from 4'-thiouridine in a one-pot process, and chemical functionalization to access new entities. This methodology delivers entry across pyrimidine and purine 4'-thionucleosides, paving a way for wider synthetic and biological exploration. We exemplify our approach by enzymatic synthesis of 5-iodo-4'-thiouridine on multi-milligram scale and from here switch to complete chemical synthesis of a novel nucleoside analogue probe, 5-ethynyl-4'-thiouridine. Finally, we demonstrate the utility of this probe to monitor RNA synthesis in proliferating HeLa cells, validating its capability as a new metabolic RNA labelling tool.


Assuntos
RNA , Tionucleosídeos , Tiouridina , Tiouridina/análogos & derivados , Tiouridina/química , Tiouridina/metabolismo , Humanos , Tionucleosídeos/química , Tionucleosídeos/metabolismo , Tionucleosídeos/síntese química , RNA/metabolismo , RNA/química , Células HeLa , Biocatálise , Estrutura Molecular
4.
Nucleic Acids Res ; 52(7): e35, 2024 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-38381903

RESUMO

Nucleoside analogues like 4-thiouridine (4sU) are used to metabolically label newly synthesized RNA. Chemical conversion of 4sU before sequencing induces T-to-C mismatches in reads sequenced from labelled RNA, allowing to obtain total and labelled RNA expression profiles from a single sequencing library. Cytotoxicity due to extended periods of labelling or high 4sU concentrations has been described, but the effects of extensive 4sU labelling on expression estimates from nucleotide conversion RNA-seq have not been studied. Here, we performed nucleotide conversion RNA-seq with escalating doses of 4sU with short-term labelling (1h) and over a progressive time course (up to 2h) in different cell lines. With high concentrations or at later time points, expression estimates were biased in an RNA half-life dependent manner. We show that bias arose by a combination of reduced mappability of reads carrying multiple conversions, and a global, unspecific underrepresentation of labelled RNA emerging during library preparation and potentially global reduction of RNA synthesis. We developed a computational tool to rescue unmappable reads, which performed favourably compared to previous read mappers, and a statistical method, which could fully remove remaining bias. All methods developed here are freely available as part of our GRAND-SLAM pipeline and grandR package.


Assuntos
RNA-Seq , Tiouridina , Tiouridina/metabolismo , Tiouridina/química , RNA-Seq/métodos , Humanos , RNA/genética , Análise de Sequência de RNA/métodos , Nucleotídeos/genética
5.
J Am Chem Soc ; 146(6): 3861-3871, 2024 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-38293747

RESUMO

2-Thiouridine (s2U) is a nucleobase modification that confers enhanced efficiency and fidelity both on modern tRNA codon translation and on nonenzymatic and ribozyme-catalyzed RNA copying. We have discovered an unusual base pair between two 2-thiouridines that stabilizes an RNA duplex to a degree that is comparable to that of a native A:U base pair. High-resolution crystal structures indicate similar base-pairing geometry and stacking interactions in duplexes containing s2U:s2U compared to those with U:U pairs. Notably, the C═O···H-N hydrogen bond in the U:U pair is replaced with a C═S···H-N hydrogen bond in the s2U:s2U base pair. The thermodynamic stability of the s2U:s2U base pair suggested that this self-pairing might lead to an increased error frequency during nonenzymatic RNA copying. However, competition experiments show that s2U:s2U base-pairing induces only a low level of misincorporation during nonenzymatic RNA template copying because the correct A:s2U base pair outcompetes the slightly weaker s2U:s2U base pair. In addition, even if an s2U is incorrectly incorporated, the addition of the next base is greatly hindered. This strong stalling effect would further increase the effective fidelity of nonenzymatic RNA copying with s2U. Our findings suggest that s2U may enhance the rate and extent of nonenzymatic copying with only a minimal cost in fidelity.


Assuntos
RNA Catalítico , RNA , Tiouridina/análogos & derivados , RNA/química , Pareamento de Bases , Tiouridina/química , RNA Catalítico/química , Conformação de Ácido Nucleico
6.
Nucleic Acids Res ; 50(19): e110, 2022 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-36018791

RESUMO

Quantitative comparisons of RNA levels from different samples can lead to new biological understanding if they are able to distinguish biological variation from variable sample preparation. These challenges are pronounced in comparisons that require complex biochemical manipulations (e.g. isolating polysomes to study translation). Here, we present Transcript Regulation Identified by Labeling with Nucleoside Analogues in Cell Culture (TILAC), an internally controlled approach for quantitative comparisons of RNA content. TILAC uses two metabolic labels, 4-thiouridine (s4U) and 6-thioguanosine (s6G), to differentially label RNAs in cells, allowing experimental and control samples to be pooled prior to downstream biochemical manipulations. TILAC leverages nucleoside recoding chemistry to generate characteristic sequencing signatures for each label and uses statistical modeling to compare the abundance of RNA transcripts between samples. We verified the performance of TILAC in transcriptome-scale experiments involving RNA polymerase II inhibition and heat shock. We then applied TILAC to quantify changes in mRNA association with actively translating ribosomes during sodium arsenite stress and discovered a set of transcripts that are translationally upregulated, including MCM2 and DDX5. TILAC is broadly applicable to uncover differences between samples leading to improved biological insights.


Assuntos
Nucleosídeos , Tiouridina , Tiouridina/química , Análise de Sequência de RNA , RNA/química , RNA Mensageiro/metabolismo
7.
Chem Commun (Camb) ; 58(9): 1322-1325, 2022 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-34985087

RESUMO

Both sequence enrichment and base resolution are essential for accurate sequencing analysis of low-abundance RNA. Yet they are hindered by the lack of molecular tools. Here we report a bifunctional chemical signature for RNA 4-thiouridine (4sU) enrichment sequencing with single-base resolution. This chemical signature is designed for specific 4sU labeling with two functional parts. One part is a distal alkynyl group for the biotin-assisted pulldown enrichment of target molecules via click chemistry crosslinking. The other part is a -NH group proximal to the pyrimidine ring of 4sU. It allows 4sU-to-cytosine transition during the polymerase-catalyzed extension reaction based on altering hydrogen-bonding patterns. Ultimately, the 4sU-containing RNA molecules can be enriched and accurately analyzed by single-base resolution sequencing. The proposed method also holds great potential to investigate transcriptome dynamics integrated with high-throughput sequencing.


Assuntos
RNA/química , Tiouridina/química , Química Click , Citosina/química , Sequenciamento de Nucleotídeos em Larga Escala , Espectrometria de Massas , Motivos de Nucleotídeos , Pirimidinas/química , Estabilidade de RNA , Análise de Sequência de RNA
8.
J Biol Chem ; 297(6): 101245, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34688660

RESUMO

RNA structure in the influenza A virus (IAV) has been the focus of several studies that have shown connections between conserved secondary structure motifs and their biological function in the virus replication cycle. Questions have arisen on how to best recognize and understand the pandemic properties of IAV strains from an RNA perspective, but determination of the RNA secondary structure has been challenging. Herein, we used chemical mapping to determine the secondary structure of segment 8 viral RNA (vRNA) of the pandemic A/California/04/2009 (H1N1) strain of IAV. Additionally, this long, naturally occurring RNA served as a model to evaluate RNA mapping with 4-thiouridine (4sU) crosslinking. We explored 4-thiouridine as a probe of nucleotides in close proximity, through its incorporation into newly transcribed RNA and subsequent photoactivation. RNA secondary structural features both universal to type A strains and unique to the A/California/04/2009 (H1N1) strain were recognized. 4sU mapping confirmed and facilitated RNA structure prediction, according to several rules: 4sU photocross-linking forms efficiently in the double-stranded region of RNA with some flexibility, in the ends of helices, and across bulges and loops when their structural mobility is permitted. This method highlighted three-dimensional properties of segment 8 vRNA secondary structure motifs and allowed to propose several long-range three-dimensional interactions. 4sU mapping combined with chemical mapping and bioinformatic analysis could be used to enhance the RNA structure determination as well as recognition of target regions for antisense strategies or viral RNA detection.


Assuntos
Reagentes de Ligações Cruzadas/química , Vírus da Influenza A/química , Influenza Humana/virologia , RNA Viral/química , Tiouridina/química , Pareamento de Bases , Sequência de Bases , Humanos , Conformação de Ácido Nucleico
9.
Bioconjug Chem ; 32(4): 721-728, 2021 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-33730486

RESUMO

Functionalized oligonucleotides (ONs) are widely applied as target binding molecules for biosensing and regulators for gene expression. Numerous efforts have been focused on developing facile methods for preparing these useful ONs carrying diverse modifications. Herein, we present a general method for postsynthetic modification of ONs via oxidative amination of 4-thio-2'-deoxyuridine (4SdU). 4SdU-containing ON can be derived by both alkyl and aromatic amines. Using this approach, ONs are successfully attached with alkyne/azide, biotin and dansylamide moieties, and these as-prepared ONs possess the expected biorthogonal reactivity, streptavidin affinity and fluorescent property, respectively. Furthermore, we also directly install fluorophores to the ON nucleobase based on oxidative amination of 4SdU, and these fluorophores exhibit distinct luminescence behaviors before and after conjugation. We believe our method will be a versatile strategy for constructing various functionalized ONs used in a wide range of nucleic acid applications.


Assuntos
Oligonucleotídeos/química , Tiouridina/análogos & derivados , Aminação , Oxirredução , Tiouridina/química
10.
Nucleic Acids Res ; 49(8): e45, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33503264

RESUMO

Crosslinking and immunoprecipitation (CLIP) methods are powerful techniques to interrogate direct protein-RNA interactions and dissect posttranscriptional gene regulatory networks. One widely used CLIP variant is photoactivatable ribonucleoside enhanced CLIP (PAR-CLIP) that involves in vivo labeling of nascent RNAs with the photoreactive nucleosides 4-thiouridine (4SU) or 6-thioguanosine (6SG), which can efficiently crosslink to interacting proteins using UVA and UVB light. Crosslinking of 4SU or 6SG to interacting amino acids changes their base-pairing properties and results in characteristic mutations in cDNA libraries prepared for high-throughput sequencing, which can be computationally exploited to remove abundant background from non-crosslinked sequences and help pinpoint RNA binding protein binding sites at nucleotide resolution on a transcriptome-wide scale. Here we present a streamlined protocol for fluorescence-based PAR-CLIP (fPAR-CLIP) that eliminates the need to use radioactivity. It is based on direct ligation of a fluorescently labeled adapter to the 3'end of crosslinked RNA on immobilized ribonucleoproteins, followed by isolation of the adapter-ligated RNA and efficient conversion into cDNA without the previously needed size fractionation on denaturing polyacrylamide gels. These improvements cut the experimentation by half to 2 days and increases sensitivity by 10-100-fold.


Assuntos
DNA Complementar/genética , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Sítios de Ligação , Linhagem Celular , Reagentes de Ligações Cruzadas/química , Eletroforese em Gel de Poliacrilamida , GTP Fosfo-Hidrolases/química , Biblioteca Gênica , Humanos , Imunoprecipitação , Oligonucleotídeos , Reação em Cadeia da Polimerase/métodos , Ligação Proteica , RNA/química , Ribonucleoproteínas/genética , Sensibilidade e Especificidade , Software , Tiouridina/química , Raios Ultravioleta
11.
Chembiochem ; 22(1): 212-216, 2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-32864814

RESUMO

Peroxidase-generated proximity labeling is in widespread use to study subcellular proteomes and the protein interaction networks in living cells, but the development of subcellular RNA labeling is limited. APEX-seq has emerged as a new method to study subcellular RNA in living cells, but the labeling of RNA still has room to improve. In this work, we describe 4-thiouridine (s4 U)-enhanced peroxidase-generated biotinylation of RNA with high efficiency. The incorporation of s4 U could introduce additional sites for RNA labeling, enhanced biotinylation was observed on monomer, model oligo RNA and total RNA. Through the s4 U metabolic approach, the in vivo RNA biotinylation efficiency by peroxidase catalysis was also dramatically increased, which will benefit RNA isolation and study for the spatial transcriptome.


Assuntos
Peroxidase/metabolismo , RNA/metabolismo , Tiouridina/farmacologia , Biotinilação , Células HEK293 , Humanos , Conformação Molecular , Tiouridina/química
12.
Methods Mol Biol ; 2192: 147-158, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33230772

RESUMO

Even though the mammalian mitochondrial genome (mtDNA) is very small and only codes for 13 proteins, all being subunits of the oxidative phosphorylation system, it requires several hundred nuclear encoded proteins for its maintenance and expression. These include replication and transcription factors, approximately 80 mitoribosomal proteins and many proteins involved in the posttranscriptional modification, processing, and stability of mitochondrial RNAs. In recent years, many of these factors have been identified and functionally characterized, but the complete mtRNA-interacting proteome is not firmly established. Shotgun proteomics has been used successfully to define whole-cell polyadenylated RNA (poly(A)-RNA) interacting proteomes using the nucleotide analogue 4-thiouridine (4SU) combined with UV crosslinking, poly(A)-RNA isolation and mass spectrometry to identify all poly(A)-RNA bound proteins. Although in this case also a considerable number of mitochondrial proteins were identified, the method was not specifically directed at the mitochondrial poly(A)-RNA bound proteome. Here we describe a method for enrichment of the mitochondrial poly(A)-RNA bound proteome based on 4SU labeling and UV crosslinking. The method can be applied either for isolated mitochondria prior to UV crosslinking or for whole-cell crosslinking followed by mitochondrial isolation.


Assuntos
Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteoma , RNA Mitocondrial/metabolismo , Proteínas de Ligação a RNA/metabolismo , DNA Mitocondrial/genética , Genoma Mitocondrial , Células HEK293 , Humanos , Espectrometria de Massas , Proteínas Mitocondriais/química , Fosforilação Oxidativa , Proteômica/métodos , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Tiouridina/química , Tiouridina/metabolismo , Raios Ultravioleta
13.
J Phys Chem Lett ; 11(15): 6337-6343, 2020 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-32701298

RESUMO

The residue 2-thiouridine (s2U) provides a remarkable example for the "modified wobble" hypothesis, which postulates that some post-transcriptional modifications at the wobble position of tRNAs restrict recognition of degenerate codons. Through extensive molecular dynamics simulations using our χIDRP force field parameters, we demonstrate how this modification shifts the conformational ensemble from a predominantly disordered, as in the case of an RNA pentamer (GUUUC), to a substantially ordered population in Gs2UUUC. Our simulations clearly showed that the van der Waals interaction of sulfur played a major role in driving the disorder-to-order transition. The conformational redistribution and the slowing down of the transition between the clusters within the population in the presence of s2U suggest ensemble allostery to be a key mechanism that may play a general role in the functioning of the wobble modifications of tRNAs.


Assuntos
RNA de Transferência/química , Tiouridina/química , Sítio Alostérico , Sequência de Bases , Códon/química , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico
14.
Chembiochem ; 21(19): 2768-2771, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32394608

RESUMO

Ribonucleic acid (RNA) is central to many life processes and, to fulfill its function, it has a substantial chemical variety in its building blocks. Enzymatic thiolation of uridine introduces 4-thiouridine (s4 U) into many bacterial transfer RNAs (tRNAs), which is used as a sensor for UV radiation. A similar modified nucleoside, 2-thiocytidine, was recently found to be sulfur-methylated especially in bacteria exposed to antibiotics and simple methylating reagents. Herein, we report the synthesis of 4-methylthiouridine (ms4 U) and confirm its presence and additional formation under stress in Escherichia coli. We used the synthetic ms4 U for isotope dilution mass spectrometry and compared its abundance to other reported tRNA damage products. In addition, we applied sophisticated stable-isotope pulse chase studies (NAIL-MS) and showed its AlkB-independent removal in vivo. Our findings reveal the complex nature of bacterial RNA damage repair.


Assuntos
Escherichia coli/metabolismo , RNA Bacteriano/metabolismo , RNA de Transferência/metabolismo , Tiouridina/metabolismo , Modelos Moleculares , Estrutura Molecular , RNA Bacteriano/química , RNA de Transferência/química , Tiouridina/síntese química , Tiouridina/química
15.
Biomolecules ; 10(2)2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32085421

RESUMO

Modifications found in the Anticodon Stem Loop (ASL) of tRNAs play important roles in regulating translational speed and accuracy. Threonylcarbamoyl adenosine (t6A37) and 5-methoxycarbonyl methyl-2-thiouridine (mcm5s2U34) are critical ASL modifications that have been linked to several human diseases. The model yeast Saccharomyces cerevisiae is viable despite the absence of both modifications, growth is however greatly impaired. The major observed consequence is a subsequent increase in protein aggregates and aberrant morphology. Proteomic analysis of the t6A-deficient strain (sua5 mutant) revealed a global mistranslation leading to protein aggregation without regard to physicochemical properties or t6A-dependent or biased codon usage in parent genes. However, loss of sua5 led to increased expression of soluble proteins for mitochondrial function, protein quality processing/trafficking, oxidative stress response, and energy homeostasis. These results point to a global function for t6A in protein homeostasis very similar to mcm5/s2U modifications.


Assuntos
Proteínas de Ligação a DNA/genética , Histona Acetiltransferases/genética , RNA de Transferência/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Anticódon/genética , Anticódon/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histona Acetiltransferases/metabolismo , Conformação de Ácido Nucleico , Fenótipo , Agregados Proteicos/fisiologia , Biossíntese de Proteínas/genética , Biossíntese de Proteínas/fisiologia , Proteínas/genética , Proteômica/métodos , RNA de Transferência/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Termodinâmica , Tiouridina/análogos & derivados , Tiouridina/química
16.
Nucleic Acids Res ; 48(2): 912-923, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31802126

RESUMO

In eukaryotic ribosomes, the conserved protein uS19, formerly known as S15, extends with its C-terminal tail to the decoding site. The cross-linking of uS19 to the A site codon has been detected using synthetic mRNAs bearing 4-thiouridine (s4U) residues. Here, we showed that the A-site tRNA prevents this cross-linking and that the P site codon does not contact uS19. Next, we focused on determining uS19-mRNA interactions in vivo by applying the photoactivatable-ribonucleoside enhancing cross-linking and immunoprecipitation method to a stable HEK293 cell line producing FLAG-tagged uS19 and grown in a medium containing s4U. We found that when translation was stopped by cycloheximide, uS19 was efficiently cross-linked to mRNA regions with a high frequency of Glu, Lys and, more rarely, Arg codons. The results indicate that the complexes, in which the A site codon is not involved in the formation of the mRNA-tRNA duplex, are present among the cycloheximide-arrested 80S complexes, which implies pausing of elongating ribosomes at the above mRNA regions. Thus, our findings demonstrate that the human ribosomal protein uS19 interacts with mRNAs during translation elongation and highlight the regions of mRNAs where ribosome pausing occurs, bringing new structural and functional insights into eukaryotic translation in vivo.


Assuntos
RNA Mensageiro/química , Proteínas Ribossômicas/química , Ribossomos/química , Códon , Eucariotos/genética , Células HEK293 , Humanos , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , RNA de Transferência/química , RNA de Transferência/genética , Proteínas Ribossômicas/genética , Ribossomos/genética , Tiouridina/química
17.
Molecules ; 24(15)2019 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-31382376

RESUMO

Radiosensitizing properties of substituted uridines are of great importance for radiotherapy. Very recently, we confirmed 5-iodo-4-thio-2'-deoxyuridine (ISdU) as an efficient agent, increasing the extent of tumor cell killing with ionizing radiation. To our surprise, a similar derivative of 4-thio-2'-deoxyuridine, 5-bromo-4-thio-2'-deoxyuridine (BrSdU), does not show radiosensitizing properties at all. In order to explain this remarkable difference, we carried out a radiolytic (stationary and pulse) and quantum chemical studies, which allowed the pathways to all radioproducts to be rationalized. In contrast to ISdU solutions, where radiolysis leads to 4-thio-2'-deoxyuridine and its dimer, no dissociative electron attachment (DEA) products were observed for BrSdU. This observation seems to explain the lack of radiosensitizing properties of BrSdU since the efficient formation of the uridine-5-yl radical, induced by electron attachment to the modified nucleoside, is suggested to be an indispensable attribute of radiosensitizing uridines. A larger activation barrier for DEA in BrSdU, as compared to ISdU, is probably responsible for the closure of DEA channel in the former system. Indeed, besides DEA, the XSdU anions may undergo competitive protonation, which makes the release of X- kinetically forbidden.


Assuntos
Halogênios/química , Radiossensibilizantes/química , Tiouridina/análogos & derivados , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Histonas/metabolismo , Humanos , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Radiossensibilizantes/farmacologia , Espectrometria de Massas em Tandem , Tiouridina/química , Tiouridina/farmacologia
18.
Nat Chem ; 11(5): 457-462, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30936523

RESUMO

Breakthroughs in the study of the origin of life have demonstrated how some of the building blocks essential to biology could have been formed under various primordial scenarios, and could therefore have contributed to the chemical evolution of life. Missing building blocks are then sometimes inferred to be products of primitive biosynthesis, which can stretch the limits of plausibility. Here, we demonstrate the synthesis of 2'-deoxy-2-thiouridine, and subsequently 2'-deoxyadenosine and 2-deoxyribose, under prebiotic conditions. 2'-Deoxy-2-thiouridine is produced by photoreduction of 2,2'-anhydro-2-thiouridine, which is in turn formed by phosphorylation of 2-thiouridine-an intermediate of prebiotic RNA synthesis. 2'-Deoxy-2-thiouridine is an effective deoxyribosylating agent and may have functioned as such in either abiotic or proto-enzyme-catalysed pathways to DNA, as demonstrated by its conversion to 2'-deoxyadenosine by reaction with adenine, and 2-deoxyribose by hydrolysis. An alternative prebiotic phosphorylation of 2-thiouridine leads to the formation of its 5'-phosphate, showing that hypotheses in which 2-thiouridine was a key component of early RNA sequences are within the bounds of synthetic credibility.


Assuntos
Evolução Química , Tiouridina/análogos & derivados , Desoxiadenosinas/síntese química , Modelos Químicos , Oxirredução , Fosforilação , Tiouridina/síntese química , Tiouridina/química , Tiouridina/efeitos da radiação , Raios Ultravioleta
19.
Nucleic Acids Res ; 47(11): 5892-5905, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-30957850

RESUMO

The phenotypic adjustments of Mycobacterium tuberculosis are commonly inferred from the analysis of transcript abundance. While mechanisms of transcriptional regulation have been extensively analysed in mycobacteria, little is known about mechanisms that shape the transcriptome by regulating RNA decay rates. The aim of the present study is to identify the core components of the RNA degradosome of M. tuberculosis and to analyse their function in RNA metabolism. Using an approach involving cross-linking to 4-thiouridine-labelled RNA, we mapped the mycobacterial RNA-bound proteome and identified degradosome-related enzymes polynucleotide phosphorylase (PNPase), ATP-dependent RNA helicase (RhlE), ribonuclease E (RNase E) and ribonuclease J (RNase J) as major components. We then carried out affinity purification of eGFP-tagged recombinant constructs to identify protein-protein interactions. This identified further interactions with cold-shock proteins and novel KH-domain proteins. Engineering and transcriptional profiling of strains with a reduced level of expression of core degradosome ribonucleases provided evidence of important pleiotropic roles of the enzymes in mycobacterial RNA metabolism highlighting their potential vulnerability as drug targets.


Assuntos
Mycobacterium tuberculosis/metabolismo , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , RNA/análise , RNA Helicases DEAD-box/metabolismo , Endorribonucleases/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Complexos Multienzimáticos , Mycobacterium smegmatis/metabolismo , Polirribonucleotídeo Nucleotidiltransferase/genética , Proteoma , Proteômica , RNA/química , RNA Helicases/metabolismo , Estabilidade de RNA , RNA Bacteriano/metabolismo , Ribonuclease III/metabolismo , Ribonucleases/metabolismo , Tiouridina/química , Transcriptoma
20.
Methods ; 155: 88-103, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30529548

RESUMO

Many open questions in RNA biology relate to the kinetics of gene expression and the impact of RNA binding regulatory factors on processing or decay rates of particular transcripts. Steady state measurements of RNA abundance obtained from RNA-seq approaches are not able to separate the effects of transcription from those of RNA decay in the overall abundance of any given transcript, instead only giving information on the (presumed steady-state) abundances of transcripts. Through the combination of metabolic labeling and high-throughput sequencing, several groups have been able to measure both transcription rates and decay rates of the entire transcriptome of an organism in a single experiment. This review focuses on the methodology used to specifically measure RNA decay at a global level. By comparing and contrasting approaches and describing the experimental protocols in a modular manner, we intend to provide both experienced and new researchers to the field the ability to combine aspects of various protocols to fit the unique needs of biological questions not addressed by current methods.


Assuntos
Química Click/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA Mensageiro/metabolismo , Coloração e Rotulagem/métodos , Transcriptoma , Animais , Biotina/análogos & derivados , Biotina/química , Bromouracila/análogos & derivados , Linhagem Celular , Humanos , Estabilidade de RNA , RNA Mensageiro/genética , Tiouracila/análogos & derivados , Tiouracila/química , Tiouracila/metabolismo , Tiouridina/química , Tiouridina/metabolismo , Uracila/análogos & derivados , Uracila/química , Uracila/metabolismo , Uridina/análogos & derivados , Uridina/química , Uridina/metabolismo
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