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[This corrects the article DOI: 10.3389/fmicb.2021.632686.].
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The Tevenvirinae viruses are some of the most common viruses on Earth. Representatives of this subfamily have long been used in the molecular biology studies as model organisms - since the emergence of the discipline. Tevenvirinae are promising agents for phage therapy in animals and humans, since their representatives have only lytic life cycle and many of their host bacteria are pathogens. As confirmed experimentally, some Tevenvirinae have non-canonical DNA bases. Non-canonical bases can play an essential role in the diversification of closely related viruses. The article performs a comparative and evolutionary analysis of Tevenvirinae genomes and components of Tevenvirinae genomes. A comparative analysis of these genomes and the genes associated with the synthesis of non-canonical bases allows us to conclude that non-canonical bases have a major influence on the divergence of Tevenvirinae viruses within the same habitats. Supposedly, Tevenvirinae developed a strategy for changing HGT frequency in individual populations, which was based on the accumulation of proteins for the synthesis of non-canonical bases and proteins that used those bases as substrates. Owing to this strategy, ancestors of Tevenvirinae with the highest frequency of HGT acquired genes that allowed them to exist in a certain niche, and ancestors with the lowest HGT frequency preserved the most adaptive of those genes. Given the origin and characteristics of genes associated with the synthesis of non-canonical bases in Tevenvirinae, one can assume that other phages may have similar strategies. The article demonstrates the dependence of genomic diversity of closely related Tevenvirinae on non-canonical bases.
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The non-natural ethynylmethylpyridone C-nucleoside (W), a thymidine (T) analogue that can be incorporated in oligonucleotides by automated synthesis, has recently been reported to form a high fidelity base pair with adenosine (A) and to be well accommodated in B-DNA duplexes. The enhanced binding affinity for A of W, as compared to T, makes it an ideal modification for biotechnological applications, such as efficient probe hybridization for the parallel detection of multiple DNA strands. In order to complement the experimental study and rationalize the impact of the non-natural W nucleoside on the structure, stability and dynamics of DNA structures, we performed quantum mechanics (QM) calculations along with molecular dynamics (MD) simulations. Consistently with the experimental study, our QM calculations show that the A:W base pair has an increased stability as compared to the natural A:T pair, due to an additional CH-π interaction. Furthermore, we show that mispairing between W and guanine (G) causes a distortion in the planarity of the base pair, thus explaining the destabilization of DNA duplexes featuring a G:W pair. MD simulations show that incorporation of single or multiple consecutive A:W pairs in DNA duplexes causes minor changes to the intra- and inter-base geometrical parameters, while a moderate widening/shrinking of the major/minor groove of the duplexes is observed. QM calculations applied to selected stacks from the MD simulations also show an increased stacking energy for W, over T, with the neighboring bases.
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RNA modifications are essential for proper RNA processing, quality control, and maturation steps. In the last decade, some eukaryotic DNA repair enzymes have been shown to have an ability to recognize and process modified RNA substrates and thereby contribute to RNA surveillance. Single-strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1) is a base excision repair enzyme that not only recognizes and removes uracil and oxidized pyrimidines from DNA but is also able to process modified RNA substrates. SMUG1 interacts with the pseudouridine synthase dyskerin (DKC1), an enzyme essential for the correct assembly of small nucleolar ribonucleoproteins (snRNPs) and ribosomal RNA (rRNA) processing. Here, we review rRNA modifications and RNA quality control mechanisms in general and discuss the specific function of SMUG1 in rRNA metabolism. Cells lacking SMUG1 have elevated levels of immature rRNA molecules and accumulation of 5-hydroxymethyluridine (5hmU) in mature rRNA. SMUG1 may be required for post-transcriptional regulation and quality control of rRNAs, partly by regulating rRNA and stability.
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Procesamiento Postranscripcional del ARN , ARN Ribosómico/metabolismo , Uracil-ADN Glicosidasa/metabolismo , Animales , Humanos , Modelos Moleculares , Estabilidad del ARN , ARN Ribosómico/químicaRESUMEN
Recent years have seen great progresses in third-generation sequencing. New commercial platforms from Oxford Nanopore Technologies (ONT) can generate ultra-long reads from single-molecule nucleic acid fragments of kilobases up to megabases, exceeding the limitation of short reads and dependency on template amplification suffered by the previous generation of sequencing technologies. Moreover, it can detect epigenetic modifications directly, as well as providing all-around field usage, being pocket-sized and low cost. It has already been applied to yeast research in many aspects, such as complete de novo genome assemblies, the phylogeny of large-brewing yeasts, gene isoform identification, and base modification detection. These applications have delivered novel insights into yeast genomic and transcriptomic analysis.
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Genoma Fúngico , Genómica , Secuenciación de Nanoporos/métodos , Schizosaccharomyces/genética , Biología Computacional/métodos , Reparación del ADN , Análisis de Datos , Perfilación de la Expresión Génica , Biblioteca Genómica , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Análisis de Secuencia de ADN , TranscriptomaRESUMEN
BACKGROUND: DNA and RNA of all cellular life forms and many viruses contain an expansive repertoire of modified bases. The modified bases play diverse biological roles that include both regulation of transcription and translation, and protection against restriction endonucleases and antibiotics. Modified bases are often recognized by dedicated protein domains. However, the elaborate networks of interactions and processes mediated by modified bases are far from being completely understood. RESULTS: We present a comprehensive census and classification of EVE domains that belong to the PUA/ASCH domain superfamily and bind various modified bases in DNA and RNA. We employ the "guilt by association" approach to make functional inferences from comparative analysis of bacterial and archaeal genomes, based on the distribution and associations of EVE domains in (predicted) operons and functional networks of genes. Prokaryotes encode two classes of EVE domain proteins, slow-evolving and fast-evolving ones. Slow-evolving EVE domains in α-proteobacteria are embedded in conserved operons, potentially involved in coupling between translation and respiration, cytochrome c biogenesis in particular, via binding 5-methylcytosine in tRNAs. In ß- and γ-proteobacteria, the conserved associations implicate the EVE domains in the coordination of cell division, biofilm formation, and global transcriptional regulation by non-coding 6S small RNAs, which are potentially modified and bound by the EVE domains. In eukaryotes, the EVE domain-containing THYN1-like proteins have been reported to inhibit PCD and regulate the cell cycle, potentially, via binding 5-methylcytosine and its derivatives in DNA and/or RNA. We hypothesize that the link between PCD and cytochrome c was inherited from the α-proteobacterial and proto-mitochondrial endosymbiont and, unexpectedly, could involve modified base recognition by EVE domains. Fast-evolving EVE domains are typically embedded in defense contexts, including toxin-antitoxin modules and type IV restriction systems, suggesting roles in the recognition of modified bases in invading DNA molecules and targeting them for restriction. We additionally identified EVE-like prokaryotic Development and Cell Death (DCD) domains that are also implicated in defense functions including PCD. This function was inherited by eukaryotes, but in animals, the DCD proteins apparently were displaced by the extended Tudor family proteins, whose partnership with Piwi-related Argonautes became the centerpiece of the Piwi-interacting RNA (piRNA) system. CONCLUSIONS: Recognition of modified bases in DNA and RNA by EVE-like domains appears to be an important, but until now, under-appreciated, common denominator in a variety of processes including PCD, cell cycle control, antivirus immunity, stress response, and germline development in animals.
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Proteínas Arqueales/metabolismo , Proteínas Bacterianas/metabolismo , ADN de Archaea/metabolismo , Genoma Arqueal , Genoma Bacteriano , ARN Bacteriano/metabolismoRESUMEN
The four canonical bases that make up genomic DNA are subject to a variety of chemical modifications in living systems. Recent years have witnessed the discovery of various new modified bases and of the enzymes responsible for their processing. Here, we review the range of DNA base modifications currently known and recent advances in chemical methodology that have driven progress in this field, in particular regarding their detection and sequencing. Elucidating the cellular functions of modifications remains an ongoing challenge; we discuss recent contributions to this area before exploring their relevance in medicine.
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ADN/química , Animales , Emparejamiento Base , Cromatografía Liquida/métodos , ADN/genética , Epigénesis Genética , Humanos , Espectrometría de Masas/métodos , Análisis de Secuencia de ADN/métodosRESUMEN
Telomerase biogenesis is a complex process where several steps remain poorly understood. Single-strand-selective uracil-DNA glycosylase (SMUG1) associates with the DKC1-containing H/ACA ribonucleoprotein complex, which is essential for telomerase biogenesis. Herein, we show that SMUG1 interacts with the telomeric RNA component (hTERC) and is required for co-transcriptional processing of the nascent transcript into mature hTERC. We demonstrate that SMUG1 regulates the presence of base modifications in hTERC, in a region between the CR4/CR5 domain and the H box. Increased levels of hTERC base modifications are accompanied by reduced DKC1 binding. Loss of SMUG1 leads to an imbalance between mature hTERC and its processing intermediates, leading to the accumulation of 3'-polyadenylated and 3'-extended intermediates that are degraded in an EXOSC10-independent RNA degradation pathway. Consequently, SMUG1-deprived cells exhibit telomerase deficiency, leading to impaired bone marrow proliferation in Smug1-knockout mice.
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Procesamiento Postranscripcional del ARN , ARN/fisiología , Telomerasa/metabolismo , Telómero/fisiología , Uracil-ADN Glicosidasa/metabolismo , Animales , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Femenino , Células HeLa , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Telomerasa/genética , Telomerasa/fisiología , Uracil-ADN Glicosidasa/genética , Uracil-ADN Glicosidasa/fisiologíaRESUMEN
5-methylcytosine (5mC) on CpG dinucleotides has been viewed as the major epigenetic modification in eukaryotes for a long time. Apart from 5mC, additional DNA modifications have been discovered in eukaryotic genomes. Many of these modifications are thought to be solely associated with DNA damage. However, growing evidence indicates that some base modifications, namely 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), 5-carboxylcytosine (5caC), and N6-methadenine (6mA), may be of biological relevance, particularly during early stages of embryo development. Although abundance of these DNA modifications in eukaryotic genomes can be low, there are suggestions that they cooperate with other epigenetic markers to affect DNA-protein interactions, gene expression, defense of genome stability and epigenetic inheritance. Little is still known about their distribution in different tissues and their functions during key stages of the animal lifecycle. This review discusses current knowledge and future perspectives of these novel DNA modifications in the mammalian genome with a focus on their dynamic distribution during early embryonic development and their potential function in epigenetic inheritance through the germ line.
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Polypeptides can fold into tertiary structures while they are synthesized by the ribosome. In addition to the amino acid sequence, protein folding is determined by several factors within the cell. Among others, the folding pathway of a nascent polypeptide can be affected by transient interactions with other proteins, ligands, or the ribosome, as well as by the translocation through membrane pores. Particularly, the translation machinery and the population of tRNA under different physiological or adaptive responses can dramatically affect protein folding. This review summarizes the scientific evidence describing the role of translation kinetics and tRNA populations on protein folding and addresses current efforts to better understand tRNA biology. It is organized into three main parts, which are focused on: (i) protein folding in the cellular context; (ii) tRNA biology and the complexity of the tRNA population; and (iii) available methods and technical challenges in the characterization of tRNA pools. In this manner, this work illustrates the ways by which functional properties of proteins may be modulated by cellular tRNA populations.
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We describe the comparative analysis of ribonucleic acid digests (CARD) approach for RNA modification analysis. This approach employs isotope labeling during RNase digestion, which allows the direct comparison of a tRNA of unknown modification status against a reference tRNA, whose sequence or modification status is known. The reference sample is labeled with 18O during RNase digestion while the candidate (unknown) sample is labeled with 16O. These RNase digestion products are combined and analyzed by mass spectrometry. Identical RNase digestion products will appear in the mass spectrum as characteristic doublets, separated by 2 Da due to the 16O/18O mass difference. Singlets arise in the mass spectrum when the sequence or modification status of a particular RNase digestion product from the reference is not matched in the candidate (unknown) sample. This CARD approach for RNA modification analysis simplifies the determination of differences between reference and candidate samples, providing a route for higher throughput screening of samples for modification profiles, including determination of tRNA methylation patterns.
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Espectrometría de Masas , ARN/química , Cromatografía Liquida , Epigénesis Genética , Perfilación de la Expresión Génica , Hidrólisis , Marcaje Isotópico , Espectrometría de Masas/métodos , ARN/metabolismo , TranscriptomaRESUMEN
We report a method for covalent modification of primers that enhances the specificity of PCR and increases the yield of specific amplification products at the end of PCR. The introduction of thermally stable covalent modifications, such as alkyl groups to the exocyclic amines of deoxyadenosine or cytosine residues at the 3'-ends of primers results in enhanced specificity of reactions. This higher specificity can result in greater sensitivity of detection by reducing competition with non-productive reactions. The reduction in the amplification of unintended byproducts is most apparent when both primers are modified at their respective 3'-ends. The T Ms of such modified primers are only slightly affected by the inclusion of these modifiers. The principal mode of action is believed to be driven by the poor enzyme extension of substrates with closely juxtaposed bulky alkyl groups, such as would result from the replication of primer dimer artifact.
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Mass spectrometry is a powerful analytical tool for identifying and characterizing structural modifications to the four canonical bases in RNA, information that is lost when using techniques such as PCR for RNA analysis. Here we described an updated method for sequence mapping of modified nucleosides in transfer RNA. This modification mapping approach utilizes knowledge of the modified nucleosides present in the sample along with the genome-derived tRNA sequence to readily locate modifications site-specifically in the tRNA sequence. The experimental approach involves isolation of the tRNA of interest followed by separate enzymatic digestion to nucleosides and oligonucleotides. Both samples are analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) and the data sets are then combined to yield the modification profile of the tRNA. Data analysis is facilitated by the use of unmodified sequence exclusion lists and new developments in software that can automate MS/MS spectral annotation. The method is illustrated using tRNA-Asn isolated from Thermus thermophilus.
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Cromatografía Liquida/métodos , ARN de Transferencia/aislamiento & purificación , Espectrometría de Masas en Tándem/métodos , Procesamiento Postranscripcional del ARN/genética , ARN de Transferencia/genética , Programas Informáticos , Thermus thermophilus/genéticaRESUMEN
In this article I discuss studies towards understanding the structure and function of DNA in the context of genomes from the perspective of a chemist. The first area I describe concerns the studies that led to the invention and subsequent development of a method for sequencing DNA on a genome scale at high speed and low cost, now known as Solexa/Illumina sequencing. The second theme will feature the four-stranded DNA structure known as a G-quadruplex with a focus on its fundamental properties, its presence in cellular genomic DNA and the prospects for targeting such a structure in cels with small molecules. The final topic for discussion is naturally occurring chemically modified DNA bases with an emphasis on chemistry for decoding (or sequencing) such modifications in genomic DNA. The genome is a fruitful topic to be further elucidated by the creation and application of chemical approaches.
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ADN/genética , ADN/química , ADN/metabolismo , G-Cuádruplex , Genoma , HumanosRESUMEN
The analysis of ribonucleic acids (RNA) by mass spectrometry has been a valuable analytical approach for more than 25 years. In fact, mass spectrometry has become a method of choice for the analysis of modified nucleosides from RNA isolated out of biological samples. This review summarizes recent progress that has been made in both nucleoside and oligonucleotide mass spectral analysis. Applications of mass spectrometry in the identification, characterization and quantification of modified nucleosides are discussed. At the oligonucleotide level, advances in modern mass spectrometry approaches combined with the standard RNA modification mapping protocol enable the characterization of RNAs of varying lengths ranging from low molecular weight short interfering RNAs (siRNAs) to the extremely large 23 S rRNAs. New variations and improvements to this protocol are reviewed, including top-down strategies, as these developments now enable qualitative and quantitative measurements of RNA modification patterns in a variety of biological systems.