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1.
Mol Cell ; 2021 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-34774131

RESUMO

Mitochondria contain a specific translation machinery for the synthesis of mitochondria-encoded respiratory chain components. Mitochondrial tRNAs (mt-tRNAs) are also generated from the mitochondrial DNA and, similar to their cytoplasmic counterparts, are post-transcriptionally modified. Here, we find that the RNA methyltransferase METTL8 is a mitochondrial protein that facilitates 3-methyl-cytidine (m3C) methylation at position C32 of the mt-tRNASer(UCN) and mt-tRNAThr. METTL8 knockout cells show a reduction in respiratory chain activity, whereas overexpression increases activity. In pancreatic cancer, METTL8 levels are high, which correlates with lower patient survival and an enhanced respiratory chain activity. Mitochondrial ribosome profiling uncovered mitoribosome stalling on mt-tRNASer(UCN)- and mt-tRNAThr-dependent codons. Further analysis of the respiratory chain complexes using mass spectrometry revealed reduced incorporation of the mitochondrially encoded proteins ND6 and ND1 into complex I. The well-balanced translation of mt-tRNASer(UCN)- and mt-tRNAThr-dependent codons through METTL8-mediated m3C32 methylation might, therefore, facilitate the optimal composition and function of the mitochondrial respiratory chain.

2.
RNA Biol ; : 1-10, 2021 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-34503375

RESUMO

Epitranscriptomic modifications of stable RNAs are dynamically regulated and specific profiles of 2'-O-methylation in rRNA have been associated with distinct cancer types. However, these observations pointed out the existence of at least two distinct groups: a rather large group with constitutive rRNA Nm residues exhibiting a stable level of methylation and a more restricted set of variable modifications, giving rise to the concept of 'specialized ribosomes'. These heterogeneous ribosomes can modulate their translational properties and be key regulatory players, depending on the physiological state of the cell. However, these conclusions were drawn from a limited set of explored human cell lines or tissues, mostly related to cancer cells of the same type. Here, we report a comprehensive analysis of human rRNA Nm modification variability observed for >15 human cell lines grown in different media and conditions. Our data demonstrate that human Nm sites can be classified into four groups, depending on their observed variability. About ⅓ of rRNA 2'-O-methylations are almost invariably modified at the same level in all tested samples (stable modifications), the second group of relatively invariant modifications (another ½ of the total) showing a slightly higher variance (low variable group) and two variable groups, showing an important heterogeneity. Mapping of these four classes on the human ribosome 3D structure shows that stably modified positions are preferentially located in the important ribosome functional sites, while variable and highly variable residues are mostly distributed to the ribosome periphery. Possible relationships of such stable and variable modifications to the ribosome functions are discussed.

3.
Methods ; 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34481083

RESUMO

Detection of RNA modified nucleotides using deep sequencing can be performed by several approaches, including antibody-driven enrichment and natural or chemically induced RT signatures. However, only very few RNA modified nucleotides generate natural RT signatures and antibody-driven enrichment heavily depends on the quality of antibodies used and may be highly biased. Thus, the use of chemically-induced RT signatures is now considered as the most trusted experimental approach. In addition, the use of chemical reagents allows inclusion of simple "mock-treated" controls, to exclude spontaneous RT arrests, SNPs and other misincorporation-prone sites. Hydrazine is a well-known RNA-specific reagent, already extensively used in the past for RNA sequencing and structural probing. Hydrazine is highly reactive to U and shows low reaction rates with ψ residues, allowing their distinction by deep sequencing-based protocols. However, other modified RNA residues also show particular behavior upon hydrazine treatment. Here we present methodological developments allowing to use HydraPsiSeq for precise quantification of RNA pseudouridylation and also detection and quantification of some other RNA modifications, in addition to ψ residues.

4.
Methods Enzymol ; 658: 25-47, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34517949

RESUMO

Precise and reliable mapping of modified nucleotides in RNA is a challenging task in epitranscriptomics analysis. Only deep sequencing-based methods are able to provide both, a single-nucleotide resolution and sufficient selectivity and sensitivity. A number of protocols employing specific chemical reagents to distinguish modified RNA nucleotides from canonical parental residues have already proven their performance. We developed a deep-sequencing analytical pipeline for simultaneous detection of several modified nucleotides of different nature (methylation, hydroxylation, reduction) in RNA. The AlkAniline-Seq protocol uses intrinsic fragility of the N-glycosidic bond present in certain modified residues (7-methylguanosine (m7G), 3-methylcytidine (m3C), dihydrouridine (D) and 5-hydroxycytidine (ho5C)) to induce cleavage under heat combined with alkaline conditions. The resulting RNA abasic site is decomposed by aniline-driven ß-elimination and creates a 5'-phosphate (5'-P) at the adjacent N+1 residue. This 5'-P is the crucial entry point for a highly selective ligation of sequencing adapters during the subsequent Illumina library preparation protocol. AlkAniline-Seq protocol has a very low background, and is both highly sensitive and specific. Applications of AlkAniline-Seq include mapping of m7G, m3C, D, and ho5C in variety of cellular RNAs, including in particular rRNAs and tRNAs.


Assuntos
Citidina , Guanosina , Citidina/análogos & derivados , Guanosina/análogos & derivados , Sequenciamento de Nucleotídeos em Larga Escala , RNA/genética , RNA de Transferência/genética , Análise de Sequência de RNA
6.
Methods Mol Biol ; 2298: 77-95, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085239

RESUMO

Epitranscriptomics is an emerging field where the development of high-throughput analytical technologies is essential to profile the dynamics of RNA modifications under different conditions. Despite important advances during the last 10 years, the number of RNA modifications detectable by next-generation sequencing is restricted to a very limited subset. Here, we describe a highly efficient and fast method called AlkAniline-Seq to map simultaneously two different RNA modifications: 7-methyl-guanosine (m7G) and 3-methyl-cytosine (m3C) in RNA. Our protocol is based on three subsequent chemical/enzymatic steps allowing the enrichment of RNA fragments ending at position n + 1 to the modified nucleotide, without any prior RNA selection. Therefore, AlkAniline-Seq demonstrates an outstanding sensitivity and specificity for these two RNA modifications. We have validated AlkAniline-Seq using bacterial, yeast, and human total RNA, and here we present, as an example, a synthetic view of the complete profiling of these RNA modifications in S. cerevisiae tRNAs.


Assuntos
Citosina/análogos & derivados , Guanosina/análogos & derivados , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA/genética , Linhagem Celular , Citosina/metabolismo , Guanosina/genética , Células HEK293 , Humanos , Metilação , Sensibilidade e Especificidade , Análise de Sequência de RNA/métodos
7.
Virologie (Montrouge) ; 25(2): 5-20, 2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33973850

RESUMO

Viral RNAs (either derived from DNA viruses and genomic/mRNAs of RNA viruses) produced and replicated in eukaryotic cells are exposed to the activity of host cell RNA modification machinery. Moreover, some complex viruses encode their own RNA modification enzymes, generally cap-related m7G-and 2'-O-methyltransferases whose expression allows specific modification of viral transcripts and modulation of viral RNA recognition by host restriction systems. Here we review current achievements in the detection of viral RNA modifications by liquid chromatography coupled to mass spectrometry (LC-MS) and deep sequencing-based approaches. The presence, origin and characterized functions of RNA modifications in viral RNAs are discussed.


Assuntos
Vírus de RNA , RNA Viral , Biologia , Metiltransferases , Vírus de RNA/genética , RNA Mensageiro , RNA Viral/genética
8.
Virologie (Montrouge) ; 25(2): 93-110, 2021 Apr 01.
Artigo em Francês | MEDLINE | ID: mdl-33973853

RESUMO

Viral RNAs (either derived from DNA viruses and genomic/mRNAs of RNA viruses) produced and replicated in eukaryotic cells are exposed to the activity of host cell RNA modification machinery. Moreover, some complex viruses encode their own RNA modification enzymes, generally cap-related m7G-and 2'-O-methyltransferases whose expression allows specific modification of viral transcripts and modulation of viral RNA recognition by host restriction systems. Here we review current achievements in the detection of viral RNA modifications by liquid chromatography coupled to mass spectrometry (LC-MS) and deep sequencing-based approaches. The presence, origin and characterized functions of RNA modifications in viral RNAs are discussed.


Assuntos
Metiltransferases , RNA Viral , Biologia , RNA Mensageiro , RNA Viral/genética
9.
Nucleic Acids Res ; 49(9): 4954-4970, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-33872355

RESUMO

Long non-coding RNAs have emerged as critical regulators of cell homeostasis by modulating gene expression at chromatin level for instance. Here, we report that the lncRNA ANRIL, associated with several pathologies, binds to thousands of loci dispersed throughout the mammalian genome sharing a 21-bp motif enriched in G/A residues. By combining ANRIL genomic occupancy with transcriptomic analysis, we established a list of 65 and 123 genes potentially directly activated and silenced by ANRIL in trans, respectively. We also found that Exon8 of ANRIL, mainly made of transposable elements, contributes to ANRIL genomic association and consequently to its trans-activity. Furthermore, we showed that Exon8 favors ANRIL's association with the FIRRE, TPD52L1 and IGFBP3 loci to modulate their expression through H3K27me3 deposition. We also investigated the mechanisms engaged by Exon8 to favor ANRIL's association with the genome. Our data refine ANRIL's trans-activity and highlight the functional importance of TEs on ANRIL's activity.


Assuntos
Elementos de DNA Transponíveis , Regulação da Expressão Gênica , RNA Longo não Codificante/química , RNA Longo não Codificante/metabolismo , DNA/química , Éxons , Loci Gênicos , Genoma Humano , Células HEK293 , Histonas/metabolismo , Humanos , RNA/química
10.
Methods Mol Biol ; 2300: 17-29, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33792868

RESUMO

Recent advances in high-throughput sequencing have shed new light on the diversity of small noncoding RNA (sncRNA) classes and their crucial roles in gene regulation and disease. One key step in sncRNA profiling consists in their quantification and assessment of their degradation extent. In this chapter, we will describe different gold standard methods used to achieve both purposes before using the sncRNAs in downstream applications.


Assuntos
Pequeno RNA não Traduzido/análise , Pequeno RNA não Traduzido/química , Eletroforese Capilar , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Estabilidade de RNA , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/isolamento & purificação , Análise de Sequência de RNA , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta
11.
Methods Mol Biol ; 2300: 165-182, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33792880

RESUMO

Extracellular RNAs (exRNAs) are secreted by nearly all cell types and are now known to play multiple physiological roles. Human plasma, a readily available sample for biomedical analysis, was reported to contain various subpopulations of exRNA, some of which are most likely components of plasma ribonucleoproteins (RNPs), while others are encapsulated into extracellular vesicles (EVs) of different size, origin, and composition. Unbiased analysis of exRNA composition can be performed with prefractionation of plasma exRNA followed by library preparation, sequencing, and bioinformatics analysis. In addition to "mature," adaptor ligation-competent RNA species (5'-P/3'-OH), human plasma contains a substantial proportion of degraded RNA fragments, featuring 5'-OH/3'-P or cyclophosphate extremities, which can be made competent for ligation using appropriate treatment. Polyethylene glycol (PEG)-based precipitation kits for EV isolation yield a fraction that is highly contaminated by large RNPs and EV-associated RNAs. Purer EV preparations are obtained by using Proteinase K and RNase A treatment, as well as by size-exclusion chromatography (SEC).


Assuntos
MicroRNAs/genética , MicroRNAs/isolamento & purificação , Plasma/química , Análise de Sequência de RNA/métodos , Fracionamento Químico , Cromatografia em Gel , Vesículas Extracelulares/genética , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Polietilenoglicóis/química , Ribonucleoproteínas/genética
12.
Genes (Basel) ; 12(2)2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33669207

RESUMO

The precise mapping and quantification of the numerous RNA modifications that are present in tRNAs, rRNAs, ncRNAs/miRNAs, and mRNAs remain a major challenge and a top priority of the epitranscriptomics field. After the keystone discoveries of massive m6A methylation in mRNAs, dozens of deep sequencing-based methods and protocols were proposed for the analysis of various RNA modifications, allowing us to considerably extend the list of detectable modified residues. Many of the currently used methods rely on the particular reverse transcription signatures left by RNA modifications in cDNA; these signatures may be naturally present or induced by an appropriate enzymatic or chemical treatment. The newest approaches also include labeling at RNA abasic sites that result from the selective removal of RNA modification or the enhanced cleavage of the RNA ribose-phosphate chain (perhaps also protection from cleavage), followed by specific adapter ligation. Classical affinity/immunoprecipitation-based protocols use either antibodies against modified RNA bases or proteins/enzymes, recognizing RNA modifications. In this survey, we review the most recent achievements in this highly dynamic field, including promising attempts to map RNA modifications by the direct single-molecule sequencing of RNA by nanopores.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Processamento Pós-Transcricional do RNA/genética , Transcriptoma/genética , DNA Complementar/genética , Humanos , Metilação , MicroRNAs/genética , RNA Mensageiro/genética , RNA de Transferência/genética
13.
Nat Commun ; 12(1): 1716, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33741917

RESUMO

Cancer stem cells (CSCs) are a small but critical cell population for cancer biology since they display inherent resistance to standard therapies and give rise to metastases. Despite accruing evidence establishing a link between deregulation of epitranscriptome-related players and tumorigenic process, the role of messenger RNA (mRNA) modifications in the regulation of CSC properties remains poorly understood. Here, we show that the cytoplasmic pool of fat mass and obesity-associated protein (FTO) impedes CSC abilities in colorectal cancer through its N6,2'-O-dimethyladenosine (m6Am) demethylase activity. While m6Am is strategically located next to the m7G-mRNA cap, its biological function is not well understood and has not been addressed in cancer. Low FTO expression in patient-derived cell lines elevates m6Am level in mRNA which results in enhanced in vivo tumorigenicity and chemoresistance. Inhibition of the nuclear m6Am methyltransferase, PCIF1/CAPAM, fully reverses this phenotype, stressing the role of m6Am modification in stem-like properties acquisition. FTO-mediated regulation of m6Am marking constitutes a reversible pathway controlling CSC abilities. Altogether, our findings bring to light the first biological function of the m6Am modification and its potential adverse consequences for colorectal cancer management.


Assuntos
Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Neoplasias Colorretais/metabolismo , Citoplasma/metabolismo , Desmetilação , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Metiltransferases/metabolismo , Proteínas Nucleares/metabolismo , RNA Mensageiro/metabolismo
14.
Crit Rev Biochem Mol Biol ; 56(2): 178-204, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33618598

RESUMO

Organisms from all domains of life invest a substantial amount of energy for the introduction of RNA modifications into nearly all transcripts studied to date. Instrumental analysis of RNA can focus on the modified residues and reveal the function of these epitranscriptomic marks. Here, we will review recent advances and breakthroughs achieved by NMR spectroscopy, sequencing, and mass spectrometry of the epitranscriptome.


Assuntos
Processamento Pós-Transcricional do RNA , RNA/genética , Animais , Epigênese Genética , Humanos , Espectrometria de Massas/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , RNA/química , Análise de Sequência de RNA/métodos , Transcriptoma
15.
Nat Commun ; 12(1): 389, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33452242

RESUMO

Recently, studies about RNA modification dynamics in human RNAs are among the most controversially discussed. As a main reason, we identified the unavailability of a technique which allows the investigation of the temporal processing of RNA transcripts. Here, we present nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS) for efficient, monoisotopic stable isotope labeling in both RNA and DNA in standard cell culture. We design pulse chase experiments and study the temporal placement of modified nucleosides in tRNAPhe and 18S rRNA. In existing RNAs, we observe a time-dependent constant loss of modified nucleosides which is masked by post-transcriptional methylation mechanisms and thus undetectable without NAIL-MS. During alkylation stress, NAIL-MS reveals an adaptation of tRNA modifications in new transcripts but not existing ones. Overall, we present a fast and reliable stable isotope labeling strategy which allows in-depth study of RNA modification dynamics in human cell culture.


Assuntos
Marcação por Isótopo/métodos , Espectrometria de Massas/métodos , Processamento Pós-Transcricional do RNA/fisiologia , RNA Ribossômico 18S/metabolismo , RNA de Transferência de Fenilalanina/metabolismo , Técnicas de Cultura de Células/métodos , Linhagem Celular , Desmetilação , Humanos , Cinética , Nucleosídeos/química , RNA Ribossômico 18S/química , RNA de Transferência de Fenilalanina/química , Reprodutibilidade dos Testes , Fatores de Tempo
16.
Genes (Basel) ; 12(1)2021 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-33435213

RESUMO

Analysis of RNA by deep-sequencing approaches has found widespread application in modern biology. In addition to measurements of RNA abundance under various physiological conditions, such techniques are now widely used for mapping and quantification of RNA modifications. Transfer RNA (tRNA) molecules are among the frequent targets of such investigation, since they contain multiple modified residues. However, the major challenge in tRNA examination is related to a large number of duplicated and point-mutated genes encoding those RNA molecules. Moreover, the existence of multiple isoacceptors/isodecoders complicates both the analysis and read mapping. Existing databases for tRNA sequencing provide near exhaustive listings of tRNA genes, but the use of such highly redundant reference sequences in RNA-seq analyses leads to a large number of ambiguously mapped sequencing reads. Here we describe a relatively simple computational strategy for semi-automatic collapsing of highly redundant tRNA datasets into a non-redundant collection of reference tRNA sequences. The relevance of the approach was validated by analysis of experimentally obtained tRNA-sequencing datasets for different prokaryotic and eukaryotic model organisms. The data demonstrate that non-redundant tRNA reference sequences allow improving unambiguous mapping of deep sequencing data.


Assuntos
Bacillus subtilis/genética , Bases de Dados de Ácidos Nucleicos , Escherichia coli/genética , RNA Bacteriano/genética , RNA de Transferência/genética , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de RNA
17.
Nucleic Acids Res ; 49(4): e23, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33313868

RESUMO

Methods for the detection of m6A by RNA-Seq technologies are increasingly sought after. We here present NOseq, a method to detect m6A residues in defined amplicons by virtue of their resistance to chemical deamination, effected by nitrous acid. Partial deamination in NOseq affects all exocyclic amino groups present in nucleobases and thus also changes sequence information. The method uses a mapping algorithm specifically adapted to the sequence degeneration caused by deamination events. Thus, m6A sites with partial modification levels of ∼50% were detected in defined amplicons, and this threshold can be lowered to ∼10% by combination with m6A immunoprecipitation. NOseq faithfully detected known m6A sites in human rRNA, and the long non-coding RNA MALAT1, and positively validated several m6A candidate sites, drawn from miCLIP data with an m6A antibody, in the transcriptome of Drosophila melanogaster. Conceptually related to bisulfite sequencing, NOseq presents a novel amplicon-based sequencing approach for the validation of m6A sites in defined sequences.


Assuntos
Adenosina/análogos & derivados , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA/química , Análise de Sequência de RNA/métodos , Adenosina/análise , Algoritmos , Animais , Cromatografia Líquida , Desaminação , Drosophila melanogaster/genética , Células HEK293 , Células HeLa , Humanos , RNA Longo não Codificante/química , RNA Mensageiro/química , RNA Ribossômico 18S/química , Alinhamento de Sequência , Espectrometria de Massas em Tandem
18.
Nucleic Acids Res ; 48(22): 12833-12844, 2020 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-33275131

RESUMO

RNA modifications are a well-recognized way of gene expression regulation at the post-transcriptional level. Despite the importance of this level of regulation, current knowledge on modulation of tRNA modification status in response to stress conditions is far from being complete. While it is widely accepted that tRNA modifications are rather dynamic, such variations are mostly assessed in terms of total tRNA, with only a few instances where changes could be traced to single isoacceptor species. Using Escherichia coli as a model system, we explored stress-induced modulation of 2'-O-methylations in tRNAs by RiboMethSeq. This analysis and orthogonal analytical measurements by LC-MS show substantial, but not uniform, increase of the Gm18 level in selected tRNAs under mild bacteriostatic antibiotic stress, while other Nm modifications remain relatively constant. The absence of Gm18 modification in tRNAs leads to moderate alterations in E. coli mRNA transcriptome, but does not affect polysomal association of mRNAs. Interestingly, the subset of motility/chemiotaxis genes is significantly overexpressed in ΔTrmH mutant, this corroborates with increased swarming motility of the mutant strain. The stress-induced increase of tRNA Gm18 level, in turn, reduced immunostimulation properties of bacterial tRNAs, which is concordant with the previous observation that Gm18 is a suppressor of Toll-like receptor 7 (TLR7)-mediated interferon release. This documents an effect of stress induced modulation of tRNA modification that acts outside protein translation.


Assuntos
Imunidade Inata/genética , Processamento Pós-Transcricional do RNA/genética , RNA de Transferência/genética , Receptor 7 Toll-Like/genética , Escherichia coli/genética , Regulação da Expressão Gênica/genética , Guanosina/genética , Guanosina/imunologia , Humanos , Interferons/genética , Interferons/imunologia , Metilação , Processamento Pós-Transcricional do RNA/imunologia , RNA de Transferência/imunologia , Receptor 7 Toll-Like/imunologia
19.
Nucleic Acids Res ; 48(19): e110, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-32976574

RESUMO

Developing methods for accurate detection of RNA modifications remains a major challenge in epitranscriptomics. Next-generation sequencing-based mapping approaches have recently emerged but, often, they are not quantitative and lack specificity. Pseudouridine (ψ), produced by uridine isomerization, is one of the most abundant RNA modification. ψ mapping classically involves derivatization with soluble carbodiimide (CMCT), which is prone to variation making this approach only semi-quantitative. Here, we developed 'HydraPsiSeq', a novel quantitative ψ mapping technique relying on specific protection from hydrazine/aniline cleavage. HydraPsiSeq is quantitative because the obtained signal directly reflects pseudouridine level. Furthermore, normalization to natural unmodified RNA and/or to synthetic in vitro transcripts allows absolute measurements of modification levels. HydraPsiSeq requires minute amounts of RNA (as low as 10-50 ng), making it compatible with high-throughput profiling of diverse biological and clinical samples. Exploring the potential of HydraPsiSeq, we profiled human rRNAs, revealing strong variations in pseudouridylation levels at ∼20-25 positions out of total 104 sites. We also observed the dynamics of rRNA pseudouridylation throughout chondrogenic differentiation of human bone marrow stem cells. In conclusion, HydraPsiSeq is a robust approach for the systematic mapping and accurate quantification of pseudouridines in RNAs with applications in disease, aging, development, differentiation and/or stress response.


Assuntos
Pseudouridina/isolamento & purificação , RNA Mensageiro , RNA Ribossômico , RNA de Transferência , Análise de Sequência de RNA/métodos , Células Cultivadas , Humanos , Células-Tronco Mesenquimais , Saccharomyces cerevisiae/genética
20.
Genes (Basel) ; 11(8)2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32824672

RESUMO

Reverse transcription of RNA templates containing modified ribonucleosides transfers modification-related information as misincorporations, arrest or nucleotide skipping events to the newly synthesized cDNA strand. The frequency and proportion of these events, merged from all sequenced cDNAs, yield a so-called RT signature, characteristic for the respective RNA modification and reverse transcriptase (RT). While known for DNA polymerases in so-called error-prone PCR, testing of four different RTs by replacing Mg2+ with Mn2+ in reaction buffer revealed the immense influence of manganese chloride on derived RT signatures, with arrest rates on m1A positions dropping from 82% down to 24%. Additionally, we observed a vast increase in nucleotide skipping events, with single positions rising from 4% to 49%, thus implying an enhanced read-through capability as an effect of Mn2+ on the reverse transcriptase, by promoting nucleotide skipping over synthesis abortion. While modifications such as m1A, m22G, m1G and m3C showed a clear influence of manganese ions on their RT signature, this effect was individual to the polymerase used. In summary, the results imply a supporting effect of Mn2+ on reverse transcription, thus overcoming blockades in the Watson-Crick face of modified ribonucleosides and improving both read-through rate and signal intensity in RT signature analysis.


Assuntos
Íons/metabolismo , Manganês/metabolismo , Transcrição Reversa , Pareamento de Bases , Íons/química , Manganês/química , RNA/genética , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/metabolismo , Ribonucleosídeos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
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