Sensitive determination of inosine RNA modification in single cell by chemical derivatization coupled with mass spectrometry analysis

Tao, Wan-Bing; Xie, Neng-Bin; Cheng, Qing-Yun; Feng, Yun; Yuan, Bi-Feng

doi:10.1016/j.cclet.2023.108243

Cited by 12 publications

(8 citation statements)

References 35 publications

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“…Sleep deprivation has been shown to affect epigenetic modifications . In our study, we observed a significant reduction of the m 6,6 A level in mitochondrial 12S rRNA in lung tissues of sleep-deprived mice.…”

Section: Resultssupporting

confidence: 63%

AlkB-Facilitated Demethylation Enables Quantitative and Site-Specific Detection of Dual Methylation of Adenosine in RNA

Guo,

Xie,

Chen

et al. 2023

Anal. Chem.

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RNA molecules undergo various chemical modifications that play critical roles in a wide range of biological processes. N 6 ,N 6 -Dimethyladenosine (m 6,6 A) is a conserved RNA modification and is essential for the processing of rRNA. To gain a deeper understanding of the functions of m 6,6 A, site-specific and accurate quantification of this modification in RNA is indispensable. In this study, we developed an AlkB-facilitated demethylation (AD-m 6,6 A) method for the site-specific detection and quantification of m 6,6 A in RNA. The N 6 ,N 6 -dimethyl groups in m 6,6 A can cause reverse transcription to stall at the m 6,6 A site, resulting in truncated cDNA. However, we found that Escherichia coli AlkB demethylase can effectively demethylate m 6,6 A in RNA, generating full-length cDNA from AlkB-treated RNA. By quantifying the amount of full-length cDNA produced using quantitative real-time PCR, we were able to achieve site-specific detection and quantification of m 6,6 A in RNA. Using the AD-m 6,6 A method, we successfully detected and quantified m 6,6 A at position 1851 of 18S rRNA and position 937 of mitochondrial 12S rRNA in human cells. Additionally, we found that the level of m 6,6 A at position 1007 of mitochondrial 12S rRNA was significantly reduced in lung tissues from sleep-deprived mice compared with control mice. Overall, the AD-m 6,6 A method provides a valuable tool for easy, accurate, quantitative, and site-specific detection of m 6,6 A in RNA, which can aid in uncovering the functions of m 6,6 A in human diseases.

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Section: Resultssupporting

confidence: 63%

AlkB-Facilitated Demethylation Enables Quantitative and Site-Specific Detection of Dual Methylation of Adenosine in RNA

Guo,

Xie,

Chen

et al. 2023

Anal. Chem.

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“…Liquid chromatography–mass spectrometry (LC–MS) is a preeminent technique that enables the detection of modifications in RNA and DNA with high specificity and sensitivity. − However, RNA is typically digested to nucleosides prior to LC–MS analysis, which allows measurement of the overall levels of modification in RNA but precludes the assignment of modifications to individual residues in RNA. − Sequencing-based approaches can tell the site information of modifications in RNA. For example, antibody-mediated immunoprecipitation or enzyme-assisted chemical labeling enrichment followed by sequencing (such as m 6 A-seq, MeRIP-seq, and m 6 A-SEAL) enables the mapping of m 6 A at approximately 100–200 nucleotide resolution.…”

Section: Introductionmentioning

confidence: 99%

Single-Base Resolution Detection of N⁶-Methyladenosine in RNA by Adenosine Deamination Sequencing

et al. 2023

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N 6 -Methyladenosine (m 6 A) is one of the most abundant and prevalent natural modifications occurring in diverse RNA species. m 6 A plays a wide range of roles in physiological and pathological processes. Revealing the functions of m 6 A relies on the faithful detection of individual m 6 A sites in RNA. However, developing a simple method for the single-base resolution detection of m 6 A is still a challenging task. Herein, we report an adenosine deamination sequencing (AD-seq) technique for the facile detection of m 6 A in RNA at single-base resolution. The ADseq approach capitalizes on the selective deamination of adenosine, but not m 6 A, by the evolved tRNA adenosine deaminase (TadA) variant of TadA8e or the dimer protein of TadA-TadA8e. In AD-seq, adenosine is deaminated by TadA8e or TadA-TadA8e to form inosine, which pairs with cytidine and is read as guanosine in sequencing. m 6 A resists deamination due to the interference of the methyl group at the N6 position of adenosine. Thus, the m 6 A base pairs with thymine and is still read as adenosine in sequencing. The differential readouts from A and m 6 A in sequencing can achieve the single-base resolution detection of m 6 A in RNA. Application of the proposed AD-seq successfully identified individual m 6 A sites in Escherichia coli 23S rRNA. Taken together, the proposed AD-seq allows simple and cost-effective detection of m 6 A at single-base resolution in RNA, which provides a valuable tool to decipher the functions of m 6 A in RNA.

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“…Mass spectrometry has been widely used in determination of modified nucleosides. [ 44‐49 ] Using metabolic labeling and mass spectrometry (MS) techniques, the Carell group confirmed that the formyl group of 5fC can be directly removed from genomic DNA. [ 50 ] They used stable isotope‐labeled 5fC ([ 13 C 5 ][ 15 N 2 ]‐5fC) and 2'‐fluorinated 5fC (F‐5fC) to demonstrate the direct deformylation of 5fC (Figure 2).…”

Section: Direct Deformylation Of 5fc and Decarboxylation Of 5cacmentioning

confidence: 99%

Recent Advances in Deciphering the Mechanisms and Biological Functions of DNA Demethylation

Feng,

Chen,

Yuan

2023

Chin. J. Chem.

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Comprehensive Summary5‐Methylcytosine (5mC) is a dynamic and reversible epigenetic modification in genomic DNA of higher eukaryotes. It has been well‐established that the demethylation of 5mC occurs through the ten‐eleven translocation (TET)‐mediated oxidation of 5mC followed by thymine DNA glycosylase (TDG)‐initiated base excision repair (BER). Recent findings also have identified an alternative pathway of DNA demethylation. In this pathway, TET enzymes directly oxidize 5mC to form 5‐formylcytosine (5fC) or 5‐carboxylcytosine (5caC) through C–C bond cleavage. These modified bases can undergo direct deformylation or decarboxylation, respectively. Additionally, DNA demethylation can also occur through the deamination of 5mC and 5hmC, resulting in the production of thymine and 5‐hydroxymethyluracil (5hmU), respectively. Various DNA demethylation pathways possess critical functional implications and roles in biological processes. This Recent Advances article will focus on the studies of mechanisms and biological functions of DNA demethylation, shedding light on the reversible nature of the epigenetic modification of 5mC.This article is protected by copyright. All rights reserved.

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Sensitive determination of inosine RNA modification in single cell by chemical derivatization coupled with mass spectrometry analysis

Cited by 12 publications

References 35 publications

AlkB-Facilitated Demethylation Enables Quantitative and Site-Specific Detection of Dual Methylation of Adenosine in RNA

AlkB-Facilitated Demethylation Enables Quantitative and Site-Specific Detection of Dual Methylation of Adenosine in RNA

Single-Base Resolution Detection of N⁶-Methyladenosine in RNA by Adenosine Deamination Sequencing

Recent Advances in Deciphering the Mechanisms and Biological Functions of DNA Demethylation

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Sensitive determination of inosine RNA modification in single cell by chemical derivatization coupled with mass spectrometry analysis

Cited by 12 publications

References 35 publications

AlkB-Facilitated Demethylation Enables Quantitative and Site-Specific Detection of Dual Methylation of Adenosine in RNA

AlkB-Facilitated Demethylation Enables Quantitative and Site-Specific Detection of Dual Methylation of Adenosine in RNA

Single-Base Resolution Detection of N6-Methyladenosine in RNA by Adenosine Deamination Sequencing

Recent Advances in Deciphering the Mechanisms and Biological Functions of DNA Demethylation

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Single-Base Resolution Detection of N⁶-Methyladenosine in RNA by Adenosine Deamination Sequencing