Identification of Flavin Mononucleotide as a Cell‐Active Artificial <i>N</i><sup>6</sup>‐Methyladenosine RNA Demethylase

Xie, Lu; Yang, Xiaoti; Wang, Rui‐Li; Li, Zhi‐Yan; Liu, Li; Mao, Lanqun; Wang, Ming; Cheng, Liang

doi:10.1002/ange.201900901

Cited by 20 publications

(12 citation statements)

References 58 publications

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“…Current methods of manipulating RNA methylation are primarily based on modulating the expression of RNA methyltransferases (Mettl3/Mettl14/WTAP complex) or demethylase (FTO and/or ALKBH5) via bioengineering methods, which cause broad epigenetic changes and activation of endogenous retroviruses 9,[11][12][13][14] . Recently, flavin mononucleotide was identified as a cell active artificial m 6 A RNA demethylase, which provided a potential and powerful small molecule for RNA demethylation 15 . However, as these methods and reagents demethylate transcriptome globally, it is difficult to study the effect of specific RNA methylation events, and there is the risk of side effects in therapeutic use.…”

Section: Introductionmentioning

confidence: 99%

Targeted mRNA demethylation using an engineered dCas13b-ALKBH5 fusion protein

Chen

et al. 2019

Preprint

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AbstractStudies on biological functions ofN6-methyladenosine (m6A) modification in mRNA have sprung up in recent years. Here we construct and characterize a CRISPR-Cas13b-based tool for the first time that targeted m6A methylation of mRNA by fusing the catalytically dead Type VI-B Cas13 enzyme from Prevotella sp.P5-125 (dPspCas13b) with the m6A demethylase ALKBH5, which is named as dm6ACRISPR. Subsequently, such system is shown to specific demethylase the m6A of target mRNA such as CYB5A to increase its mRNA stability. In addition, the dm6ACRISPR system appeared to afford efficient demethylation of the target genes with tenuous off-target effects. Together, we provide a programmable andin vivomanipulation tool to study mRNA modification and its potential biological functions of specific gene.

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Section: Introductionmentioning

confidence: 99%

Targeted mRNA demethylation using an engineered dCas13b-ALKBH5 fusion protein

Chen

et al. 2019

Preprint

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“…26 However, this method, along with other labelling reactions, can not distinguish f 5 C from another formyl containing f 5 U. In our continuous effort to explore opto-bioorthogonal chemistry of RNA modifications, [33][34][35][36][37][38] we envisioned that a 4,5-type cyclization with both N 4 -amino and C5formyl functionalities would be an ideal solution to this. We firstly explored a bunch of nucleophiles that could selectively react with the carbonyl substituent (Table S1).…”

Section: Results and Disscusionsmentioning

confidence: 99%

“…MALDI-TOF analysis (Figure S18) and enzymatic cleavages (Figure 2F) also confirmed that the chemical recognition and functionalization with Wittig reagent 1 showed no sequence or conformation preference but a high selectivity for f 5 C modification, which is consistent with our previously developed chemical modulation of RNA epigenetics with small organic molecules. 37,38,54 with Wittig reagent 1 before (hν ˗ ) and after light irradiation (hν + ). HPLC chromatograms with a wavelength of 260 nm and 350 nm were recorded respectively.…”

Section: Results and Disscusionsmentioning

confidence: 99%

Photo‐Facilitated Detection and Sequencing of 5‐Formylcytidine RNA

et al. 2022

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The simultaneous detection of a specific post-transcriptional modification that decorates the natural RNA can provide comprehensive information to better understand cellular and molecular functions of this variation. 5-Formylcytidine f5C is one of the natural nucleotides that was discovered in the tRNA of many species during the oxidative demethylation of 5-methylcytidine m5C. Despite the evident importance of f5C in playing an important role in gene expression regulation, little is known about the exact amount and positions of this modified cytidine in RNAs, as well as its exact functions. To capture this type of information, we performed a site-specific dual functionalization of f5C with semi-stabilized ylide cyanomethylene triphenylphosphorane that can simultaneously seizure the C5-formyl and N4-amino groups in the cytidine residue under light irradiation. The photochemical labelling with f5C-containing oligonucleotides imparts a high selectivity towards f5C and allows distinction from structurally similar 5-formyluridine f5U. We implemented a detection strategy of f5C modification in RNA based on the fluorescence signal of the cyclization product 4,5-pyridin-2-amine-cytidine paC, which exhibited a relatively high fluorescence quantum yield. The results could clearly identify f5C with a limit of detection LOD at 0.58 nM. The chemical labelling was capable of altering the hydrogen bonding activity of the parent cytidine and enabled the modulation of the reverse transcription signature of f5C in sequencing profile. We showed that using this Pan-Seq strategy, f5C can be detected from tRNA segments with a single-base resolution. Taken together, this approach represents a sensitive, robust, antibody-free and applicable detection ad sequencing to f5C RNA.

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“…So far, only 3‐deazaadenosine has been proven to inhibit METTL3, but it has a broad‐spectrum efficacy and inhibits the activity of all m 6 A “readers” 140 . Furthermore, chemical oxidation eliminates the m 6 A modification of mRNA 141,142 . Although several m 6 A demethylase agents have been reported in the literature, their specificity or efficacy cannot achieve the goal of precise and suboptimal treatment.…”

Section: M6a and Related Inhibitorsmentioning

confidence: 99%

N⁶‐methyladenosine (m⁶A) RNA modification in human cancer

2020

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N6‐methyladenosine (m6A) RNA modification, first discovered in 1974, is the most prevalent, abundant and penetrating messenger RNA (mRNA) modification in eukaryotes. This governs the fate of modified transcripts, regulates RNA metabolism and biological processes, and participates in pathogenesis of numerous human diseases, especially in cancer through the reciprocal regulation of m6A methyltransferases (“writers”) and demethylases (“erasers”) and the binding proteins decoding m6A methylation (“readers”). Accumulating evidence indicates a complicated regulation network of m6A modification involving multiple m6A‐associated regulatory proteins whose biological functions have been further analysed. This review aimed to summarize the current knowledge on the potential significance and molecular mechanisms of m6A RNA modification in the initiation and progression of cancer.

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Identification of Flavin Mononucleotide as a Cell‐Active Artificial N⁶‐Methyladenosine RNA Demethylase

Cited by 20 publications

References 58 publications

Targeted mRNA demethylation using an engineered dCas13b-ALKBH5 fusion protein

Targeted mRNA demethylation using an engineered dCas13b-ALKBH5 fusion protein

Photo‐Facilitated Detection and Sequencing of 5‐Formylcytidine RNA

N⁶‐methyladenosine (m⁶A) RNA modification in human cancer

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Identification of Flavin Mononucleotide as a Cell‐Active Artificial N6‐Methyladenosine RNA Demethylase

Cited by 20 publications

References 58 publications

Targeted mRNA demethylation using an engineered dCas13b-ALKBH5 fusion protein

Targeted mRNA demethylation using an engineered dCas13b-ALKBH5 fusion protein

Photo‐Facilitated Detection and Sequencing of 5‐Formylcytidine RNA

N6‐methyladenosine (m6A) RNA modification in human cancer

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Product

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Identification of Flavin Mononucleotide as a Cell‐Active Artificial N⁶‐Methyladenosine RNA Demethylase

N⁶‐methyladenosine (m⁶A) RNA modification in human cancer