Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome

Linder, Bastian; Grozhik, Anya V.; Olarerin-George, Anthony O.; Meydan, Cem; Mason, Christopher E.; Jaffrey, Samie R.

doi:10.1038/nmeth.3453

Cited by 1,196 publications

(1,507 citation statements)

References 33 publications

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“…While this manuscript was in preparation, data from Jaffrey and colleagues was published showing that FTO does not specifically demethylate m 6 A RNA, but instead prefers to demethylate m 6 A m (48 6 A relied on an antibody against m 6 A, but this antibody was shown to detect both m 6 A and m 6 A m (49). In addition, while the approach we used for m 6 A-seq did not provide us with the precise locations of demethylated adenosines in RNA from Gsk-3 DKO ESCs, we nonetheless observed hundreds of mRNAs had regions largely devoid of methylated adenosines.…”

Section: Discussionmentioning

confidence: 99%

“…6B) are both reduced by approximately 40% with respect to input mRNA in Gsk-3 DKO ESCs, providing independent validation of our m 6 A-seq results. Our approach for detecting m 6 A relied on an antibody against m 6 A, but this antibody was recently shown to detect both m 6 A and N 6 , 2'-Odimethyladenosine (m 6 A m ) (48), making it impossible for us to discriminate between these two RNA modifications using this technique (49). However, since m 6 A m is only found directly adjacent to the 7-methylguanosine cap on mRNA (50), we are confident that most of the changes we observe are m 6 A, but we cannot discount the possibility of changes in m 6 A m as well.…”

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confidence: 99%

See 1 more Smart Citation

Glycogen synthase kinase-3 (GSK-3) activity regulates mRNA methylation in mouse embryonic stem cells

Faulds

Egelston

Sedivy

et al. 2018

Journal of Biological Chemistry

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Glycogen synthase kinase-3 (Gsk-3) activity regulates multiple signal transduction pathways, and is also a key component of the network responsible for maintaining stem cell pluripotency. Genetic deletion of Gsk-3α and Gsk-3β or inhibition of Gsk-3 activity via small molecules promotes stem cell pluripotency, yet the mechanism underlying the role for Gsk-3 in this process remains ambiguous. Another cellular process that has been shown to affect stem cell pluripotency is mRNA methylation (m 6 A). Here, we describe an intersection between these components -the regulation of m 6 A by Gsk-3. We find that protein levels for the RNA demethylase, FTO (fat mass and obesity-associated protein), are elevated in Gsk-3α;Gsk-3β-deficient mouse embryonic stem cells (ESCs). FTO is normally phosphorylated by Gsk-3, and mass spectrometry identified the sites on FTO that are phosphorylated in a Gsk-3-dependent fashion. Gsk-3 phosphorylation of FTO leads to polyubiquitination, but in Gsk-3 knockout ESCs, that process is impaired, resulting in elevated levels of FTO protein. As a consequence of altered FTO protein levels, mRNAs in Gsk-3 knockout ESCs have 50% less m 6 A than wild-type ESCs, and m 6 A-seq analysis reveals the specific mRNAs that have reduced m 6 A modifications. Taken together, we provide the first evidence for how m 6 A demethylation is regulated in mammalian cells, and sheds light onto a possible novel mechanism by which Gsk-3 activity regulates stem cell pluripotency.Glycogen synthase kinase-3 (Gsk-3) activity is an important regulator of numerous signal transduction pathways (1). Gsk-3 activity is the sum of two largely redundant proteins, Gsk-3α and Gsk-3β, and in general, Gsk-3 is a negative regulator of cellular signaling (2). Rare among kinases, Gsk-3 is active at a basal state, while pathway activation from upstream signaling cascades results in the inhibition of Gsk-3 activity (2). Gsk-3α and Gsk-3β together regulate signal transduction pathways such as Wnt, protein kinase A (PKA), Hedgehog, transforming growth factor-β (TGF-β), nuclear factor of activated T-cells (NF-AT) and phosphatidylinositol 3-kinase (PI3K)-dependent insulin signaling in a variety of biological settings (3)(4)(5).Gsk-3 activity can be inhibited through the use of small molecule inhibitors, such as SB- (6)(7)(8), and the clinically relevant mood-stabilizer lithium (9,10); however, a drawback to the use of small molecules to study Gsk-3 function is the potential for off-target effects (11). Cells in which Gsk-3α and Gsk-3β have been genetically deleted allows for a more confident assessment of Gsk-3-specific functions. Therefore, we utilize mouse embryonic stem cells (ESCs) deficient in both Gsk-3α and Gsk-3β (Gsk-3α -/-; Gsk-3β -/-), i.e., Gsk-3 double knockout (DKO), to assess Gsk-3-specific functions (12,13). One prominent phenotype of Gsk-3 DKO ESCs is their persistent pluripotency, assessed by their inability to differentiate into all three germs layers in a teratoma assay, as well as by analysis of global gene expression ...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Glycogen synthase kinase-3 (GSK-3) activity regulates mRNA methylation in mouse embryonic stem cells

Faulds

Egelston

Sedivy

et al. 2018

Journal of Biological Chemistry

View full text Add to dashboard Cite

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“…Another RNA modification is 5-methylcytosine (5-mC) which is also an epigenetic mark. The details of the methods utilized for the understanding the different types RNA modifications are summarized in Table 1 [33][34][35][36][37][38][39][40][41][42].…”

Section: Rna Methylationmentioning

confidence: 99%

Recent Advancement in Methodology for Understanding Epigenetic Modifications

Tiwari¹

2017

J Clin Epigenet

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“…These features insure that the nucleotide modiications are accurately assigned to the appropriate RNA and not falsely atributed to homologous genes or RNA contaminates [6]. Now, several high-throughput NGS-based technologies, including RNA-seq, have been established to proile and quantitate RNA modiications (m A marks at singlenucleotide resolution [38]. As illustrated in Figure 1B Chemical-based methods rely on the misincorporation of nucleotide or nucleotide conversion to truncate or stop RNA products during reverse transcription.…”

Section: Ngs-based Rna Modiication Techniquesmentioning

confidence: 99%

Epitranscriptomics for Biomedical Discovery

Xiong¹,

Heruth²,

Jiang³

et al. 2017

Applications of RNA-Seq and Omics Strategies - From Microorganisms to Human Health

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Epitranscriptomics is a newly burgeoning ield pertaining to the complete delineation and elucidation of chemical modiications of nucleotides found within all classes of RNA that do not involve a change in the ribonucleotide sequence. More than 140 diverse and distinct nucleotide modiications have been identiied in RNA, dwaring the number of nucleotide modiications found in DNA. The majority of epitranscriptomic modiications have been identiied in ribosomal RNA (rRNA), transfer RNA (tRNA), and small nuclear RNA (snRNA). However, in total, the knowledge of the occurrence, and speciically the function, of RNA modiications remains scarce. Recently, the rapid advancement of next-generation sequencing and mass spectrometry technologies have allowed for the identiication and functional characterization of nucleotide modiications in both protein-coding and non-coding RNA on a global, transcriptome scale. In this chapter, we will introduce the concepts of nucleotide modiication, summarize transcriptome-wide RNA modiication mapping techniques, highlight recent studies exploring the functions of RNA modiications and their association to disease, and inally ofer insight into the future progression of epitranscriptomics.

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Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome

Cited by 1,196 publications

References 33 publications

Glycogen synthase kinase-3 (GSK-3) activity regulates mRNA methylation in mouse embryonic stem cells

Glycogen synthase kinase-3 (GSK-3) activity regulates mRNA methylation in mouse embryonic stem cells

Recent Advancement in Methodology for Understanding Epigenetic Modifications

Epitranscriptomics for Biomedical Discovery

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