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N(6)-Methyladenosine: a conformational marker that regulates the substrate specificity of human demethylases FTO and ALKBH5.

ABSTRACT: N(6)-Methyladenosine (m6A) is currently one of the most intensively studied post-transcriptional modifications in RNA. Due to its critical role in epigenetics and physiological links to several human diseases, it is also of tremendous biological and medical interest. The m6A mark is dynamically reversed by human demethylases FTO and ALKBH5, however the mechanism by which these enzymes selectively recognise their target transcripts remains unclear. Here, we report combined biophysical and biochemical studies on the specificity determinants of m6A demethylases, which led to the identification of an m6A-mediated substrate discrimination mechanism. Our results reveal that m6A itself serves as a 'conformational marker', which induces different conformational outcomes in RNAs depending on sequence context. This critically impacts its interactions with several m6A-recognising proteins, including FTO and ALKBH5. Remarkably, through the RNA-remodelling effects of m6A, the demethylases were able to discriminate substrates with very similar nucleotide sequences. Our findings provide novel insights into the biological functions of m6A modifications. The mechanism identified in this work is likely of significance to other m6A-recognising proteins.

SUBMITTER: Zou S

PROVIDER: S-EPMC4860565 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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N(6)-Methyladenosine: a conformational marker that regulates the substrate specificity of human demethylases FTO and ALKBH5.

Zou Shui S Toh Joel D W JD Wong Kendra H Q KH Gao Yong-Gui YG Hong Wanjin W Woon Esther C Y EC

Scientific reports 20160509

N(6)-Methyladenosine (m6A) is currently one of the most intensively studied post-transcriptional modifications in RNA. Due to its critical role in epigenetics and physiological links to several human diseases, it is also of tremendous biological and medical interest. The m6A mark is dynamically reversed by human demethylases FTO and ALKBH5, however the mechanism by which these enzymes selectively recognise their target transcripts remains unclear. Here, we report combined biophysical and biochem ...[more]

PMID: 27156733

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Project description:Translation is one of the main gene expression steps targeted by cellular stress, commonly referred to as translational stress, which includes treatment with anticancer drugs. While translational stress blocks the translation initiation of bulk mRNAs, it nonetheless activates the translation of specific mRNAs known as short upstream open reading frames (uORFs)-mRNAs. Among these, the ATF4 mRNA encodes a transcription factor that reprograms gene expression in cells responding to various stresses. Although the stress-induced translation of the ATF4 mRNA relies on the presence of uORFs (upstream to the main ATF4 ORF), the mechanisms mediating this effect, particularly during chemoresistance, remain elusive. Here, we report that ALKBH5 (AlkB Homolog 5) and FTO (FTO: Fat mass and obesity-associated protein), the two RNA demethylating enzymes, promote the translation of ATF4 mRNA in a transformed liver cell line (Hep3B) treated with the chemotherapeutic drug sorafenib. Using the in vitro luciferase reporter translational assay, we found that depletion of both enzymes reduced the translation of the reporter ATF4 mRNA upon drug treatment. Consistently, depletion of either protein abrogates the loading of the ATF3 mRNA into translating ribosomes as assessed by polyribosome assays coupled to RT-qPCR. Collectively, these results indicate that the ALKBH5 and FTO-mediated translation of the ATF4 mRNA is regulated at its initiation step. Using in vitro methylation assays, we found that ALKBH5 is required for the inhibition of the methylation of a reporter ATF4 mRNA at a conserved adenosine (A235) site located at its uORF2, suggesting that ALKBH5-mediated translation of ATF4 mRNA involves demethylation of its A235. Preventing methylation of A235 by introducing an A/G mutation into an ATF4 mRNA reporter renders its translation insensitive to ALKBH5 depletion, supporting the role of ALKBH5 demethylation activity in translation. Finally, targeting either ALKBH5 or FTO sensitizes Hep3B to sorafenib-induced cell death, contributing to their resistance. In summary, our data show that ALKBH5 and FTO are novel factors that promote resistance to sorafenib treatment, in part by mediating the translation of ATF4 mRNA.

Project description:ContextN(6)-methyladenosine (m(6)A) modification plays a fundamental role in the epigenetic regulation of the mammalian transcriptome. m(6)A can be demethylated by fat mass- and obesity-associated (FTO) protein and α-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5) protein. However, the importance of m(6)A alteration in type 2 diabetes mellitus (T2DM) has not been explored.ObjectiveThe objective of the study was to investigate whether m(6)A content was reduced in T2DM patients and whether m(6)A content was correlated with the mRNA expression levels of the FTO and ALKBH5 genes.MethodsIn this case-control study, peripheral blood samples were obtained from 88 T2DM patients and 92 healthy controls. For the diabetic animal model experiment, blood samples were obtained from seven diabetic and eight nondiabetic rats. A sensitive liquid chromatography-electrospray ionization-tandem mass spectrometry method was developed for the determination of the m(6)A content in RNA, quantitative real-time PCR was used to examine the mRNA expression levels of the FTO and ALKBH5 genes, and high-resolution melting and DNA sequencing were used to detect FTO single-nucleotide polymorphisms.ResultsOur results showed that the m(6)A contents in the RNA from T2DM patients and diabetic rats were significantly lower compared with the control groups (P = 2.6 × 10(-24) for T2DM patients; P = .001 for diabetic rats, respectively), and T2DM can be characterized by the content of m(6)A. The mRNA expression level of FTO was significantly higher in T2DM patients than that of the controls (P = .0007) and was associated with the risk of T2DM (odds ratio 2.797, 95% confidence interval 1.452-5.389, P = .002). Moreover, the m(6)A contents were correlated with FTO mRNA expression.ConclusionsThese data suggest that the increased mRNA expression of FTO could be responsible for the reduction of m(6)A in T2DM, which may further increase the risk of complications of T2DM. Low m(6)A should be investigated further as a novel potential biomarker of T2DM.

Dataset Information

N(6)-Methyladenosine: a conformational marker that regulates the substrate specificity of human demethylases FTO and ALKBH5.

Publications

N(6)-Methyladenosine: a conformational marker that regulates the substrate specificity of human demethylases FTO and ALKBH5.

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