MRNA-Seq of mouse Mettl3 cKO and Fto cKO hippocampus after fear conditioning
ABSTRACT: N6-Methyladenosine (m6A) and N6,2′-O-dimethyladenosine (m6Am) are abundant mRNA modifications that regulate transcript processing and translation. The role of both, here termed m6A/m, in the stress response in the adult brain in vivo are currently unknown. Here, we investigated the effect of gene deletion of Mettl3, a m6A methyltransferase, and Fto, a m6A and m6Am demethlyase, induced in adulthood in excitatory neurons of the CA1 and CA3 in the hippocampus (Nex-CreERT2 Mettl3 or Fto cKO) on the transcriptome of CA1 and CA3 as well as the transcriptomic response of the CA1 and CA3 transcriptome to fear conditioning. Overall design: 5 biological replicates each for CA1/3 of Nex-CreERT2 Mettl3 wild type ("WT") and conditional knock-out mice ("cKO") or Nex-CreERT2 Fto WT and cKO, each at baseline("Box") or 24 h after fear conditioning ("FC").
Project description:N6-Methyladenosine (m6A) and N6,2′-O-dimethyladenosine (m6Am) are abundant mRNA modifications that regulate transcript processing and translation. The role of both, here termed m6A/m, in the stress response in the adult brain in vivo are currently unknown. Here, we investigated the effect of gene deletion of Mettl3, a m6A methyltransferase, and Fto, a m6A and m6Am demethlyase, induced in adulthood in excitatory neurons of the neocortex and hippocampus (Camk2a-Cre Mettl3 or Fto cKO) on the cortical epitranscriptome. PolyA-RNA-fragments from 3-5 replicates per group were processed both as m6A/m-sample (RNA immunoprecipiation RIP with an m6A and m6Am antibody) and RNA-input sample. Overall design: 5 replicates of WT mice (from both lines) as well as 3 replicates of Mettl3 cKO and 3 replicates of Fto cKO mice (cortex sample from 3 mice pooled per replicate) were processed as m6A/m-immunoprecipitation-sample ("m6A") or as input RNA samples ("RNA"). Additionally, 1 RNA sample (equimolar mixed from all samples) was processed as IgG control.
Project description:FTO, the first RNA demethylase discovered, mediates the demethylation of internal N6-methyladenosine (m6A) and N6, 2-O-dimethyladenosine (m6Am) at the +1 position from the 5' cap in mRNA. Here we demonstrate that the cellular distribution of FTO is distinct among different cell lines, affecting the access of FTO to different RNA substrates. We find that FTO binds multiple RNA species, including mRNA, snRNA, and tRNA, and can demethylate internal m6A and cap m6Am in mRNA, internal m6A in U6 RNA, internal and cap m6Am in snRNAs, and N1-methyladenosine (m1A) in tRNA. FTO-mediated demethylation has a greater effect on the transcript levels of mRNAs possessing internal m6A than the ones with cap m6Am in the tested cells. We also show that FTO can directly repress translation by catalyzing m1A tRNA demethylation. Collectively, FTO-mediated RNA demethylation occurs to m6A and m6Am in mRNA and snRNA as well as m1A in tRNA.
Project description:FTO demethylates internal N 6-methyladenosine (m6A) and N 6,2'-O-dimethyladenosine (m6Am; at the cap +1 position) in mRNA, m6A and m6Am in snRNA, and N 1-methyladenosine (m1A) in tRNA in vivo, and in vitro evidence supports that it can also demethylate N 6-methyldeoxyadenosine (6mA), 3-methylthymine (3mT), and 3-methyluracil (m3U). However, it remains unclear how FTO variously recognizes and catalyzes these diverse substrates. Here we demonstrate-in vitro and in vivo-that FTO has extensive demethylation enzymatic activity on both internal m6A and cap m6Am Considering that 6mA, m6A, and m6Am all share the same nucleobase, we present a crystal structure of human FTO bound to 6mA-modified ssDNA, revealing the molecular basis of the catalytic demethylation of FTO toward multiple RNA substrates. We discovered that (i) N 6-methyladenine is the most favorable nucleobase substrate of FTO, (ii) FTO displays the same demethylation activity toward internal m6A and m6Am in the same RNA sequence, suggesting that the substrate specificity of FTO primarily results from the interaction of residues in the catalytic pocket with the nucleobase (rather than the ribose ring), and (iii) the sequence and the tertiary structure of RNA can affect the catalytic activity of FTO. Our findings provide a structural basis for understanding the catalytic mechanism through which FTO demethylates its multiple substrates and pave the way forward for the structure-guided design of selective chemicals for functional studies and potential therapeutic applications.
Project description:N6-methyladenosine (m6A) and N6,2'-O-dimethyladenosine (m6Am) are abundant mRNA modifications that regulate transcript processing and translation. The role of both, here termed m6A/m, in the stress response in the adult brain in vivo is currently unknown. Here, we provide a detailed analysis of the stress epitranscriptome using m6A/m-seq, global and gene-specific m6A/m measurements. We show that stress exposure and glucocorticoids region and time specifically alter m6A/m and its regulatory network. We demonstrate that deletion of the methyltransferase Mettl3 or the demethylase Fto in adult neurons alters the m6A/m epitranscriptome, increases fear memory, and changes the transcriptome response to fear and synaptic plasticity. Moreover, we report that regulation of m6A/m is impaired in major depressive disorder patients following glucocorticoid stimulation. Our findings indicate that brain m6A/m represents a novel layer of complexity in gene expression regulation after stress and that dysregulation of the m6A/m response may contribute to the pathophysiology of stress-related psychiatric disorders.
Project description:N6-Methyladenosine (m6A), the most prevalent modification in mammalian mRNA, plays important roles in numerous biological processes. Several m6A associated proteins such as methyltransferase like 3 (METTL3), methyltransferase like 14 (METTL14), α-ketoglutarate-dependent dioxygenase AlkB homolog 5 (ALKBH5) and YTH domain containing 2 (YTHDC2) are involved in the regulation of spermatogenesis and oogenesis. However, the role of the first detected m6A demethylase, fat mass and obesity associate protein (FTO), in germ cells remains elusive. Elucidation of FTO roles in the regulation of germ cell fate will provide novel insights into the mammalian reproduction. Mouse GC-1 spg cells were treated with the ester form of meclofenamic acid (MA2) to inhibit the demethylase activity of FTO. The cellular m6A and m6Am level were analyzed through high performance liquid chromatography combined with tandem mass spectrometry (HPLC/MS-MS). The cell apoptosis was detected via TUNEL and flow cytometry. The cell proliferation was detected through EdU and western blot. The mRNA level of core cyclin dependent kinases (CDKs) was quantified via q-PCR. RNA decay assay were performed to detect RNA stability. Dual fluorescence assay was conducted to study whether MA2 affects the expression of CDK2 dependent on the m6A modification at 3'UTR. MA2 significantly increased the cellular m6A level and down-regulated the expression of CDK1, CDK2, CDK6 and CdC25a, resulting in arrest of G1/S transition and decrease of cell proliferation. MA2 downregulated CDK2 mRNA stability. Additionally, mutation of the predicted m6A sites in the Cdk2-3'UTR could mitigated the degradation of CDK2 mRNA after MA2 treatment. MA2 affected CDKs expression through the m6A-dependent mRNA degradation pathway, and thus repressed spermatogonial proliferation.
Project description:Various methyltransferases and demethylases catalyse methylation and demethylation of N6-methyladenosine (m6A) and N6,2'-O-dimethyladenosine (m6Am) but precise methylomes uniquely mediated by each methyltransferase/demethylase are still lacking. Here, we develop m6A-Crosslinking-Exonuclease-sequencing (m6ACE-seq) to map transcriptome-wide m6A and m6Am at quantitative single-base-resolution. This allows for the generation of a comprehensive atlas of distinct methylomes uniquely mediated by every individual known methyltransferase or demethylase. Our atlas reveals METTL16 to indirectly impact manifold methylation targets beyond its consensus target motif and highlights the importance of precision in mapping PCIF1-dependent m6Am. Rather than reverse RNA methylation, we find that both ALKBH5 and FTO instead maintain their regulated sites in an unmethylated steady-state. In FTO's absence, anomalous m6Am disrupts snRNA interaction with nuclear export machinery, potentially causing aberrant pre-mRNA splicing events.
Project description:Internal bases in mRNA can be subjected to modifications that influence the fate of mRNA in cells. One of the most prevalent modified bases is found at the 5' end of mRNA, at the first encoded nucleotide adjacent to the 7-methylguanosine cap. Here we show that this nucleotide, N6,2'-O-dimethyladenosine (m6Am), is a reversible modification that influences cellular mRNA fate. Using a transcriptome-wide map of m6Am we find that m6Am-initiated transcripts are markedly more stable than mRNAs that begin with other nucleotides. We show that the enhanced stability of m6Am-initiated transcripts is due to resistance to the mRNA-decapping enzyme DCP2. Moreover, we find that m6Am is selectively demethylated by fat mass and obesity-associated protein (FTO). FTO preferentially demethylates m6Am rather than N6-methyladenosine (m6A), and reduces the stability of m6Am mRNAs. Together, these findings show that the methylation status of m6Am in the 5' cap is a dynamic and reversible epitranscriptomic modification that determines mRNA stability.
Project description:RNA modifications play critical roles in important biological processes. However, the functions of N6-methyladenosine (m6A) mRNA modification in cancer biology and cancer stem cells remain largely unknown. Here, we show that m6A mRNA modification is critical for glioblastoma stem cell (GSC) self-renewal and tumorigenesis. Knockdown of METTL3 or METTL14, key components of the RNA methyltransferase complex, dramatically promotes human GSC growth, self-renewal, and tumorigenesis. In contrast, overexpression of METTL3 or inhibition of the RNA demethylase FTO suppresses GSC growth and self-renewal. Moreover, inhibition of FTO suppresses tumor progression and prolongs lifespan of GSC-grafted mice substantially. m6A sequencing reveals that knockdown of METTL3 or METTL14 induced changes in mRNA m6A enrichment and altered mRNA expression of genes (e.g., ADAM19) with critical biological functions in GSCs. In summary, this study identifies the m6A mRNA methylation machinery as promising therapeutic targets for glioblastoma.
Project description:N6-methyladenosine (m6A) RNA methylation is the most abundant modification on mRNAs and plays important roles in various biological processes. The formation of m6A is catalyzed by a methyltransferase complex including methyltransferase-like 3 (METTL3) as a key factor. However, the in vivo functions of METTL3 and m6A modification in mammalian development remain unclear. Here, we show that specific inactivation of Mettl3 in mouse nervous system causes severe developmental defects in the brain. Mettl3 conditional knockout (cKO) mice manifest cerebellar hypoplasia caused by drastically enhanced apoptosis of newborn cerebellar granule cells (CGCs) in the external granular layer (EGL). METTL3 depletion-induced loss of m6A modification causes extended RNA half-lives and aberrant splicing events, consequently leading to dysregulation of transcriptome-wide gene expression and premature CGC death. Our findings reveal a critical role of METTL3-mediated m6A in regulating the development of mammalian cerebellum.
Project description:Although it has been proved that the epigenetic modification of DNA and histones is involved in the pathogenesis of systemic lupus erythematosus (SLE), there is no study to explore whether the modification of N6-methyladenosine (m6A) in RNA is involved. In this study, the mRNA levels of m6A "writers" (METTL3, MTEEL14, and WTAP), "erasers" (FTO and ALKBH5), and "readers" (YTHDF2) in peripheral blood were determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The results demonstrated that the mRNA levels of METTL3, WTAP, FTO, ALKBH5, and YTHDF2 in peripheral blood from SLE patients were significantly decreased. The levels of ALKBH5 mRNA in SLE patients were associated with anti-dsDNA, antinucleosome, rash, and ulceration. Multivariate logistic regression analysis showed that the level of ALKBH5 mRNA in peripheral blood is a risk factor of SLE (P < 0.001). Moreover, our results suggested that there was a positive correlation between m6A"writers" (METTL3 and WTAP), "erasers" (FTO and ALKBH5), and "readers" (YTHDF2) in SLE patients. This study suggests that the mRNA level of ALKBH5 in peripheral blood may be involved in the pathogenesis of SLE.