Structural insights into FTO's catalytic mechanism for the demethylation of multiple RNA substrates.
ABSTRACT: 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: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: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:FTO, the first RNA demethylase discovered, mediates the demethylation of N6-methyladenosine (m6A), installed internally on messenger RNA, and N6,2′-O-dimethyladenosine (m6Am), occurring at the +1 position from the 5’ cap. Despite extensive recent research on FTO, its physiological impact on cellular processes has yet to be fully elucidated. Here, we demonstrate that the cellular distribution of FTO is distinct among different cell lines, which critically affects the access of FTO to different RNA substrates. FTO binds multiple RNA substrates, including mRNA, U6 RNA, and tRNA. It mainly targets internal m6A when located in the cell nucleus and preferentially demethylates m6Am when residing in the cytoplasm. The expression levels of transcripts containing internal m6A are associated with the alteration of the FTO more so than transcripts containing m6Am. We also discover that N1-methyladenosine (m1A) in tRNA is a main substrate of FTO, with the FTO-catalyzed demethylation of target tRNAs repressing protein synthesis. Collectively, FTO-mediated RNA demethylation affects both mRNA level and translation through distinct pathways. Overall design: Profile the binding RNA of ALKBH1 by CLIP-seq in 3T3-L1 cells transfected with FLAG-tagged FTO. RNA-seq in HeLa cells transfected with FTO siRNA or control siRNA
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:The posttranscriptional modification of messenger RNA (mRNA) and transfer RNA (tRNA) provides an additional layer of regulatory complexity during gene expression. Here, we show that a tRNA methyltransferase, TRMT10A, interacts with an mRNA demethylase FTO (ALKBH9), both in vitro and inside cells. TRMT10A installs N 1-methylguanosine (m1G) in tRNA, and FTO performs demethylation on N 6-methyladenosine (m6A) and N 6,2'-O-dimethyladenosine (m6Am) in mRNA. We show that TRMT10A ablation not only leads to decreased m1G in tRNA but also significantly increases m6A levels in mRNA. Cross-linking and immunoprecipitation, followed by high-throughput sequencing results show that TRMT10A shares a significant overlap of associated mRNAs with FTO, and these mRNAs have accelerated decay rates potentially through the regulation by a specific m6A reader, YTHDF2. Furthermore, transcripts with increased m6A upon TRMT10A ablation contain an overrepresentation of m1G9-containing tRNAs codons read by tRNAGln(TTG), tRNAArg(CCG), and tRNAThr(CGT) These findings collectively reveal the presence of coordinated mRNA and tRNA methylations and demonstrate a mechanism for regulating gene expression through the interactions between mRNA and tRNA modifying enzymes.
Project description:N 6-methyladenosine (m6A) is the most common internal modification in eukaryotic mRNA and associated with numerous cellular processes in health and disease. Up- and down-regulation of its "writer" or "eraser" proteins alter the global m6A level; however, modifying distinct m6A sites has remained elusive. We genetically fused the dioxygenase FTO responsible for m6A demethylation to RCas9 as an RNA-targeting module. The resulting RCas9-FTO retained demethylation activity and bound to RNA in a sequence-specific manner depending on the sgRNA and PAMmer. Using SCARLET analysis, we quantified the m6A level at a specific site and analyzed the effect of the PAM-to-m6A distance on activity. Sequence-specific demethylation by RCas9-FTO was tested on different RNA combinations and showed up to 15-fold sequence preference for target RNA compared to off-target RNA. Taken together, RCas9-FTO represents a new tool for sequence-specific demethylation of m6A in RNA that can be readily adapted to any given RNA sequence and opens the door to studying the function of distinct m6A sites.
Project description:BACKGROUND:Despite its functional importance in various fundamental bioprocesses, studies of N6-methyladenosine (m6A) in the heart are lacking. Here, we show that the FTO (fat mass and obesity-associated protein), an m6A demethylase, plays a critical role in cardiac contractile function during homeostasis, remodeling, and regeneration. METHODS:We used clinical human samples, preclinical pig and mouse models, and primary cardiomyocyte cell cultures to study the functional role of m6A and FTO in the heart and in cardiomyocytes. We modulated expression of FTO by using adeno-associated virus serotype 9 (in vivo), adenovirus (both in vivo and in vitro), and small interfering RNAs (in vitro) to study its function in regulating cardiomyocyte m6A, calcium dynamics and contractility, and cardiac function postischemia. We performed methylated (m6A) RNA immunoprecipitation sequencing to map transcriptome-wide m6A, and methylated (m6A) RNA immunoprecipitation quantitative polymerase chain reaction assays to map and validate m6A in individual transcripts, in healthy and failing hearts, and in myocytes. RESULTS:We discovered that FTO has decreased expression in failing mammalian hearts and hypoxic cardiomyocytes, thereby increasing m6A in RNA and decreasing cardiomyocyte contractile function. Improving expression of FTO in failing mouse hearts attenuated the ischemia-induced increase in m6A and decrease in cardiac contractile function. This is performed by the demethylation activity of FTO, which selectively demethylates cardiac contractile transcripts, thus preventing their degradation and improving their protein expression under ischemia. In addition, we demonstrate that FTO overexpression in mouse models of myocardial infarction decreased fibrosis and enhanced angiogenesis. CONCLUSIONS:Collectively, our study demonstrates the functional importance of the FTO-dependent cardiac m6A methylome in cardiac contraction during heart failure and provides a novel mechanistic insight into the therapeutic mechanisms of FTO.
Project description:N6-methyladenosine (m6A) is the most abundant base modification found in messenger RNAs (mRNAs). The discovery of FTO as the first m6A mRNA demethylase established the concept of reversible RNA modification. Here, we present a comprehensive transcriptome-wide analysis of RNA demethylation and uncover FTO as a potent regulator of nuclear mRNA processing events such as alternative splicing and 3? end mRNA processing. We show that FTO binds preferentially to pre-mRNAs in intronic regions, in the proximity of alternatively spliced (AS) exons and poly(A) sites. FTO knockout (KO) results in substantial changes in pre-mRNA splicing with prevalence of exon skipping events. The alternative splicing effects of FTO KO anti-correlate with METTL3 knockdown suggesting the involvement of m6A. Besides, deletion of intronic region that contains m6A-linked DRACH motifs partially rescues the FTO KO phenotype in a reporter system. All together, we demonstrate that the splicing effects of FTO are dependent on the catalytic activity in vivo and are mediated by m6A. Our results reveal for the first time the dynamic connection between FTO RNA binding and demethylation activity that influences several mRNA processing events.
Project description:N6-methyladenosine (m6A) is a reversible modification in mRNA and has been shown to regulate processing, translation and decay of mRNA. However, the roles of m6A modification in neuronal development are still not known. Here, we found that the m6A eraser FTO is enriched in axons and can be locally translated. Axon-specific inhibition of FTO by rhein, or compartmentalized siRNA knockdown of Fto in axons led to increases of m6A levels. GAP-43 mRNA is modified by m6A and is a substrate of FTO in axons. Loss-of-function of this non-nuclear pool of FTO resulted in increased m6A modification and decreased local translation of axonal GAP-43 mRNA, which eventually repressed axon elongation. Mutation of a predicted m6A site in GAP-43 mRNA eliminated its m6A modification and exempted regulation of its local translation by axonal FTO. This work showed an example of dynamic internal m6A demethylation of non-nuclear localized mRNA by the demethylase FTO. Regulation of m6A modification of axonal mRNA by axonal FTO might be a general mechanism to control their local translation in neuronal development.
Project description:mRNAs are regulated by nucleotide modifications that influence their cellular fate. Two of the most abundant modified nucleotides are N6-methyladenosine (m6A), found within mRNAs, and N6,2'-O-dimethyladenosine (m6Am), which is found at the first transcribed nucleotide. Distinguishing these modifications in mapping studies has been difficult. Here, we identify and biochemically characterize PCIF1, the methyltransferase that generates m6Am. We find that PCIF1 binds and is dependent on the m7G cap. By depleting PCIF1, we generated transcriptome-wide maps that distinguish m6Am and m6A. We find that m6A and m6Am misannotations arise from mRNA isoforms with alternative transcription start sites (TSSs). These isoforms contain m6Am that maps to "internal" sites, increasing the likelihood of misannotation. We find that depleting PCIF1 does not substantially affect mRNA translation but is associated with reduced stability of a subset of m6Am-annotated mRNAs. The discovery of PCIF1 and our accurate mapping technique will facilitate future studies to characterize m6Am's function.