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Information on EC 1.14.11.53 - mRNA N6-methyladenine demethylase and Organism(s) Homo sapiens and UniProt Accession Q9C0B1
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1.14.11.53 mRNA N6-methyladenine demethylaseIUBMB Comments
Contains iron(II). Catalyses oxidative demethylation of mRNA N6-methyladenine. The FTO enzyme from human can also demethylate N3-methylthymine from single stranded DNA and N3-methyluridine from single stranded RNA [1,2] with low activity .
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Word Map
- 1.14.11.53
-
demethylation
- demethylases
- mettl14
-
reader
-
writer
-
erasers
-
ythdf1
- methyltransferases
-
epitranscriptomic
-
m6a-related
-
fto-mediated
-
m6a-dependent
-
hnrnpa2b1
-
methyltransferase-like
-
hnrnpc
-
lasso
-
m6a-modified
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igf2bp1
-
merip
-
merip-seq
-
m6a-binding
The taxonomic range for the selected organisms is: Homo sapiens
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Reaction Schemes
N6-methyladenine in mRNA
+
+
=
adenine in mRNA
+
+
+
Synonyms
alkbh5, fat mass and obesity-associated protein, alkb homolog 5, alkbh10b, n6-methyladenosine demethylase, m6a rna demethylase, alkylation repair homolog protein 5, alkbh9b, fat mass and obesity-associated enzyme, alkbh5 demethylase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
ALKBH5
FTO
ALKBH5
-
-
SYSTEMATIC NAME
IUBMB Comments
mRNA-N6-methyladenosine,2-oxoglutarate:oxygen oxidoreductase (formaldehyde-forming)
Contains iron(II). Catalyses oxidative demethylation of mRNA N6-methyladenine. The FTO enzyme from human can also demethylate N3-methylthymine from single stranded DNA and N3-methyluridine from single stranded RNA [1,2] with low activity [3].
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
biotin-AAGCTCCCATGTTAGGm6ATCAGTGTCTCGAG-biotin + 2-oxoglutarate + O2
biotin-AAGCTCCCATGTTAGGATCAGTGTCTCGAG-biotin + formaldehyde + succinate + CO2
-
-
-
?
CATGTTAGGATCAGTG + 2-oxoglutarate + O2
CATGTTAGGm6ATCAGTG + formaldehyde + succinate + CO2
-
-
-
?
N3-methylcytosine in single-stranded DNA + 2-oxoglutarate + O2
cytosine in single-stranded DNA + formaldehyde + succinate + CO2
strong preference toward N3-methylthymine over N3-methylcytosine in single-stranded DNA
-
-
?
N3-methylthymine in single-stranded DNA + 2-oxoglutarate + O2
thymine in single-stranded DNA + formaldehyde + succinate + CO2
strong preference toward N3-methylthymine over N3-methylcytosine in single-stranded DNA. Negligible activities against N3-methylthymine in double-stranded DNA
-
-
?
N3-methyluracil in single-stranded mRNA + 2-oxoglutarate + O2
uracil in single-stranded mRNA + formaldehyde + succinate + CO2
2-fold preference for N3-methyluracil in single-stranded mRNA as the substrate over N3-methylthymine in single-stranded DNA. Slightly higher efficiency over that of N3-methylthymine in single-stranded DNA
-
-
?
N6-methyladenine in mRNA + 2-oxoglutarate + O2
adenine in mRNA + formaldehyde + succinate + CO2
5'-CUCGAUACG(m6A)UCCGGUCAAA-3' + 2-oxoglutarate + O2
5'-CUCGAUACGAUCCGGUCAAA-3' + formaldehyde + succinate + CO2
-
-
-
?
5'-UACACUCGAUCUGG(m6A)CUAAAGCUGCUC-3'-biotin + 2-oxoglutarate + O2
5'-UACACUCGAUCUGGCUAAAGCUGCUC-3'-biotin + formaldehyde + succinate + CO2
-
-
-
-
?
biotin-AAGCTCCCATGTTAGGm6ATCAGTGTCTCGAG-biotin + 2-oxoglutarate + O2
biotin-AAGCTCCCATGTTAGGATCAGTGTCTCGAG-biotin + formaldehyde + succinate + CO2
-
-
-
?
CATGTTAGGATCAGTG + 2-oxoglutarate + O2
CATGTTAGGm6ATCAGTG + formaldehyde + succinate + CO2
-
-
-
?
CCCC(m6A)CCCCCCCCC + 2-oxoglutarate + O2
? + formaldehyde + succinate + CO2
-
30% demethylation
-
-
?
GA(m6A)CA + 2-oxoglutarate + O2
GAACA + formaldehyde + succinate + CO2
-
39% demethylation
-
-
?
GCGG(m6A)CUCCAGAUG + 2-oxoglutarate + O2
GCGGACUCCAGAUG + formaldehyde + succinate + CO2
-
31% demethylation
-
-
?
GG(m6A)CU + 2-oxoglutarate + O2
GGACU + formaldehyde + succinate + CO2
-
37% demethylation
-
-
?
N6-methyladenine in mRNA + 2-oxoglutarate + O2
adenine in mRNA + formaldehyde + succinate + CO2
N6-methyladenine in NANOG mRNA + 2-oxoglutarate + O2
adenine in NANOG mRNA + formaldehyde + succinate + CO2
-
-
-
-
?
N6-methyladenine in NEAT1 + 2-oxoglutarate + O2
adenine in NEAT1 + formaldehyde + succinate + CO2
substrate i.e. nuclear paraspeckle assembly transcript 1
-
-
?
N6-methyladenine in single-stranded DNA + 2-oxoglutarate + O2
adenine in single-stranded DNA + formaldehyde + succinate + CO2
the ALKBH5 catalytic domain (residues 74294) is active and can demethylate ssDNA and ssRNA with similar activity. m6A ssDNA may not be a physiologically relevant ALKBH5 substrate
-
-
?
N6-methyladenine in single-stranded DNA oligonucleotide + 2-oxoglutarate + O2
adenine in single-stranded DNA oligonucleotide + formaldehyde + succinate + CO2
-
-
-
?
N6-methyladenine in ZNF333 mRNA + 2-oxoglutarate + O2
adenine in ZNF333 mRNA + formaldehyde + succinate + CO2
-
-
-
?
N6-methyladenine in mRNA + 2-oxoglutarate + O2
adenine in mRNA + formaldehyde + succinate + CO2
-
-
-
?
N6-methyladenine in mRNA + 2-oxoglutarate + O2
adenine in mRNA + formaldehyde + succinate + CO2
m6A in nuclear RNA is the physiological substrate of the enzyme
-
-
?
N6-methyladenine in mRNA + 2-oxoglutarate + O2
adenine in mRNA + formaldehyde + succinate + CO2
15-mer m6A-containing ssRNA or 15-mer m3U-containing ssRNA. Over 50-fold preference of the enzyme for m6A (at pH 7.0) over m3U (qat pH 6.0)
-
-
?
N6-methyladenine in mRNA + 2-oxoglutarate + O2
adenine in mRNA + formaldehyde + succinate + CO2
-
-
-
-
?
N6-methyladenine in mRNA + 2-oxoglutarate + O2
adenine in mRNA + formaldehyde + succinate + CO2
-
-
-
?
N6-methyladenine in mRNA + 2-oxoglutarate + O2
adenine in mRNA + formaldehyde + succinate + CO2
-
-
-
-
?
N6-methyladenine in mRNA + 2-oxoglutarate + O2
adenine in mRNA + formaldehyde + succinate + CO2
-
m6A modification preferentially appears after G in the conserved motif RRm6ACH (R is A/G and H is A/C/U)
-
-
?
N6-methyladenine in mRNA + 2-oxoglutarate + O2
adenine in mRNA + formaldehyde + succinate + CO2
the ALKBH5 catalytic domain (residues 74294) is active and can demethylate ssDNA and ssRNA with similar activity
-
-
?
additional information
?
-
very low activity toward repairing N1-methyladenine in 49 mer single-stranded DNA
-
-
?
additional information
?
-
negligible activity with m6A-containing dsDNA and dsRNA. Treatment with one molar equivalent of enzyme at 16°C overnight results in a demethylation yield of 40% for dsDNA and 24% for dsRNA, respectively
-
-
?
additional information
?
-
-
negligible activity with m6A-containing dsDNA and dsRNA. Treatment with one molar equivalent of enzyme at 16°C overnight results in a demethylation yield of 40% for dsDNA and 24% for dsRNA, respectively
-
-
?
additional information
?
-
-
the enzyme binds preferentially to pre-mRNAs in intronic regions, in the proximity of alternatively spliced exons and poly(A) sites
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
N6-methyladenine in mRNA + 2-oxoglutarate + O2
adenine in mRNA + formaldehyde + succinate + CO2
N6-methyladenine in mRNA + 2-oxoglutarate + O2
adenine in mRNA + formaldehyde + succinate + CO2
N6-methyladenine in NANOG mRNA + 2-oxoglutarate + O2
adenine in NANOG mRNA + formaldehyde + succinate + CO2
-
-
-
-
?
additional information
?
-
-
the enzyme binds preferentially to pre-mRNAs in intronic regions, in the proximity of alternatively spliced exons and poly(A) sites
-
-
?
N6-methyladenine in mRNA + 2-oxoglutarate + O2
adenine in mRNA + formaldehyde + succinate + CO2
-
-
-
?
N6-methyladenine in mRNA + 2-oxoglutarate + O2
adenine in mRNA + formaldehyde + succinate + CO2
m6A in nuclear RNA is the physiological substrate of the enzyme
-
-
?
N6-methyladenine in mRNA + 2-oxoglutarate + O2
adenine in mRNA + formaldehyde + succinate + CO2
-
-
-
-
?
N6-methyladenine in mRNA + 2-oxoglutarate + O2
adenine in mRNA + formaldehyde + succinate + CO2
-
-
-
?
N6-methyladenine in mRNA + 2-oxoglutarate + O2
adenine in mRNA + formaldehyde + succinate + CO2
-
-
-
-
?
N6-methyladenine in mRNA + 2-oxoglutarate + O2
adenine in mRNA + formaldehyde + succinate + CO2
-
m6A modification preferentially appears after G in the conserved motif RRm6ACH (R is A/G and H is A/C/U)
-
-
?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Fe2+
Mn2+
the enzyme requires binding of the cosubstrate 2-oxoglutarate and Fe2+ for catalysis. In the crystallization trial, Fe2+ is replaced with Mn2+ to obtain a catalytically inactive form of the enzyme
Fe2+
the enzyme requires binding of the cosubstrate 2-oxoglutarate and Fe2+ for catalysis
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
2-[(1-hydroxy-2-oxo-2-phenylethyl)sulfanyl]acetic acid
cell proliferation of cell lines HL-60, CCRF-CEM, and K562 is suppressed at low micromolar concentrations. The effect is low or negligible in Jurkat cells and glioblastoma cell line A-172
-
4-[[(furan-2-yl)methyl]amino]-1,2-diazinane-3,6-dione
cell proliferation of cell lines HL-60, CCRF-CEM, and K562 is suppressed at low micromolar concentrations. The effect is low or negligible in Jurkat cells and glioblastoma cell line A-172
-
additional information
-
the presence of S-adenosyl-L-homocysteine, NaCl, MgCl2, dimethylsulfoxide, and Triton X-100 does not influence enzyme activity
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.00166
N6-methyladenine in mRNA
-
pH and temperature not specified in the publication
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0029
N6-methyladenine in mRNA
-
pH and temperature not specified in the publication
-
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
1.75
N6-methyladenine in mRNA
-
pH and temperature not specified in the publication
-
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.00084
2-[(1-hydroxy-2-oxo-2-phenylethyl)sulfanyl]acetic acid
Homo sapiens
pH not specified in the publication, temperature not specified in the publication
-
0.00179
4-[[(furan-2-yl)methyl]amino]-1,2-diazinane-3,6-dione
Homo sapiens
pH not specified in the publication, temperature not specified in the publication
-
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
6
demethylation of N3-methylthymine in single-stranded DNA and N3-methyluracil in single-stranded RNA
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
20
21
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
SwissProt
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
of ovaries, N6-methyladenosine increases in granulosa cells of human aged ovaries , while FTO-knockdown granulosa cells show faster aging-related phenotypes
m6A demethylase FTO is downregulated and N6-methyladenosine increases in granulosa cells of human aged ovaries
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
localised in nuclear speckles, which are subnuclear structures that are enriched in pre-messenger RNA splicing factors
localised in nuclear speckles, which are subnuclear structures that are enriched in pre-messenger RNA splicing factors
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
metabolism
comparison of isoforms FTO and ALKBH5. FTO follows a traditional oxidative N-demethylation pathway to catalyze conversion of m6A to hm6A with subsequent slow release of adenosine and formaldehyde. FTO behaves like a classical nonheme Fe(II)-2OG-dependent dioxygenase by performing stepwise oxidation, whereas ALKBH5 catalyzes a unique direct 6-methyladenosine-to-adeosine conversion. FTO gives 6-hydroxymethyladenosine as a major product and 6-formyladenosine as a minorproduct
physiological function
malfunction
metabolism
physiological function
physiological function
FTO acts as a senescence-retarding protein via N6-methyladenosine, and transcription factor subunit FOS knockdown significantly alleviates the aging of FTO-knockdown granulosa cells. FTO-knockdown granulosa cells show faster aging-related phenotypes, and increased N6-methyladenosine levels are found in the FOS-mRNA 3'UTR
physiological function
FTO loss-of-function mutations reduce the proliferation rate of cancer cells. FTO knockdown also inhibits the colony formation ability of lung cancer cells. FTO knockdown reduces lung cancer cells growth in vivo. FTO decreases the m6A level and increases mRNA stability of ubiquitin-specific protease (USP7), which relies on the demethylase activity of FTO
physiological function
HCT-116 cells show high expression of both FTO and programmed cell death-ligand 1 (PD-L1) proteins. The knockdown of FTO decreases mRNA and protein levels of PD-L1 in HCT-116 cells. Upon depletion of FTO in HCT-116 cells in the presence of IFN-gamma to upregulate PD-L1 expression, PD-L1 expression is reduced in an IFN-gamma signaling-independent manner. PD-L1 mRNA is m6A-modified and FTO binds to the PD-L1 mRNA in HCT-116 cells
physiological function
knockdown of FTO increases m6A methylation in critical protumorigenic melanoma cell-intrinsic genes including PD-1, CXCR4, and SOX10, leading to increased RNA decay through the m6A reader YTHDF2. Knockdown of FTO sensitizes melanoma cells to interferon gamma
malfunction
Alkbh5-deficient male mice have increased m6A in mRNA and are characterized by impaired fertility resulting from apoptosis that affects meiotic metaphase stage spermatocytes
malfunction
-
enzyme knockdown in MDAMB-231 human breast cancer cells significantly reduces their capacity for tumor initiation as a result of reduced numbers of breast cancer stem cells
metabolism
N6-methylation of adenosine is the most ubiquitous and abundant modification of nucleoside in eukaryotic mRNA and long non-coding RNA. This modification plays an essential role in the regulation of mRNA translation and RNA metabolism
metabolism
-
the enzyme enhances NANOG mRNA stability by catalyzing m6A demethylation
metabolism
comparison of isoforms FTO and ALKBH5. FTO behaves like a classical nonheme Fe(II)-2OG-dependent dioxygenase by performing stepwise oxidation, whereas ALKBH5 catalyzes a unique direct 6-methyladenosine-to-adenosine conversion with rapid release of formaldehyde. A catalytic R130/K132/Y139 triad within ALKBH5 facilitates release of formaldehyde via an covalent-based demethylation mechanism with direct detection of a covalent intermediate. In a mechanistic model for ALKBH5, K132 promotes Schiff base formation on hm6A, which may then undergo subsequent nucleophilic attack by K132 or Y139. Y139 may alternatively play a role in nucleobase recognition via hydrogen bonding to the N6 nitrogen. Formation of a methylene bridge between K132 and Y139 is a probable intermediate prior to hydrolysis and may facilitate release of adeosine
physiological function
-
enzyme expression promotes m6A RNA demethylation in hypoxic breast cancer cells
physiological function
-
the enzyme FTO plays a role in human obesity and energy utilization. FTO is involved in various diseases including cardiovascular diseases,Alzheimer's disease, type II diabetes, breast cancer, and end-stage renal disease. The enzyme ALKHB5 is involoved in sperm development
physiological function
-
the enzyme triggers inclusion of alternatively spliced exons and regulates expression of last exons
physiological function
ALKBH5 demethylates zinc finger protein ZNF333 mRNA, leading to enhanced ZNF333 expression by abolishing m6A-YTHDF2-dependent mRNA degradation. ALKBH5 activates CDX2 and downstream intestinal markers by targeting the ZNF333/CYLD axis and activating NF-kappaB signaling. p65, the key transcription factor of the canonical NF-kappaB pathway, enhances the transcription activity of ALKBH5 in the nucleus. ALKBH5 levels are positively correlated with ZNF333 and CDX2 levels in gastric intestinal metaplasia tissues
physiological function
ROS significantly induces global mRNA N6-methyladenosine levels by modulating ALKBH5 post-translational modifications, leading to the rapid and efficient induction of thousands of genes. DNA damage repair genes are ALKBH5 downstream targets induced by ROS. ROS promotes ALKBH5 SUMOylation through activating ERK/JNK signaling, leading to inhibition of ALKBH5m6A demethylase activity by blocking substrate accessibility
physiological function
substrate NEAT1 is a potential binding long noncoding lncRNA of ALKBH5. NEAT1 is overexpressed in gastric cancer cells and tissue. Knockdown of NEAT1 significantly represses invasion and metastasis of gastric cancer cells. ALKBH5 affects the m6A level of NEAT1. The binding of ALKBH5 and NEAT1 influences the expression of EZH2 (a subunit of the polycomb repressive complex) and thus affects gastric cancer invasion and metastasis
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). FTO_HUMAN
505
0
58282
Swiss-Prot
other Location (Reliability: 2)
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
sumoylation
ROS promotes ALKBH5 sumoylation, which blocks m6A demethylase activity by inhibition of substrate accessibility. Overexpression of sumoylation-deficient ALKBH5 blocks ROS-induced mRNA m6A methylation, leading to a significant delay of DNA repair and increase of cell apoptosis
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
hanging-drop vapour-diffusion method, crystal structure of FTO in complex with the mononucleotide 3-methylthymine
ALKBH566292 is crystallized in sitting drops at 20°C by the vapour diffusion method in the presence of Mn2+ and (1-chloro-4-hydroxyisoquinoline-3-carbonyl)glycine. Crystallization drops contain 0.2 ml of a protein solution containing a final concentration of 10 mg/ml hexahistidine-tagged ALKBH566292, 0.5 mM MnCl2 and 2 mM IOX3 mixed with 0.1 ml of well solution containing 125 mM potassium nitrate and 15% (w/v) polyethylene glycol 3350. Crystals (size 100 x 50 x 50 mM) appeared after 3 months. Crystals are harvested using nylon loops and cryoprotected using well solution diluted with 25% (v/v) glycerol and flashcooled in liquid nitrogen. Crystal structure of human ALKBH5 (residues 66-292) to 2.0 A resolution. ALKBH566292 has a double-stranded beta-helix core fold. The active site metal is octahedrally coordinated by an HXD...H motif (comprising residues His204, Asp206 and His266) and three water molecules. ALKBH5 shares a nucleotide recognition lid and conserved active site residues with other ferrous iron-dependent nucleic acid oxygenase
crystallizations are performed at 24 and 4°C using both the hanging drop and sitting drop vapor diffusion methods. Five high resolution crystal structures of the catalytic core of Alkbh5 in complex with different ligands. These findings provide a structural basis for understanding the substrate recognition specificity of Alkbh5 and offer a foundation for selective drug design against AlkB members
hanging drop vapor diffusion method at 18 °C. The ALKBH52-oxoglutarate-Mn2+ is crystallized in a buffer containing 0.2 M ammonium dihydrogen phosphate, 20% PEG 3350. The ALKBH5 is crystallized with citrate in a buffer containing ammonium citrate, 20% PEG 3350. Before flashfreezing crystals in liquid nitrogen, crystals are soaked in a cryoprotectant consisting mother liquor plus 12% glycerol
hanging drop vapor diffusion method, using 0.2 M ammonium citrate dibasic pH 5.1, 20% (w/v) PEG 3350
-
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
R316Q
mutation abolishes 80% of the wild-type activity towards m6A demethylation in vitro
269A/Q271A
double mutation shows no impact on the repair efficiency of Alkbh5
F232D/Q233D/F234E
the mutant enzyme displays a severe loss of activity, demonstrating only 13.5% of wild-type activity
K132Q
mutant furnishes the ALKBH5 enzyme with an m6A demethylation profile that resembles that of isoform FTO
K231A/K235A
double mutation shows no impact on the repair capacity of Alkbh5
K231E/K235E
double mutation shows no impact on the repair capacity of Alkbh5
K86R/K321R
ROS-induced global mRNA m6A modification is completely blocked by mutant K86R/K321R overexpression
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
the purified recombinant mRNA N6-methyladenine demethylase appears to be more stable than the purified recombinant murine mRNA N6-methyladenine demethylase in vitro
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
DE52 column chromatography, Ni-NTA column chromatography, and Superdex 200 gel filtration
-
Ni affinity column chromatography, HiTrap Q column chromatography, and Superdex 75 gel filtration
-
Ni-NTA His-Bind resin column chromatography, Source 15S column chromatography, and Superdex 200 gel filtration
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
Wild-type and various mutant enzymes cloned into pET-15b are expressed in Escherichia coli strain BL21
a plasmid with the vector backbone pNIC28-Bsa4 encoding a hexahistidine-tagged ALKBH566292 construct is transformed into Escherichia coli BL21 (DE3) cells
mutants of human Alkbh5 are amplified by PCR and subcloned into a modified pET-28a (Novagen) vector encoding a tobacco etch virus protease recognition site. The final clones are verified by DNA sequencing. All of the recombinant plasmids are transformed into Escherichia coli strain BL21(DE3)
the human ALKBH5 catalytic AlkB domain (residues 74294) is subcloned into pET28a-MHL vector and expressed. The cloned vector is transformed into Escherichia coli BL21-V2R-pRARE2. Recombinant protein is produced at 16°C as an N-terminal His-tagged protein
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
ALKBH5 mRNA levels are significantly increased under hypoxic conditions by greater than 1.6fold in SUM-159 and MDA-MB-435 cells and greater than 2fold in MDA-MB-231 and MCF-7 cells
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
analysis
development of a single-quantum-dot-based fluorescence resonance energy transfer (FRET) sensor for the identification of specific FTO demethylase inhibitors. The FTO-mediated demethylation of m6A can induce the cleavage of demethylated DNA to generate the biotinylated DNA fragments, which may function as capture probes to assemble the Cy5-labeled reporter probes onto the quantum dot surface, enabling the occurrence of FRET between the quantum dot and Cy5
medicine
analysis
development of a single-quantum-dot-based fluorescence resonance energy transfer (FRET) sensor for the identification of specific FTO demethylase inhibitors. The FTO-mediated demethylation of m6A can induce the cleavage of demethylated DNA to generate the biotinylated DNA fragments, which may function as capture probes to assemble the Cy5-labeled reporter probes onto the quantum dot surface, enabling the occurrence of FRET between the quantum dot and Cy5
medicine
substrate NEAT1 is a potential binding long noncoding lncRNA of ALKBH5. NEAT1 is overexpressed in gastric cancer cells and tissue. Knockdown of NEAT1 significantly represses invasion and metastasis of gastric cancer cells. ALKBH5 affects the m6A level of NEAT1. The binding of ALKBH5 and NEAT1 influences the expression of EZH2 (a subunit of the polycomb repressive complex) and thus affects gastric cancer invasion and metastasis
medicine
FTO is up-regulated in human breast cancer. High level of FTO is significantly associated with lower survival rates in patients with breast cancer. FTO promotes breast cancer cell proliferation, colony formation and metastasis in vitro and in vivo. BNIP3, a pro-apoptosis gene, is a downstream target of FTO-mediated m6A modification. BNIP3 acts as a tumor suppressor and is negatively correlated with FTO expression in clinical breast cancer patients. BNIP3 dramatically alleviates FTO-dependent tumor growth retardation and metastasis
medicine
FTO level is increased in human melanoma. Knockdown of FTO increases m6A methylation in critical protumorigenic melanoma cell-intrinsic genes including PD-1, CXCR4, and SOX10, leading to increased RNA decay through the m6A reader YTHDF2. Knockdown of FTO sensitizes melanoma cells to interferon gamma
medicine
FTO mRNA and protein levels are overexpressed in non-small cell lung cancer tissues and cell lines, associated with a reduced m6A content. FTO loss-of-function mutations reduce the proliferation rate of cancer cells. FTO knockdown also inhibits the colony formation ability of lung cancer cells. FTO knockdown reduces lung cancer cells growth in vivo. FTO decreases the m6A level and increases mRNA stability of ubiquitin-specific protease (USP7), which relies on the demethylase activity of FTO. USP7 mRNA level is overexpressed in human lung cancer tissues and USP7 expression was positively correlated with FTO mRNA level
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Jia, G.; Yang, C.G.; Yang, S.; Jian, X.; Yi, C.; Zhou, Z.; He, C.
Oxidative demethylation of 3-methylthymine and 3-methyluracil in single-stranded DNA and RNA by mouse and human FTO
FEBS Lett.
582
3313-3319
2008
Mus musculus (Q8BGW1), Homo sapiens (Q9C0B1)
Aik, W.; Scotti, J.S.; Choi, H.; Gong, L.; Demetriades, M.; Schofield, C.J.; McDonough, M.A.
Structure of human RNA N6-methyladenine demethylase ALKBH5 provides insights into its mechanisms of nucleic acid recognition and demethylation
Nucleic Acids Res.
42
4741-4754
2014
Homo sapiens (Q6P6C2), Homo sapiens
Zhou, B.; Han, Z.
Crystallization and preliminary X-ray diffraction of the RNA demethylase ALKBH5
Acta Crystallogr. Sect. F
69
1231-1234
2013
Homo sapiens
Zhu, C.; Yi, C.
Switching demethylation activities between AlkB family RNA/DNA demethylases through exchange of active-site residues
Angew. Chem. Int. Ed. Engl.
53
3659-3662
2014
Homo sapiens
Liu, K.; Ding, Y.; Ye, W.; Liu, Y.; Yang, J.; Liu, J.; Qi, C.
Structural and functional characterization of the proteins responsible for N6-methyladenosine modification and recognition
Curr. Protein Pept. Sci.
17
306-318
2016
Homo sapiens
Landfors, M.; Nakken, S.; Fusser, M.; Dahl, J.A.; Klungland, A.; Fedorcsak, P.
Sequencing of FTO and ALKBH5 in men undergoing infertility work-up identifies an infertility-associated variant and two missense mutations
Fertil. Steril.
105
1170-1179
2016
Homo sapiens
Feng, C.; Liu, Y.; Wang, G.; Deng, Z.; Zhang, Q.; Wu, W.; Tong, Y.; Cheng, C.; Chen, Z.
Crystal structures of the human RNA demethylase Alkbh5 reveal basis for substrate recognition
J. Biol. Chem.
289
11571-11583
2014
Homo sapiens (Q6P6C2), Homo sapiens
Xu, C.; Liu, K.; Tempel, W.; Demetriades, M.; Aik, W.; Schofield, C.J.; Min, J.
Structures of human ALKBH5 demethylase reveal a unique binding mode for specific single-stranded N6-methyladenosine RNA demethylation
J. Biol. Chem.
289
17299-17311
2014
Homo sapiens (Q6P6C2), Homo sapiens
Li, F.; Kennedy, S.; Hajian, T.; Gibson, E.; Seitova, A.; Xu, C.; Arrowsmith, C.H.; Vedadi, M.
A Radioactivity-based assay for screening human m6A-RNA methyltransferase, METTL3-METTL14 complex, and demethylase ALKBH5
J. Biomol. Screen.
21
290-297
2016
Homo sapiens
Shen, F.; Huang, W.; Huang, J.T.; Xiong, J.; Yang, Y.; Wu, K.; Jia, G.F.; Chen, J.; Feng, Y.Q.; Yuan, B.F.; Liu, S.M.
Decreased N6-methyladenosine in peripheral blood RNA from diabetic patients is associated with FTO expression rather than ALKBH5
J. Clin. Endocrinol. Metab.
100
E148-E154
2015
Homo sapiens
Zheng, G.
Dahl, J.A.; Niu, Y.; Fedorcsak, P.; Huang, C.M.; Li, C.J.; Vagbo, C.B.; Shi, Y.; Wang, W.L.; Song, S.H.; Lu, Z.; Bosmans, R.P.; Dai, Q.; Hao, Y.J.; Yang, X.; Zhao, W.M.; Tong, W.M.; Wang, X.J.; Bogdan, F.; Furu, K.; Fu, Y.; Jia, G.; Zhao, X.; Liu, J.; Krokan, H.E.; Klungland, A.; Yang, Y.G.; He, C.: ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility
Mol. Cell
49
18-29
2013
Homo sapiens (Q6P6C2), Mus musculus (Q3TSG4), Mus musculus
Jia, G.; Fu, Y.; Zhao, X.; Dai, Q.; Zheng, G.; Yang, Y.; Yi, C.; Lindahl, T.; Pan, T.; Yang, Y.G.; He, C.
N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO
Nat. Chem. Biol.
7
885-887
2011
Homo sapiens (Q9C0B1), Homo sapiens
Han, Z.; Niu, T.; Chang, J.; Lei, X.; Zhao, M.; Wang, Q.; Cheng, W.; Wang, J.; Feng, Y.; Chai, J.
Crystal structure of the FTO protein reveals basis for its substrate specificity
Nature
464
1205-1209
2010
Homo sapiens (Q9C0B1)
Huang, Y.; Yan, J.; Li, Q.; Li, J.; Gong, S.; Zhou, H.; Gan, J.; Jiang, H.; Jia, G.F.; Luo, C.; Yang, C.G.
Meclofenamic acid selectively inhibits FTO demethylation of m6A over ALKBH5
Nucleic Acids Res.
43
373-384
2015
Homo sapiens (Q6P6C2), Homo sapiens
Zhang, C.; Samanta, D.; Lu, H.; Bullen, J.W.; Zhang, H.; Chen, I.; He, X.; Semenza, G.L.
Hypoxia induces the breast cancer stem cell phenotype by HIF-dependent and ALKBH5-mediated m6A-demethylation of NANOG mRNA
Proc. Natl. Acad. Sci. USA
113
E2047-E2056
2016
Homo sapiens
Zou, S.; Toh, J.D.; Wong, K.H.; Gao, Y.G.; Hong, W.; Woon, E.C.
N6-methyladenosine: a conformational marker that regulates the substrate specificity of human demethylases FTO and ALKBH5
Sci. Rep.
6
25677
2016
Homo sapiens
Chandola, U.; Das, R.; Panda, B.
Role of the N6-methyladenosine RNA mark in gene regulation and its implications on development and disease
Brief. Funct. Genomics
14
169-179
2015
Homo sapiens
Liu, K.; Ding, Y.; Ye, W.; Liu, Y.; Yang, J.; Liu, J.; Qi, C.
Structural and functional characterization of the proteins responsible for N6-methyladenosine modification and recognition
Curr. Protein Pept. Sci.
17
306-318
2015
Homo sapiens
Ye, F.; Zhang, L.; Jin, L.; Zheng, M.; Jiang, H.; Luo, C.
Repair of methyl lesions in RNA by oxidative demethylation
MedChemComm
5
1797-1803
2014
Homo sapiens
-
Bartosovic, M.; Molares, H.C.; Gregorova, P.; Hrossova, D.; Kudla, G.; Vanacova, S.
N6-methyladenosine demethylase FTO targets pre-mRNAs and regulates alternative splicing and 3-end processing
Nucleic Acids Res.
45
11356-11370
2017
Homo sapiens
Berulava, T.; Rahmann, S.; Rademacher, K.; Klein-Hitpass, L.; Horsthemke, B.
N6-adenosine methylation in miRNAs
PLoS ONE
10
e0118438
2015
Homo sapiens
Yang, Y.; Huang, W.; Huang, J.T.; Shen, F.; Xiong, J.; Yuan, E.F.; Qin, S.S.; Zhang, M.; Feng, Y.Q.; Yuan, B.F.; Liu, S.M.
Increased N6-methyladenosine in human sperm RNA as a risk factor for asthenozoospermia
Sci. Rep.
6
24345
2016
Homo sapiens
Selberg, S.; Seli, N.; Kankuri, E.; Karelson, M.
Rational design of novel anticancer small-molecule RNA m6A demethylase ALKBH5 inhibitors
ACS Omega
6
13310-13320
2021
Homo sapiens (Q6P6C2)
Zhang, Y.; Li, Q.N.; Zhou, K.; Xu, Q.; Zhang, C.Y.
Identification of specific N6-methyladenosine RNA demethylase FTO inhibitors by single-quantum-dot-based FRET nanosensors
Anal. Chem.
92
13936-13944
2020
Homo sapiens (Q6P6C2), Homo sapiens (Q9C0B1), Homo sapiens
Li, J.; Han, Y.; Zhang, H.; Qian, Z.; Jia, W.; Gao, Y.; Zheng, H.; Li, B.
The m6A demethylase FTO promotes the growth of lung cancer cells by regulating the m6A level of USP7 mRNA
Biochem. Biophys. Res. Commun.
512
479-485
2019
Homo sapiens (Q9C0B1)
Tsuruta, N.; Tsuchihashi, K.; Ohmura, H.; Yamaguchi, K.; Ito, M.; Ariyama, H.; Kusaba, H.; Akashi, K.; Baba, E.
RNA N6-methyladenosine demethylase FTO regulates PD-L1 expression in colon cancer cells
Biochem. Biophys. Res. Commun.
530
235-239
2020
Homo sapiens (Q9C0B1)
Jiang, Z.X.; Wang, Y.N.; Li, Z.Y.; Dai, Z.H.; He, Y.; Chu, K.; Gu, J.Y.; Ji, Y.X.; Sun, N.X.; Yang, F.; Li, W.
The m6A mRNA demethylase FTO in granulosa cells retards FOS-dependent ovarian aging
Cell Death Dis.
12
744
2021
Homo sapiens (Q9C0B1), Homo sapiens
Zhang, J.; Guo, S.; Piao, H.; Wang, Y.; Wu, Y.; Meng, X.; Yang, D.; Zheng, Z.; Zhao, Y.
ALKBH5 promotes invasion and metastasis of gastric cancer by decreasing methylation of the lncRNA NEAT1
J. Physiol. Biochem.
75
379-389
2019
Homo sapiens (Q6P6C2)
Niu, Y.; Lin, Z.; Wan, A.; Chen, H.; Liang, H.; Sun, L.; Wang, Y.; Li, X.; Xiong, X.F.; Wei, B.; Wu, X.; Wan, G.
RNA N6-methyladenosine demethylase FTO promotes breast tumor progression through inhibiting BNIP3
Mol. Cancer
18
46
2019
Homo sapiens (Q9C0B1), Homo sapiens
Yue, B.; Cui, R.; Zheng, R.; Jin, W.; Song, C.; Bao, T.; Wang, M.; Yu, F.; Zhao, E.
Essential role of ALKBH5-mediated RNA demethylation modification in bile acid-induced gastric intestinal metaplasia
Mol. Ther. Nucleic Acids
26
458-472
2021
Homo sapiens (Q6P6C2)
Yang, S.; Wei, J.; Cui, Y.H.; Park, G.; Shah, P.; Deng, Y.; Aplin, A.E.; Lu, Z.; Hwang, S.; He, C.; He, Y.Y.
m6A mRNA demethylase FTO regulates melanoma tumorigenicity and response to anti-PD-1 blockade
Nat. Commun.
10
2782
2019
Homo sapiens (Q9C0B1), Homo sapiens, Mus musculus (Q8BGW1)
Yu, F.; Wei, J.; Cui, X.; Yu, C.; Ni, W.; Bungert, J.; Wu, L.; He, C.; Qian, Z.
Post-translational modification of RNA m6A demethylase ALKBH5 regulates ROS-induced DNA damage response
Nucleic Acids Res.
49
5779-5797
2021
Homo sapiens (Q6P6C2)
Toh, J.; Crossley, S.; Bruemmer, K.; Ge, E.; He, D.; Iovan, D.; Chang, C.
Distinct RNA N-demethylation pathways catalyzed by nonheme iron ALKBH5 and FTO enzymes enable regulation of formaldehyde release rates
Proc. Natl. Acad. Sci. USA
117
25284-25292
2020
Homo sapiens (Q6P6C2), Homo sapiens (Q9C0B1)
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