NAR Breakthrough Article: Structure of human RNA N6-methyladenine demethylase ALKBH5 provides insights into its mechanisms of nucleic acid recognition and demethylation

ALKBH5 is a 2-oxoglutarate (2OG) and ferrous iron-dependent nucleic acid oxygenase (NAOX) that catalyzes the demethylation of N⁶-methyladenine in RNA. ALKBH5 is upregulated under hypoxia and plays a role in spermatogenesis. We describe a crystal structure of human ALKBH5 (residues 66–292) to 2.0 Å resolution. ALKBH5_66–292 has a double-stranded β-helix core fold as observed in other 2OG and iron-dependent oxygenase family members. 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 NAOXs. A large loop (βIV–V) in ALKBH5 occupies a similar region as the L1 loop of the fat mass and obesity-associated protein that is proposed to confer single-stranded RNA selectivity. Unexpectedly, a small molecule inhibitor, IOX3, was observed covalently attached to the side chain of Cys200 located outside of the active site. Modelling substrate into the active site based on other NAOX–nucleic acid complexes reveals conserved residues important for recognition and demethylation mechanisms. The structural insights will aid in the development of inhibitors selective for NAOXs, for use as functional probes and for therapeutic benefit.     

N6-methyladenosine demethylase ALKBH5:a novel regulator of proliferation and differentiation of chicken preadipocytes

Previous studies have reported that the N6-methyladenosine demethylase ALKBH5 can regulate adipogenesis in humans.However,its function in birds remains unclear....     

N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO

We report here that fat mass and obesity-associated protein (FTO) has efficient oxidative demethylation activity targeting the abundant N6-methyladenosine (m(6)A) residues in RNA in vitro. FTO knockdown with siRNA led to increased amounts of m(6)A in mRNA, whereas overexpression of FTO resulted in decreased amounts of m(6)A in human cells. We further show the partial colocalization of FTO with nuclear speckles, which supports the notion that m(6)A in nuclear RNA is a major physiological substrate of FTO.     

Mechanism of noncoding RNA-associated N6-methyladenosine recognition by an RNA processing complex during IgH DNA recombination

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Analysis of mRNA 5'-terminal cap structures and internal N6-methyladenosine by reversed-phase high-performance liquid chromatography

A simple procedure for determining the complete methylation profile of an mRNA molecule in a single chromatographic separation is described. The mRNA is selectively hydrolyzed to its component nucleosides leaving its cap 0 (m⁷GpppN′) or cap 1 (m⁷GpppN′m) structure intact. The hydrolysis products, which can include cap 0, cap 1, 2′-O-methylnucleosides (N″m) of cap 2 (m⁷GpppN′mpN″m) and internal N⁶-methyladenosine, are separated on an octadecyl reverse-phase column using a mobile phase containing acetonitrile and ammonium formate, a weak ion-pairing reagent. methyl-³H-labeled poly(A)-containing mRNA is used to demonstrate the efficacy of the procedure.     

Influenza viral mRNA contains internal N6-methyladenosine and 5''-terminal 7-methylguanosine in cap structures.

Influenza viral complementary RNA (cRNA), i.e., viral mRNA was radioactive when purified from the cytoplasmic fraction of cordycepin-treated canine kidney cells that were incubated with [methyl-3H]methionine during infection. Approximately 55 to 60% of the methyl-3H radioactivity was in internal N6-methyladenosine, a feature distinguishing this mRNA from those viral mRNA's that are known to be synthesized in the cytoplasm. The remaining methyl-3H radioactivity was in 5'-terminal cap structures that consisted of 7-methylguanosine in pyrophosphate linkage to 2'-o-methyladenosine, N6, 2'-O-dimethyladenosine, or 2'-O-methylguanosine. Methylated adenosine was the predominant penultimate nucleoside in caps, suggesting that cRNA synthesis in infected cells initiates preferentially with adenosine at the 5' end. In contrast to cRNA, influenza virion RNA segments extracted from purified virus contained mainly 5'-terminal ppA and no detectable cap structures.     

Specificity determinants of human cathepsin s revealed by crystal structures of complexes

Cathepsin S, a lysosomal cysteine protease of the papain superfamily, has been implicated in the preparation of MHC class II alphabeta-heterodimers for antigen presentation to CD4+ T lymphocytes and is considered a potential target for autoimmune-disease therapy. Selective inhibition of this enzyme may be therapeutically useful for attenuating the hyperimmune responses in a number of disorders. We determined the three-dimensional crystal structures of human cathepsin S in complex with potent covalent inhibitors, the aldehyde inhibitor 4-morpholinecarbonyl-Phe-(S-benzyl)Cys-Psi(CH=O), and the vinyl sulfone irreversible inhibitor 4-morpholinecarbonyl-Leu-Hph-Psi(CH=CH-SO(2)-phenyl) at resolutions of 1.8 and 2.0 A, respectively. In the structure of the cathepsin S-aldehyde complex, the tetrahedral thiohemiacetal adduct favors the S-configuration, in which the oxygen atom interacts with the imidazole group of the active site His164 rather than with the oxyanion hole. The present structures provide a detailed map of noncovalent intermolecular interactions established in the substrate-binding subsites S3 to S1' of cathepsin S. In the S2 pocket, which is the binding affinity hot spot of cathepsin S, the Phe211 side chain can assume two stable conformations that accommodate either the P2-Leu or a bulkier P2-Phe side chain. This structural plasticity of the S2 pocket in cathepsin S explains the selective inhibition of cathepsin S over cathepsin K afforded by inhibitors with the P2-Phe side chain. Comparison with the structures of cathepsins K, V, and L allows delineation of local intermolecular contacts that are unique to cathepsin S.     

Structures of Human ALKBH5 Demethylase Reveal a Unique Binding Mode for Specific Single-stranded N6-Methyladenosine RNA Demethylation

N⁶-Methyladenosine (m⁶A) is the most prevalent internal RNA modification in eukaryotes. ALKBH5 belongs to the AlkB family of dioxygenases and has been shown to specifically demethylate m⁶A in single-stranded RNA. Here we report crystal structures of ALKBH5 in the presence of either its cofactors or the ALKBH5 inhibitor citrate. Catalytic assays demonstrate that the ALKBH5 catalytic domain can demethylate both single-stranded RNA and single-stranded DNA. We identify the TCA cycle intermediate citrate as a modest inhibitor of ALKHB5 (IC₅₀, ∼488 μm). The structural analysis reveals that a loop region of ALKBH5 is immobilized by a disulfide bond that apparently excludes the binding of dsDNA to ALKBH5. We identify the m⁶A binding pocket of ALKBH5 and the key residues involved in m⁶A recognition using mutagenesis and ITC binding experiments.     

Identification of recognition residues for ligation-based detection and quantitation of pseudouridine and N6-methyladenosine

Over 100 chemical types of RNA modifications have been identified in thousands of sites in all three domains of life. Recent data suggest that modifications function synergistically to mediate biological function, and that cells may coordinately modulate modification levels for regulatory purposes. However, this area of RNA biology remains largely unexplored due to the lack of robust, high-throughput methods to quantify the extent of modification at specific sites. Recently, we developed a facile enzymatic ligation-based method for detection and quantitation of methylated 2′-hydroxyl groups within RNA. Here we exploit the principles of molecular recognition and nucleic acid chemistry to establish the experimental parameters for ligation-based detection and quantitation of pseudouridine (Ψ) and N⁶-methyladenosine (m⁶A), two abundant modifications in eukaryotic rRNA/tRNA and mRNA, respectively. Detection of pseudouridylation at several sites in the large subunit rRNA derived from yeast demonstrates the feasibility of the approach for analysis of pseudouridylation in biological RNA samples.     

Diverse substrate recognition mechanism revealed by Thermotoga maritima Cel5A structures in complex with cellotetraose, cellobiose and mannotriose

The hyperthermophilic endoglucanase Cel5A from Thermotoga maritima can find applications in lignocellulosic biofuel production, because it catalyzes the hydrolysis of glucan- and mannan-based polysaccharides. Here, we report the crystal structures in apo-form and in complex with three ligands, cellotetraose, cellobiose and mannotriose, at 1.29? to 2.40? resolution. The open carbohydrate-binding cavity which can accommodate oligosaccharide substrates with extensively branched chains explained the dual specificity of the enzyme. Combining our structural information and the previous kinetic data, it is suggested that this enzyme prefers β-glucosyl and β-mannosyl moieties at the reducing end and uses two conserved catalytic residues, E253 (nucleophile) and E136 (general acid/base), to hydrolyze the glycosidic bonds. Moreover, our results also suggest that the wide spectrum of Tm_Cel5A substrates might be due to the lack of steric hindrance around the C2-hydroxyl group of the glucose or mannose unit from active-site residues.     

Identification and mapping of N6-methyladenosine containing sequences in simian virus 40 RNA.

Late SV40 16S and 19S mRNAs were found to contain an average of three m6A residues per mRNA molecule. The methylated residues of both the viral and cellular mRNAs occur in two sequences; Gpm6ApC and (Ap)nm6ApC, where n = 1-4. More than 60% of the m6A residues in SV40 16S and 19S mRNAs occur in Gpm6ApC even though there are twice as many (A)nAC than GAC sequences in these messengers. The m6A containing oligonucleotides of late SV40 MRNAs were localized in the viral messengers. In the 16S mRNA two m6A oligonucleotides were located at the 5' coding region between 0.95--0.0 map units. The third m6A residue was mapped between 0.0--0.14 map units in the translated portion of this mRNA. The overall pattern of internal methylation in the 19S mRNA is similar. However, some differences between 16S and 19S mRNAs were observed in both the content and location of the longer (Ap)n m6AC nucleotides. These results provide the first example of precise localization of internal methylation sequences in mRNA species with defined coding specificity. It implies that a) location of m6A residues is not random but specific to a particular region of the RNA, b) apart from sequence specificity other structural features of the mRNA may influence internal methylation and c) m6A residues are present in coding regions of SV40 mRNAs.     

Regulation of RNA N6-methyladenosine modification and its emerging roles in skeletal muscle development

N⁶-methyladenosine (m⁶A) is one of the most widespread and highly conserved chemical modifications in cellular RNAs of eukaryotic genomes. Owing to the development of high-throughput m⁶A sequencing, the functions and mechanisms of m⁶A modification in development and diseases have been revealed. Recent studies have shown that RNA m⁶A methylation plays a critical role in skeletal muscle development, which regulates myoblast proliferation and differentiation, and muscle regeneration. Exploration of the functions of m⁶A modification and its regulators provides a deeper understanding of the regulatory mechanisms underlying skeletal muscle development. In the present review, we aim to summarize recent breakthroughs concerning the global landscape of m⁶A modification in mammals and examine the biological functions and mechanisms of enzymes regulating m⁶A RNA methylation. We describe the interplay between m⁶A and other epigenetic modifications and highlight the regulatory roles of m⁶A in development, especially that of skeletal muscle. m⁶A and its regulators are expected to be targets for the treatment of human muscle-related diseases and novel epigenetic markers for animal breeding in meat production.     

Acceptor substrate binding revealed by crystal structure of human glucosamine-6-phosphate N-acetyltransferase 1

Glucosamine-6-phosphate (GlcN6P) N-acetyltransferase 1 (GNA1) is a key enzyme in the pathway toward biosynthesis of UDP-N-acetylglucosamine, an important donor substrate for N-linked glycosylation. GNA1 catalyzes the formation of N-acetylglucosamine-6-phosphate (GlcNAc6P) from acetyl-CoA (AcCoA) and the acceptor substrate GlcN6P. Here, we report crystal structures of human GNA1, including apo GNA1, the GNA1-GlcN6P complex and an E156A mutant. Our work showed that GlcN6P binds to GNA1 without the help of AcCoA binding. Structural analyses and mutagenesis studies have shed lights on the charge distribution in the GlcN6P binding pocket, and an important role for Glu156 in the substrate binding. Hence, these findings have broadened our knowledge of structural features required for the substrate affinity of GNA1. STRUCTURED SUMMARY:     

Shared and specific functions of Arfs 1–5 at the Golgi revealed by systematic knockouts

ADP-ribosylation factors (Arfs) are small GTPases regulating membrane traffic in the secretory pathway. They are closely related and appear to have overlapping functions, regulators, and effectors. The functional specificity of individual Arfs and the extent of redundancy are still largely unknown. We addressed these questions by CRISPR/Cas9-mediated genomic deletion of the human class I (Arf1/3) and class II (Arf4/5) Arfs, either individually or in combination. Most knockout cell lines were viable with slight growth defects only when lacking Arf1 or Arf4. However, Arf1+4 and Arf4+5 could not be deleted simultaneously. Class I Arfs are nonessential, and Arf4 alone is sufficient for viability. Upon Arf1 deletion, the Golgi was enlarged, and recruitment of vesicle coats decreased, confirming a major role of Arf1 in vesicle formation at the Golgi. Knockout of Arf4 caused secretion of ER-resident proteins, indicating specific defects in coatomer-dependent ER protein retrieval by KDEL receptors. The knockout cell lines will be useful tools to study other Arf-dependent processes.     

Substrate shape determines specificity of recognition for HIV-1 protease: analysis of crystal structures of six substrate complexes

The homodimeric HIV-1 protease is the target of some of the most effective antiviral AIDS therapy, as it facilitates viral maturation by cleaving ten asymmetric and nonhomologous sequences in the Gag and Pol polyproteins. Since the specificity of this enzyme is not easily determined from the sequences of these cleavage sites alone, we solved the crystal structures of complexes of an inactive variant (D25N) of HIV-1 protease with six peptides that correspond to the natural substrate cleavage sites. When the protease binds to its substrate and buries nearly 1000 A2 of surface area, the symmetry of the protease is broken, yet most internal hydrogen bonds and waters are conserved. However, no substrate side chain hydrogen bond is conserved. Specificity of HIV-1 protease appears to be determined by an asymmetric shape rather than a particular amino acid sequence.     

Enzyme-substrate interactions revealed by the crystal structures of the archaeal Sulfolobus PTP-fold phosphatase and its phosphopeptide complexes

The P-loop-containing protein phos-phatases are important regulators in signal transduction. These enzymes have structural and functional similarity with a conserved sequence of Dx(25-41)HCxxGxxR(T/S) essential for catalysis. The singular protein tyrosine phosphatase (PTP) from archaeal Sulfolobus solfataricus is one of the smallest known PTPs with extreme thermostability. Here, we report the crystal structure of this phosphatase and its complexes with two tyrosyl phosphopeptides A-(p)Y-R and N-K-(p)Y-G-N. The structure suggests the minimal structural motif of the PTP family, having two variable sequences inserted between the beta2-beta3 and beta3-beta4 strands, respectively. The phosphate of both phosphopeptide substrates is bound to the P-loop through several hydrogen bonds. Comparison of several phosphatase-substrate complexes revealed that Gln135 on the Q-loop has different modes of recognition toward phosphopeptides. The substrate specificity of SsoPTP is primarily localized at the phosphotyrosine, suggesting that this phosphatase may be a prototypical PTP.