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Nature ; 593(7860): 597-601, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33902106


N6-methyladenosine (m6A) is an abundant internal RNA modification1,2 that is catalysed predominantly by the METTL3-METTL14 methyltransferase complex3,4. The m6A methyltransferase METTL3 has been linked to the initiation and maintenance of acute myeloid leukaemia (AML), but the potential of therapeutic applications targeting this enzyme remains unknown5-7. Here we present the identification and characterization of STM2457, a highly potent and selective first-in-class catalytic inhibitor of METTL3, and a crystal structure of STM2457 in complex with METTL3-METTL14. Treatment of tumours with STM2457 leads to reduced AML growth and an increase in differentiation and apoptosis. These cellular effects are accompanied by selective reduction of m6A levels on known leukaemogenic mRNAs and a decrease in their expression consistent with a translational defect. We demonstrate that pharmacological inhibition of METTL3 in vivo leads to impaired engraftment and prolonged survival in various mouse models of AML, specifically targeting key stem cell subpopulations of AML. Collectively, these results reveal the inhibition of METTL3 as a potential therapeutic strategy against AML, and provide proof of concept that the targeting of RNA-modifying enzymes represents a promising avenue for anticancer therapy.

Nat Commun ; 11(1): 926, 2020 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-32066737


The field of epitranscriptomics continues to reveal how post-transcriptional modification of RNA affects a wide variety of biological phenomena. A pivotal challenge in this area is the identification of modified RNA residues within their sequence contexts. Mass spectrometry (MS) offers a comprehensive solution by using analogous approaches to shotgun proteomics. However, software support for the analysis of RNA MS data is inadequate at present and does not allow high-throughput processing. Existing software solutions lack the raw performance and statistical grounding to efficiently handle the numerous modifications found on RNA. We present a free and open-source database search engine for RNA MS data, called NucleicAcidSearchEngine (NASE), that addresses these shortcomings. We demonstrate the capability of NASE to reliably identify a wide range of modified RNA sequences in four original datasets of varying complexity. In human tRNA, we characterize over 20 different modification types simultaneously and find many cases of incomplete modification.

Epigenômica/métodos , Ensaios de Triagem em Larga Escala/métodos , Processamento Pós-Transcricional do RNA/genética , Ferramenta de Busca , Espectrometria de Massas em Tandem/métodos , Sequência de Bases/genética , Bases de Dados Factuais/estatística & dados numéricos , Conjuntos de Dados como Assunto , Humanos , Oligonucleotídeos/química , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência/metabolismo , Reprodutibilidade dos Testes
Nucleic Acids Res ; 47(19): 10267-10281, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31665743


Post-transcriptional RNA modifications, the epitranscriptome, play important roles in modulating the functions of RNA species. Modifications of rRNA are key for ribosome production and function. Identification and characterization of enzymes involved in epitranscriptome shaping is instrumental for the elucidation of the functional roles of specific RNA modifications. Ten modified sites have been thus far identified in the mammalian mitochondrial rRNA. Enzymes responsible for two of these modifications have not been characterized. Here, we identify METTL15, show that it is the main N4-methylcytidine (m4C) methyltransferase in human cells and demonstrate that it is responsible for the methylation of position C839 in mitochondrial 12S rRNA. We show that the lack of METTL15 results in a reduction of the mitochondrial de novo protein synthesis and decreased steady-state levels of protein components of the oxidative phosphorylation system. Without functional METTL15, the assembly of the mitochondrial ribosome is decreased, with the late assembly components being unable to be incorporated efficiently into the small subunit. We speculate that m4C839 is involved in the stabilization of 12S rRNA folding, therefore facilitating the assembly of the mitochondrial small ribosomal subunits. Taken together our data show that METTL15 is a novel protein necessary for efficient translation in human mitochondria.

Metiltransferases/genética , Mitocôndrias/genética , Ribossomos Mitocondriais/química , RNA Ribossômico/genética , Citidina/genética , Humanos , Metilação , Mitocôndrias/química , Fosforilação Oxidativa , Biossíntese de Proteínas/genética , Dobramento de RNA/genética , Processamento Pós-Transcricional do RNA/genética , RNA Ribossômico/química
Mol Cell ; 74(6): 1278-1290.e9, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31031083


7-methylguanosine (m7G) is present at mRNA caps and at defined internal positions within tRNAs and rRNAs. However, its detection within low-abundance mRNAs and microRNAs (miRNAs) has been hampered by a lack of sensitive detection strategies. Here, we adapt a chemical reactivity assay to detect internal m7G in miRNAs. Using this technique (Borohydride Reduction sequencing [BoRed-seq]) alongside RNA immunoprecipitation, we identify m7G within a subset of miRNAs that inhibit cell migration. We show that the METTL1 methyltransferase mediates m7G methylation within miRNAs and that this enzyme regulates cell migration via its catalytic activity. Using refined mass spectrometry methods, we map m7G to a single guanosine within the let-7e-5p miRNA. We show that METTL1-mediated methylation augments let-7 miRNA processing by disrupting an inhibitory secondary structure within the primary miRNA transcript (pri-miRNA). These results identify METTL1-dependent N7-methylation of guanosine as a new RNA modification pathway that regulates miRNA structure, biogenesis, and cell migration.

Guanosina/análogos & derivados , Metiltransferases/genética , MicroRNAs/genética , Processamento Pós-Transcricional do RNA , Células A549 , Sequência de Bases , Bioensaio , Células CACO-2 , Movimento Celular , Proliferação de Células , Guanosina/metabolismo , Células HEK293 , Humanos , Metilação , Metiltransferases/metabolismo , MicroRNAs/metabolismo , Conformação de Ácido Nucleico
Cell ; 174(2): 325-337.e14, 2018 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-29887380


Multiple proteins act co-operatively in mammalian clathrin-mediated endocytosis (CME) to generate endocytic vesicles from the plasma membrane. The principles controlling the activation and organization of the actin cytoskeleton during mammalian CME are, however, not fully understood. Here, we show that the protein FCHSD2 is a major activator of actin polymerization during CME. FCHSD2 deletion leads to decreased ligand uptake caused by slowed pit maturation. FCHSD2 is recruited to endocytic pits by the scaffold protein intersectin via an unusual SH3-SH3 interaction. Here, its flat F-BAR domain binds to the planar region of the plasma membrane surrounding the developing pit forming an annulus. When bound to the membrane, FCHSD2 activates actin polymerization by a mechanism that combines oligomerization and recruitment of N-WASP to PI(4,5)P2, thus promoting pit maturation. Our data therefore describe a molecular mechanism for linking spatiotemporally the plasma membrane to a force-generating actin platform guiding endocytic vesicle maturation.

Citoesqueleto de Actina/fisiologia , Proteínas de Transporte/metabolismo , Clatrina/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/química , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Membrana Celular/química , Membrana Celular/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Endocitose , Células HeLa , Humanos , Lipossomos/química , Lipossomos/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Microscopia de Fluorescência , Modelos Moleculares , Mutagênese Sítio-Dirigida , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/química , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Domínios de Homologia de src
Proc Natl Acad Sci U S A ; 110(47): 18934-9, 2013 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-24191001


Mitochondrial respiratory complex I is a product of both the nuclear and mitochondrial genomes. The integration of seven subunits encoded in mitochondrial DNA into the inner membrane, their association with 14 nuclear-encoded membrane subunits, the construction of the extrinsic arm from 23 additional nuclear-encoded proteins, iron-sulfur clusters, and flavin mononucleotide cofactor require the participation of assembly factors. Some are intrinsic to the complex, whereas others participate transiently. The suppression of the expression of the NDUFA11 subunit of complex I disrupted the assembly of the complex, and subcomplexes with masses of 550 and 815 kDa accumulated. Eight of the known extrinsic assembly factors plus a hydrophobic protein, C3orf1, were associated with the subcomplexes. The characteristics of C3orf1, of another assembly factor, TMEM126B, and of NDUFA11 suggest that they all participate in constructing the membrane arm of complex I.

Complexo I de Transporte de Elétrons/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas de Membrana/metabolismo , Membranas Mitocondriais/metabolismo , Multimerização Proteica/fisiologia , Western Blotting , Linhagem Celular Tumoral , Eletroforese em Gel de Poliacrilamida , Humanos , Espectrometria de Massas , Microscopia Confocal , Proteínas de Transporte da Membrana Mitocondrial , Consumo de Oxigênio/fisiologia