RESUMO
N(6)-methyladenosine (m(6)A) is the most prevalent internal modification of messenger RNA (mRNA) in higher eukaryotes. Here we report ALKBH5 as another mammalian demethylase that oxidatively reverses m(6)A in mRNA in vitro and in vivo. This demethylation activity of ALKBH5 significantly affects mRNA export and RNA metabolism as well as the assembly of mRNA processing factors in nuclear speckles. Alkbh5-deficient male mice have increased m(6)A in mRNA and are characterized by impaired fertility resulting from apoptosis that affects meiotic metaphase-stage spermatocytes. In accordance with this defect, we have identified in mouse testes 1,551 differentially expressed genes that cover broad functional categories and include spermatogenesis-related mRNAs involved in the p53 functional interaction network. The discovery of this RNA demethylase strongly suggests that the reversible m(6)A modification has fundamental and broad functions in mammalian cells.
Assuntos
Dioxigenases/metabolismo , Proteínas de Membrana/metabolismo , Oxirredutases N-Desmetilantes/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Homólogo AlkB 5 da RNA Desmetilase , Animais , Sequência de Bases , Núcleo Celular/metabolismo , Dioxigenases/química , Dioxigenases/genética , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Infertilidade Masculina/enzimologia , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Tamanho do Órgão , Oxirredutases N-Desmetilantes/química , Oxirredutases N-Desmetilantes/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico , Interferência de RNA , RNA Mensageiro/química , Espermatogênese/genética , Testículo/enzimologia , Testículo/patologia , TranscriptomaRESUMO
Uranyl (UO2(2+)), the predominant aerobic form of uranium, is present in the ocean at a concentration of ~3.2 parts per 10(9) (13.7 nM); however, the successful enrichment of uranyl from this vast resource has been limited by the high concentrations of metal ions of similar size and charge, which makes it difficult to design a binding motif that is selective for uranyl. Here we report the design and rational development of a uranyl-binding protein using a computational screening process in the initial search for potential uranyl-binding sites. The engineered protein is thermally stable and offers very high affinity and selectivity for uranyl with a Kd of 7.4 femtomolar (fM) and >10,000-fold selectivity over other metal ions. We also demonstrated that the uranyl-binding protein can repeatedly sequester 30-60% of the uranyl in synthetic sea water. The chemical strategy employed here may be applied to engineer other selective metal-binding proteins for biotechnology and remediation applications.
Assuntos
Nanopartículas Metálicas/química , Proteínas/química , Urânio/química , Sítios de Ligação , Modelos Moleculares , Engenharia de Proteínas , Proteínas/metabolismoRESUMO
N(6)-methyladenosine is a prevalent internal modification in messenger RNA and non-coding RNA affecting various cellular pathways. Here we report the discovery of two additional modifications, N(6)-hydroxymethyladenosine (hm(6)A) and N(6)-formyladenosine (f(6)A), in mammalian messenger RNA. We show that Fe(II)- and α-ketoglutarate-dependent fat mass and obesity-associated (FTO) protein oxidize N(6)-methyladenosine to generate N(6)-hydroxymethyladenosine as an intermediate modification, and N(6)-formyladenosine as a further oxidized product. N(6)-hydroxymethyladenosine and N(6)-formyladenosine have half-life times of ~3 h in aqueous solution under physiological relevant conditions, and are present in isolated messenger RNA from human cells as well as mouse tissues. These previously unknown modifications derived from the prevalent N(6)-methyladenosine in messenger RNA, formed through oxidative RNA demethylation, may dynamically modulate RNA-protein interactions to affect gene expression regulation.
Assuntos
Adenosina/análogos & derivados , Mamíferos/metabolismo , Proteínas/metabolismo , RNA/metabolismo , Adenosina/química , Adenosina/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato , Animais , Humanos , Cinética , Metilação , Camundongos , Modelos Biológicos , Simulação de Dinâmica Molecular , Oxirredução , Ligação Proteica , RNA/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Especificidade por SubstratoRESUMO
ALKBH2 is a direct DNA repair dioxygenase guarding the mammalian genome against N(1)-methyladenine, N(3)-methylcytosine and 1,N(6)-ethenoadenine damage. A prerequisite for repair is to identify these lesions in the genome. Here we present crystal structures of human ALKBH2 bound to different duplex DNAs. Together with computational and biochemical analyses, our results suggest that DNA interrogation by ALKBH2 has two previously unknown features: (i) ALKBH2 probes base-pair stability and detects base pairs with reduced stability, and (ii) ALKBH2 does not have nor need a damage-checking site, which is critical for preventing spurious base cleavage for several glycosylases. The demethylation mechanism of ALKBH2 insures that only cognate lesions are oxidized and reversed to normal bases, and that a flipped, non-substrate base remains intact in the active site. Overall, the combination of duplex interrogation and oxidation chemistry allows ALKBH2 to detect and process diverse lesions efficiently and correctly.