RESUMO
RNA viruses have evolved elaborate strategies to protect their genomes, including 5' capping. However, until now no RNA 5' cap has been identified for hepatitis C virus1,2 (HCV), which causes chronic infection, liver cirrhosis and cancer3. Here we demonstrate that the cellular metabolite flavin adenine dinucleotide (FAD) is used as a non-canonical initiating nucleotide by the viral RNA-dependent RNA polymerase, resulting in a 5'-FAD cap on the HCV RNA. The HCV FAD-capping frequency is around 75%, which is the highest observed for any RNA metabolite cap across all kingdoms of life4-8. FAD capping is conserved among HCV isolates for the replication-intermediate negative strand and partially for the positive strand. It is also observed in vivo on HCV RNA isolated from patient samples and from the liver and serum of a human liver chimeric mouse model. Furthermore, we show that 5'-FAD capping protects RNA from RIG-I mediated innate immune recognition but does not stabilize the HCV RNA. These results establish capping with cellular metabolites as a novel viral RNA-capping strategy, which could be used by other viruses and affect anti-viral treatment outcomes and persistence of infection.
Assuntos
Flavina-Adenina Dinucleotídeo , Hepacivirus , Capuzes de RNA , RNA Viral , Animais , Humanos , Camundongos , Quimera/virologia , Flavina-Adenina Dinucleotídeo/metabolismo , Hepacivirus/genética , Hepacivirus/imunologia , Hepatite C/virologia , Reconhecimento da Imunidade Inata , Fígado/virologia , Estabilidade de RNA , RNA Viral/química , RNA Viral/genética , RNA Viral/imunologia , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Replicação Viral/genética , Capuzes de RNA/metabolismoRESUMO
Activation-induced cytidine deaminase (AID) interacts with replication protein A (RPA), the major ssDNA-binding protein, to promote deamination of cytosine to uracil in transcribed immunoglobulin (Ig) genes. Uracil-DNA glycosylase (UNG) acts in concert with AID during Ig diversification. In addition, UNG preserves genome integrity by base-excision repair (BER) in the overall genome. How UNG is regulated to support both mutagenic processing and error-free repair remains unknown. UNG is expressed as two isoforms, UNG1 and UNG2, which both contain an RPA-binding helix that facilitates uracil excision from RPA-coated ssDNA. However, the impact of this interaction in antibody diversification and genome maintenance has not been investigated. Here, we generated B-cell clones with targeted mutations in the UNG RPA-binding motif, and analysed class switch recombination (CSR), mutation frequency (5' Ig Sµ), and genomic uracil in clones representing seven Ung genotypes. We show that the UNG:RPA interaction plays a crucial role in both CSR and repair of AID-induced uracil at the Ig loci. By contrast, the interaction had no significant impact on total genomic uracil levels. Thus, RPA coordinates UNG during CSR and pre-replicative repair of mutagenic uracil in ssDNA but is not essential in post-replicative and canonical BER of uracil in dsDNA.
Assuntos
Proteína de Replicação A , Uracila-DNA Glicosidase , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Reparo do DNA/genética , DNA de Cadeia Simples/genética , Switching de Imunoglobulina/genética , Isotipos de Imunoglobulinas/genética , Imunoglobulinas/genética , Mutagênicos , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Uracila/metabolismo , Uracila-DNA Glicosidase/genética , Uracila-DNA Glicosidase/metabolismo , Humanos , Animais , CamundongosRESUMO
DNA repair is essential to prevent the cytotoxic or mutagenic effects of various types of DNA lesions, which are sensed by distinct pathways to recruit repair factors specific to the damage type. Although biochemical mechanisms for repairing several forms of genomic insults are well understood, the upstream signalling pathways that trigger repair are established for only certain types of damage, such as double-stranded breaks and interstrand crosslinks. Understanding the upstream signalling events that mediate recognition and repair of DNA alkylation damage is particularly important, since alkylation chemotherapy is one of the most widely used systemic modalities for cancer treatment and because environmental chemicals may trigger DNA alkylation. Here we demonstrate that human cells have a previously unrecognized signalling mechanism for sensing damage induced by alkylation. We find that the alkylation repair complex ASCC (activating signal cointegrator complex) relocalizes to distinct nuclear foci specifically upon exposure of cells to alkylating agents. These foci associate with alkylated nucleotides, and coincide spatially with elongating RNA polymerase II and splicing components. Proper recruitment of the repair complex requires recognition of K63-linked polyubiquitin by the CUE (coupling of ubiquitin conjugation to ER degradation) domain of the subunit ASCC2. Loss of this subunit impedes alkylation adduct repair kinetics and increases sensitivity to alkylating agents, but not other forms of DNA damage. We identify RING finger protein 113A (RNF113A) as the E3 ligase responsible for upstream ubiquitin signalling in the ASCC pathway. Cells from patients with X-linked trichothiodystrophy, which harbour a mutation in RNF113A, are defective in ASCC foci formation and are hypersensitive to alkylating agents. Together, our work reveals a previously unrecognized ubiquitin-dependent pathway induced specifically to repair alkylation damage, shedding light on the molecular mechanism of X-linked trichothiodystrophy.
Assuntos
Enzimas AlkB/metabolismo , Adutos de DNA/metabolismo , Reparo do DNA , Complexos Multiproteicos/metabolismo , Transdução de Sinais , Síndromes de Tricotiodistrofia/genética , Ubiquitina/metabolismo , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/metabolismo , Alquilantes/farmacologia , Alquilação , Sequência de Aminoácidos , Adutos de DNA/química , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Retículo Endoplasmático/metabolismo , Genes Ligados ao Cromossomo X , Humanos , Cinética , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Poliubiquitina/metabolismo , RNA Polimerase II/metabolismo , Splicing de RNA , Síndromes de Tricotiodistrofia/metabolismo , Síndromes de Tricotiodistrofia/patologia , UbiquitinaçãoRESUMO
RNA methylations are essential both for RNA structure and function, and are introduced by a number of distinct methyltransferases (MTases). In recent years, N6-methyladenosine (m6A) modification of eukaryotic mRNA has been subject to intense studies, and it has been demonstrated that m6A is a reversible modification that regulates several aspects of mRNA function. However, m6A is also found in other RNAs, such as mammalian 18S and 28S ribosomal RNAs (rRNAs), but the responsible MTases have remained elusive. 28S rRNA carries a single m6A modification, found at position A4220 (alternatively referred to as A4190) within a stem-loop structure, and here we show that the MTase ZCCHC4 is the enzyme responsible for introducing this modification. Accordingly, we found that ZCCHC4 localises to nucleoli, the site of ribosome assembly, and that proteins involved in RNA metabolism are overrepresented in the ZCCHC4 interactome. Interestingly, the absence of m6A4220 perturbs codon-specific translation dynamics and shifts gene expression at the translational level. In summary, we establish ZCCHC4 as the enzyme responsible for m6A modification of human 28S rRNA, and demonstrate its functional significance in mRNA translation.
Assuntos
Adenosina/análogos & derivados , Metiltransferases/genética , RNA Mensageiro/genética , RNA Ribossômico 28S/genética , Adenosina/química , Adenosina/genética , Catálise , Humanos , Metilação , Metiltransferases/química , Ligação Proteica/genética , RNA Ribossômico 28S/químicaRESUMO
BACKGROUND: Reversible enzymatic methylation of mammalian mRNA is widespread and serves crucial regulatory functions, but little is known to what degree chemical alkylators mediate overlapping modifications and whether cells distinguish aberrant from canonical methylations. METHODS: Here we use quantitative mass spectrometry to determine the fate of chemically induced methylbases in the mRNA of human cells. Concomitant alteration in the mRNA binding proteome was analyzed by SILAC mass spectrometry. RESULTS: MMS induced prominent direct mRNA methylations that were chemically identical to endogenous methylbases. Transient loss of 40S ribosomal proteins from isolated mRNA suggests that aberrant methylbases mediate arrested translational initiation and potentially also no-go decay of the affected mRNA. Four proteins (ASCC3, YTHDC2, TRIM25 and GEMIN5) displayed increased mRNA binding after MMS treatment. ASCC3 is a binding partner of the DNA/RNA demethylase ALKBH3 and was recently shown to promote disassembly of collided ribosomes as part of the ribosome quality control (RQC) trigger complex. We find that ASCC3-deficient cells display delayed removal of MMS-induced 1-methyladenosine (m1A) and 3-methylcytosine (m3C) from mRNA and impaired formation of MMS-induced P-bodies. CONCLUSIONS: Our findings conform to a model in which ASCC3-mediated disassembly of collided ribosomes allows demethylation of aberrant m1A and m3C by ALKBH3. Our findings constitute first evidence of selective sanitation of aberrant mRNA methylbases over their endogenous counterparts and warrant further studies on RNA-mediated effects of chemical alkylators commonly used in the clinic.
Assuntos
Citosina , Ribossomos , Adenosina/análogos & derivados , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato , Animais , Citosina/análogos & derivados , DNA Helicases , Humanos , RNA Helicases , RNA Mensageiro/genética , Fatores de Transcrição , Proteínas com Motivo Tripartido , Ubiquitina-Proteína LigasesRESUMO
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
The Escherichia coli AlkB protein (EcAlkB) is a DNA repair enzyme which reverses methylation damage such as 1-methyladenine (1-meA) and 3-methylcytosine (3-meC). The mammalian AlkB homologues ALKBH2 and ALKBH3 display EcAlkB-like repair activity in vitro, but their substrate specificities are different, and ALKBH2 is the main DNA repair enzyme for 1-meA in vivo. The genome of the model plant Arabidopsis thaliana encodes several AlkB homologues, including the yet uncharacterized protein AT2G22260, which displays sequence similarity to both ALKBH2 and ALKBH3. We have here characterized protein AT2G22260, by us denoted ALKBH2, as both our functional studies and bioinformatics analysis suggest it to be an orthologue of mammalian ALKBH2. The Arabidopsis ALKBH2 protein displayed in vitro repair activities towards methyl and etheno adducts in DNA, and was able to complement corresponding repair deficiencies of the E. coli alkB mutant. Interestingly, alkbh2 knock-out plants were sensitive to the methylating agent methylmethanesulphonate (MMS), and seedlings from these plants developed abnormally when grown in the presence of MMS. The present study establishes ALKBH2 as an important enzyme for protecting Arabidopsis against methylation damage in DNA, and suggests its homologues in other plants to have a similar function.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Reparo do DNA , Dioxigenases/metabolismo , Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato , Alquilantes/farmacologia , Sequência de Aminoácidos , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Dano ao DNA , Metilação de DNA , Dioxigenases/genética , Proteínas de Escherichia coli/genética , Genoma de Planta , Metanossulfonato de Metila/farmacologia , Oxigenases de Função Mista/genética , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
The intracellular ATP-ribosyltransferases PARP1 and PARP2, contribute to DNA base excision repair (BER) and DNA demethylation and have been implicated in epigenetic programming in early mammalian development. Recently, proteomic analyses identified BER proteins to be covalently poly-ADP-ribosylated by PARPs. The role of this posttranslational modification in the BER process is unknown. Here, we show that PARP1 senses AP-sites and SSBs generated during TET-TDG mediated active DNA demethylation and covalently attaches PAR to each BER protein engaged. Covalent PARylation dissociates BER proteins from DNA, which accelerates the completion of the repair process. Consistently, inhibition of PARylation in mESC resulted both in reduced locus-specific TET-TDG-targeted DNA demethylation, and in reduced general repair of random DNA damage. Our findings establish a critical function of covalent protein PARylation in coordinating molecular processes associated with dynamic DNA methylation.
Assuntos
Reparo do DNA , Reparo por Excisão , Animais , Poli ADP Ribosilação , Desmetilação do DNA , Proteômica , Poli(ADP-Ribose) Polimerase-1/metabolismo , Dano ao DNA , DNA/genética , DNA/metabolismo , Mamíferos/genéticaRESUMO
Recently, 5-hydroxymethylcytosine (5hmC) was identified in mammalian genomic DNA. The biological role of this modification remains unclear; however, identifying the genomic location of this modified base will assist in elucidating its function. We describe a method for the rapid and inexpensive identification of genomic regions containing 5hmC. This method involves the selective glucosylation of 5hmC residues by the ß-glucosyltransferase from T4 bacteriophage creating ß-glucosyl-5-hydroxymethylcytosine (ß-glu-5hmC). The ß-glu-5hmC modification provides a target that can be efficiently and selectively pulled down by J-binding protein 1 coupled to magnetic beads. DNA that is precipitated is suitable for analysis by quantitative PCR, microarray or sequencing. Furthermore, we demonstrate that the J-binding protein 1 pull down assay identifies 5hmC at the promoters of developmentally regulated genes in human embryonic stem cells. The method described here will allow for a greater understanding of the temporal and spatial effects that 5hmC may have on epigenetic regulation at the single gene level.
Assuntos
Citosina/análogos & derivados , DNA/química , Genômica/métodos , 5-Metilcitosina/análogos & derivados , Proteínas de Transporte , Citosina/análise , Citosina/metabolismo , DNA/isolamento & purificação , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , Genoma Humano , Glucosiltransferases/metabolismo , Humanos , Regiões Promotoras Genéticas , Proteínas de ProtozoáriosRESUMO
Uridine at the wobble position of tRNA is usually modified, and modification is required for accurate and efficient protein translation. In eukaryotes, wobble uridines are modified into 5-methoxycarbonylmethyluridine (mcm(5)U), 5-carbamoylmethyluridine (ncm(5)U) or derivatives thereof. Here, we demonstrate, both by in vitro and in vivo studies, that the Arabidopsis thaliana methyltransferase AT1G31600, denoted by us AtTRM9, is responsible for the final step in mcm(5)U formation, thus representing a functional homologue of the Saccharomyces cerevisiae Trm9 protein. We also show that the enzymatic activity of AtTRM9 depends on either one of two closely related proteins, AtTRM112a and AtTRM112b. Moreover, we demonstrate that AT1G36310, denoted AtALKBH8, is required for hydroxylation of mcm(5)U to (S)-mchm(5)U in tRNA(Gly)(UCC), and has a function similar to the mammalian dioxygenase ALKBH8. Interestingly, atalkbh8 mutant plants displayed strongly increased levels of mcm(5)U, and also of mcm(5)Um, its 2'-O-ribose methylated derivative. This suggests that accumulated mcm(5)U is prone to further ribose methylation by a non-specialized mechanism, and may challenge the notion that the existence of mcm(5)U- and mcm(5)Um-containing forms of the selenocysteine-specific tRNA(Sec) in mammals reflects an important regulatory process. The present study reveals a role in for several hitherto uncharacterized Arabidopsis proteins in the formation of modified wobble uridines.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Dioxigenases/metabolismo , Oxigenases de Função Mista/metabolismo , Uridina/metabolismo , tRNA Metiltransferases/metabolismo , Homólogo AlkB 8 da RNAt Metiltransferase , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Dioxigenases/química , Dioxigenases/genética , Humanos , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Dados de Sequência Molecular , Mutação , RNA de Transferência/química , RNA de Transferência/metabolismo , RNA de Transferência de Glicina/química , RNA de Transferência de Glicina/metabolismo , Alinhamento de Sequência , tRNA Metiltransferases/química , tRNA Metiltransferases/genéticaRESUMO
Mechanistic and functional studies by gene disruption or editing approaches often suffer from confounding effects like compensatory cellular adaptations generated by clonal selection. These issues become particularly relevant when studying factors directly involved in genetic or epigenetic maintenance. To provide a genetic tool for functional and mechanistic investigation of DNA-repair mediated active DNA demethylation, we generated experimental models in mice and murine embryonic stem cells (ESCs) based on a minigene of the thymine-DNA glycosylase (TDG). The loxP-flanked miniTdg is rapidly and reliably excised in mice and ESCs by tamoxifen-induced Cre activation, depleting TDG to undetectable levels within 24 hours. We describe the functionality of the engineered miniTdg in mouse and ESCs (TDGiKO ESCs) and validate the pluripotency and differentiation potential of TDGiKO ESCs as well as the phenotype of induced TDG depletion. The controlled and rapid depletion of TDG allows for a precise manipulation at any point in time of multistep experimental procedures as presented here for neuronal differentiation in vitro. Thus, we provide a tested and well-controlled genetic tool for the functional and mechanistic investigation of TDG in active DNA (de)methylation and/or DNA repair with minimal interference from adaptive effects and clonal selection.
Assuntos
Timina DNA Glicosilase , Animais , Metilação de DNA , Reparo do DNA , Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Camundongos , Timina DNA Glicosilase/genética , Timina DNA Glicosilase/metabolismoRESUMO
Plasma cell leukemia is a rare and aggressive plasma cell neoplasm that may either originate de novo (primary PCL) or by leukemic transformation of multiple myeloma (MM) to secondary PCL (sPCL). The prognosis of sPCL is very poor, and currently no standard treatment is available due to lack of prospective clinical studies. In an attempt to elucidate factors contributing to transformation, we have performed super-SILAC quantitative proteome profiling of malignant plasma cells collected from the same patient at both the MM and sPCL stages of the disease. 795 proteins were found to be differentially expressed in the MM and sPCL samples. Gene ontology analysis indicated a metabolic shift towards aerobic glycolysis in sPCL as well as marked down-regulation of enzymes involved in glycan synthesis, potentially mediating altered glycosylation of surface receptors. There was no significant change in overall genomic 5-methylcytosine or 5-hydroxymethylcytosine at the two stages, indicating that epigenetic dysregulation was not a major driver of transformation to sPCL. The present study constitutes the first attempt to provide a comprehensive map of the altered protein expression profile accompanying transformation of MM to sPCL in a single patient, identifying several candidate proteins that can be targeted by currently available small molecule drugs. Our dataset furthermore constitutes a reference dataset for further proteomic analysis of sPCL transformation.
Assuntos
Biomarcadores Tumorais/metabolismo , Transformação Celular Neoplásica/patologia , Regulação Leucêmica da Expressão Gênica , Leucemia Plasmocitária/patologia , Mieloma Múltiplo/patologia , Proteoma/análise , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Metilação de DNA , Perfilação da Expressão Gênica , Humanos , Leucemia Plasmocitária/genética , Leucemia Plasmocitária/metabolismo , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Proteômica/métodos , Células Tumorais CultivadasRESUMO
Cytosine methylation in CpG dinucleotides is an epigenetic DNA modification dynamically established and maintained by DNA methyltransferases and demethylases. Molecular mechanisms of active DNA demethylation began to surface only recently with the discovery of the 5-methylcytosine (5mC)-directed hydroxylase and base excision activities of ten-eleven translocation (TET) proteins and thymine DNA glycosylase (TDG). This implicated a pathway operating through oxidation of 5mC by TET proteins, which generates substrates for TDG-dependent base excision repair (BER) that then replaces 5mC with C. Yet, direct evidence for a productive coupling of TET with BER has never been presented. Here we show that TET1 and TDG physically interact to oxidize and excise 5mC, and proof by biochemical reconstitution that the TET-TDG-BER system is capable of productive DNA demethylation. We show that the mechanism assures a sequential demethylation of symmetrically methylated CpGs, thereby avoiding DNA double-strand break formation but contributing to the mutability of methylated CpGs.
Assuntos
Metilação de DNA , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Timina DNA Glicosilase/metabolismo , Ilhas de CpG , Citosina/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/genética , Epigênese Genética , Escherichia coli/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas Proto-Oncogênicas/genética , Timina DNA Glicosilase/genéticaRESUMO
Well-known epigenetic DNA modifications in mammals include the addition of a methyl group and a hydroxyl group to cytosine, resulting in 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) respectively. In contrast, the abundance and the functional implications of these modifications in invertebrate model organisms such as the honey bee (Apis mellifera) and the fruit fly (Drosophila melanogaster) are not well understood. Here we show that both adult honey bees and fruit flies contain 5mC and also 5hmC. Using a highly sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) technique, we quantified 5mC and 5hmC in different tissues of adult honey bee worker castes and in adult fruit flies. A comparison of our data with reports from human and mouse shed light on notable differences in 5mC and 5hmC levels between tissues and species.
RESUMO
BACKGROUND: Hematopoietic stem cell renewal and differentiation are regulated through epigenetic processes. The conversion of 5-methylcytosine into 5-hydroxymethylcytosine (5hmC) by ten-eleven-translocation enzymes provides new insights into the epigenetic regulation of gene expression during development. Here, we studied the potential gene regulatory role of 5hmC during human hematopoiesis. RESULTS: We used reduced representation of 5-hydroxymethylcytosine profiling (RRHP) to characterize 5hmC distribution in CD34+ cells, CD4+ T cells, CD19+ B cells, CD14+ monocytes and granulocytes. In all analyzed blood cell types, the presence of 5hmC at gene bodies correlates positively with gene expression, and highest 5hmC levels are found around transcription start sites of highly expressed genes. In CD34+ cells, 5hmC primes for the expression of genes regulating myeloid and lymphoid lineage commitment. Throughout blood cell differentiation, intragenic 5hmC is maintained at genes that are highly expressed and required for acquisition of the mature blood cell phenotype. Moreover, in CD34+ cells, the presence of 5hmC at enhancers associates with increased binding of RUNX1 and FLI1, transcription factors essential for hematopoiesis. CONCLUSIONS: Our study provides a comprehensive genome-wide overview of 5hmC distribution in human hematopoietic cells and new insights into the epigenetic regulation of gene expression during human hematopoiesis.
RESUMO
Ogg1 and Mutyh DNA glycosylases cooperate to prevent mutations caused by 8-oxoG, a major premutagenic DNA lesion associated with cognitive decline. We have examined behavior and cognitive function in mice deficient of these glycosylases. Ogg1(-/-)Mutyh(-/-) mice were more active and less anxious, with impaired learning ability. In contrast, Mutyh(-/-) mice showed moderately improved memory. We observed no apparent change in genomic 8-oxoG levels, suggesting that Ogg1 and Mutyh play minor roles in global repair in adult brain. Notably, transcriptome analysis of hippocampus revealed that differentially expressed genes in the mutants belong to pathways known to be involved in anxiety and cognition. Esr1 targets were upregulated, suggesting a role of Ogg1 and Mutyh in repression of Esr1 signaling. Thus, beyond their involvement in DNA repair, Ogg1 and Mutyh regulate hippocampal gene expression related to cognition and behavior, suggesting a role for the glycosylases in regulating adaptive behavior.
Assuntos
Ansiedade/enzimologia , DNA Glicosilases/metabolismo , Animais , Ansiedade/genética , Ansiedade/metabolismo , DNA Glicosilases/deficiência , DNA Glicosilases/genética , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Espécies Reativas de Oxigênio/metabolismoRESUMO
The ALKBH family of Fe(II) and 2-oxoglutarate dependent oxygenases comprises enzymes that display sequence homology to AlkB from E. coli, a DNA repair enzyme that uses an oxidative mechanism to dealkylate methyl and etheno adducts on the nucleobases. Humans have nine different ALKBH proteins, ALKBH1-8 and FTO. Mammalian and plant ALKBH8 are tRNA hydroxylases targeting 5-methoxycarbonylmethyl-modified uridine (mcm5U) at the wobble position of tRNAGly(UCC). In contrast, the genomes of some bacteria encode a protein with strong sequence homology to ALKBH8, and robust DNA repair activity was previously demonstrated for one such protein. To further explore this apparent functional duality of the ALKBH8 proteins, we have here enzymatically characterized a panel of such proteins, originating from bacteria, protozoa and mimivirus. All the enzymes showed DNA repair activity in vitro, but, interestingly, two protozoan ALKBH8s also catalyzed wobble uridine modification of tRNA, thus displaying a dual in vitro activity. Also, we found the modification status of tRNAGly(UCC) to be unaltered in an ALKBH8 deficient mutant of Agrobacterium tumefaciens, indicating that bacterial ALKBH8s have a function different from that of their eukaryotic counterparts. The present study provides new insights on the function and evolution of the ALKBH8 family of proteins.
Assuntos
Reparo do DNA , Dioxigenases/metabolismo , Proteínas de Protozoários/metabolismo , RNA de Transferência/metabolismo , tRNA Metiltransferases/metabolismo , Agrobacterium tumefaciens/enzimologia , Agrobacterium tumefaciens/genética , Sequência de Aminoácidos , Biologia Computacional , Dano ao DNA , Metilação de DNA , Dioxigenases/química , Dioxigenases/genética , Ativação Enzimática , Humanos , Dados de Sequência Molecular , Mutação , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , tRNA Metiltransferases/química , tRNA Metiltransferases/genéticaRESUMO
Considerable progress has been made in understanding the origins of genomic uracil and its role in genome stability and host defense; however, the main question concerning the basal level of uracil in DNA remains disputed. Results from assays designed to quantify genomic uracil vary by almost three orders of magnitude. To address the issues leading to this inconsistency, we explored possible shortcomings with existing methods and developed a sensitive LC/MS/MS-based method for the absolute quantification of genomic 2'-deoxyuridine (dUrd). To this end, DNA was enzymatically hydrolyzed to 2'-deoxyribonucleosides and dUrd was purified in a preparative HPLC step and analyzed by LC/MS/MS. The standard curve was linear over four orders of magnitude with a quantification limit of 5 fmol dUrd. Control samples demonstrated high inter-experimental accuracy (94.3%) and precision (CV 9.7%). An alternative method that employed UNG2 to excise uracil from DNA for LC/MS/MS analysis gave similar results, but the intra-assay variability was significantly greater. We quantified genomic dUrd in Ung(+/+) and Ung(-/-) mouse embryonic fibroblasts and human lymphoblastoid cell lines carrying UNG mutations. DNA-dUrd is 5-fold higher in Ung(-/-) than in Ung(+/+) fibroblasts and 11-fold higher in UNG2 dysfunctional than in UNG2 functional lymphoblastoid cells. We report approximately 400-600 dUrd per human or murine genome in repair-proficient cells, which is lower than results using other methods and suggests that genomic uracil levels may have previously been overestimated.
Assuntos
DNA/química , Nucleotídeos de Uracila/química , Animais , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , DNA/genética , DNA/isolamento & purificação , Contaminação por DNA , Genoma Humano , Humanos , Hidrólise , Limite de Detecção , Camundongos , Camundongos Knockout , Padrões de Referência , Espectrometria de Massas em Tandem/normas , Nucleotídeos de Uracila/genética , Nucleotídeos de Uracila/isolamento & purificação , Uracila-DNA Glicosidase/genética , Uracila-DNA Glicosidase/metabolismoRESUMO
Mammals have nine different homologues (ALKBH1-9) of the Escherichia coli DNA repair demethylase AlkB. ALKBH2 is a genuine DNA repair enzyme, but the in vivo function of the other ALKBH proteins has remained elusive. It was recently shown that ALKBH8 contains an additional transfer RNA (tRNA) methyltransferase domain, which generates the wobble nucleoside 5-methoxycarbonylmethyluridine (mcm(5)U) from its precursor 5-carboxymethyluridine (cm(5)U). In this study, we report that (R)- and 5-methoxycarbonylhydroxymethyluridine (mchm(5)U), hydroxylated forms of mcm(5)U, are present in mammalian tRNA-Arg(UCG), and tRNA-Gly(UCC), respectively, representing the first example of a diastereomeric pair of modified RNA nucleosides. Through in vitro and in vivo studies, we show that both diastereomers of mchm(5)U are generated from mcm(5)U, and that the AlkB domain of ALKBH8 specifically hydroxylates mcm(5)U into (S)-mchm(5)U in tRNA-Gly(UCC). These findings expand the function of the ALKBH oxygenases beyond nucleic acid repair and increase the current knowledge on mammalian wobble uridine modifications and their biogenesis.
Assuntos
Códon/genética , Biossíntese de Proteínas/fisiologia , RNA Mensageiro/metabolismo , RNA de Transferência de Glicina/química , Uridina/análogos & derivados , tRNA Metiltransferases/genética , Homólogo AlkB 8 da RNAt Metiltransferase , Animais , Bovinos , Cromatografia Líquida , Biologia Computacional , Camundongos , Estrutura Molecular , Especificidade da Espécie , Espectrometria de Massas em Tandem , Uridina/química , Uridina/metabolismo , tRNA Metiltransferases/química , tRNA Metiltransferases/metabolismoRESUMO
Uridines in the wobble position of tRNA are almost invariably modified. Modifications can increase the efficiency of codon reading, but they also prevent mistranslation by limiting wobbling. In mammals, several tRNAs have 5-methoxycarbonylmethyluridine (mcm5U) or derivatives thereof in the wobble position. Through analysis of tRNA from Alkbh8-/- mice, we show here that ALKBH8 is a tRNA methyltransferase required for the final step in the biogenesis of mcm5U. We also demonstrate that the interaction of ALKBH8 with a small accessory protein, TRM112, is required to form a functional tRNA methyltransferase. Furthermore, prior ALKBH8-mediated methylation is a prerequisite for the thiolation and 2'-O-ribose methylation that form 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) and 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um), respectively. Despite the complete loss of all of these uridine modifications, Alkbh8-/- mice appear normal. However, the selenocysteine-specific tRNA (tRNASec) is aberrantly modified in the Alkbh8-/- mice, and for the selenoprotein Gpx1, we indeed observed reduced recoding of the UGA stop codon to selenocysteine.