RESUMO
Ribosomal RNAs (rRNAs) act as scaffolds and ribozymes in ribosomes, and these functions are modulated by post-transcriptional modifications. However, the biological role of base methylation, a well-conserved modification of rRNA, is poorly understood. Here, we demonstrate that a nucleolar factor, nucleomethylin (NML; also known as RRP8), is required for the N(1)-methyladenosine (m(1)A) modification in 28S rRNAs of human and mouse cells. NML also contributes to 60S ribosomal subunit formation. Intriguingly, NML depletion increases 60S ribosomal protein L11 (RPL11) levels in the ribosome-free fraction and protein levels of p53 through an RPL11-MDM2 complex, which activates the p53 pathway. Consequently, the growth of NML-depleted cells is suppressed in a p53-dependent manner. These observations reveal a new biological function of rRNA base methylation, which links ribosomal subunit formation to p53-dependent inhibition of cell proliferation in mammalian cells.
Assuntos
Metiltransferases/metabolismo , Proteínas Nucleares/metabolismo , RNA Ribossômico/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Sequência de Bases , Proliferação de Células , Técnicas de Silenciamento de Genes , Células HCT116 , Células HeLa , Humanos , Metilação , Camundongos Endogâmicos C57BL , Proteínas de Ligação a RNA , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismoRESUMO
Tetramerization of p53 is crucial to exert its biological activity, and nucleolar disruption is sufficient to activate p53. We previously demonstrated that nucleolar stress induces translocation of the nucleolar protein MYBBP1A from the nucleolus to the nucleoplasm and enhances p53 activity. However, whether and how MYBBP1A regulates p53 tetramerization in response to nucleolar stress remain unclear. In this study, we demonstrated that MYBBP1A enhances p53 tetramerization, followed by acetylation under nucleolar stress. We found that MYBBP1A has two regions that directly bind to lysine residues of the p53 C-terminal regulatory domain. MYBBP1A formed a self-assembled complex that provided a molecular platform for p53 tetramerization and enhanced p300-mediated acetylation of the p53 tetramer. Moreover, our results show that MYBBP1A functions to enhance p53 tetramerization that is necessary for p53 activation, followed by cell death with actinomycin D treatment. Thus, we suggest that MYBBP1A plays a pivotal role in the cellular stress response.
Assuntos
Nucléolo Celular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Multimerização Proteica , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Sítios de Ligação , Linhagem Celular Tumoral , Proteínas de Ligação a DNA , Proteína p300 Associada a E1A/metabolismo , Humanos , Modelos Biológicos , Proteínas Nucleares/química , Proteínas de Transporte Nucleocitoplasmático/química , Regiões Promotoras Genéticas/genética , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição , Proteína Supressora de Tumor p53/genéticaRESUMO
Estrogen receptor alpha (ERα) expression is a risk factor for breast cancer. HDAC inhibitors have been demonstrated to down-regulate ERα expression in ERα-positive breast cancer cell lines, but the molecular mechanisms are poorly understood. Here, we showed that HDAC inhibitors decrease the stability of ERα mRNA, and that knockdown of HDAC3 decreases the stability of ERα mRNA and suppresses estrogen-dependent proliferation of ERα-positive MCF-7 breast cancer cells. In the Oncomine database, expression levels of HDAC3 in ERα-positive tumors are higher than those in ERα-negative tumors, thus suggesting that HDAC3 is necessary for ERα mRNA stability, and is involved in the estrogen-dependent proliferation of ERα-positive tumors.
Assuntos
Neoplasias da Mama/enzimologia , Receptor alfa de Estrogênio/biossíntese , Histona Desacetilases/metabolismo , Estabilidade de RNA , RNA Mensageiro/química , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Receptor alfa de Estrogênio/genética , Feminino , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , HumanosRESUMO
Nucleolar dynamics are important for cellular stress response. We previously demonstrated that nucleolar stress induces nucleolar protein Myb-binding protein 1A (MYBBP1A) translocation from the nucleolus to the nucleoplasm and enhances p53 activity. However, the underlying molecular mechanism is understood to a lesser extent. Here we demonstrate that MYBBP1A interacts with lysine residues in the C-terminal regulatory domain region of p53. MYBBP1A specifically interacts with nonacetylated p53 and induces p53 acetylation. We propose that MYBBP1A dissociates from acetylated p53 because MYBBP1A did not interact with acetylated p53 and because MYBBP1A was not recruited to the p53 target promoter. Therefore, once p53 is acetylated, MYBBP1A dissociates from p53 and interacts with nonacetylated p53, which enables another cycle of p53 activation. Based on our observations, this MYBBP1A-p53 binding property can account for efficient p53-activation by MYBBP1A under nucleolar stress. Our results support the idea that MYBBP1A plays catalytic roles in p53 acetylation and activation.
Assuntos
Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Ativação Transcricional , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Primers do DNA , Proteínas de Ligação a DNA , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proteínas de Ligação a RNA , Fatores de Transcrição , Proteína Supressora de Tumor p53/químicaRESUMO
Nucleomethylin (NML) has been shown to contribute to ribosome formation through regulating transcription and post-transcriptional modification of rRNA. Based on the observation that NML-/- mice are frequently embryonic lethal, we analyzed NML-/- embryos to clarify the role of NML in embryogenesis. We found that NML deficiency leads to lethality at the time point between E10.5 and E12.5. Most of E10.5 NML-/- embryos exhibited growth retardation and/or malformation with marked impairment of erythropoiesis. Consistent with a previous study, the m1A in 28S rRNA was dramatically reduced in NML-/- foetal liver (FL) cells. Because the previous study demonstrated p53-dependent apoptosis of NML-knockdown cells, and because we observed upregulation of p21, one of the p53 target genes, in NML-/- FL cells, we tested whether p53 disruption cancelled the NML-deficient phenotypes. Contrary to our expectation, suppression of p53 did not rescue the lethality or impaired erythropoiesis of NML-/- embryos, suggesting that p53-independent mechanisms underlie the NML-deficient phenotypes. These results clarify an essential role of NML during embryogenesis, particularly in erythropoiesis. We surmise that embryonic erythropoiesis is particularly sensitive to impaired protein synthesis, which is caused by the defective methylation of rRNA and consequent failure of ribosome formation.
Assuntos
Eritropoese , Feto/metabolismo , Fígado/metabolismo , Metiltransferases/deficiência , Proteínas Nucleares/deficiência , Animais , Feminino , Feto/citologia , Fígado/citologia , Masculino , Metilação , Metiltransferases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/metabolismo , RNA Ribossômico/metabolismoRESUMO
We describe the mechanism, substituent effects, and origins of the selectivity of the nickel-catalyzed four-component coupling reactions of alkyl fluorides, aryl Grignard reagents, and two molecules of 1,3-butadiene that affords a 1,6-octadiene carbon framework bearing alkyl and aryl groups at the 3- and 8-positions, respectively, and the competing cross-coupling reaction. Both the four-component coupling reaction and the cross-coupling reaction are triggered by the formation of anionic nickel complexes, which are generated by the oxidative dimerization of two molecules of 1,3-butadiene on Ni(0) and the subsequent complexation with the aryl Grignard reagents. The C-C bond formation of the alkyl fluorides with the γ-carbon of the anionic nickel complexes leads to the four-component coupling product, whereas the cross-coupling product is yielded via nucleophilic attack of the Ni center toward the alkyl fluorides. These steps are found to be the rate-determining and selectivity-determining steps of the whole catalytic cycle, in which the C-F bond of the alkyl fluorides is activated by the Mg cation rather than a Li or Zn cation. ortho-Substituents of the aryl Grignard reagents suppressed the cross-coupling reaction leading to the selective formation of the four-component products. Such steric effects of the ortho-substituents were clearly demonstrated by crystal structure characterizations of ate complexes and DFT calculations. The electronic effects of the para-substituent of the aryl Grignard reagents on both the selectivity and reaction rates are thoroughly discussed. The present mechanistic study offers new insight into anionic complexes, which are proposed as the key intermediates in catalytic transformations even though detailed mechanisms are not established in many cases, and demonstrates their synthetic utility as promising intermediates for C-C bond forming reactions, providing useful information for developing efficient and straightforward multicomponent reactions.
RESUMO
RNAs are post-transcriptionally modified in all kingdoms of life. Of these modifications, base methylations are highly conserved in eukaryote ribosomal RNA (rRNA). Recently, rRNA processing protein 8 (Rrp8) and nucleomethylin (NML) were identified as factors of N1-methyladenosine (m1A) modification in yeast 25 S and mammalian 28 S rRNA, respectively. However, m1A modification of rRNA is still poorly understood in Caenorhabditis elegans (C. elegans). Here, using the liquid chromatography/tandem mass spectrometry analysis and RNA immunoprecipitation assay, we have identified that the m1A modification is located around position 674 (A674) of 26 S rRNA in C. elegans. Furthermore, quantitative PCR-based analysis revealed that T07A9.8, a C. elegans homolog of yeast Rrp8 and human NML, is responsible for m1A modification at A674 of 26 S rRNA. This m1A modification site in C. elegans corresponds to those in yeast 25 S rRNA and human 28 S rRNA. Intriguingly, T07A9.8 is not associated with pre-rRNA transcription under normal nutrient conditions. Since the m1A modification of 26 S rRNA requires T07A9.8 in C. elegans, we designated the gene as rRNA adenine methyltransferase-1 (rram-1).
Assuntos
Adenina/metabolismo , Caenorhabditis elegans/genética , RNA Ribossômico/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Animais , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/metabolismo , Metilação , RNA Ribossômico/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genéticaRESUMO
An anionic Ni complex was isolated and its structure determined by X-ray crystallography. With such an anionic complex as a key intermediate, a regio- and stereoselective multicomponent coupling reaction of perfluoroarenes, aryl Grignard reagents, and 1,3-butadiene in a 1:1:2 ratio was achieved, resulting in the formation of 1,6-octadiene derivatives containing two aryl groups, one from the perfluoroarene and the other from the aryl Grignard reagent, at the 3- and 8-positions, respectively.
RESUMO
Ribosome biosynthesis is a major intracellular energy-consuming process. We previously identified a nucleolar factor, nucleomethylin (NML), which regulates intracellular energy consumption by limiting rRNA transcription. Here, we show that, in livers of obese mice, the recruitment of NML to rRNA gene loci is increased to repress rRNA transcription. To clarify the relationship between obesity and rRNA transcription, we generated NML-null (NML-KO) mice. NML-KO mice show elevated rRNA level, reduced ATP concentration, and reduced lipid accumulation in the liver. Furthermore, in high-fat-diet (HFD)-fed NML-KO mice, hepatic rRNA levels are not decreased. Both weight gain and fat accumulation in HFD-fed NML-KO mice are significantly lower than those in HFD-fed wild-type mice. These findings indicate that rRNA transcriptional activation promotes hepatic energy consumption, which alters hepatic lipid metabolism. Namely, hepatic rRNA transcriptional repression by HFD feeding is essential for energy storage.