RESUMO
Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm caused by the presence of tyrosine kinase BCR-ABL1 fusion protein, which deregulate transcription and mRNA translation. Tyrosine kinase inhibitors (TKIs) are the first-choice treatment. However, resistance to TKIs remains a challenge to cure CML patients. Here, we reveal that the m6A methyltransferase complex METTL3/METTL14 is upregulated in CML patients and that is required for proliferation of primary CML cells and CML cell lines sensitive and resistant to the TKI imatinib. We demonstrate that depletion of METTL3 strongly impairs global translation efficiency. In particular, our data show that METTL3 is crucial for the expression of genes involved in ribosome biogenesis and translation. Specifically, we found that METTL3 directly regulates the level of PES1 protein identified as an oncogene in several tumors. We propose a model in which nuclear METTL3/METTL14 methyltransferase complex modified nascent transcripts whose translation is enhanced by cytoplasmic localization of METTL3, independently from its catalytic activity. In conclusion, our results point to METTL3 as a novel relevant oncogene in CML and as a promising therapeutic target for TKI resistant CML.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Metiltransferases/metabolismo , Biossíntese de Proteínas , Adenosina/análogos & derivados , Adenosina/metabolismo , Catálise , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células , Sobrevivência Celular , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Mesilato de Imatinib/farmacologia , Mesilato de Imatinib/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Modelos Biológicos , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Regulação para CimaRESUMO
The Wilms tumor 1 (WT1)-associated protein (WTAP) is upregulated in many tumors, including, acute myeloid leukemia (AML), where it plays an oncogenic role by interacting with different proteins involved in RNA processing and cell proliferation. In addition, WTAP is also a regulator of the nuclear complex required for the deposition of N6-methyladenosine (m6A) into mRNAs, containing the METTL3 methyltransferase. However, it is not clear if WTAP may have m6A-independent regulatory functions that might contribute to its oncogenic role. Here, we show that both knockdown and overexpression of METTL3 protein results in WTAP protein upregulation, indicating that METTL3 levels are critical for WTAP protein homeostasis. However, we show that WTAP upregulation is not sufficient to promote cell proliferation in the absence of a functional METTL3. Therein, these data indicate that the reported oncogenic function of WTAP is strictly connected to a functional m6A methylation complex.
Assuntos
Metiltransferases/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Metiltransferases/antagonistas & inibidores , Metiltransferases/genética , Proteínas Nucleares/genética , Proteostase , Interferência de RNA , Fatores de Processamento de RNA , RNA Interferente Pequeno/metabolismo , Ribossomos/metabolismoRESUMO
Accumulating evidences indicate that different long non-coding RNAs (lncRNAs) might play a relevant role in tumorigenesis, with their expression and function already associated to cancer development and progression. CCAAT/enhancer-binding protein-α (CEBPA) is a critical regulator of myeloid differentiation whose inactivation contributes to the development of acute myeloid leukemia (AML). Mutations in C/EBPα occur in around 10% of AML cases, leading to the expression of a 30-kDa dominant negative isoform (C/EBPα-p30). In this study, we identified the oncogenic urothelial carcinoma associated 1 (UCA1) lncRNA as a novel target of the C/EBPα-p30. We show that wild-type C/EBPα and C/EBPα-p30 isoform can bind the UCA1 promoter but have opposite effects on UCA1 expression. While wild-type C/EBPα represses, C/EBPα-p30 can induce UCA1 transcription. Notably, we also show that UCA1 expression increases in cytogenetically normal AML cases carrying biallelic CEBPA mutations. Furthermore, we demonstrate that UCA1 sustains proliferation of AML cells by repressing the expression of the cell cycle regulator p27kip1. Thus, we identified, for the first time, an oncogenic lncRNA functioning in concert with the dominant negative isoform of C/EBPα in AML.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/genética , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide/genética , RNA Longo não Codificante/genética , Doença Aguda , Biomarcadores Tumorais/genética , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Proliferação de Células/genética , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Humanos , Immunoblotting , Células K562 , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patologia , Mutação , Regiões Promotoras Genéticas/genética , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Musashi1 is an RNA binding protein that controls the neural cell fate, being involved in maintaining neural progenitors in their proliferative state. In particular, its downregulation is needed for triggering early neural differentiation programs. In this study, we profiled microRNA expression during the transition from neural progenitors to differentiated astrocytes and underscored 2 upregulated microRNAs, miR-23a and miR-125b, that sinergically act to restrain Musashi1 expression, thus creating a regulatory module controlling neural progenitor proliferation.
Assuntos
Proliferação de Células , Embrião de Mamíferos/citologia , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/genética , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/citologia , Proteínas de Ligação a RNA/metabolismo , Animais , Western Blotting , Diferenciação Celular , Células Cultivadas , Embrião de Mamíferos/metabolismo , Técnicas Imunoenzimáticas , Camundongos , Proteínas do Tecido Nervoso/genética , Células-Tronco Neurais/metabolismo , Neurogênese/fisiologia , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ativação TranscricionalRESUMO
The first step in microRNA (miRNA) biogenesis occurs in the nucleus and is mediated by the Microprocessor complex containing the RNase III-like enzyme Drosha and its cofactor DGCR8. Here we show that the 5'-->3' exonuclease Xrn2 associates with independently transcribed miRNAs and, in combination with Drosha processing, attenuates transcription in downstream regions. We suggest that, after Drosha cleavage, a torpedo-like mechanism acts on nascent long precursor miRNAs, whereby Xrn2 exonuclease degrades the RNA polymerase II-associated transcripts inducing its release from the template. While involved in primary transcript termination, this attenuation effect does not restrict clustered miRNA expression, which, in the majority of cases, is separated by short spacers. We also show that transcripts originating from a miRNA promoter are retained on the chromatin template and are more efficiently processed than those produced from mRNA or snRNA Pol II-dependent promoters. These data imply that coupling between transcription and processing promotes efficient expression of independently transcribed miRNAs.
Assuntos
MicroRNAs/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , RNA Nuclear Pequeno/metabolismo , Ribonuclease III/metabolismo , Linhagem Celular Tumoral , Exorribonucleases/genética , Exorribonucleases/metabolismo , Células HeLa , Humanos , MicroRNAs/genética , Regiões Promotoras Genéticas/fisiologia , Proteínas/genética , Proteínas/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , RNA Nuclear Pequeno/genética , Proteínas de Ligação a RNA , Ribonuclease III/genéticaRESUMO
betaTrCP mediates the ubiquitination and subsequent degradation of several key molecules thereby playing a relevant role in different cellular processes during development and in the adult. In Xenopus embryo, betaTrCP acts as a negative regulator of Wnt signaling by interacting with beta-catenin. In this paper, we report results of the study on expression and regulation of the Xenopus betaTrCP gene. We found that xbetaTrCP is expressed in Xenopus oocytes as three transcripts, which very likely correspond to the previously identified localized mRNAs, and four isoforms. The xbetaTrCP promoter functional and structural analysis showed the presence of elements target of positive transcriptional control. Among them, we have identified a beta-catenin/Tcf signaling responsive region and a 45-bp element containing a sequence motif conforming to the SRF binding site, closer to the transcription initiation sites. There are also elements of transcriptional negative control.
Assuntos
Sequências Reguladoras de Ácido Nucleico/genética , Proteínas de Xenopus/genética , Xenopus laevis/genética , Proteínas Contendo Repetições de beta-Transducina/genética , Região 5'-Flanqueadora/genética , Animais , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Linhagem Celular Tumoral , Sondas de DNA/genética , Sondas de DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Regulação da Expressão Gênica , Humanos , Luciferases/genética , Luciferases/metabolismo , Dados de Sequência Molecular , Oócitos/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Elementos de Resposta/genética , Sítio de Iniciação de TranscriçãoRESUMO
beta TrCP plays a relevant role in the control of stability of several key protein factors. In Xenopus, beta TrCP acts as an inhibitor of Wnt signaling and dorsal axis formation. We determined the primary structure of the frog beta TrCP gene, which consists of 14 exons and 13 introns, spanning over 34 kb. Isoforms of x-beta TrCP have been found, which show differences in the NH(2) and COOH regions. NH(2) isoforms differ for the presence or absence of a 30 aa sequence, coded by exon III. In COOH isoforms, 19 C-terminal amino acids are replaced by three different amino acids. Occurrence of two 5' splice donor sites for splicing of intron XIII provides an explanation for these isoforms, based on alternative splicing. The DNA region of the putative beta TrCP promoter contains several TATA elements, one GCCAAT box, and putative binding sites for Ets, Tcf/Lef and NF-kappa B transcription factors. Two transcription initiation sites have been mapped downstream of TATA boxes proximal to ATG for start of translation. Comparison of the Xenopus and human beta TrCP genes indicates high conservation of exon nucleotide and amino acid sequences, size and organization; differences are limited to exons coding for N- and C-terminal regions.