RESUMO
Ewing sarcoma cells depend on the EWS-FLI1 fusion transcription factor for cell survival. Using an assay of EWS-FLI1 activity and genome-wide RNAi screening, we have identified proteins required for the processing of the EWS-FLI1 pre-mRNA. We show that Ewing sarcoma cells harboring a genomic breakpoint that retains exon 8 of EWSR1 require the RNA-binding protein HNRNPH1 to express in-frame EWS-FLI1. We also demonstrate the sensitivity of EWS-FLI1 fusion transcripts to the loss of function of the U2 snRNP component, SF3B1. Disrupted splicing of the EWS-FLI1 transcript alters EWS-FLI1 protein expression and EWS-FLI1-driven expression. Our results show that the processing of the EWS-FLI1 fusion RNA is a potentially targetable vulnerability in Ewing sarcoma cells.
Assuntos
Proteínas de Fusão Oncogênica/metabolismo , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteína EWS de Ligação a RNA/metabolismo , Sequência de Bases , Sítios de Ligação , Proteínas de Ligação a Calmodulina/antagonistas & inibidores , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Éxons , Regulação Neoplásica da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/metabolismo , Humanos , Proteínas dos Microfilamentos/antagonistas & inibidores , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Proteínas de Fusão Oncogênica/genética , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteína Proto-Oncogênica c-fli-1/antagonistas & inibidores , Proteína Proto-Oncogênica c-fli-1/genética , Interferência de RNA , Precursores de RNA/metabolismo , Splicing de RNA , Fatores de Processamento de RNA , RNA Interferente Pequeno/metabolismo , Proteína EWS de Ligação a RNA/antagonistas & inibidores , Proteína EWS de Ligação a RNA/genética , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Ribonucleoproteína Nuclear Pequena U2/antagonistas & inibidores , Ribonucleoproteína Nuclear Pequena U2/genética , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Sarcoma de Ewing/patologia , Transativadores , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The tristetraprolin (TTP) family of zinc-finger proteins, TTP, BRF1 and BRF2, regulate the stability of a subset of mRNAs containing 3'UTR AU-rich elements (AREs), including mRNAs coding for cytokines, transcription factors, and proto-oncogenes. To better understand the mechanism by which TTP-family proteins control mRNA stability in mammalian cells, we aimed to identify TTP- and BRF1-interacting proteins as potential TTP-family co-factors. This revealed hnRNP F as a prominent interactor of TTP and BRF1. While TTP, BRF1 and hnRNP F are all RNA binding proteins (RBPs), the interaction of hnRNP F with TTP and BRF1 is independent of RNA. Depletion of hnRNP F impairs the decay of a subset of TTP-substrate ARE-mRNAs by a mechanism independent of the extent of hnRNP F binding to the mRNA. Taken together, these findings implicate hnRNP F as a co-factor in a subset of TTP/BRF-mediated mRNA decay and highlight the importance of RBP cooperativity in mRNA regulation.
Assuntos
Regiões 3' não Traduzidas , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/metabolismo , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Fator de Transcrição TFIIIB/metabolismo , Tristetraprolina/metabolismo , Animais , Linhagem Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Células HEK293 , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/genética , Humanos , Luciferases/genética , Luciferases/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Células NIH 3T3 , Estabilidade de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fatores Associados à Proteína de Ligação a TATA/genética , Fator de Transcrição TFIIIB/genética , Tristetraprolina/genéticaRESUMO
Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a cytoplasmic protein that plays a critical role in the maintenance of energy homeostasis. However, its role in the nucleus is still largely unknown. Here, we showed that AMPKα2 translocated into the nucleus during muscle differentiation. We also showed that upon treatment with 5-aminoimidazole-4-carboxy-amide-1-d-ribofuranoside (AICAR), an AMPK activator, AMPK rapidly translocated into the nucleus in rat myoblast L6 cells. On the other hand, the AMPKα2 phosphorylation-defective mutant did not translocate into the nucleus. Knockdown of AMPKα2 suppressed the differentiation-induced expression of myogenin, a differentiation marker. A physiological AMPK activator, metformin, also induced the translocation of AMPKα2 into the nucleus. Both inhibition and knockdown of AMPKα2 suppressed metformin-mediated glucose uptake. In addition, AMPKα2 was shown to directly interact with the heterogeneous nuclear ribonucleoprotein H (hnRNP H). AICAR treatment increased the phosphorylation of hnRNP H. Metformin increased the interaction between AMPKα2 and hnRNP H in the nucleus. Knockdown of hnRNP H blocked metformin-induced glucose uptake. In summary, these results demonstrate that AMPKα2 translocates into the nucleus via phosphorylation, AMPKα2 interacts with and phosphorylates hnRNP H in the nucleus, and such a protein-protein interaction modulates metformin-mediated glucose uptake.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Núcleo Celular/metabolismo , Glucose/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/metabolismo , Metformina/farmacologia , Transcitose/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/genética , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Diferenciação Celular , Linhagem Celular , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/genética , Músculo Esquelético/citologia , Fosforilação/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Ribonucleotídeos/farmacologiaRESUMO
A-Raf belongs to the family of oncogenic Raf kinases that are involved in mitogenic signaling by activating the mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK pathway. Low kinase activity of A-Raf toward MEK suggested that A-Raf might have alternative functions. Here, we show that A-Raf prevents cancer cell apoptosis contingent on the expression of the heterogeneous nuclear ribonucleoprotein H (hnRNP H) splice factor, which is required for the correct transcription and expression of a-raf. Apoptosis was prevented by A-Raf through sequestration and inactivation of the proapoptotic MST2 kinase. Small interfering RNA-mediated knockdown of hnRNP H or A-Raf resulted in MST2-dependent apoptosis. In contrast, enforced expression of either hnRNP H or A-Raf partially counteracted apoptosis induced by etoposide. In vivo expression studies of colon specimens corroborated the overexpression of hnRNP H in malignant tissues and its correlation with A-Raf levels. Our findings define a novel mechanism that is usurped in tumor cells to escape naturally imposed apoptotic signals.
Assuntos
Apoptose/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/fisiologia , Neoplasias/genética , Neoplasias/patologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas A-raf/genética , Apoptose/efeitos dos fármacos , Células Cultivadas , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/genética , Humanos , Modelos Biológicos , Neoplasias/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas A-raf/metabolismo , RNA Interferente Pequeno/farmacologia , Serina-Treonina Quinase 3 , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
The human thrombopoietin (THPO) gene displays a series of alternative splicing events that provide valuable models for studying splicing mechanisms. The THPO region spanning exon 1-4 presents both alternative splicing of exon 2 and partial intron 2 (IVS2) retention following the activation of a cryptic 3' splice site 85 nt upstream of the authentic acceptor site. IVS2 is particularly rich in stretches of 3-5 guanosines (namely, G1-G10) and we have characterized the role of these elements in the processing of this intron. In vivo studies show that runs G7-G10 work in a combinatorial way to control the selection of the proper 3' splice site. In particular, the G7 element behaves as the splicing hub of intron 2 and its interaction with hnRNP H1 is critical for the splicing process. Removal of hnRNP H1 by RNA interference promoted the usage of the cryptic 3' splice site so providing functional evidence that this factor is involved in the selection of the authentic 3' splice site of THPO IVS2.