Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 25
Filtrar
1.
iScience ; 26(1): 105784, 2023 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-36590164

RESUMO

THOC5, a member of the THO complex, is essential for the 3'processing of some inducible genes, the export of a subset of mRNAs and stem cell survival. Here we show that THOC5 depletion results in altered 3'cleavage of >50% of mRNAs and changes in RNA polymerase II binding across genes. THOC5 is recruited close to high-density polymerase II sites, suggesting that THOC5 is involved in transcriptional elongation. Indeed, measurement of elongation rates in vivo demonstrated decreased rates in THOC5-depleted cells. Furthermore, THOC5 is preferentially recruited to its target genes in slow polymerase II cells compared with fast polymerase II cells. Importantly chromatin-associated THOC5 interacts with CDK12 (a modulator of transcription elongation) and RNA helicases DDX5, DDX17, and THOC6 only in slow polymerase II cells. The CDK12/THOC5 interaction promotes CDK12 recruitment to R-loops in a THOC6-dependent manner. These data demonstrate a novel function of THOC5 in transcription elongation.

2.
Oncogenesis ; 10(3): 31, 2021 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-33731669

RESUMO

In most human cancers, a large number of proteins with driver mutations are involved in tumor development, implying that multiple fine tuners are involved in cancer formation and/or maintenance. A useful strategy for cancer therapy may therefore be to target multiple cancer type-specific fine tuners. Furthermore, genome-wide association studies of tumor samples have identified a large number of long noncoding (lnc)RNA associated with various types of tumor. In this context we have previously found that C20orf204 (a splice variant of Linc00176) RNA contains a 189 amino acid (AA) long open reading frame (C20orf204-189AA) that is expressed predominantly in hepatocellular carcinoma (HCC). We report here that a protein, C20orf204-189AA, was detected in the nucleus of 14 out of 20 primary HCC, but not in control livers. Strikingly, overexpression of C20orf204-189AA enhanced cell proliferation and ribosomal RNA transcription. C20orf204-189AA is co-localized, and interacted with nucleolin via the C-terminal and with ribosomal RNA via the N-terminal domain. Furthermore, the expression of C20orf204-189AA upregulates the protein level of nucleolin. Nucleolin and C20orf204 mRNA levels in HCC are correlated with tumor differentiation grade and patient survival, suggesting that C20orf204-189AA is a cancer type-specific fine tuner in some HCC that presents itself for potential targeting therapy and cancer biomarker. Thus, cancer cells exhibit remarkable transcriptome alterations partly by adopting cancer-specific splicing isoforms of noncoding RNAs and may participate in tumor development.

3.
Int J Mol Sci ; 23(1)2021 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-35008483

RESUMO

Identification of cancer-specific target molecules and biomarkers may be useful in the development of novel treatment and immunotherapeutic strategies. We have recently demonstrated that the expression of long noncoding (lnc) RNAs can be cancer-type specific due to abnormal chromatin remodeling and alternative splicing. Furthermore, we identified and determined that the functional small protein C20orf204-189AA encoded by long intergenic noncoding RNA Linc00176 that is expressed predominantly in hepatocellular carcinoma (HCC), enhances transcription of ribosomal RNAs and supports growth of HCC. In this study we combined RNA-sequencing and polysome profiling to identify novel micropeptides that originate from HCC-specific lncRNAs. We identified nine lncRNAs that are expressed exclusively in HCC cells but not in the liver or other normal tissues. Here, DNase-sequencing data revealed that the altered chromatin structure plays a key role in the HCC-specific expression of lncRNAs. Three out of nine HCC-specific lncRNAs contain at least one open reading frame (ORF) longer than 50 amino acid (aa) and enriched in the polysome fraction, suggesting that they are translated. We generated a peptide specific antibody to characterize one candidate, NONHSAT013026.2/Linc013026. We show that Linc013026 encodes a 68 amino acid micropeptide that is mainly localized at the perinuclear region. Linc013026-68AA is expressed in a subset of HCC cells and plays a role in cell proliferation, suggesting that Linc013026-68AA may be used as a HCC-specific target molecule. Our finding also sheds light on the role of the previously ignored 'dark proteome', that originates from noncoding regions in the maintenance of cancer.


Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , RNA Longo não Codificante/genética , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Redes Reguladoras de Genes/genética , Células HeLa , Células Hep G2 , Humanos , Fases de Leitura Aberta/genética , Peptídeos , Análise de Sequência de RNA/métodos
4.
Sci Rep ; 9(1): 17319, 2019 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-31754186

RESUMO

The Myc gene has been implicated in the pathogenesis of most types of human cancerous tumors. Myc/Max activates large numbers of pro-tumor genes; however it also induces anti-proliferation genes. When anti-proliferation genes are activated by Myc, cancer cells can only survive if they are downregulated. Hepatocellular carcinoma (HCC) specific intronic long noncoding antisense (lnc-AS) RNA, the EVA1A-AS gene, is located within the second intron (I2) of the EVA1A gene (EVA-1 homolog A) that encodes an anti-proliferation factor. Indeed, EVA1A, but not EVA1A-AS, is expressed in normal liver. Depletion of EVA1A-AS suppressed cell proliferation of HepG2 cells by upregulation of EVA1A. Overexpression of EVA1A caused cell death at the G2/M phase via microtubule catastrophe. Furthermore, suppressed EVA1A expression levels are negatively correlated with differentiation grade in 365 primary HCCs, while EVA1A-AS expression levels are positively correlated with patient survival. Notably, both EVA1A and EVA1A-AS were activated by the Myc/Max complex. Eva1A-AS is transcribed in the opposite direction near the 3'splice site of EVA1A I2. The second intron did not splice out in a U2 dependent manner and EVA1A mRNA is not exported. Thus, the Myc/Max dependent anti-proliferating gene, EVA1A, is controlled by Myc/Max dependent anti-sense noncoding RNA for HCC survival.


Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Proteínas de Membrana/genética , RNA Longo não Codificante/metabolismo , RNA Nuclear Pequeno/metabolismo , Apoptose/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Proliferação de Células/genética , Conjuntos de Dados como Assunto , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Íntrons/genética , Fígado/patologia , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Longo não Codificante/genética , RNA Interferente Pequeno/metabolismo , RNA-Seq , Regulação para Cima
5.
Cell Signal ; 28(12): 1872-1880, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27619201

RESUMO

Over 100 putative driver genes that are associated with multiple recurrently altered pathways were detected in hepatocellular carcinoma (HCC), suggesting that multiple pathways will need to be inhibited for any therapeutic method to be effective. In this context, functional modification of the RNA regulating protein, tristetraprolin (TTP) that regulates approximately 2500 genes represents a promising strategy in HCC therapy. Since overexpression of TTP induces cell death in all cell types, it would be useful to target the regulator of TTP. In this study, we applied an inhibitor to MAPKAP2 (MK2) that suppresses TTP function. Importantly, cBIOportal for HCC genomics shows that expression level of the MK2 gene correlates with clinical outcome of HCC. We show that upon treatment with MK2 inhibitor, all 5 HCC cell lines, namely HepG2, Huh7, Hep3B, HLE and HLF, reduced cell growth, especially HepG2 and Hep3B cells underwent apoptosis. Simultaneously, TTP target genes such as c-Myc, IER3 or AKT-1 were downregulated. Depletion of the TTP gene rescued cells from apoptosis and restored the TTP-target mRNA expression in the presence of MK2 inhibitor. Furthermore, MK2 was activated in primary HCC that express TTP at high level. The TTP gene was induced upon treatment with DNA methylation inhibitor, 5-aza dC or interferon in three other cell lines, Huh7, HLE or HLF. Upon treatment with MK2 inhibitor and 5-aza dC or interferon these cells underwent apoptosis. The depletion of TTP in these cells partially rescued them from apoptosis, suggesting that the MK2/TTP pathway plays a role in proliferation and maintenance of HCCs. Notably, under the same conditions human hepatocyte cells (THLE-2) did not undergo apoptosis. These data also suggest that MK2 inhibitor with 5-aza dC or interferon may be a useful tool for therapy against HCC.


Assuntos
Apoptose/efeitos dos fármacos , Azacitidina/farmacologia , Carcinoma Hepatocelular/enzimologia , Metilação de DNA/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Neoplasias Hepáticas/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Tristetraprolina/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Interferon-alfa/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Antígeno Ki-67/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Resultado do Tratamento , Tristetraprolina/genética
6.
Cell Signal ; 28(5): 438-447, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26876787

RESUMO

Differentiated hepatocytes are long-lived and normally do not undergo cell division, however they have the unique capacity to autonomously decide their replication fate after liver injury. In this context, the key players of liver regeneration immediately after injury have not been adequately studied. Using an in vitro liver culture system, we show that after liver injury, p38 mitogen-activated protein kinase (p38MAPK), mitogen-activated protein kinase-activated protein kinase 2 (MK2) and extracellular-signal regulated kinase (Erk)1/2 were activated within 15 min and continued to be phosphorylated for more than 2h. Both p38MAPK and Erk1/2 were activated at the edge of the cut as well as on the liver surface where the mesothelial cell sheet expresses several cytokines. Notably, in human liver Erk1/2 was also activated under the mesothelial cell sheet shortly after liver resections. Furthermore, in in vitro liver slice culture immediate early genes (IEGs) were upregulated within 1-2 h and the S phase marker proliferation-cell-nuclear-antigen (PCNA) appeared 24 h after injury. Although Erk1/2 was activated after injury, in MK2 depleted liver a set of IEGs, such as Dusp1, Cox2, or c-Myc and proliferation marker gene Ki67 were not induced. In addition, in immortalized hepatocyte cells, THLE-2, the same subset of genes was upregulated upon stimulation with lipopolysaccharide (LPS), but not in the presence of MK2 inhibitor. The protein level of tristetraprolin (TTP), a substrate for MK2 that plays a role in mRNA degradation, was increased in the presence of MK2 inhibitor. In this context, the depletion of TTP gene rescued Dusp1, Cox2, or c-Myc upregulation in the presence of MK2 inhibitor. These data imply that MK2 pathway is positively involved in Erk1/2 induced IEG response after liver injury. These data also suggest that in vitro liver culture may be a useful tool for measuring the proliferation potential of hepatocytes in individual liver.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Genes Precoces , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Fígado/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas Serina-Treonina Quinases/fisiologia , Tristetraprolina/fisiologia , Animais , Células Cultivadas , Hepatócitos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Fígado/enzimologia , Fígado/lesões , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Técnicas de Cultura de Órgãos , Antígeno Nuclear de Célula em Proliferação/análise , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Ativação Transcricional , Transcriptoma , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
7.
Cancer Lett ; 373(2): 222-6, 2016 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-26828015

RESUMO

Recent evidence indicates that mRNA export is selective, giving priority to a subset of mRNAs that control diverse biological processes including cell proliferation, differentiation, stress response, and cell survival as well as tumor development. The depletion of a member of the mRNA export complex, the THO complex, impairs the expression of only a subset of genes, but causes dramatic changes in phenotype, such as cell cycle inhibition, abnormal differentiation, and importantly apoptosis of stem cells and cancer cells but not normal epithelial cells, hepatocytes, or fibroblasts. Recent exosome sequence data revealed that over 100 driver gene mutations with a number of signaling pathways are involved in human cancer formation, indicating that multiple signaling pathways will need to be inhibited for cancer therapy. In this review we firstly describe a basic feature and function of the mRNA export complex, THO, secondly, the biological alteration upon depletion of a member of the THO complex in normal and cancer cells, and thirdly, identification of its target genes. Finally we describe our recent data on selection of targeting candidates from THOC5 dependent genes for application in cancer therapy.


Assuntos
Neoplasias/terapia , Proteínas Nucleares/fisiologia , Animais , Humanos , Neoplasias/genética , Transporte de RNA , RNA Mensageiro/metabolismo
8.
Cell Commun Signal ; 12: 73, 2014 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-25376987

RESUMO

BACKGROUND: One of the most insidious characteristics of cancer is its spread to and ability to compromise distant organs via the complex process of metastasis. Communication between cancer cells and organ-resident cells via cytokines/chemokines and direct cell-cell contacts are key steps for survival, proliferation and invasion of metastasized cancer cells in organs. Precision-cut liver slices (PCLS) are considered to closely reflect the in vivo situation and are potentially useful for studying the interaction of cancer cells with liver-resident cells as well as being a potentially useful tool for screening anti-cancer reagents. Application of the PCLS technique in the field of cancer research however, has not yet been well developed. RESULTS: We established the mouse PCLS system using perfluorodecalin (PFD) as an artificial oxygen carrier. Using this system we show that the adherence of green fluorescent protein (GFP) labeled MDA-MB-231 (highly invasive) cells to liver tissue in the PCLS was 5-fold greater than that of SK-BR-3 (less invasive) cells. In addition, we generated PCLS from THOC5, a member of transcription/export complex (TREX), knockout (KO) mice. The PCLS still expressed Gapdh or Albumin mRNAs at normal levels, while several chemokine/growth factor or metalloprotease genes, such as Cxcl12, Pdgfa, Tgfb, Wnt11, and Mmp1a genes were downregulated more than 2-fold. Interestingly, adhesion of cancer cells to THOC5 KO liver slices was far less (greater than 80% reduction) than to wild-type liver slices. CONCLUSION: Mouse PCLS cultures in the presence of PFD may serve as a useful tool for screening local adherence and invasiveness of individual cancer cells, since single cells can be observed. This method may also prove useful for identification of genes in liver-resident cells that support cancer invasion by using PCLS from transgenic liver.


Assuntos
Fígado/metabolismo , Neoplasias/patologia , Microambiente Tumoral , Trifosfato de Adenosina/metabolismo , Animais , Adesão Celular , Linhagem Celular Tumoral , Fluorocarbonos , Proteínas de Fluorescência Verde , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Invasividade Neoplásica , Neoplasias/metabolismo , Ratos Wistar , Transdução de Sinais , Triglicerídeos/metabolismo
9.
Nucleic Acids Res ; 42(19): 12249-60, 2014 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-25274738

RESUMO

Transcription of immediate early genes (IEGs) in response to extrinsic and intrinsic signals is tightly regulated at multiple stages. It is known that untranslated regions of the RNA can play a role in these processes. Here we show that THOC5, a member of the TREX (transcription/export) complex, plays a role in expression of only a subset of constitutively active genes, however transcriptome analysis reveals that more than 90% of IEG were not induced by serum in THOC5 depleted cells. Furthermore, THOC5 depletion does not influence the expression of the most rapidly induced IEGs, e.g. Fos and Jun. One group of THOC5 target genes, including Id1, Id3 and Wnt11 transcripts, were not released from chromatin in THOC5 depleted cells. Genes in another group, including Myc and Smad7 transcripts, were released with shortening of 3'UTR by alternative cleavage, and were spliced but export was impaired in THOC5 depleted cells. By interactome analysis using THOC5 as bait, we show that upon stimulation with serum THOC5 forms a complex with polyadenylation-specific factor 100 (CPSF100). THOC5 is required for recruitment of CPSF100 to 3'UTR of THOC5 target genes. These data suggest the presence of a novel mechanism for the control of IEG response by THOC5 via 3'end-processing.


Assuntos
Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Genes Precoces , Proteínas Nucleares/metabolismo , Processamento de Terminações 3' de RNA , Regiões 3' não Traduzidas , Animais , Linhagem Celular , Camundongos , Células NIH 3T3 , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Splicing de RNA , Proteína Smad7/genética , Proteína Smad7/metabolismo , Transcrição Gênica
10.
Cell Commun Signal ; 12: 3, 2014 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-24410813

RESUMO

Cell growth, differentiation, and commitment to a restricted lineage are guided by a timely expressed set of growth factor/cytokine receptors and their down-stream transcription factor genes. Transcriptional control mechanisms of gene expression during differentiation have been mainly studied by focusing on the cis- and trans-elements in promoters however, the role of mRNA export machinery during differentiation has not been adequately examined. THO (Suppressors of the transcriptional defects of hpr1 delta by overexpression) complex 5 (THOC5) is a member of THO complex which is a subcomplex of the transcription/export complex (TREX). THOC5 is evolutionarily conserved in higher eukaryotes, however the exact roles of THOC5 in transcription and mRNA export are still unclear. In this review, we focus on recently uncovered aspects of the role of THOC5 in signal transduction induced by extracellular stimuli. THOC5 is phosphorylated by several protein kinases at multiple residues upon extracellular stimuli. These include stimulation with growth factors/cytokines/chemokines, or DNA damage reagents. Furthermore, THOC5 is a substrate for several oncogenic tyrosine kinases, suggesting that THOC5 may be involved in cancer development. Recent THOC5 knockout mouse data reveal that THOC5 is an essential element in the maintenance of stem cells and growth factor/cytokine-mediated differentiation/proliferation. Furthermore, depletion of THOC5 influences less than 1% of total mRNA export in the steady state, however it influences more than 90% of growth factor/cytokine induced genes. THOC5, thereby contributes to the 3' processing and/or export of immediate-early genes induced by extracellular stimuli. These studies bring new insight into the link between the mRNA export complex and immediate-early gene response. The data from these studies also suggest that THOC5 may be a useful tool for studying stem cell biology, for modifying the differentiation processes and for cancer therapy.


Assuntos
Diferenciação Celular , Proliferação de Células , Proteínas Nucleares/metabolismo , Transporte de RNA , Transdução de Sinais , Sequência de Aminoácidos , Animais , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
11.
BMC Cell Biol ; 14: 51, 2013 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-24267292

RESUMO

BACKGROUND: THO (Suppressors of the transcriptional defects of hpr1 delta by overexpression) complex 5 (THOC5), an mRNA export protein, is involved in the expression of only 1% of all genes. Using an interferon inducible knockout mouse system, we have previously shown that THOC5 is an essential element in the maintenance of hematopoietic stem cells and cytokine-mediated hematopoiesis in adult mice. Here we interrogate THOC5 function in cell differentiation beyond the hematopoietic system and study pathological changes caused by THOC5 deficiency. RESULTS: To examine whether THOC5 plays a role in general differentiation processes, we generated tamoxifen inducible THOC5 knockout mice. We show here that the depletion of THOC5 impaired not only hematopoietic differentiation, but also differentiation and self renewal of the gut epithelium. Depletion of the THOC5 gene did not cause pathological alterations in liver or kidney. We further show that THOC5 is indispensable for processing of mRNAs induced by Wnt (wingless/integrated) signaling which play key roles in epithelial cell differentiation/proliferation. A subset of Wnt target mRNAs, SRY-box containing gene 9 (Sox9), and achaete-scute complex homolog 2 (Ascl2), but not Fibronectin 1 (Fn1), were down-regulated in THOC5 knockout intestinal cells. The down-regulated Wnt target mRNAs were able to bind to THOC5. Furthermore, pathological alterations in the gastrointestinal tract induced translocation of intestinal bacteria and caused sepsis in mice. The bacteria translocation may cause Toll-like receptor activation. We identified one of the Toll-like receptor inducible genes, prostaglandin-endoperoxidase synthase 2 (Ptgs2 or COX2) transcript as THOC5 target mRNA. CONCLUSION: THOC5 is indispensable for processing of only a subset of mRNAs, but plays a key role in processing of mRNAs inducible by Wnt signals. Furthermore, THOC5 is dispensable for general mRNA export in terminally differentiated organs, indicating that multiple mRNA export pathways exist. These data imply that THOC5 may be a useful tool for studying intestinal stem cells, for modifying the differentiation processes and for cancer therapy.


Assuntos
Células Epiteliais/metabolismo , Infecções por Escherichia coli/genética , Mucosa Intestinal/metabolismo , Proteínas Nucleares/genética , RNA Mensageiro/genética , Sepse/genética , Proteínas Wnt/genética , Animais , Translocação Bacteriana , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proliferação de Células , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Escherichia coli/crescimento & desenvolvimento , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Fibronectinas/genética , Fibronectinas/metabolismo , Regulação da Expressão Gênica , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Camundongos , Camundongos Knockout , Proteínas Nucleares/deficiência , Ligação Proteica , Transporte de RNA , RNA Mensageiro/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Sepse/metabolismo , Sepse/microbiologia , Transdução de Sinais , Proteínas Wnt/metabolismo
12.
RNA ; 17(11): 1957-66, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21937706

RESUMO

In response to DNA damage, transcription is blocked by inhibition of RNA polymerase II activity. The regulation of a preexisting pool of mRNAs, therefore, plays a key role in DNA repair, cell cycle arrest, or inhibition of differentiation. THOC5 is a member of the THO complex and plays a role in the export of a subset of mRNA, which plays an important role in hematopoiesis and maintaining primitive cells. Since three serine residues in the PEST domain of THOC5 have been shown to be directly phosphorylated by ataxia-telangiectasia-mutated (ATM) kinase, we examined the THOC5-dependent mRNA export under DNA damage. We show here that DNA damage drastically decreased the cytoplasmic pool of a set of THOC5-dependent mRNAs and impaired the THOC5/mRNA complex formation. The mRNP complex formed with nonphosphorylation mutant (S307/312/314A) THOC5, but not with a C-terminal deletion mutant after DNA damage, suggesting that the C-terminal domain of THOC5, but not its phosphorylation in the PEST domain, is necessary for the regulation of the mRNA-binding potency of THOC5. The cytoplasmic THOC5-dependent mRNAs were recovered by treatment with ATM kinase-specific or p53-specific siRNA, as well as by treatment with ATM kinase inhibitor, KU55933, under DNA damage conditions, suggesting that the ATM-kinase-p53 pathway is involved in this response to the DNA damage. Furthermore, the treatment with KU55933 blocked DNA damage-induced THOC5mRNP complex dissociation, indicating that activation of ATM kinase suppresses the ability of THOC5 to bind to its target mRNAs.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Mutação , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/genética , Células Cultivadas , Proteínas de Ligação a DNA/genética , Camundongos , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas de Ligação a RNA/genética , Proteínas Supressoras de Tumor/genética
13.
RNA ; 17(6): 1048-56, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21525145

RESUMO

The TREX (transcription/export) complex has been conserved throughout evolution from yeast to man and is required for coupled transcription elongation and nuclear export of mRNAs. The TREX complex in mammals and Drosophila is composed of the THO subcomplex (THOC1, THOC2, THOC5, THOC6, and THOC7), THOC3, UAP56, and Aly/THOC4. In human and Drosophila, various studies have shown that THO is required for the export of heat shock mRNAs, but nothing is known about other mRNAs. Our previous study using conditional THOC5 (or FMIP) knockout mice revealed that the presence of THOC5 is critical in hematopoietic cells but not for terminally differentiated cells. In this study, we describe the establishment of a mouse embryo fibroblast cell line (MEF), THOC5 flox/flox. Four days after infection of MEF THOC5 flox/flox with adenovirus carrying Cre-recombinase gene (Ad-GFP-Cre), THOC5 is down-regulated >95% at the protein level, and cell growth is strongly suppressed. Transcriptome analysis using cytoplasmic RNA isolated from cells lacking functional THOC5 reveals that only 2.9% of all genes were down-regulated more than twofold. Although we examined these genes in fibroblasts, one-fifth of all down-regulated genes (including HoxB3 and polycomb CBX2) are known to play a key role in hematopoietic development. We further identified 10 genes that are spliced but not exported to the cytoplasm in the absence of THOC5. These mRNAs were copurified with THOC5. Furthermore, Hsp70 mRNA was exported in the absence of THOC5 at 37°C, but not under heat shock condition (42°C), suggesting that THOC5 may be required for mRNA export under stress and/or upon signaling-induced conditions.


Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas Nucleares/fisiologia , Proteínas de Transporte Nucleocitoplasmático/fisiologia , Splicing de RNA/genética , RNA Mensageiro/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Regulação para Baixo , Embrião de Mamíferos/metabolismo , Fibroblastos/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Resposta ao Choque Térmico , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Transporte de RNA , RNA Mensageiro/química
14.
BMC Genomics ; 12: 196, 2011 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-21501463

RESUMO

BACKGROUND: Nerve growth factor (NGF) is a potent growth factor that plays a key role in neuronal cell differentiation and may also play a role in hematopoietic differentiation. It has been shown that NGF induced synergistic action for the colony formation of CD34 positive hematopoietic progenitor cells treated with macrophage-colony stimulating factor (M-CSF or CSF-1), or stem cell factor (SCF). However, the exact role of NGF in hematopoietic system is unclear. It is also not clear whether NGF mediated signals in hematopoietic cells are identical to those in neuronal cells. RESULTS: To study the signal transduction pathways induced by NGF treatment in hematopoietic cells, we utilized the mastocytoma cell line HMC-1(V560G c-Kit) which expresses the NGF receptor, tropomyosin-receptor-kinase (Trk)A, as well as the constitutively activated SCF receptor, V560G c-Kit, which can be inhibited completely by treatment with the potent tyrosine kinase inhibitor imatinib mesylate (imatinib). NGF rescues HMC-1(V560G c-Kit) cells from imatinib mediated cell death and promotes proliferation. To examine the NGF mediated proliferation and survival in these cells, we compared the NGF mediated upregulated genes (30 and 120 min after stimulation) to the downregulated genes by imatinib treatment (downregulation of c-Kit activity for 4 h) by transcriptome analysis. The following conclusions can be drawn from the microarray data: Firstly, gene expression profiling reveals 50% overlap of genes induced by NGF-TrkA with genes expressed downstream of V560G c-Kit. Secondly, NGF treatment does not enhance expression of genes involved in immune related functions that were down regulated by imatinib treatment. Thirdly, more than 55% of common upregulated genes are involved in cell proliferation and survival. Fourthly, we found Kruppel-like factor (KLF) 2 and Smad family member 7 (SMAD7) as the NGF mediated novel downstream genes in hematopoietic cells. Finally, the downregulation of KLF2 gene enhanced imatinib induced apoptosis. CONCLUSION: NGF does not induce genes which are involved in immune related functions, but induces proliferation and survival signals in HMC-1(V560G c-Kit) cells. Furthermore, the current data provide novel candidate genes, KLF2 and SMAD7 which are induced by NGF/TrkA activation in hematopoietic cells. Since the depletion of KLF2 causes enhanced apoptosis of HMC-1(V560G c-Kit), KLF2 may play a role in the NGF mediated survival signal.


Assuntos
Perfilação da Expressão Gênica , Fator de Crescimento Neural/farmacologia , Apoptose , Benzamidas , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Regulação para Baixo , Humanos , Mesilato de Imatinib , Fatores de Transcrição Kruppel-Like/antagonistas & inibidores , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Mastocitoma , Piperazinas/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Pirimidinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptor de Fator de Crescimento Neural/genética , Receptor de Fator de Crescimento Neural/metabolismo , Receptor trkA/genética , Receptor trkA/metabolismo , Transdução de Sinais , Proteína Smad7/genética , Proteína Smad7/metabolismo
15.
BMC Biol ; 8: 1, 2010 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-20051105

RESUMO

BACKGROUND: The transcription/export complex is evolutionarily conserved from yeast to man and is required for coupled transcription elongation and nuclear export of mRNAs. FMIP(Fms interacting protein) is a member of the THO (suppressors of the transcriptional defects of hpr1delta by overexpression) complex which is a subcomplex of the transcription/export complex. THO complex (THOC) components are not essential for bulk poly (A)+ RNA export in higher eukaryotes, but for the nuclear export of subset of mRNAs, however, their exact role is still unclear. RESULTS: To study the role of THOC5/Fms interacting protein in vivo, we generated THOC5/Fms interacting protein knockout mice. Since these mice are embryonic lethal, we then generated interferon inducible conditional THOC5/Fms interacting protein knockout mice. After three poly injections all of the mice died within 14 days. No pathological alterations, however, were observed in liver, kidney or heart. Thus we considered the hematopoietic system and found that seven days after poly injection, the number of blood cells in peripheral blood decreased drastically. Investigation of bone marrow cells showed that these became apoptotic within seven days after poly injection. Committed myeloid progenitor cells and cells with long term reconstituting potential were lost from bone marrow within four days after poly injection. Furthermore, infusion of normal bone marrow cells rescued mice from death induced by loss of THOC5/Fms interacting protein. CONCLUSION: THOC5/Fms interacting protein is an essential element in the maintenance of hematopoiesis. Furthermore, mechanistically depletion of THOC5/Fms interacting protein causes the down-regulation of its direct interacting partner, THOC1 which may contribute to altered THO complex function and cell death.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Ligação a RNA/metabolismo , Anemia/metabolismo , Animais , Apoptose/fisiologia , Células Sanguíneas/fisiologia , Células da Medula Óssea/fisiologia , Transplante de Medula Óssea , Sobrevivência Celular/fisiologia , Hepatócitos/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Leucócitos/fisiologia , Leucopenia/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/metabolismo
16.
Cell Signal ; 21(2): 309-16, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19015024

RESUMO

THOC5 is a nuclear/cytoplasmic protein member of the spliceosome complex which potentiates C/EBP expression in adipocyte differentiation. As C/EBP family members are important regulators of myelopoiesis and THOC5 is highly expressed in neutrophil/macrophage progenitor cells we assessed the role of THOC5 in cytokine-stimulated monocytic development. M-CSF stimulated maturation of the NFS60 cell line was associated with enhanced THOC5 expression and phosphorylation. THOC5 was also shown to form a complex with C/EBPbeta. Ectopic expression of THOC5 mimicked M-CSF mediated cell maturation and enhanced protein expression of the myeloid transcription factors C/EBPbeta, C/EBPalpha, Pu-1 and also GAB2 (a PI-3 Kinase and macrophage development regulator). Increased THOC5 expression also mimicked M-CSF stimulated increases in the lipid second messenger PtdInsP(3). Inhibition of THOC5-induced increases in PtdInsP(3) levels abrogated the elevated levels of C/EBPbeta. Thus THOC5 expression can potentiate receptor signalling to transcription factor expression and monocyte differentiation.


Assuntos
Fator Estimulador de Colônias de Macrófagos/farmacologia , Proteínas Nucleares/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Linhagem Celular , Humanos , Fator Estimulador de Colônias de Macrófagos/metabolismo , Camundongos , Monócitos/citologia , Monócitos/metabolismo , Proteínas Nucleares/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Transdução de Sinais , Fatores de Transcrição/genética , Transfecção
17.
FEBS Lett ; 583(1): 13-8, 2009 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-19059247

RESUMO

THOC7 and Fms-interacting protein (FMIP) are members of the THO complex that associate with the mRNA export apparatus. FMIP is a nucleocytoplasmic shuttling protein with a nuclear localization signal (NLS), whereas THOC7 does not contain a typical NLS motif. We show here that THOC7 (50-137, amino acid numbers) binds to the N-terminal portion (1-199) of FMIP directly. FMIP is detected mainly in the nucleus. In the absence of exogenous FMIP, THOC7 resides mainly in the cytoplasm, while in the presence of FMIP, THOC7 is transported into the nucleus with FMIP. Furthermore, THOC7 lacking the FMIP binding site does not co-localize with FMIP, indicating that THOC7/FMIP interaction is required for nuclear localization of THOC7.


Assuntos
Núcleo Celular/metabolismo , Proteínas Nucleares/metabolismo , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Motivos de Aminoácidos/genética , Animais , Humanos , Camundongos , Mutação , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Proteínas Nucleares/genética , Mapeamento de Interação de Proteínas , Proteínas de Ligação a RNA/genética , Xenopus , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo
18.
Br J Haematol ; 141(5): 641-50, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18373705

RESUMO

The fusion protein TEL/PDGFRB is associated with chronic myelomonocytic leukaemia and has intrinsic tyrosine kinase activity. The effects of TEL/PDGFRB were assessed using the multipotent haemopoietic cell line FDCP-Mix. In the absence of growth factors, TEL/PDGFRB expression increased survival that was associated with elevated levels of phosphatidylinositol 3,4,5 trisphosphate (PIP3). Whilst TEL/PDGFRB had subtle effects on the growth factor requirements it had a profound effect on differentiation. The cells became refractory to cytokine-stimulated development, showing limited maturation but failing to produce fully mature cells. We have previously identified the spliceosome protein THOC5 as a target in macrophage colony-stimulating factor signalling and a protein involved in the regulation of transcription factor expression. TEL/PDGFRB expression increased the expression and phosphorylation of THOC5. Elevated expression of THOC5 increased PIP3 levels and decreased apoptosis. Mass spectrometry was used to identify a site for TEL/PDGFRB-mediated phosphorylation on THOC5, which was shown to be a target for a number of other leukaemogenic tyrosine kinases. Thus, THOC5 is a novel target for modulation of signal transduction with a potential role in leukaemogenesis.


Assuntos
Células-Tronco Hematopoéticas/metabolismo , Inositol/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Fusão Oncogênica/fisiologia , Apoptose/fisiologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular , Sobrevivência Celular/fisiologia , Substâncias de Crescimento/farmacologia , Células-Tronco Hematopoéticas/citologia , Humanos , Proteínas Nucleares/fisiologia , Proteínas de Fusão Oncogênica/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilação , Transdução de Sinais/fisiologia
19.
Ind Health ; 44(3): 441-4, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16922188

RESUMO

A thermal manikin is a useful tool to evaluate thermal environments such as clothing, houses and others. A manikin needs to have the following properties: shape and size, heat production, skin temperature, sweat rate, physiological responses and others. So far, no manikin satisfying all these criteria has been available. In the present study, a two-layer movable sweating thermal manikin was newly developed as a trial to compensate for the differences between conventional thermal manikins and the human body. The manikin consisted of two layers, a core section in the trunk and a shell section divided into 17 parts over the whole body, the temperature and heat supply of which could be independently controlled. The manikin is possible to change the posture and to walk. The sweat rate from 180 pores on the skin was controlled by peristaltic pumps. The temperature performance of the manikin was investigated using thermograph. As a result, the surface temperature of the manikin decreased more in the extremities than in the trunk when the manikin was exposed to 18 degrees C after being exposed to 28 degrees C for 20 min. This is analogous to the skin temperature decline in the extremities of the human body.


Assuntos
Manequins , Sudorese , Exposição Ambiental , Humanos , Japão , Modelos Psicológicos , Temperatura Cutânea
20.
Blood ; 107(3): 1133-40, 2006 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-16195327

RESUMO

We have investigated the role of tyrosine phosphorylation of the cyclin-dependent kinase (cdk) inhibitor p27Kip1 using the acute promyelocytic leukemia cell line NB4 together with granulocyte colony-stimulating factor (G-CSF). Short-term G-CSF stimulation resulted in a rapid tyrosine dephosphorylation of p27Kip1 accompanied by a change in its binding preferences to cdks. On G-CSF stimulation, p27Kip1 dissociated from cdk4 and associated with cdk2. Binding assays with recombinant p27Kip1 confirmed that tyrosine-phosphorylated p27Kip1 preferentially bound to cdk4, whereas unphosphorylated protein preferentially associated with cdk2. In addition, studies with p27Kip1 point mutations revealed a decisive role of Tyr88 and Tyr89 in binding to cdk4. Furthermore, phosphorylation of Tyr88 and Tyr89 was accompanied by strong nuclear translocation of p27Kip1. Taken together, this report provides the first evidence that tyrosine phosphorylation of p27Kip1 plays a crucial role in binding to cdks and its subcellular localization. Moreover, both effects are mediated by application of G-CSF.


Assuntos
Núcleo Celular/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Fator Estimulador de Colônias de Granulócitos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucemia Promielocítica Aguda/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Fator Estimulador de Colônias de Granulócitos/metabolismo , Humanos , Fosforilação , Ligação Proteica , Tirosina/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA