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1.
Cancer Sci ; 110(10): 3132-3144, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31390121

RESUMO

Alternative splicing, regulated by DEAD-Box Helicase (DDX) families, plays an important role in cancer. However, the relationship between the DDX family and cancer has not been fully elucidated. In the present study, we identified a candidate oncogene DDX56 on Ch.7p by a bioinformatics approach using The Cancer Genome Atlas (TCGA) dataset of colorectal cancer (CRC). DDX56 expression was measured by RT-qPCR and immunochemical staining in 108 CRC patients. Clinicopathological and survival analyses were carried out using three CRC datasets. Biological roles of DDX56 were explored by gene set enrichment analysis (GSEA), and cell proliferation in vitro and in vivo, cell cycle assays, and using DDX56-knockdown or overexpressed CRC cells. RNA sequencing was carried out to elucidate the effect of DDX56 on mRNA splicing. We found that DDX56 expression was positively correlated with the amplification of DDX56 and was upregulated in CRC cells. High DDX56 expression was associated with lymphatic invasion and distant metastasis and was an independent poor prognostic factor. In vitro analysis, in vivo analysis and GSEA showed that DDX56 promoted proliferation ability through regulating the cell cycle. DDX56 knockdown reduced intron retention and tumor suppressor WEE1 expression, which functions as a G2-M DNA damage checkpoint. We have identified DDX56 as a novel oncogene and prognostic biomarker of CRC that promotes alternative splicing of WEE1.


Assuntos
Proteínas de Ciclo Celular/genética , Neoplasias Colorretais/patologia , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Amplificação de Genes , Proteínas Nucleares/genética , Proteínas Tirosina Quinases/genética , Regulação para Cima , Idoso , Animais , Ciclo Celular , Linhagem Celular Tumoral , Cromossomos Humanos Par 7/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Invasividade Neoplásica , Estadiamento de Neoplasias , Transplante de Neoplasias , Prognóstico , Processamento de RNA , Análise de Sequência de RNA , Análise de Sobrevida
2.
Nat Commun ; 10(1): 3085, 2019 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-31300642

RESUMO

DEAD-box helicases (DDXs) regulate RNA processing and metabolism by unwinding short double-stranded (ds) RNAs. Sharing a helicase core composed of two RecA-like domains (D1D2), DDXs function in an ATP-dependent, non-processive manner. As an attractive target for cancer and AIDS treatment, DDX3X and its orthologs are extensively studied, yielding a wealth of biochemical and biophysical data, including structures of apo-D1D2 and post-unwound D1D2:single-stranded RNA complex, and the structure of a D2:dsRNA complex that is thought to represent a pre-unwound state. However, the structure of a pre-unwound D1D2:dsRNA complex remains elusive, and thus, the mechanism of DDX action is not fully understood. Here, we describe the structure of a D1D2 core in complex with a 23-base pair dsRNA at pre-unwound state, revealing that two DDXs recognize a 2-turn dsRNA, each DDX mainly recognizes a single RNA strand, and conformational changes induced by ATP binding unwinds the RNA duplex in a cooperative manner.


Assuntos
RNA Helicases DEAD-box/ultraestrutura , RNA de Cadeia Dupla/metabolismo , RNA Helicases DEAD-box/isolamento & purificação , RNA Helicases DEAD-box/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Espalhamento a Baixo Ângulo , Especificidade por Substrato , Difração de Raios X
3.
Environ Pollut ; 253: 538-551, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31330346

RESUMO

The effect of fluoride as an ongoing topic has attracted much attentions due to the decline in overall human fertility worldwide. However, whether fluorine causes a temporary stimulus or permanent damage to the male reproductive system, as well as the mechanism of fluoride influencing spermatogenesis remained unclear. 48 adult male rats were randomly divided into four groups (twelve each). Control group received the distilled water, while the other three groups were treated with 25, 50, 100 mg/L NaF via drinking water for 8 weeks. Six rats from each group were selected randomly to detect the levels of various indices related to spermatogenesis. The remaining rats were given only distilled water and left for recovery of a period of 2 weeks. Results showed that the levels of serum CK, ALP, CHE, BUN, UA, and Cr, testis morphology and the ultrastructure of sperm acrosome and chromatoid body (CB) were significantly changed by fluoride. Interestingly, the elongated spermatid counts, spermatids elongation ratio, and mRNA expressions of Prm1/2 and MIWI, TDRD1, TDRD 6, TDRD7, PABP, and Hsp72 related to CB decreased markedly in fluoride treatment groups compared to the control. Furthermore, the expression levels of DDX25 and associated regulatory proteins like CRM1, HMG2, H4, TP2, and PGK2 were down-regulated by fluoride. After 2-weeks withdrawal period, out of the 19 altered spermatogenesis indicators, 15 indicators in 100 mg/L group and 3 indicators in 50 mg/L group still exhibited a significant change, while none showed change in 25 mg/L group. These results proved that the reversibility of fluoride toxicity is dose-dependent on the male reproductive system. Meanwhile, fluoride caused unrestored arrest during the haploid period of spermatogenesis, where reduced DDX25 and associated regulatory proteins play a crucial role in this process, which could provide the underlying insights to the toxic mechanism of fluoride induced male reproductive toxicity.


Assuntos
RNA Helicases DEAD-box/metabolismo , Fluoretos/toxicidade , Espermatogênese/efeitos dos fármacos , Testículo/enzimologia , Animais , RNA Helicases DEAD-box/genética , Água Potável , Masculino , Camundongos Knockout , Ratos , Espermatozoides/efeitos dos fármacos , Testículo/efeitos dos fármacos
4.
Artigo em Inglês | MEDLINE | ID: mdl-31176867

RESUMO

In rice field eel (Monopterus albus), germ cell development in the developing gonad has been revealed in detail. However, it is unclear how primordial germ cells (PGCs) migrate to the somatic part of the gonad (genital ridge). This study visualized PGC migration by injecting a chimeric mRNA containing a fluorescent protein fused to the 3' untranslated region (3'UTR) of three different genes, nanos3 of zebrafish (Danio rerio) and dead end (dnd) and vasa of rice field eel. The mRNAs were injected either alone or in pairs into embryos at the one-cell stage. The results showed that mRNAs containing nanos3 and dnd 3'UTRs labeled PGCs over a wider time frame than those containing vasa 3'UTR, suggesting that nanos3 and dnd 3'UTRs are suitable for visualizing PGCs in rice field eel. Using this direct visualization method, the normal migration route of PGCs was observed from the 50%-epiboly stage to hatching stage for the first time, and the ectopic PGCs were also visualized during this period in rice field eel. These findings extend our knowledge of germ cell development, and lay a foundation for further research on the relationship between PGCs and sex differentiation, and on incubation conditions for embryos in rice field eel.


Assuntos
Células Germinativas/metabolismo , Smegmamorpha/embriologia , Regiões 3' não Traduzidas , Sequência de Aminoácidos , Animais , Movimento Celular/genética , Movimento Celular/fisiologia , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Feminino , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Células Germinativas/citologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , RNA/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Smegmamorpha/genética , Smegmamorpha/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
5.
Nat Commun ; 10(1): 2421, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160600

RESUMO

Translation efficiency can be affected by mRNA stability and secondary structures, including G-quadruplex structures (G4s). The highly conserved DEAH-box helicase DHX36/RHAU resolves G4s on DNA and RNA in vitro, however a systems-wide analysis of DHX36 targets and function is lacking. We map globally DHX36 binding to RNA in human cell lines and find it preferentially interacting with G-rich and G4-forming sequences on more than 4500 mRNAs. While DHX36 knockout (KO) results in a significant increase in target mRNA abundance, ribosome occupancy and protein output from these targets decrease, suggesting that they were rendered translationally incompetent. Considering that DHX36 targets, harboring G4s, preferentially localize in stress granules, and that DHX36 KO results in increased SG formation and protein kinase R (PKR/EIF2AK2) phosphorylation, we speculate that DHX36 is involved in resolution of rG4 induced cellular stress.


Assuntos
RNA Helicases DEAD-box/metabolismo , Quadruplex G , RNA Mensageiro/metabolismo , Regiões não Traduzidas , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Fosforilação , Biossíntese de Proteínas , Ribossomos/metabolismo , Estresse Fisiológico , eIF-2 Quinase/metabolismo
6.
Int J Exp Pathol ; 100(3): 184-191, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31090156

RESUMO

The expression of microRNA in eukaryotic cells is subject to tightly regulated processing. The altered expression of microRNAs in a number of cancers suggests their contribution to disease pathogenesis, where processing pathways may be involved in disease pathogenesis. In the present study, we evaluated changes in the profile of two main components of microRNA biogenesis, AGO2 and DICER, and assessed their correlation with disease progression in childhood acute lymphoblastic leukaemia (ALL). To achieve this aim, 25 patients afflicted with ALL were included in the study along with 25 healthy subjects as control. The expression level of AGO2 and DICER was evaluated by real-time PCR. The results revealed an increase in the expression of DICER and a decrease in AGO2 in patients. The correlation between the alteration levels of these genes with pathologic events was also studied. This increase or decrease proved to be directly correlated with the progression of the disease particularly in L1 to L2. According to the obtained results, it can be deduced that dysregulation in transcription of DICER and AGO2, involved in the formation of mature microRNAs in cytoplasm of ALL cancer cells, is a part of the pathological molecular mechanism implicated in the exacerbation of this malignancy. Therefore, the genes involved in microRNAs biogenesis that have been studied here could be considered as candidate prognostic markers especially in childhood ALL which will help towards a better understanding of the molecular basis of ALL.


Assuntos
Proteínas Argonauta/metabolismo , Linfócitos B/citologia , Linhagem da Célula/imunologia , RNA Helicases DEAD-box/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Ribonuclease III/metabolismo , Adolescente , Criança , Pré-Escolar , Progressão da Doença , Feminino , Humanos , Masculino , MicroRNAs/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos
7.
Nat Commun ; 10(1): 2278, 2019 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-31123254

RESUMO

Mammalian spermatogenesis is sustained by mitotic germ cells with self-renewal potential known as undifferentiated spermatogonia. Maintenance of undifferentiated spermatogonia and spermatogenesis is dependent on tightly co-ordinated transcriptional and post-transcriptional mechanisms. The RNA helicase DDX5 is expressed by spermatogonia but roles in spermatogenesis are unexplored. Using an inducible knockout mouse model, we characterise an essential role for DDX5 in spermatogonial maintenance and show that Ddx5 is indispensable for male fertility. We demonstrate that DDX5 regulates appropriate splicing of key genes necessary for spermatogenesis. Moreover, DDX5 regulates expression of cell cycle genes in undifferentiated spermatogonia post-transcriptionally and is required for cell proliferation and survival. DDX5 can also act as a transcriptional co-activator and we demonstrate that DDX5 interacts with PLZF, a transcription factor required for germline maintenance, to co-regulate select target genes. Combined, our data reveal a critical multifunctional role for DDX5 in regulating gene expression programmes and activity of undifferentiated spermatogonia.


Assuntos
RNA Helicases DEAD-box/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Processamento de RNA/fisiologia , Espermatogênese/genética , Espermatogônias/metabolismo , Animais , Ciclo Celular/genética , Proliferação de Células/genética , Técnicas de Cocultura , RNA Helicases DEAD-box/genética , Embrião de Mamíferos , Fertilidade/genética , Fibroblastos , Regulação da Expressão Gênica/fisiologia , Masculino , Camundongos , Camundongos Knockout , Modelos Animais , Cultura Primária de Células , Testículo/citologia
8.
RNA ; 25(8): 1020-1037, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31110137

RESUMO

Stable recognition of the intron branchpoint (BP) by the U2 snRNP to form the pre-spliceosome is the first ATP-dependent step of splicing. Genetic and biochemical data from yeast indicate that Cus2 aids U2 snRNA folding into the stem IIa conformation prior to pre-spliceosome formation. Cus2 must then be removed by an ATP-dependent function of Prp5 before assembly can progress. However, the location from which Cus2 is displaced and the nature of its binding to the U2 snRNP are unknown. Here, we show that Cus2 contains a conserved UHM (U2AF homology motif) that binds Hsh155, the yeast homolog of human SF3b1, through a conserved ULM (U2AF ligand motif). Mutations in either motif block binding and allow pre-spliceosome formation without ATP. A 2.0 Å resolution structure of the Hsh155 ULM in complex with the UHM of Tat-SF1, the human homolog of Cus2, and complementary binding assays show that the interaction is highly similar between yeast and humans. Furthermore, we show that Tat-SF1 can replace Cus2 function by enforcing ATP dependence of pre-spliceosome formation in yeast extracts. Cus2 is removed before pre-spliceosome formation, and both Cus2 and its Hsh155 ULM binding site are absent from available cryo-EM structure models. However, our data are consistent with the apparent location of the disordered Hsh155 ULM between the U2 stem-loop IIa and the HEAT repeats of Hsh155 that interact with Prp5. We propose a model in which Prp5 uses ATP to remove Cus2 from Hsh155 such that extended base-pairing between U2 snRNA and the intron BP can occur.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U2/química , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Motivos de Aminoácidos , Sítios de Ligação , Sequência Conservada , Cristalografia por Raios X , RNA Helicases DEAD-box/metabolismo , Humanos , Modelos Moleculares , Mutação , Ligação Proteica , Processamento de RNA , Proteínas de Ligação a RNA/genética , Ribonucleoproteína Nuclear Pequena U2/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
9.
Theriogenology ; 131: 61-71, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30947076

RESUMO

The analysis of early gonadogenesis during larval development requires a molecular marker that is specifically expressed in the germ cell lineage, such as the vasa gene. In this study, we cloned and characterized vasa in the striped catfish (Pangasianodon hypophthalmus), and designated this as Phy-vasa. Phy-vasa contained all of the predicted consensus motifs that are shared among the vasa genes in other fish species, including RG and RGG repeats, ATPase motifs, and a DEAD-box, and phylogenetic analysis using various DEAD-box family proteins demonstrated that the Phy-vasa protein clustered within the Vasa family. Reverse transcription polymerase chain reaction (RT-PCR) indicated that Phy-vasa mRNA only occurred in the testis and ovary, and in situ hybridization showed that the gene was expressed only in the germ cells, with strong expression in the spermatogonia and oogonia. To investigate early gonadogenesis in catfish larvae, we undertook histological characterization and in situ hybridization using a Phy-vasa probe, which showed that migration of the primordial germ cells (PGCs) most commonly occurred in larvae at 2-10 days post-fertilization (dpf), the PGCs started to be surrounded by gonadal somatic cells at around 10-20 dpf, and rapid proliferation of the PGCs had begun by 30 dpf. These findings provide a valuable insight into early gonadal development in the striped catfish.


Assuntos
Peixes-Gato/metabolismo , RNA Helicases DEAD-box/metabolismo , Proteínas de Peixes/metabolismo , Células Germinativas/metabolismo , Gônadas/metabolismo , Animais , Peixes-Gato/genética , RNA Helicases DEAD-box/química , Proteínas de Peixes/química , Filogenia , RNA/metabolismo , Alinhamento de Sequência , Análise de Sequência de Proteína
10.
Int J Mol Sci ; 20(7)2019 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-30959742

RESUMO

Gastric cancer (GC) is one of the most common cancers worldwide. In the clinical setting, the identification of HER2 overexpression in GC was a significant finding, as trastuzumab, an anti-HER2 drug, provides a survival advantage to HER2-positive GC patients. In HER2-postive GC, the dysregulation of PI3K/AKT and MAPK/ERK signaling pathways has been reported, and inhibition of these pathways is an important therapeutic strategy. MiR-143 is known to act as a tumor suppressor in several cancers, such as bladder cancer, breast cancer, colorectal cancer, and gastric cancer. In the current study, we developed a novel chemically-modified miR-143 and explored the functions of this synthetic miR-143 (syn-miR-143) in HER2-positive gastric cancer. The expression level of miR-143 was down-regulated in GC cell lines, including HER2-positive GC cell lines, MKN7, and KATO-III. The ectopic expression of miR-143 in those cell lines suppressed cell growth through systemic silencing of KRAS and its effector signaling molecules, AKT and ERK. Furthermore, syn-miR-143 indirectly down-regulated the expression of HER2, an upstream molecule of KRAS, through silencing DEAD/H-box RNA helicase 6 (DDX6), RNA helicase, which enhanced HER2 protein expression at the translational step in HER2-positive GC cells. These findings suggested that syn-miR-143 acted as a tumor suppressor through the impairment of KRAS networks including the DDX6.


Assuntos
RNA Helicases DEAD-box/metabolismo , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptor ErbB-2/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Animais , Antagomirs/metabolismo , Apoptose/genética , Sequência de Bases , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Camundongos Nus , MicroRNAs/genética , Modelos Biológicos , Transdução de Sinais , Regulação para Cima/genética , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Cell Physiol Biochem ; 52(5): 984-1002, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30977984

RESUMO

BACKGROUND/AIMS: The epithelial sodium channel (ENaC) expressed in alveolar epithelial cells plays a major role in lung liquid clearance at birth and lung edema resorption in adulthood. We showed previously that αENaC mRNA expression is downregulated in part via posttranscriptional regulation of mRNA stability. In the present work, the role of the αENaC 3' untranslated region (3'UTR) in the regulation of mRNA stability was studied further. METHODS: Quantitative reverse transcription PCR (qRT-PCR) was performed to investigate the expression of αENaC in alveolar epithelial cells. The role of the αENaC 3'UTR was evaluated through sequential deletions. RNA affinity chromatography and mass spectrometry were achieved to investigate the nature of the proteins that could bind this sequence. The function of these proteins was assessed through knockdown and overexpression in vitro. RESULTS: First, we found that αENaC mRNA half-life was much shorter than expected when using a transcriptionally controlled plasmid expression system compared to Actinomycin D treatment. Sequential deletions of the αENaC 3'UTR revealed that the αENaC 3'UTR plays an important role in the modulation of αENaC mRNA stability, and that there is a complex stabilizing and destabilizing interplay between different regions of the 3'UTR that modulate this process. Finally, we identified RNA-binding proteins that interact with the αENaC 3'UTR and showed that Dhx36 and Tial1 are involved in the decrease in αENaC mRNA stability via the proximal region of its 3'UTR. CONCLUSION: Taken together, these findings indicate that the αENaC 3'UTR plays an important role in modulating transcript levels, and Dhx36 and Tial1 seem to be involved in posttranscriptional regulation of αENaC expression in alveolar epithelial cells.


Assuntos
Regiões 3' não Traduzidas , Células Epiteliais/metabolismo , Canais Epiteliais de Sódio/biossíntese , Regulação da Expressão Gênica , Alvéolos Pulmonares/metabolismo , Estabilidade de RNA , Animais , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Células Epiteliais/citologia , Canais Epiteliais de Sódio/genética , Masculino , Alvéolos Pulmonares/citologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ratos , Ratos Sprague-Dawley
12.
Nat Commun ; 10(1): 1855, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015431

RESUMO

DHX36 is a DEAH-box helicase that resolves parallel G-quadruplex structures formed in DNA and RNA. The recent co-crystal structure of DHX36 bound G4-DNA revealed an intimate contact, but did not address the role of ATP hydrolysis in G4 resolving activity. Here, we demonstrate that unlike on G4-DNA, DHX36 displays ATP-independent unfolding of G4-RNA followed by ATP-dependent refolding, generating a highly asymmetric pattern of activity. Interestingly, DHX36 refolds G4-RNA in several steps, reflecting the discrete steps in forming the G4 structure. We show that the ATP-dependent activity of DHX36 arises from the RNA tail rather than the G4. Mutations that perturb G4 contact result in quick dissociation of the protein from RNA upon ATP hydrolysis, while mutations that interfere with binding the RNA tail induce dysregulated activity. We propose that the ATP-dependent activity of DHX36 may be useful for dynamically resolving various G4-RNA structures in cells.


Assuntos
Trifosfato de Adenosina/metabolismo , RNA Helicases DEAD-box/metabolismo , Quadruplex G , Dobramento de RNA , RNA/metabolismo , RNA Helicases DEAD-box/genética , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Microscopia de Fluorescência/métodos , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica/genética , RNA/química , Imagem Individual de Molécula/métodos
13.
Proc Natl Acad Sci U S A ; 116(12): 5687-5692, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30842276

RESUMO

Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) is a multifunctional cytokine displaying broad-spectrum anticancer activity in vitro or in vivo in preclinical animal cancer models and in a phase 1/2 clinical trial in patients with advanced cancers. mda-7/IL-24 targets specific miRNAs, including miR-221 and miR-320, for down-regulation in a cancer-selective manner. We demonstrate that mda-7/IL-24, administered through a replication incompetent type 5 adenovirus (Ad.mda-7) or with His-MDA-7/IL-24 protein, down-regulates DICER, a critical regulator in miRNA processing. This effect is specific for mature miR-221, as it does not affect Pri-miR-221 expression, and the DICER protein, as no changes occur in other miRNA processing cofactors, including DROSHA, PASHA, or Argonaute. DICER is unchanged by Ad.mda-7/IL-24 in normal immortal prostate cells, whereas Ad.mda-7 down-regulates DICER in multiple cancer cells including glioblastoma multiforme and prostate, breast, lung, and liver carcinoma cells. MDA-7/IL-24 protein down-regulates DICER expression through canonical IL-20/IL-22 receptors. Gain- and loss-of-function studies confirm that overexpression of DICER rescues deregulation of miRNAs by mda-7/IL-24, partially rescuing cancer cells from mda-7/IL-24-mediated cell death. Stable overexpression of DICER in cancer cells impedes Ad.mda-7 or His-MDA-7/IL-24 inhibition of cell growth, colony formation, PARP cleavage, and apoptosis. In addition, stable overexpression of DICER renders cancer cells more resistant to Ad.mda-7 inhibition of primary and secondary tumor growth. MDA-7/IL-24-mediated regulation of DICER is reactive oxygen species-dependent and mediated by melanogenesis-associated transcription factor. Our research uncovers a distinct role of mda-7/IL-24 in the regulation of miRNA biogenesis through alteration of the MITF-DICER pathway.


Assuntos
RNA Helicases DEAD-box/metabolismo , Interleucinas/metabolismo , MicroRNAs/metabolismo , Fator de Transcrição Associado à Microftalmia/metabolismo , Ribonuclease III/metabolismo , Animais , Apoptose/fisiologia , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , RNA Helicases DEAD-box/biossíntese , RNA Helicases DEAD-box/genética , Regulação para Baixo , Genes Supressores de Tumor , Humanos , Interleucinas/genética , Masculino , Camundongos , Camundongos Nus , MicroRNAs/biossíntese , Fator de Transcrição Associado à Microftalmia/genética , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/parasitologia , Espécies Reativas de Oxigênio/metabolismo , Ribonuclease III/biossíntese , Ribonuclease III/genética , Transdução de Sinais , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
14.
Genetics ; 212(1): 153-174, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30902808

RESUMO

RNA helicases are a class of enzymes that unwind RNA duplexes in vitro but whose cellular functions are largely enigmatic. Here, we provide evidence that the DEAD-box protein Dbp2 remodels RNA-protein complex (RNP) structure to facilitate efficient termination of transcription in Saccharomyces cerevisiae via the Nrd1-Nab3-Sen1 (NNS) complex. First, we find that loss of DBP2 results in RNA polymerase II accumulation at the 3' ends of small nucleolar RNAs and a subset of mRNAs. In addition, Dbp2 associates with RNA sequence motifs and regions bound by Nrd1 and can promote its recruitment to NNS-targeted regions. Using Structure-seq, we find altered RNA/RNP structures in dbp2∆ cells that correlate with inefficient termination. We also show a positive correlation between the stability of structures in the 3' ends and a requirement for Dbp2 in termination. Taken together, these studies provide a role for RNA remodeling by Dbp2 and further suggests a mechanism whereby RNA structure is exploited for gene regulation.


Assuntos
RNA Helicases DEAD-box/metabolismo , RNA Mensageiro/metabolismo , RNA Nucleolar Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Terminação da Transcrição Genética , DNA Helicases/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas Nucleares/metabolismo , RNA Helicases/metabolismo , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Análise de Sequência de RNA
15.
RNA ; 25(6): 685-701, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30910870

RESUMO

Eukaryotic ribosome biogenesis is a highly orchestrated process involving numerous assembly factors including ATP-dependent RNA helicases. The DEAH helicase DHX37 (Dhr1 in yeast) is activated by the ribosome biogenesis factor UTP14A to facilitate maturation of the small ribosomal subunit. We report the crystal structure of DHX37 in complex with single-stranded RNA, revealing a canonical DEAH ATPase/helicase architecture complemented by a structurally unique carboxy-terminal domain (CTD). Structural comparisons of the nucleotide-free DHX37-RNA complex with DEAH helicases bound to RNA and ATP analogs reveal conformational changes resulting in a register shift in the bound RNA, suggesting a mechanism for ATP-dependent 3'-5' RNA translocation. We further show that a conserved sequence motif in UTP14A interacts with and activates DHX37 by stimulating its ATPase activity and enhancing RNA binding. In turn, the CTD of DHX37 is required, but not sufficient, for interaction with UTP14A in vitro and is essential for ribosome biogenesis in vivo. Together, these results shed light on the mechanism of DHX37 and the function of UTP14A in controlling its recruitment and activity during ribosome biogenesis.


Assuntos
Adenosina Trifosfatases/química , Trifosfato de Adenosina/análogos & derivados , RNA Helicases DEAD-box/química , Biogênese de Organelas , RNA Helicases/química , RNA/química , Ribonucleoproteínas Nucleolares Pequenas/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cinética , Camundongos , Modelos Moleculares , Ligação Proteica , Biossíntese de Proteínas , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/genética , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Especificidade por Substrato
17.
Plant Physiol ; 179(4): 1525-1536, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30700540

RESUMO

In eukaryotes, the regulated transport of mRNAs from the nucleus to the cytosol through nuclear pore complexes represents an important step in the expression of protein-coding genes. In plants, the mechanism of nucleocytosolic mRNA transport and the factors involved are poorly understood. The Arabidopsis (Arabidopsis thaliana) genome encodes two likely orthologs of UAP56-interacting factor, which acts as mRNA export factor in mammalian cells. In yeast and plant cells, both proteins interact directly with the mRNA export-related RNA helicase UAP56 and the interaction was mediated by an N-terminal UAP56-binding motif. Accordingly, the two proteins were termed UAP56-INTERACTING EXPORT FACTOR1 and 2 (UIEF1/2). Despite lacking a known RNA-binding motif, recombinant UIEF1 interacted with RNA, and the C-terminal part of UIEF1 mainly contributed to the RNA interaction. Mutation of UIEF1, UIEF2, or both in the double-mutant 2xuief caused modest growth defects. A cross between the 2xuief and 4xaly (defective in the four ALY1-4 mRNA export factors) mutants produced the sextuple mutant 4xaly 2xuief, which displayed more severe growth impairment than the 4xaly plants. Developmental defects including delayed bolting and reduced seed set were observed in the 4xaly but not the 2xuief plants. Analysis of the cellular distribution of polyadenylated mRNAs revealed more pronounced nuclear mRNA accumulation in 4xaly 2xuief than in 2xuief and 4xaly cells. In conclusion, the results indicate that UIEF1 and UIEF2 act as mRNA export factors in plants and that they cooperate with ALY1-ALY4 to mediate efficient nucleocytosolic mRNA transport.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , RNA Helicases DEAD-box/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/fisiologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Genoma de Planta , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
18.
Genes Cells ; 24(4): 272-283, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30721563

RESUMO

A multiprotein complex, THO/TREX, couples the transcription, 3'-end formation and nuclear export of mRNAs. In this study, we report that crucial factors for mRNA processing, such as XRN2, DDX5/DDX17 and CstF64, are copurified with human THO (hTHO). Using chromatin immunoprecipitation, we found increased cross-linking of XRN2 and CstF64 to the RNA polymerase II (RNAP II) pause site of the HSPA1A gene upon down-regulation of THOC5, a metazoan-specific component of hTHO. As observed in THOC5-depleted cells, knockdown of XRN2 blocked HSP70 transcript release and increased the amount of CstF64 at the pause site. In addition, our data indicate that DDX5/DDX17 is also required for HSP70 transcript release. As the degradation of read-through transcripts, but not cleavage at polyadenylation sites per se, was hindered upon THOC5 or DDX5/DDX17 down-regulation, these factors appear to influence transcriptional termination. Interestingly, over-expression of RNase H suppressed the accumulation of HSP70 transcripts in nuclear foci in THOC5- or DDX5/DDX17-depleted cells. Thus, we propose a model in which hTHO, along with DDX5/DDX17, restricts the formation of R-loops, thereby facilitating the XRN2-mediated transcriptional termination and release of the mature transcript from the HSPA1A locus.


Assuntos
Proteínas de Choque Térmico HSP70/genética , Proteínas Nucleares/metabolismo , RNA Mensageiro/genética , Terminação da Transcrição Genética , RNA Helicases DEAD-box/metabolismo , Exorribonucleases/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Células HeLa , Humanos , Ligação Proteica , RNA Polimerase II/metabolismo , RNA Mensageiro/metabolismo
19.
J Reprod Dev ; 65(2): 121-128, 2019 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-30613052

RESUMO

About 10% of male infertile patients show abnormalities in spermatogenesis. The microdeletion of azoospermia factor a (AZFa) region of the Y chromosome is thought to be a cause of spermatogenic failure. However, candidate gene responsible for the spermatogenic failure in AZFa deleted patients has not been elucidated yet. Using mice, we explored the function of Ddx3y, a strong candidate gene in the Azfa region, and Ddx3x, a Ddx3y paralog on the X chromosome, in spermatogenesis. We first generated Ddx3y KO male mice using CRISPR/Cas9 and found that the Ddx3y KO male mice show normal spermatogenesis, produce morphologically normal spermatozoa, and sire healthy offspring. Because Ddx3x KO males were embryonic lethal, we next generated chimeric mice, which contain Ddx3x and Ddx3y double KO (dKO) germ cells, and found that the dKO germ cells can differentiate into spermatozoa and transmit their mutant alleles to offspring by normal mating. We conclude that Ddx3x and Ddx3y are dispensable for spermatogenesis at least in mice. Unlike human, mice have an additional Ddx3y paralog D1pas1, that has been reported to be essential for spermatogenesis. These findings suggest that human and mouse DDX3 related proteins have distinct differences in their functions.


Assuntos
Azoospermia/genética , RNA Helicases DEAD-box/genética , Fertilidade/genética , Células Germinativas/metabolismo , Antígenos de Histocompatibilidade Menor/genética , RNA Helicases/genética , Animais , RNA Helicases DEAD-box/metabolismo , Desenvolvimento Embrionário/genética , Feminino , Fertilização In Vitro , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Antígenos de Histocompatibilidade Menor/metabolismo , RNA Helicases/metabolismo , Homologia de Sequência , Espermatogênese/genética , Espermatozoides/metabolismo
20.
BMC Plant Biol ; 19(1): 17, 2019 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-30626336

RESUMO

BACKGROUND: Despite increasing characterization of DEAD-box RNA helicases (RHs) in chloroplast gene expression regulation at posttranscriptional levels in plants, their functional roles in growth responses of crops, including rice (Oryza sativa), to abiotic stresses are yet to be characterized. In this study, rice OsRH58 (LOC_Os01g73900), a chloroplast-localized DEAD-box RH, was characterized for its expression patterns upon stress treatment and its functional roles using transgenic Arabidopsis plants under normal and abiotic stress conditions. RESULTS: Chloroplast localization of OsRH58 was confirmed by analyzing the expression of OsRH58-GFP fusion proteins in tobacco leaves. Expression of OsRH58 in rice was up-regulated by salt, drought, or heat stress, whereas its expression was decreased by cold, UV, or ABA treatment. The OsRH58-expressing Arabidopsis plants were taller and had more seeds than the wild type under favorable conditions. The transgenic plants displayed faster seed germination, better seedling growth, and a higher survival rate than the wild type under high salt or drought stress. Importantly, levels of several chloroplast proteins were increased in the transgenic plants under salt or dehydration stress. Notably, OsRH58 harbored RNA chaperone activity. CONCLUSIONS: These findings suggest that the chloroplast-transported OsRH58 possessing RNA chaperone activity confers stress tolerance by increasing translation of chloroplast mRNAs.


Assuntos
Arabidopsis/metabolismo , Cloroplastos/metabolismo , RNA Helicases DEAD-box/metabolismo , Secas , Oryza/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Cloreto de Sódio/farmacologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cloroplastos/efeitos dos fármacos , Cloroplastos/genética , RNA Helicases DEAD-box/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/genética , Oryza/genética , Plantas Geneticamente Modificadas/efeitos dos fármacos , Estresse Fisiológico
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