Browsing by Author "Tsai, Tsung-Yuan"

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Critical roles of RNA helicase DDX3 and its interactions with eIF4E/PABP1 in stress granule assembly and stress response
(2012-01-01)
Shih, Jing-Wen
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Wang, Wei-Ting
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Tsai, Tsung-Yuan
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Kuo, Chu-Yun
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Li, Hao-Kang
Upon environmental insults, SGs (stress granules) aid cell survival by serving as sites of translational silencing. RNA helicase DDX3 was reported to associate with SGs. However, its role in SG physiology remains undefined. We have demonstrated previously that DDX3 acts as an eIF4E (eukaryotic initiation factor 4E)-inhibitory protein to suppress translation. In the present study, we indentified the SG marker PABP1 [poly(A)-binding protein 1] as another direct interaction partner of DDX3. We established various stimuli as novel stressors that direct DDX3 with eIF4E and PABP1 into SGs, but not to processing bodies. Interestingly, down-regulation of DDX3 interfered with SG assembly, led to nuclear accumulation of PABP1 and reduced cell viability following stress. Conversely, supplementation with a shRNA (short hairpin RNA)-resistant DDX3 restored SG formation, the translocation of PABP1 into SGs and cell survival. Notably, the SG-inducing capacity of DDX3 is independent of its ATPase and helicase activities, but mapped to the eIF4E-binding region. Moreover, the eIF4E-binding-defective mutant DDX3 was impaired in its SG-inducing ability and protective effect on cell survival under adverse conditions. All together, the present study has characterized DDX3 as a pivotal SG-nucleating factor and illustrates co-ordinative roles for DDX3, eIF4E and PABP1 in integrating environmental stress with translational regulation.
DDX3 localizes to the centrosome and prevents multipolar mitosis by epigenetically and translationally modulating p53 expression
(2017-08-25)
Chen, Wei-Ju
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Wang, Wei-Ting
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Tsai, Tsung-Yuan
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Li, Hao-Kang
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Lee, Yan-Hwa Wu
The DEAD-box RNA helicase DDX3 plays divergent roles in tumorigenesis, however, its function in mitosis is unclear. Immunofluorescence indicated that DDX3 localized to centrosome throughout the cell cycle and colocalized with centrosome-associated p53 during mitosis in HCT116 and U2OS cells. DDX3 depletion promoted chromosome misalignment, segregation defects and multipolar mitosis, eventually leading to G2/M delay and cell death. DDX3 prevented multipolar mitosis by inactivation and coalescence of supernumerary centrosomes. DDX3 silencing suppressed Ser(15) phosphorylation of p53 which is required for p53 centrosomal localization. Additionally, knockout of p53 dramatically diminished the association of DDX3 with centrosome, which was rescued by overexpression of the centrosomal targeting-defective p53 S15A mutant, indicating that centrosomal localization of DDX3 is p53 dependent but not through centrosomal location of p53. Furthermore, DDX3 knockdown suppressed p53 transcription through activation of DNA methyltransferases (DNMTs) along with hypermethylation of p53 promoter and promoting the binding of repressive histone marks to p53 promoter. Moreover, DDX3 modulated p53 mRNA translation. Taken together, our study suggests that DDX3 regulates epigenetic transcriptional and translational activation of p53 and colocalizes with p53 at centrosome during mitosis to ensure proper mitotic progression and genome stability, which supports the tumor-suppressive role of DDX3.
RNA helicase DDX3 maintains lipid homeostasis through upregulation of the microsomal triglyceride transfer protein by interacting with HNF4 and SHP
(2017-01-27)
Tsai, Tsung-Yuan
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Wang, Wei-Ting
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Li, Hao-Kang
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Chen, Wei-Ju
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Tsai, Yu-Hong
Multifunctional RNA helicase DDX3 participates in HCV infection, one of the major causes of hepatic steatosis. Here, we investigated the role of DDX3 in hepatic lipid metabolism. We found that HCV infection severely reduced DDX3 expression. Analysis of intracellular triglyceride and secreted ApoB indicated that lipid accumulations were increased while ApoB secretion were decreased in DDX3 knockdown HuH7 and HepG2 cell lines. Down-regulation of DDX3 significantly decreased protein and transcript expression of microsomal triglyceride transfer protein (MTP), a key regulator of liver lipid homeostasis. Moreover, DDX3 interacted with hepatocyte nuclear factor 4 (HNF4) and small heterodimer partner (SHP), and synergistically up-regulated HNF4-mediated transactivation of MTP promoter via its ATPase activity. Further investigation revealed that DDX3 interacted with CBP/p300 and increased the promoter binding affinity of HNF4 by enhancing HNF4 acetylation. Additionally, DDX3 partially relieved the SHP-mediated suppression on MTP promoter by competing with SHP for HNF4 binding which disrupted the inactive HNF4/SHP heterodimer while promoted the formation of the active HNF4 homodimer. Collectively, these results imply that DDX3 regulates MTP gene expression and lipid homeostasis through interplay with HNF4 and SHP, which may also reveal a novel mechanism of HCV-induced steatosis.
Targeted inactivation of murine Ddx3x: essential roles of Ddx3x in placentation and embryogenesis
(2016-07-15)
Chen, Chia-Yu
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Chan, Chieh-Hsiang
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Chen, Chun-Ming
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Tsai, Yin-Shuan
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Tsai, Tsung-Yuan
The X-linked DEAD-box RNA helicase DDX3 (DDX3X) is a multifunctional protein that has been implicated in gene regulation, cell cycle control, apoptosis, and tumorigenesis. However, the precise physiological function of Ddx3x during development remains unknown. Here, we show that loss of Ddx3x results in an early post-implantation lethality in male mice. The size of the epiblast marked by Oct3/4 is dramatically reduced in embryonic day 6.5 (E6.5) Ddx3x(-)/Y embryos. Preferential paternal X chromosome inactivation (XCI) in extraembryonic tissues of Ddx3x heterozygous (Ddx3x(-/+)) female mice with a maternally inherited null allele leads to placental abnormalities and embryonic lethality during development. In the embryonic tissues, Ddx3x exhibits developmental-and tissue-specific differences in escape from XCI. Targeted Ddx3x ablation in the epiblast leads to widespread apoptosis and abnormal growth, which causes embryonic lethality in the Sox2-cre/+; Ddx3x(flox)/Y mutant around E11.5. The observation of significant increases in gamma H2AX and p-p53(Ser15) indicates DNA damage, which suggests that loss of Ddx3x leads to higher levels of genome damage. Significant upregulation of p21(WAF1/Cip1) and p15(Ink4b) results in cell cycle arrest and apoptosis in Ddx3x-deficient cells. These results have uncovered that mouse Ddx3x is essential for both embryo and extraembryonic development.
Targeted inactivation of murine Ddx3x: essential roles of Ddx3x in placentation and embryogenesis
(2016-07-15)
Chen, Chia-Yu
;
Chan, Chieh-Hsiang
;
Chen, Chun-Ming
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Tsai, Yin-Shuan
;
Tsai, Tsung-Yuan
The X-linked DEAD-box RNA helicase DDX3 (DDX3X) is a multifunctional protein that has been implicated in gene regulation, cell cycle control, apoptosis, and tumorigenesis. However, the precise physiological function of Ddx3x during development remains unknown. Here, we show that loss of Ddx3x results in an early post-implantation lethality in male mice. The size of the epiblast marked by Oct3/4 is dramatically reduced in embryonic day 6.5 (E6.5) Ddx3x(-)/Y embryos. Preferential paternal X chromosome inactivation (XCI) in extraembryonic tissues of Ddx3x heterozygous (Ddx3x(-/+)) female mice with a maternally inherited null allele leads to placental abnormalities and embryonic lethality during development. In the embryonic tissues, Ddx3x exhibits developmental-and tissue-specific differences in escape from XCI. Targeted Ddx3x ablation in the epiblast leads to widespread apoptosis and abnormal growth, which causes embryonic lethality in the Sox2-cre/+; Ddx3x(flox)/Y mutant around E11.5. The observation of significant increases in gamma H2AX and p-p53(Ser15) indicates DNA damage, which suggests that loss of Ddx3x leads to higher levels of genome damage. Significant upregulation of p21(WAF1/Cip1) and p15(Ink4b) results in cell cycle arrest and apoptosis in Ddx3x-deficient cells. These results have uncovered that mouse Ddx3x is essential for both embryo and extraembryonic development.
Y-Box Binding Protein 1 Stabilizes Hepatitis C Virus NS5A via Phosphorylation-Mediated Interaction with NS5A To Regulate Viral Propagation
(2015-11-01)
Wang, Wei-Ting
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Tsai, Tsung-Yuan
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Chao, Chi-Hong
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Lai, Bo-Ying
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Lee, Yan-Hwa Wu
Replication of hepatitis C virus (HCV) is dependent on virus-encoded proteins and numerous cellular factors. DDX3 is a well-known host cofactor of HCV replication. In this study, we investigated the role of a DDX3-interacting protein, Y-box binding protein 1 (YB-1), in the HCV life cycle. Both YB-1 and DDX3 interacted with the viral nonstructural protein NS5A. During HCV infection, YB-1 partially colocalized with NS5A and the HCV replication intermediate double-stranded RNA (dsRNA) in HCV-infected Huh-7.5.1 cells. Despite sharing the same interacting partners, YB-1 participated in HCV RNA replication but was dispensable in steady-state HCV RNA replication, different from the action of DDX3. Moreover, knockdown of YB-1 in HCV-infected cells prevented infectious virus production and reduced the ratio of hyperphosphorylated (p58) to hypophosphorylated (p56) forms of NS5A, whereas DDX3 silencing did not affect the ratio of the p58 and p56 phosphoforms of NS5A. Interestingly, silencing of YB-1 severely reduced NS5A protein stability in NS5A-ectopically expressing, replicon-containing, and HCV-infected cells. Furthermore, mutations of serine 102 of YB-1 affected both YB-1-NS5A interaction and NS5A-stabilizing activity of YB-1, indicating that this Akt phosphorylation site of YB-1 plays an important role in stabilizing NS5A. Collectively, our results support a model in which the event of YB-1 phosphorylation-mediated interaction with NS5A results in stabilizing NS5A to sustain HCV RNA replication and infectious HCV production. Overall, our study may reveal a new aspect for the development of novel anti-HCV drugs. IMPORTANCE Chronic hepatitis C virus (HCV) infection induces liver cirrhosis and hepatocellular carcinoma. The viral nonstructural protein NS5A co-opting various cellular signaling pathways and cofactors to support viral genome replication and virion assembly is a new strategy for anti-HCV drug development. NS5A phosphorylation is believed to modulate switches between different stages of the HCV life cycle. In this study, we identified the cellular protein YB-1 as a novel NS5A-interacting protein. YB-1 is a multifunctional protein participating in oncogenesis and is an oncomarker of hepatocellular carcinoma (HCC). We found that YB-1 protects NS5A from degradation and likely regulates NS5A phosphorylation through its phosphorylation-dependent interaction with NS5A, which might be controlled by HCV-induced signaling pathways. Our observations suggest a model in which HCV modulates NS5A level and the ratio of the p58 and p56 phosphoforms for efficient viral propagation via regulation of cellular signaling inducing YB-1 phosphorylation. Our finding may provide new aspects for developing novel anti-HCV drugs.