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The DEAD-box helicase DDX3 supports the assembly of functional 80S ribosomes.

Geissler R, Golbik RP, Behrens SE - Nucleic Acids Res. (2012)

Bottom Line: DDX3 was found to interact in an RNA-independent manner with defined components of the translational pre-initiation complex and to specifically associate with newly assembling 80S ribosomes.DDX3 knock down and in vitro reconstitution experiments revealed a significant function of the protein in the formation of 80S translation initiation complexes.Our study implies that DDX3 assists the 60S subunit joining process to assemble functional 80S ribosomes.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry and Biotechnology, Faculty of Life Sciences (NFI), Martin Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, D-06120 Halle/Saale, Germany.

ABSTRACT
The DEAD-box helicase DDX3 has suggested functions in innate immunity, mRNA translocation and translation, and it participates in the propagation of assorted viruses. Exploring initially the role of DDX3 in the life cycle of hepatitis C virus, we observed the protein to be involved in translation directed by different viral internal ribosomal entry sites. Extension of these studies revealed a general supportive role of DDX3 in translation initiation. DDX3 was found to interact in an RNA-independent manner with defined components of the translational pre-initiation complex and to specifically associate with newly assembling 80S ribosomes. DDX3 knock down and in vitro reconstitution experiments revealed a significant function of the protein in the formation of 80S translation initiation complexes. Our study implies that DDX3 assists the 60S subunit joining process to assemble functional 80S ribosomes.

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Related in: MedlinePlus

DDX3 is involved in translation. (A) Schematic representation of HCV replicons used in this study. The subgenomic viral RNAs encode all RNA elements and viral non-structural (NS) proteins (processed by the viral protease NS3/4A) enabling autonomous replication in the cytoplasm of transfected host cells. The UTRs are drawn as lines displaying the proposed RNA secondary structures; genetic units are boxed. (Upper panel) Bi-cistronic RNA replicon. Expression of the NEO selection marker enables the generation of G418-resistant replicon-containing cell lines (45). (Lower panel) Mono-cistronic RNA replicon. ΔC: HCV core-coding sequence element that supports efficient HCV IRES-mediated translation. ubi: ubiquitin-coding sequence enabling processing of the NS3 N-terminus by ubiquitincarboxyhydrolase. (B) Replication of the bi-cistronic HCV replicon in stably transfected Huh7 cells at 72 h post-transfection of two different anti-DDX3 siRNAs (siDDX3-1, siDDX3-2) and a control siRNA (siGFP), respectively. The viral RNA levels were measured by qRT–PCR. (C) Representative western blots showing the levels of DDX3, Neomycine phosphotransferase II and HCV NS5A in the cell cytoplasm after treatment with the indicated siRNAs. The vinculin levels served as controls. (D) Replication of the mono-cistronic HCV replicon in transiently transfected Huh7 cells at 72 h post-transfection of the indicated siRNAs. (E) Representative western blots of indicated proteins in cytoplasmic extracts of Huh7 cells that had been transfected with the indicated siRNAs and HCV mono-cistronic RNA replicon. (F, G) (Right) Schematic representation of the (F) mRNA and (G) HCV luciferase reporter constructs. The UTRs are drawn as lines with proposed RNA structures; the genetic unit encoding firefly luciferase is boxed. (Left) Luciferase activity assays. The reporter RNA transcripts were transfected into Huh7 cells 72 h after previous transfection with the indicated siRNAs. One hour post-transfection, the luciferase activity was determined in the cell lysates. The activity measured after transfection of the respective transcripts into control cells (that had been previously transfected with siGFP RNA) was set 100%. Error bars indicate standard deviation of four independent experiments.
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gks070-F2: DDX3 is involved in translation. (A) Schematic representation of HCV replicons used in this study. The subgenomic viral RNAs encode all RNA elements and viral non-structural (NS) proteins (processed by the viral protease NS3/4A) enabling autonomous replication in the cytoplasm of transfected host cells. The UTRs are drawn as lines displaying the proposed RNA secondary structures; genetic units are boxed. (Upper panel) Bi-cistronic RNA replicon. Expression of the NEO selection marker enables the generation of G418-resistant replicon-containing cell lines (45). (Lower panel) Mono-cistronic RNA replicon. ΔC: HCV core-coding sequence element that supports efficient HCV IRES-mediated translation. ubi: ubiquitin-coding sequence enabling processing of the NS3 N-terminus by ubiquitincarboxyhydrolase. (B) Replication of the bi-cistronic HCV replicon in stably transfected Huh7 cells at 72 h post-transfection of two different anti-DDX3 siRNAs (siDDX3-1, siDDX3-2) and a control siRNA (siGFP), respectively. The viral RNA levels were measured by qRT–PCR. (C) Representative western blots showing the levels of DDX3, Neomycine phosphotransferase II and HCV NS5A in the cell cytoplasm after treatment with the indicated siRNAs. The vinculin levels served as controls. (D) Replication of the mono-cistronic HCV replicon in transiently transfected Huh7 cells at 72 h post-transfection of the indicated siRNAs. (E) Representative western blots of indicated proteins in cytoplasmic extracts of Huh7 cells that had been transfected with the indicated siRNAs and HCV mono-cistronic RNA replicon. (F, G) (Right) Schematic representation of the (F) mRNA and (G) HCV luciferase reporter constructs. The UTRs are drawn as lines with proposed RNA structures; the genetic unit encoding firefly luciferase is boxed. (Left) Luciferase activity assays. The reporter RNA transcripts were transfected into Huh7 cells 72 h after previous transfection with the indicated siRNAs. One hour post-transfection, the luciferase activity was determined in the cell lysates. The activity measured after transfection of the respective transcripts into control cells (that had been previously transfected with siGFP RNA) was set 100%. Error bars indicate standard deviation of four independent experiments.

Mentions: Earlier RNA–protein interaction studies and siRNA knock down screens of others and us showed that DDX3 interacts in a yet unknown manner with the UTRs of the HCV RNA genome, and that the protein is indispensable for viral replication (18–21). This study was originally aimed at further investigating the function of DDX3 in the HCV life cycle, and it was initiated by testing the effect of a siRNA-directed intracellular depletion of DDX3 (‘DDX3 knock down’) on HCV replication. For this purpose, we first used Huh7 cells that were persistently transfected with a subgenomic, ‘bi-cistronic’ HCV replicon RNA [HCV subtype 2A ‘JFH’; (22); Figure 2A]. In the bi-cistronic HCV replicon, the HCV IRES directs the translation of a neomycine/geneticin (Neo) resistance gene while synthesis of the viral replicase proteins is enabled by the IRES of encephalomyocarditis virus (EMCV). The DDX3 knock down was achieved by transfection of the Huh7/HCV replicon cells with two different siRNAs, each causing a reduction of cytoplasmic DDX3 to ∼15–25% of the original protein level at 72 h post-transfection (Figure 2C). Importantly, this moderate depletion of DDX3 had no impact on the cellular growth rate. DDX3 knock down with each of the two siRNAs did not affect the replication of the bi-cistronic HCV replicon in comparison to control cells (Figure 2B). However, immunoblotting revealed that the expression levels of Neomycin phosphotransferase as well as that of the HCV-encoded proteins (Figure 2C; reduction of HCV proteins exemplified by NS5A) were considerably lowered in both types of DDX3 knock down cells.Figure 2.


The DEAD-box helicase DDX3 supports the assembly of functional 80S ribosomes.

Geissler R, Golbik RP, Behrens SE - Nucleic Acids Res. (2012)

DDX3 is involved in translation. (A) Schematic representation of HCV replicons used in this study. The subgenomic viral RNAs encode all RNA elements and viral non-structural (NS) proteins (processed by the viral protease NS3/4A) enabling autonomous replication in the cytoplasm of transfected host cells. The UTRs are drawn as lines displaying the proposed RNA secondary structures; genetic units are boxed. (Upper panel) Bi-cistronic RNA replicon. Expression of the NEO selection marker enables the generation of G418-resistant replicon-containing cell lines (45). (Lower panel) Mono-cistronic RNA replicon. ΔC: HCV core-coding sequence element that supports efficient HCV IRES-mediated translation. ubi: ubiquitin-coding sequence enabling processing of the NS3 N-terminus by ubiquitincarboxyhydrolase. (B) Replication of the bi-cistronic HCV replicon in stably transfected Huh7 cells at 72 h post-transfection of two different anti-DDX3 siRNAs (siDDX3-1, siDDX3-2) and a control siRNA (siGFP), respectively. The viral RNA levels were measured by qRT–PCR. (C) Representative western blots showing the levels of DDX3, Neomycine phosphotransferase II and HCV NS5A in the cell cytoplasm after treatment with the indicated siRNAs. The vinculin levels served as controls. (D) Replication of the mono-cistronic HCV replicon in transiently transfected Huh7 cells at 72 h post-transfection of the indicated siRNAs. (E) Representative western blots of indicated proteins in cytoplasmic extracts of Huh7 cells that had been transfected with the indicated siRNAs and HCV mono-cistronic RNA replicon. (F, G) (Right) Schematic representation of the (F) mRNA and (G) HCV luciferase reporter constructs. The UTRs are drawn as lines with proposed RNA structures; the genetic unit encoding firefly luciferase is boxed. (Left) Luciferase activity assays. The reporter RNA transcripts were transfected into Huh7 cells 72 h after previous transfection with the indicated siRNAs. One hour post-transfection, the luciferase activity was determined in the cell lysates. The activity measured after transfection of the respective transcripts into control cells (that had been previously transfected with siGFP RNA) was set 100%. Error bars indicate standard deviation of four independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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gks070-F2: DDX3 is involved in translation. (A) Schematic representation of HCV replicons used in this study. The subgenomic viral RNAs encode all RNA elements and viral non-structural (NS) proteins (processed by the viral protease NS3/4A) enabling autonomous replication in the cytoplasm of transfected host cells. The UTRs are drawn as lines displaying the proposed RNA secondary structures; genetic units are boxed. (Upper panel) Bi-cistronic RNA replicon. Expression of the NEO selection marker enables the generation of G418-resistant replicon-containing cell lines (45). (Lower panel) Mono-cistronic RNA replicon. ΔC: HCV core-coding sequence element that supports efficient HCV IRES-mediated translation. ubi: ubiquitin-coding sequence enabling processing of the NS3 N-terminus by ubiquitincarboxyhydrolase. (B) Replication of the bi-cistronic HCV replicon in stably transfected Huh7 cells at 72 h post-transfection of two different anti-DDX3 siRNAs (siDDX3-1, siDDX3-2) and a control siRNA (siGFP), respectively. The viral RNA levels were measured by qRT–PCR. (C) Representative western blots showing the levels of DDX3, Neomycine phosphotransferase II and HCV NS5A in the cell cytoplasm after treatment with the indicated siRNAs. The vinculin levels served as controls. (D) Replication of the mono-cistronic HCV replicon in transiently transfected Huh7 cells at 72 h post-transfection of the indicated siRNAs. (E) Representative western blots of indicated proteins in cytoplasmic extracts of Huh7 cells that had been transfected with the indicated siRNAs and HCV mono-cistronic RNA replicon. (F, G) (Right) Schematic representation of the (F) mRNA and (G) HCV luciferase reporter constructs. The UTRs are drawn as lines with proposed RNA structures; the genetic unit encoding firefly luciferase is boxed. (Left) Luciferase activity assays. The reporter RNA transcripts were transfected into Huh7 cells 72 h after previous transfection with the indicated siRNAs. One hour post-transfection, the luciferase activity was determined in the cell lysates. The activity measured after transfection of the respective transcripts into control cells (that had been previously transfected with siGFP RNA) was set 100%. Error bars indicate standard deviation of four independent experiments.
Mentions: Earlier RNA–protein interaction studies and siRNA knock down screens of others and us showed that DDX3 interacts in a yet unknown manner with the UTRs of the HCV RNA genome, and that the protein is indispensable for viral replication (18–21). This study was originally aimed at further investigating the function of DDX3 in the HCV life cycle, and it was initiated by testing the effect of a siRNA-directed intracellular depletion of DDX3 (‘DDX3 knock down’) on HCV replication. For this purpose, we first used Huh7 cells that were persistently transfected with a subgenomic, ‘bi-cistronic’ HCV replicon RNA [HCV subtype 2A ‘JFH’; (22); Figure 2A]. In the bi-cistronic HCV replicon, the HCV IRES directs the translation of a neomycine/geneticin (Neo) resistance gene while synthesis of the viral replicase proteins is enabled by the IRES of encephalomyocarditis virus (EMCV). The DDX3 knock down was achieved by transfection of the Huh7/HCV replicon cells with two different siRNAs, each causing a reduction of cytoplasmic DDX3 to ∼15–25% of the original protein level at 72 h post-transfection (Figure 2C). Importantly, this moderate depletion of DDX3 had no impact on the cellular growth rate. DDX3 knock down with each of the two siRNAs did not affect the replication of the bi-cistronic HCV replicon in comparison to control cells (Figure 2B). However, immunoblotting revealed that the expression levels of Neomycin phosphotransferase as well as that of the HCV-encoded proteins (Figure 2C; reduction of HCV proteins exemplified by NS5A) were considerably lowered in both types of DDX3 knock down cells.Figure 2.

Bottom Line: DDX3 was found to interact in an RNA-independent manner with defined components of the translational pre-initiation complex and to specifically associate with newly assembling 80S ribosomes.DDX3 knock down and in vitro reconstitution experiments revealed a significant function of the protein in the formation of 80S translation initiation complexes.Our study implies that DDX3 assists the 60S subunit joining process to assemble functional 80S ribosomes.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry and Biotechnology, Faculty of Life Sciences (NFI), Martin Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, D-06120 Halle/Saale, Germany.

ABSTRACT
The DEAD-box helicase DDX3 has suggested functions in innate immunity, mRNA translocation and translation, and it participates in the propagation of assorted viruses. Exploring initially the role of DDX3 in the life cycle of hepatitis C virus, we observed the protein to be involved in translation directed by different viral internal ribosomal entry sites. Extension of these studies revealed a general supportive role of DDX3 in translation initiation. DDX3 was found to interact in an RNA-independent manner with defined components of the translational pre-initiation complex and to specifically associate with newly assembling 80S ribosomes. DDX3 knock down and in vitro reconstitution experiments revealed a significant function of the protein in the formation of 80S translation initiation complexes. Our study implies that DDX3 assists the 60S subunit joining process to assemble functional 80S ribosomes.

Show MeSH
Related in: MedlinePlus