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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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DDX3 promotes the assembly of functional 80S ribosomes. (A) Western blot analyses indicating the amounts of vinculin and of DDX3 in S10 extracts of Huh7 cells that were pre-treated with siGFP or siDDX3-2. (B–E) Translation initiation assays performed with mock-depleted and DDX3-depleted S10 extracts; formation of the 48S and 80S translation initiation complexes was monitored on [32P]-labeled substrate RNA (Figure 5). (B) 48S and (C) 80S complex assembly on 5′cap-sORF-poly A at 20 s and 10 min, respectively (top gradient fractions omitted for better resolution). (D) 48S and (E) 80S complex assembly on HCV 5′-UTR-sORF-3′-UTR RNA at 1 and 10 min, respectively. Dashed line/triangles: data obtained with mock-depleted extract. Continuous line/squares: data obtained with DDX3-depleted extract. Error bars represent standard deviation of six independent experiments, (*) P ≤ 0.05, (**) P ≤ 0.01, (***) P ≤ 0.001. Differences in the amounts of formed 48S and 80S translation initiation complexes were estimated by comparing the measured radioactivity in the 48S and 80S peak fractions. (F and G) In vitro assembly of translation initiation complexes on [32P]-labeled HCV RNA. Following the protocol of Terenin et al. (31) (see also Supplementary Figure S9), 48S complexes were assembled on the template using purified eIF3, Met–tRNAiMet and 40S subunits in the presence and absence of purified Flag-DDX3, respectively (F). For the assembly of 80S complexes (G), the 48S complexes were further incubated with purified eIF5B587–1220 and 60S ribosomal subunits, respectively. The assembled 48S and 80S complexes were separated by centrifugation through a 5–25% linear sucrose density gradient and the positions are indicated. One representative experiment is shown.
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gks070-F7: DDX3 promotes the assembly of functional 80S ribosomes. (A) Western blot analyses indicating the amounts of vinculin and of DDX3 in S10 extracts of Huh7 cells that were pre-treated with siGFP or siDDX3-2. (B–E) Translation initiation assays performed with mock-depleted and DDX3-depleted S10 extracts; formation of the 48S and 80S translation initiation complexes was monitored on [32P]-labeled substrate RNA (Figure 5). (B) 48S and (C) 80S complex assembly on 5′cap-sORF-poly A at 20 s and 10 min, respectively (top gradient fractions omitted for better resolution). (D) 48S and (E) 80S complex assembly on HCV 5′-UTR-sORF-3′-UTR RNA at 1 and 10 min, respectively. Dashed line/triangles: data obtained with mock-depleted extract. Continuous line/squares: data obtained with DDX3-depleted extract. Error bars represent standard deviation of six independent experiments, (*) P ≤ 0.05, (**) P ≤ 0.01, (***) P ≤ 0.001. Differences in the amounts of formed 48S and 80S translation initiation complexes were estimated by comparing the measured radioactivity in the 48S and 80S peak fractions. (F and G) In vitro assembly of translation initiation complexes on [32P]-labeled HCV RNA. Following the protocol of Terenin et al. (31) (see also Supplementary Figure S9), 48S complexes were assembled on the template using purified eIF3, Met–tRNAiMet and 40S subunits in the presence and absence of purified Flag-DDX3, respectively (F). For the assembly of 80S complexes (G), the 48S complexes were further incubated with purified eIF5B587–1220 and 60S ribosomal subunits, respectively. The assembled 48S and 80S complexes were separated by centrifugation through a 5–25% linear sucrose density gradient and the positions are indicated. One representative experiment is shown.

Mentions: The following experiments were aimed at dissecting if DDX3 is involved in 48S or 80S complex formation of the translation initiation process. For this purpose, we prepared cytoplasmic S10 extracts from Huh7 cells where the level of DDX3 had been significantly reduced to ∼25% of the original level by prior treatment of the cells with one of the DDX3-specific siRNAs (Figure 7A). In accordance with the earlier cell transfection studies (Figure 2), in vitro translation experiments with such ‘DDX3-depleted’ extracts and reporter mRNAs showed a translation activity that was ∼35–50% lower than that of mock-depleted extracts (Supplementary Figure S7).Figure 7.


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

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

DDX3 promotes the assembly of functional 80S ribosomes. (A) Western blot analyses indicating the amounts of vinculin and of DDX3 in S10 extracts of Huh7 cells that were pre-treated with siGFP or siDDX3-2. (B–E) Translation initiation assays performed with mock-depleted and DDX3-depleted S10 extracts; formation of the 48S and 80S translation initiation complexes was monitored on [32P]-labeled substrate RNA (Figure 5). (B) 48S and (C) 80S complex assembly on 5′cap-sORF-poly A at 20 s and 10 min, respectively (top gradient fractions omitted for better resolution). (D) 48S and (E) 80S complex assembly on HCV 5′-UTR-sORF-3′-UTR RNA at 1 and 10 min, respectively. Dashed line/triangles: data obtained with mock-depleted extract. Continuous line/squares: data obtained with DDX3-depleted extract. Error bars represent standard deviation of six independent experiments, (*) P ≤ 0.05, (**) P ≤ 0.01, (***) P ≤ 0.001. Differences in the amounts of formed 48S and 80S translation initiation complexes were estimated by comparing the measured radioactivity in the 48S and 80S peak fractions. (F and G) In vitro assembly of translation initiation complexes on [32P]-labeled HCV RNA. Following the protocol of Terenin et al. (31) (see also Supplementary Figure S9), 48S complexes were assembled on the template using purified eIF3, Met–tRNAiMet and 40S subunits in the presence and absence of purified Flag-DDX3, respectively (F). For the assembly of 80S complexes (G), the 48S complexes were further incubated with purified eIF5B587–1220 and 60S ribosomal subunits, respectively. The assembled 48S and 80S complexes were separated by centrifugation through a 5–25% linear sucrose density gradient and the positions are indicated. One representative experiment is shown.
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gks070-F7: DDX3 promotes the assembly of functional 80S ribosomes. (A) Western blot analyses indicating the amounts of vinculin and of DDX3 in S10 extracts of Huh7 cells that were pre-treated with siGFP or siDDX3-2. (B–E) Translation initiation assays performed with mock-depleted and DDX3-depleted S10 extracts; formation of the 48S and 80S translation initiation complexes was monitored on [32P]-labeled substrate RNA (Figure 5). (B) 48S and (C) 80S complex assembly on 5′cap-sORF-poly A at 20 s and 10 min, respectively (top gradient fractions omitted for better resolution). (D) 48S and (E) 80S complex assembly on HCV 5′-UTR-sORF-3′-UTR RNA at 1 and 10 min, respectively. Dashed line/triangles: data obtained with mock-depleted extract. Continuous line/squares: data obtained with DDX3-depleted extract. Error bars represent standard deviation of six independent experiments, (*) P ≤ 0.05, (**) P ≤ 0.01, (***) P ≤ 0.001. Differences in the amounts of formed 48S and 80S translation initiation complexes were estimated by comparing the measured radioactivity in the 48S and 80S peak fractions. (F and G) In vitro assembly of translation initiation complexes on [32P]-labeled HCV RNA. Following the protocol of Terenin et al. (31) (see also Supplementary Figure S9), 48S complexes were assembled on the template using purified eIF3, Met–tRNAiMet and 40S subunits in the presence and absence of purified Flag-DDX3, respectively (F). For the assembly of 80S complexes (G), the 48S complexes were further incubated with purified eIF5B587–1220 and 60S ribosomal subunits, respectively. The assembled 48S and 80S complexes were separated by centrifugation through a 5–25% linear sucrose density gradient and the positions are indicated. One representative experiment is shown.
Mentions: The following experiments were aimed at dissecting if DDX3 is involved in 48S or 80S complex formation of the translation initiation process. For this purpose, we prepared cytoplasmic S10 extracts from Huh7 cells where the level of DDX3 had been significantly reduced to ∼25% of the original level by prior treatment of the cells with one of the DDX3-specific siRNAs (Figure 7A). In accordance with the earlier cell transfection studies (Figure 2), in vitro translation experiments with such ‘DDX3-depleted’ extracts and reporter mRNAs showed a translation activity that was ∼35–50% lower than that of mock-depleted extracts (Supplementary Figure S7).Figure 7.

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