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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

Estimating the amount of DDX3 that associates with newly assembled 80S ribosomes. (A) (Upper panel) Representative immunoblot of gradient fractions of a translation initiation reaction on capped mRNA at 15 min that was used to estimate the amounts of rpS3 and DDX3 in the 80S fractions. At this time all rpS3 sediments at 80S. (Lower panel) The molar amounts of rpS3 and of DDX3 in 48S and 80S fractions were established by comparing the western blot signals in the corresponding gradient fractions with signals obtained with known quantities of purified 40S subunits and recombinant DDX3, respectively. The recombinant DDX3 contains an additional tag and hence is slightly bigger than the natural DDX3. (B, C and D) Data from gradients of three independent translation initiation experiments were used to estimate the percentile (B) of newly assembled 80S translation initiation complexes, (C) the calculated molar amount of 80S ribosomal complexes at 15 min translation initiation, and (D) the calculated molar amount of DDX3 in newly assembled 80S complexes. (B) Translation initiation reactions were carried out for 20 s and the amounts of rpS3 (representing the 40S subunits) determined in the 48S and 80S peak fractions (fractions 7, 8 and 12), respectively (see Figure 5B for a representative gradient). The bar graph represents the percentage of 40S subunits in 48S complexes (i.e. the proportion that is capable of assembly into new 80S complexes) in relation to the sum of 40S subunits detectable in the 48S and 80S fractions. (C) Bar graph showing the molar amount of 40S subunits that was assessed to be present in the 80S fractions at 15 min translation initiation (i.e. total number of 80S complexes in fractions 12–14 of Figure 6A). (D) Bar graph representing the molar amount of DDX3 that was found to be associated with newly assembled 80S translation initiation complexes at 15 min translation initiation. Considering that ∼35% (according to Figure 6B), i.e. ∼0.6 pmol (according to Figure 6C) of the 80S complexes corresponded to newly assembled 80S translation initiation complexes, DDX3 interacted with 0.3 pmol (∼50%) of these complexes (giving a 1:1 molar ratio of ribosomes:DDX3). Error bars indicate standard deviation of three independent experiments.
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gks070-F6: Estimating the amount of DDX3 that associates with newly assembled 80S ribosomes. (A) (Upper panel) Representative immunoblot of gradient fractions of a translation initiation reaction on capped mRNA at 15 min that was used to estimate the amounts of rpS3 and DDX3 in the 80S fractions. At this time all rpS3 sediments at 80S. (Lower panel) The molar amounts of rpS3 and of DDX3 in 48S and 80S fractions were established by comparing the western blot signals in the corresponding gradient fractions with signals obtained with known quantities of purified 40S subunits and recombinant DDX3, respectively. The recombinant DDX3 contains an additional tag and hence is slightly bigger than the natural DDX3. (B, C and D) Data from gradients of three independent translation initiation experiments were used to estimate the percentile (B) of newly assembled 80S translation initiation complexes, (C) the calculated molar amount of 80S ribosomal complexes at 15 min translation initiation, and (D) the calculated molar amount of DDX3 in newly assembled 80S complexes. (B) Translation initiation reactions were carried out for 20 s and the amounts of rpS3 (representing the 40S subunits) determined in the 48S and 80S peak fractions (fractions 7, 8 and 12), respectively (see Figure 5B for a representative gradient). The bar graph represents the percentage of 40S subunits in 48S complexes (i.e. the proportion that is capable of assembly into new 80S complexes) in relation to the sum of 40S subunits detectable in the 48S and 80S fractions. (C) Bar graph showing the molar amount of 40S subunits that was assessed to be present in the 80S fractions at 15 min translation initiation (i.e. total number of 80S complexes in fractions 12–14 of Figure 6A). (D) Bar graph representing the molar amount of DDX3 that was found to be associated with newly assembled 80S translation initiation complexes at 15 min translation initiation. Considering that ∼35% (according to Figure 6B), i.e. ∼0.6 pmol (according to Figure 6C) of the 80S complexes corresponded to newly assembled 80S translation initiation complexes, DDX3 interacted with 0.3 pmol (∼50%) of these complexes (giving a 1:1 molar ratio of ribosomes:DDX3). Error bars indicate standard deviation of three independent experiments.

Mentions: 40S and 60S ribosomal subunits, eIF2, recombinant eIF1, eIF1A, eIF5, eIF5B587–1220 and Escherichia coli methionyl tRNA synthetase were purified essentially as described by Pisarev et al. (13). During purification of eIF1A, eIF5 and E. coli methionyl tRNA synthetase, MonoQ HR 5/5 was replaced by ResourceQ (Amersham Biosciences). During purification of eIF1 and eIF5B587–1220, MonoS HR 5/5 and MonoQ HR 5/5 were replaced by HiTrap Heparin HP (GE Healthcare). All proteins were finally applied to HiLoad 16/60 Superdex 75 (GE Healthcare) equilibrated with 20 mM Tris pH 7.5, 100 mM KCl, 1 mM DTT, 0.1 mM EDTA and 10% glycerol. eIF2 and eIF3 were purified from HeLa extract as described by Damoc et al. (14) and Pisarev et al. (13), respectively. Flag-DDX3 was purified 36 h after transfection of HEK293T-REx cells with pcDNA5/TO Flag-DDX3 using ANTI-FLAG M2 Affinity Gel (Sigma). Recombinant DDX3 (ε = 74260/M cm) was purified from E. coli inclusion bodies according to Stoyan et al. (15). The recombinant protein was not applicable in functional assays; it was used for quantification purposes (Figure 6).


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

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

Estimating the amount of DDX3 that associates with newly assembled 80S ribosomes. (A) (Upper panel) Representative immunoblot of gradient fractions of a translation initiation reaction on capped mRNA at 15 min that was used to estimate the amounts of rpS3 and DDX3 in the 80S fractions. At this time all rpS3 sediments at 80S. (Lower panel) The molar amounts of rpS3 and of DDX3 in 48S and 80S fractions were established by comparing the western blot signals in the corresponding gradient fractions with signals obtained with known quantities of purified 40S subunits and recombinant DDX3, respectively. The recombinant DDX3 contains an additional tag and hence is slightly bigger than the natural DDX3. (B, C and D) Data from gradients of three independent translation initiation experiments were used to estimate the percentile (B) of newly assembled 80S translation initiation complexes, (C) the calculated molar amount of 80S ribosomal complexes at 15 min translation initiation, and (D) the calculated molar amount of DDX3 in newly assembled 80S complexes. (B) Translation initiation reactions were carried out for 20 s and the amounts of rpS3 (representing the 40S subunits) determined in the 48S and 80S peak fractions (fractions 7, 8 and 12), respectively (see Figure 5B for a representative gradient). The bar graph represents the percentage of 40S subunits in 48S complexes (i.e. the proportion that is capable of assembly into new 80S complexes) in relation to the sum of 40S subunits detectable in the 48S and 80S fractions. (C) Bar graph showing the molar amount of 40S subunits that was assessed to be present in the 80S fractions at 15 min translation initiation (i.e. total number of 80S complexes in fractions 12–14 of Figure 6A). (D) Bar graph representing the molar amount of DDX3 that was found to be associated with newly assembled 80S translation initiation complexes at 15 min translation initiation. Considering that ∼35% (according to Figure 6B), i.e. ∼0.6 pmol (according to Figure 6C) of the 80S complexes corresponded to newly assembled 80S translation initiation complexes, DDX3 interacted with 0.3 pmol (∼50%) of these complexes (giving a 1:1 molar ratio of ribosomes:DDX3). Error bars indicate standard deviation of three independent experiments.
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Related In: Results  -  Collection

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Show All Figures
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gks070-F6: Estimating the amount of DDX3 that associates with newly assembled 80S ribosomes. (A) (Upper panel) Representative immunoblot of gradient fractions of a translation initiation reaction on capped mRNA at 15 min that was used to estimate the amounts of rpS3 and DDX3 in the 80S fractions. At this time all rpS3 sediments at 80S. (Lower panel) The molar amounts of rpS3 and of DDX3 in 48S and 80S fractions were established by comparing the western blot signals in the corresponding gradient fractions with signals obtained with known quantities of purified 40S subunits and recombinant DDX3, respectively. The recombinant DDX3 contains an additional tag and hence is slightly bigger than the natural DDX3. (B, C and D) Data from gradients of three independent translation initiation experiments were used to estimate the percentile (B) of newly assembled 80S translation initiation complexes, (C) the calculated molar amount of 80S ribosomal complexes at 15 min translation initiation, and (D) the calculated molar amount of DDX3 in newly assembled 80S complexes. (B) Translation initiation reactions were carried out for 20 s and the amounts of rpS3 (representing the 40S subunits) determined in the 48S and 80S peak fractions (fractions 7, 8 and 12), respectively (see Figure 5B for a representative gradient). The bar graph represents the percentage of 40S subunits in 48S complexes (i.e. the proportion that is capable of assembly into new 80S complexes) in relation to the sum of 40S subunits detectable in the 48S and 80S fractions. (C) Bar graph showing the molar amount of 40S subunits that was assessed to be present in the 80S fractions at 15 min translation initiation (i.e. total number of 80S complexes in fractions 12–14 of Figure 6A). (D) Bar graph representing the molar amount of DDX3 that was found to be associated with newly assembled 80S translation initiation complexes at 15 min translation initiation. Considering that ∼35% (according to Figure 6B), i.e. ∼0.6 pmol (according to Figure 6C) of the 80S complexes corresponded to newly assembled 80S translation initiation complexes, DDX3 interacted with 0.3 pmol (∼50%) of these complexes (giving a 1:1 molar ratio of ribosomes:DDX3). Error bars indicate standard deviation of three independent experiments.
Mentions: 40S and 60S ribosomal subunits, eIF2, recombinant eIF1, eIF1A, eIF5, eIF5B587–1220 and Escherichia coli methionyl tRNA synthetase were purified essentially as described by Pisarev et al. (13). During purification of eIF1A, eIF5 and E. coli methionyl tRNA synthetase, MonoQ HR 5/5 was replaced by ResourceQ (Amersham Biosciences). During purification of eIF1 and eIF5B587–1220, MonoS HR 5/5 and MonoQ HR 5/5 were replaced by HiTrap Heparin HP (GE Healthcare). All proteins were finally applied to HiLoad 16/60 Superdex 75 (GE Healthcare) equilibrated with 20 mM Tris pH 7.5, 100 mM KCl, 1 mM DTT, 0.1 mM EDTA and 10% glycerol. eIF2 and eIF3 were purified from HeLa extract as described by Damoc et al. (14) and Pisarev et al. (13), respectively. Flag-DDX3 was purified 36 h after transfection of HEK293T-REx cells with pcDNA5/TO Flag-DDX3 using ANTI-FLAG M2 Affinity Gel (Sigma). Recombinant DDX3 (ε = 74260/M cm) was purified from E. coli inclusion bodies according to Stoyan et al. (15). The recombinant protein was not applicable in functional assays; it was used for quantification purposes (Figure 6).

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