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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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Models of cap- and HCV IRES-directed translation; suggested participation of DDX3 in translation initiation. Canonical (cap-mediated) translation is depicted in four stages; ➀ and ➁ represent the translation initiation process (see text). ➀ Binding of the 43S PIC and initiation factors to the 5′-end of mRNA and formation of the 48S complex (5′–3′ interactions of the mRNA not shown). ➁ Joining of 60S and assembly of the 80S translation initiation complex. ➂ Elongation and termination phase resulting in 80S post-termination complexes. ➃ Recycling of 80S post-termination complexes and of free 80S ribosomes. In HCV IRES-directed translation initiation, the 43S PIC directly binds to the AUG initiation codon in the absence of eIF4F and without scanning to assemble the 48S complex ➀. Our data suggest that DDX3 joins the 43S PIC via direct (non-RNA mediated) interactions with eIF3 and the 40S subunit. This complex assembles with the mRNA to generate the 48S complex. DDX3 then is involved in conformational changes that favor the release of translation initiation factors and the joining of the 60S subunit. DDX3 remains bound to the 80S translation initiation complex but disassembles prior to the elongation process.
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gks070-F1: Models of cap- and HCV IRES-directed translation; suggested participation of DDX3 in translation initiation. Canonical (cap-mediated) translation is depicted in four stages; ➀ and ➁ represent the translation initiation process (see text). ➀ Binding of the 43S PIC and initiation factors to the 5′-end of mRNA and formation of the 48S complex (5′–3′ interactions of the mRNA not shown). ➁ Joining of 60S and assembly of the 80S translation initiation complex. ➂ Elongation and termination phase resulting in 80S post-termination complexes. ➃ Recycling of 80S post-termination complexes and of free 80S ribosomes. In HCV IRES-directed translation initiation, the 43S PIC directly binds to the AUG initiation codon in the absence of eIF4F and without scanning to assemble the 48S complex ➀. Our data suggest that DDX3 joins the 43S PIC via direct (non-RNA mediated) interactions with eIF3 and the 40S subunit. This complex assembles with the mRNA to generate the 48S complex. DDX3 then is involved in conformational changes that favor the release of translation initiation factors and the joining of the 60S subunit. DDX3 remains bound to the 80S translation initiation complex but disassembles prior to the elongation process.

Mentions: Eukaryotic protein synthesis involves translation initiation, elongation, termination and the recycling of ribosomes. The canonical translation initiation process on capped mRNAs consists of two intricate steps that eventually lead to the assembly of 80S initiation complexes where the anticodon of an aminoacylated initiator tRNA (mostly Met–tRNAi) is base-paired with the mRNA’s initiation codon in the ribosomal P(peptidyl)-site. In the current model [(1,2) and references herein; Figure 1], a pre-assembled 43S pre-initiation complex (PIC) that consists of a small 40S ribosomal subunit, the translation initiation factors eIF1, eIF1A, eIF3, conceivably eIF5 and the ternary eIF2-GTP-Met-tRNAi complex (TC), first associates with the 5′-region of the mRNA. During this process, structures in the mRNA’s 5′-untranslated region (5′-UTR) are unwound in an ATP-dependent manner involving eIF4F and eIF4B. eIF4F is a protein complex that includes the cap-binding protein eIF4E, the helicase eIF4A and the ‘scaffold’ protein eIF4G, the latter of which binds to eIF3, eIF4E, eIF4A and the poly A binding protein PABP. Interactions of eIF4F with the cap (via eIF4E) and with PABP and the poly A tail (via eIF4G) generates a loop structure of the mRNA. The PIC then scans in 5′–3′ direction for translation initiation codons. A matching contact of an initiation codon with the anticodon in Met–tRNAi switches the scanning complex to a ‘closed conformation’ that is discernible as 48S complex. Subsequent dislocation of eIF1 leads to eIF5-mediated GTP-hydrolysis in the TC. In the second step of translation initiation, eIF5B triggers the attachment of the 60S subunit to the 48S complex and the simultaneous displacement of eIF2–GDP, eIF1, eIF3, eIF4A, eIF4B, eIF4G and eIF5. The initiation process is concluded by eIF5B-mediated GTP hydrolysis and release of eIF1A and GDP-bound eIF5B from the assembled, elongation-competent 80S ribosome. The next codon-complementary aminoacyl–tRNA may now associate into the A(aminoacyl)-site of the ribosome and the first peptide bond may be produced.Figure 1.


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

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

Models of cap- and HCV IRES-directed translation; suggested participation of DDX3 in translation initiation. Canonical (cap-mediated) translation is depicted in four stages; ➀ and ➁ represent the translation initiation process (see text). ➀ Binding of the 43S PIC and initiation factors to the 5′-end of mRNA and formation of the 48S complex (5′–3′ interactions of the mRNA not shown). ➁ Joining of 60S and assembly of the 80S translation initiation complex. ➂ Elongation and termination phase resulting in 80S post-termination complexes. ➃ Recycling of 80S post-termination complexes and of free 80S ribosomes. In HCV IRES-directed translation initiation, the 43S PIC directly binds to the AUG initiation codon in the absence of eIF4F and without scanning to assemble the 48S complex ➀. Our data suggest that DDX3 joins the 43S PIC via direct (non-RNA mediated) interactions with eIF3 and the 40S subunit. This complex assembles with the mRNA to generate the 48S complex. DDX3 then is involved in conformational changes that favor the release of translation initiation factors and the joining of the 60S subunit. DDX3 remains bound to the 80S translation initiation complex but disassembles prior to the elongation process.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3367175&req=5

gks070-F1: Models of cap- and HCV IRES-directed translation; suggested participation of DDX3 in translation initiation. Canonical (cap-mediated) translation is depicted in four stages; ➀ and ➁ represent the translation initiation process (see text). ➀ Binding of the 43S PIC and initiation factors to the 5′-end of mRNA and formation of the 48S complex (5′–3′ interactions of the mRNA not shown). ➁ Joining of 60S and assembly of the 80S translation initiation complex. ➂ Elongation and termination phase resulting in 80S post-termination complexes. ➃ Recycling of 80S post-termination complexes and of free 80S ribosomes. In HCV IRES-directed translation initiation, the 43S PIC directly binds to the AUG initiation codon in the absence of eIF4F and without scanning to assemble the 48S complex ➀. Our data suggest that DDX3 joins the 43S PIC via direct (non-RNA mediated) interactions with eIF3 and the 40S subunit. This complex assembles with the mRNA to generate the 48S complex. DDX3 then is involved in conformational changes that favor the release of translation initiation factors and the joining of the 60S subunit. DDX3 remains bound to the 80S translation initiation complex but disassembles prior to the elongation process.
Mentions: Eukaryotic protein synthesis involves translation initiation, elongation, termination and the recycling of ribosomes. The canonical translation initiation process on capped mRNAs consists of two intricate steps that eventually lead to the assembly of 80S initiation complexes where the anticodon of an aminoacylated initiator tRNA (mostly Met–tRNAi) is base-paired with the mRNA’s initiation codon in the ribosomal P(peptidyl)-site. In the current model [(1,2) and references herein; Figure 1], a pre-assembled 43S pre-initiation complex (PIC) that consists of a small 40S ribosomal subunit, the translation initiation factors eIF1, eIF1A, eIF3, conceivably eIF5 and the ternary eIF2-GTP-Met-tRNAi complex (TC), first associates with the 5′-region of the mRNA. During this process, structures in the mRNA’s 5′-untranslated region (5′-UTR) are unwound in an ATP-dependent manner involving eIF4F and eIF4B. eIF4F is a protein complex that includes the cap-binding protein eIF4E, the helicase eIF4A and the ‘scaffold’ protein eIF4G, the latter of which binds to eIF3, eIF4E, eIF4A and the poly A binding protein PABP. Interactions of eIF4F with the cap (via eIF4E) and with PABP and the poly A tail (via eIF4G) generates a loop structure of the mRNA. The PIC then scans in 5′–3′ direction for translation initiation codons. A matching contact of an initiation codon with the anticodon in Met–tRNAi switches the scanning complex to a ‘closed conformation’ that is discernible as 48S complex. Subsequent dislocation of eIF1 leads to eIF5-mediated GTP-hydrolysis in the TC. In the second step of translation initiation, eIF5B triggers the attachment of the 60S subunit to the 48S complex and the simultaneous displacement of eIF2–GDP, eIF1, eIF3, eIF4A, eIF4B, eIF4G and eIF5. The initiation process is concluded by eIF5B-mediated GTP hydrolysis and release of eIF1A and GDP-bound eIF5B from the assembled, elongation-competent 80S ribosome. The next codon-complementary aminoacyl–tRNA may now associate into the A(aminoacyl)-site of the ribosome and the first peptide bond may be produced.Figure 1.

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