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Two RNA-binding motifs in eIF3 direct HCV IRES-dependent translation.

Sun C, Querol-Audí J, Mortimer SA, Arias-Palomo E, Doudna JA, Nogales E, Cate JH - Nucleic Acids Res. (2013)

Bottom Line: Mutations in the RNA-binding motif of subunit eIF3a weaken eIF3 binding to the HCV IRES and the 40S ribosomal subunit, thereby suppressing eIF2-dependent recognition of the start codon.Mutations in the eIF3c RNA-binding motif also reduce 40S ribosomal subunit binding to eIF3, and inhibit eIF5B-dependent steps downstream of start codon recognition.These results provide the first connection between the structure of the central translation initiation factor eIF3 and recognition of the HCV genomic RNA start codon, molecular interactions that likely extend to the human transcriptome.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA, Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA, Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA, Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA and Department of Chemistry, University of California, Berkeley, CA 94720, USA.

ABSTRACT
The initiation of protein synthesis plays an essential regulatory role in human biology. At the center of the initiation pathway, the 13-subunit eukaryotic translation initiation factor 3 (eIF3) controls access of other initiation factors and mRNA to the ribosome by unknown mechanisms. Using electron microscopy (EM), bioinformatics and biochemical experiments, we identify two highly conserved RNA-binding motifs in eIF3 that direct translation initiation from the hepatitis C virus internal ribosome entry site (HCV IRES) RNA. Mutations in the RNA-binding motif of subunit eIF3a weaken eIF3 binding to the HCV IRES and the 40S ribosomal subunit, thereby suppressing eIF2-dependent recognition of the start codon. Mutations in the eIF3c RNA-binding motif also reduce 40S ribosomal subunit binding to eIF3, and inhibit eIF5B-dependent steps downstream of start codon recognition. These results provide the first connection between the structure of the central translation initiation factor eIF3 and recognition of the HCV genomic RNA start codon, molecular interactions that likely extend to the human transcriptome.

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Binding of wild-type and mutant eIF3 complexes to the 40S ribosomal subunit. Native agarose gel showing binding of the eIF3 wild-type and mutant 12-mer eIF3 to the 40S ribosomal subunit, monitored by UV absorbance of the 40S subunit rRNA. The nanomolar concentrations of the eIF3 complexes are indicated. The 40S ribosomal subunit was used at a concentration of 10 nM. The binding of the 40S subunit to natively purified human eIF3 or 12-mer is shown as a control (lane 1).
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gkt510-F4: Binding of wild-type and mutant eIF3 complexes to the 40S ribosomal subunit. Native agarose gel showing binding of the eIF3 wild-type and mutant 12-mer eIF3 to the 40S ribosomal subunit, monitored by UV absorbance of the 40S subunit rRNA. The nanomolar concentrations of the eIF3 complexes are indicated. The 40S ribosomal subunit was used at a concentration of 10 nM. The binding of the 40S subunit to natively purified human eIF3 or 12-mer is shown as a control (lane 1).

Mentions: The Proteasome, COP9, eIF3/Mpr1-Pad1 N terminal (PCI/MPN) octameric core of eIF3 binds the 40S ribosomal subunit with only 2–3-fold weaker affinity compared with native eIF3 (16). Considering the importance of the N-termini of eIF3 subunits a* and c* for HCV IRES binding, as well as their proximity to the 40S subunit (14), we tested whether mutations in the HLH motifs in subunits a and c affect the interaction of eIF3 with the 40S ribosomal subunit. Remarkably, eIF3 octameric core complexes with mutations in the HLH motifs bound the 40S ribosomal subunit with severely reduced affinity (Supplementary Figure S4). The addition of subunits b, d, g and i restored 40S ribosomal subunit binding of eIF3 complexes with mutations in the HLH motifs to some degree (Figure 4). These results indicate that the HLH motifs in eIF3a and eIF3c are important for eIF3 binding to the 40S ribosomal subunit, as well as to the HCV IRES.Figure 4.


Two RNA-binding motifs in eIF3 direct HCV IRES-dependent translation.

Sun C, Querol-Audí J, Mortimer SA, Arias-Palomo E, Doudna JA, Nogales E, Cate JH - Nucleic Acids Res. (2013)

Binding of wild-type and mutant eIF3 complexes to the 40S ribosomal subunit. Native agarose gel showing binding of the eIF3 wild-type and mutant 12-mer eIF3 to the 40S ribosomal subunit, monitored by UV absorbance of the 40S subunit rRNA. The nanomolar concentrations of the eIF3 complexes are indicated. The 40S ribosomal subunit was used at a concentration of 10 nM. The binding of the 40S subunit to natively purified human eIF3 or 12-mer is shown as a control (lane 1).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3753635&req=5

gkt510-F4: Binding of wild-type and mutant eIF3 complexes to the 40S ribosomal subunit. Native agarose gel showing binding of the eIF3 wild-type and mutant 12-mer eIF3 to the 40S ribosomal subunit, monitored by UV absorbance of the 40S subunit rRNA. The nanomolar concentrations of the eIF3 complexes are indicated. The 40S ribosomal subunit was used at a concentration of 10 nM. The binding of the 40S subunit to natively purified human eIF3 or 12-mer is shown as a control (lane 1).
Mentions: The Proteasome, COP9, eIF3/Mpr1-Pad1 N terminal (PCI/MPN) octameric core of eIF3 binds the 40S ribosomal subunit with only 2–3-fold weaker affinity compared with native eIF3 (16). Considering the importance of the N-termini of eIF3 subunits a* and c* for HCV IRES binding, as well as their proximity to the 40S subunit (14), we tested whether mutations in the HLH motifs in subunits a and c affect the interaction of eIF3 with the 40S ribosomal subunit. Remarkably, eIF3 octameric core complexes with mutations in the HLH motifs bound the 40S ribosomal subunit with severely reduced affinity (Supplementary Figure S4). The addition of subunits b, d, g and i restored 40S ribosomal subunit binding of eIF3 complexes with mutations in the HLH motifs to some degree (Figure 4). These results indicate that the HLH motifs in eIF3a and eIF3c are important for eIF3 binding to the 40S ribosomal subunit, as well as to the HCV IRES.Figure 4.

Bottom Line: Mutations in the RNA-binding motif of subunit eIF3a weaken eIF3 binding to the HCV IRES and the 40S ribosomal subunit, thereby suppressing eIF2-dependent recognition of the start codon.Mutations in the eIF3c RNA-binding motif also reduce 40S ribosomal subunit binding to eIF3, and inhibit eIF5B-dependent steps downstream of start codon recognition.These results provide the first connection between the structure of the central translation initiation factor eIF3 and recognition of the HCV genomic RNA start codon, molecular interactions that likely extend to the human transcriptome.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA, Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA, Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA, Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA and Department of Chemistry, University of California, Berkeley, CA 94720, USA.

ABSTRACT
The initiation of protein synthesis plays an essential regulatory role in human biology. At the center of the initiation pathway, the 13-subunit eukaryotic translation initiation factor 3 (eIF3) controls access of other initiation factors and mRNA to the ribosome by unknown mechanisms. Using electron microscopy (EM), bioinformatics and biochemical experiments, we identify two highly conserved RNA-binding motifs in eIF3 that direct translation initiation from the hepatitis C virus internal ribosome entry site (HCV IRES) RNA. Mutations in the RNA-binding motif of subunit eIF3a weaken eIF3 binding to the HCV IRES and the 40S ribosomal subunit, thereby suppressing eIF2-dependent recognition of the start codon. Mutations in the eIF3c RNA-binding motif also reduce 40S ribosomal subunit binding to eIF3, and inhibit eIF5B-dependent steps downstream of start codon recognition. These results provide the first connection between the structure of the central translation initiation factor eIF3 and recognition of the HCV genomic RNA start codon, molecular interactions that likely extend to the human transcriptome.

Show MeSH
Related in: MedlinePlus