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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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Identification of the eIF3 binding sites in the HCV IRES. SHAPE reactivity differences mapped onto the secondary structure of the IRES IIIabc domain in the presence of eIF3 8-mer mutated in the subunit c HLH motif (8-Cm), 8-mer or 10-mer, indicated from left to right by colored circles. Nucleotides in the HCV IRES that show greater (red) or reduced (blue) SHAPE reactivity in the presence of the eIF3 subcomplexes are indicated.
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gkt510-F3: Identification of the eIF3 binding sites in the HCV IRES. SHAPE reactivity differences mapped onto the secondary structure of the IRES IIIabc domain in the presence of eIF3 8-mer mutated in the subunit c HLH motif (8-Cm), 8-mer or 10-mer, indicated from left to right by colored circles. Nucleotides in the HCV IRES that show greater (red) or reduced (blue) SHAPE reactivity in the presence of the eIF3 subcomplexes are indicated.

Mentions: To identify the regions within the HCV IRES that contact the predicted HLH motifs in eIF3 subunits a* and c*, we used SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) chemistry to analyze the flexibility of each nucleotide in the HCV IRES in the absence and presence of wild-type eIF3 octamer, 10-mer or with mutations in the predicted HLH motif of subunit c* (19) (Supplementary Materials and Methods). The eIF3 octamer or 10-subunit complex strongly protected the HCV IRES IIIb stem-loop from SHAPE reactivity (Figure 3), consistent with the IIIb region serving as the main binding site for eIF3 (9). The eIF3 octamer with mutations in the HLH motif of subunit eIF3c, which slightly reduced IRES binding (Figure 3), also protected the IIIb stem-loop from SHAPE reactivity, albeit less strongly (Figure 3). Together with the mutational analysis above (Figure 2), these results indicate that the HLH motif of subunit eIF3a makes the most extensive contacts with the HCV IRES by binding the IRES IIIb stem loop. Subunit eIF3c may augment this primary interaction rather than contacting an entirely separate region of the IRES, whereas subunits eIF3b and eIF3d may contribute to additional contacts near the lower portions of domain III and the pseudoknot regions of the IRES (Figure 3).Figure 3.


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)

Identification of the eIF3 binding sites in the HCV IRES. SHAPE reactivity differences mapped onto the secondary structure of the IRES IIIabc domain in the presence of eIF3 8-mer mutated in the subunit c HLH motif (8-Cm), 8-mer or 10-mer, indicated from left to right by colored circles. Nucleotides in the HCV IRES that show greater (red) or reduced (blue) SHAPE reactivity in the presence of the eIF3 subcomplexes are indicated.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt510-F3: Identification of the eIF3 binding sites in the HCV IRES. SHAPE reactivity differences mapped onto the secondary structure of the IRES IIIabc domain in the presence of eIF3 8-mer mutated in the subunit c HLH motif (8-Cm), 8-mer or 10-mer, indicated from left to right by colored circles. Nucleotides in the HCV IRES that show greater (red) or reduced (blue) SHAPE reactivity in the presence of the eIF3 subcomplexes are indicated.
Mentions: To identify the regions within the HCV IRES that contact the predicted HLH motifs in eIF3 subunits a* and c*, we used SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) chemistry to analyze the flexibility of each nucleotide in the HCV IRES in the absence and presence of wild-type eIF3 octamer, 10-mer or with mutations in the predicted HLH motif of subunit c* (19) (Supplementary Materials and Methods). The eIF3 octamer or 10-subunit complex strongly protected the HCV IRES IIIb stem-loop from SHAPE reactivity (Figure 3), consistent with the IIIb region serving as the main binding site for eIF3 (9). The eIF3 octamer with mutations in the HLH motif of subunit eIF3c, which slightly reduced IRES binding (Figure 3), also protected the IIIb stem-loop from SHAPE reactivity, albeit less strongly (Figure 3). Together with the mutational analysis above (Figure 2), these results indicate that the HLH motif of subunit eIF3a makes the most extensive contacts with the HCV IRES by binding the IRES IIIb stem loop. Subunit eIF3c may augment this primary interaction rather than contacting an entirely separate region of the IRES, whereas subunits eIF3b and eIF3d may contribute to additional contacts near the lower portions of domain III and the pseudoknot regions of the IRES (Figure 3).Figure 3.

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