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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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The roles of the HLH motifs in eIF3 subunits a and c subunits in translation initiation complex formation. (A) Mutations in the HLH motif of eIF3a greatly weaken eIF3 binding to the HCV IRES and 40S ribosomal subunit, whereas mutations in the HLH motif of eIF3c primarily affect eIF3 binding to the 40S ribosomal subunit. (B) Mutations in the HLH motif of eIF3a disrupt eIF2-mediated Met-tRNAi loading into preinitiation complexes, whereas mutations in the eIF3c HLH motif impair eIF5B exchange with eIF2 after start codon recognition.
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gkt510-F6: The roles of the HLH motifs in eIF3 subunits a and c subunits in translation initiation complex formation. (A) Mutations in the HLH motif of eIF3a greatly weaken eIF3 binding to the HCV IRES and 40S ribosomal subunit, whereas mutations in the HLH motif of eIF3c primarily affect eIF3 binding to the 40S ribosomal subunit. (B) Mutations in the HLH motif of eIF3a disrupt eIF2-mediated Met-tRNAi loading into preinitiation complexes, whereas mutations in the eIF3c HLH motif impair eIF5B exchange with eIF2 after start codon recognition.

Mentions: Based on the experiments in translation extracts, the HLH motif in eIF3a serves as a lynchpin in assembling translation initiation complexes on the HCV IRES. The HLH motif in eIF3a contributes to early steps of HCV IRES-mediated translation initiation dependent on eIF2, before start codon selection, whereas the eIF3c HLH motif is important for later events dependent on eIF5B, after start codon selection but before 60S subunit joining (Figure 6). Intriguingly, the HLH motif in eIF3a is highly conserved across metazoans and is present in most eukaryotes with available genome sequences (Figure 1C, Supplementary Figure S2D), suggesting that this motif may play a role in loading of the eIF2–GTP–Met-tRNAi ternary complex into translation preinitiation complexes on cellular mRNAs as well as the HCV genomic RNA. The HLH motif in eIF3c is highly conserved in all eukaryotes (Figure 1C), implying that its role in eIF5B exchange with eIF2 after start codon selection to catalyze 60S ribosomal subunit joining (33,37) may be a nearly universal feature of eukaryotic translation initiation. Interestingly, the conformation of the eIF3 octameric core closely resembles the proteasome lid subcomplex once it is bound to the 26S proteasome holoenzyme, which causes the N-terminus of Rpn5 to adopt an extended conformation, similar to the conformation seen here for eIF3a (38). The a* and c* arms are the most mobile elements in the eIF3 core, and correspond to the eIF3 regions in closest proximity to the mRNA binding cleft of the 40S subunit (14,28). The flexibility of these arms in eIF3 may be important for their function in translation initiation complex assembly. Based on its location in eIF3, the eIF3c HLH motif would be positioned near the head domain of the 40S ribosomal subunit (14,28). Contacts between the eIF3c HLH domain and the head of the 40S subunit, which is thought to adopt a closed conformation after start codon selection (11,39), may serve to stimulate eIF2 exchange with eIF5B after start codon recognition (40).


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)

The roles of the HLH motifs in eIF3 subunits a and c subunits in translation initiation complex formation. (A) Mutations in the HLH motif of eIF3a greatly weaken eIF3 binding to the HCV IRES and 40S ribosomal subunit, whereas mutations in the HLH motif of eIF3c primarily affect eIF3 binding to the 40S ribosomal subunit. (B) Mutations in the HLH motif of eIF3a disrupt eIF2-mediated Met-tRNAi loading into preinitiation complexes, whereas mutations in the eIF3c HLH motif impair eIF5B exchange with eIF2 after start codon recognition.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt510-F6: The roles of the HLH motifs in eIF3 subunits a and c subunits in translation initiation complex formation. (A) Mutations in the HLH motif of eIF3a greatly weaken eIF3 binding to the HCV IRES and 40S ribosomal subunit, whereas mutations in the HLH motif of eIF3c primarily affect eIF3 binding to the 40S ribosomal subunit. (B) Mutations in the HLH motif of eIF3a disrupt eIF2-mediated Met-tRNAi loading into preinitiation complexes, whereas mutations in the eIF3c HLH motif impair eIF5B exchange with eIF2 after start codon recognition.
Mentions: Based on the experiments in translation extracts, the HLH motif in eIF3a serves as a lynchpin in assembling translation initiation complexes on the HCV IRES. The HLH motif in eIF3a contributes to early steps of HCV IRES-mediated translation initiation dependent on eIF2, before start codon selection, whereas the eIF3c HLH motif is important for later events dependent on eIF5B, after start codon selection but before 60S subunit joining (Figure 6). Intriguingly, the HLH motif in eIF3a is highly conserved across metazoans and is present in most eukaryotes with available genome sequences (Figure 1C, Supplementary Figure S2D), suggesting that this motif may play a role in loading of the eIF2–GTP–Met-tRNAi ternary complex into translation preinitiation complexes on cellular mRNAs as well as the HCV genomic RNA. The HLH motif in eIF3c is highly conserved in all eukaryotes (Figure 1C), implying that its role in eIF5B exchange with eIF2 after start codon selection to catalyze 60S ribosomal subunit joining (33,37) may be a nearly universal feature of eukaryotic translation initiation. Interestingly, the conformation of the eIF3 octameric core closely resembles the proteasome lid subcomplex once it is bound to the 26S proteasome holoenzyme, which causes the N-terminus of Rpn5 to adopt an extended conformation, similar to the conformation seen here for eIF3a (38). The a* and c* arms are the most mobile elements in the eIF3 core, and correspond to the eIF3 regions in closest proximity to the mRNA binding cleft of the 40S subunit (14,28). The flexibility of these arms in eIF3 may be important for their function in translation initiation complex assembly. Based on its location in eIF3, the eIF3c HLH motif would be positioned near the head domain of the 40S ribosomal subunit (14,28). Contacts between the eIF3c HLH domain and the head of the 40S subunit, which is thought to adopt a closed conformation after start codon selection (11,39), may serve to stimulate eIF2 exchange with eIF5B after start codon recognition (40).

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