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Structural analysis of an eIF3 subcomplex reveals conserved interactions required for a stable and proper translation pre-initiation complex assembly.

Herrmannová A, Daujotyte D, Yang JC, Cuchalová L, Gorrec F, Wagner S, Dányi I, Lukavsky PJ, Valásek LS - Nucleic Acids Res. (2011)

Bottom Line: Mutating these interactions displays severe growth defects and eliminates association of eIF3i/TIF34 and strikingly also eIF3g/TIF35 with eIF3 and 40S subunits in vivo.Leaky scanning is also partially suppressed by eIF1, one of the key regulators of AUG recognition, and its mutant sui1(G107R) but the mechanism differs.We conclude that the C-terminus of eIF3b/PRT1 orchestrates co-operative recruitment of eIF3i/TIF34 and eIF3g/TIF35 to the 40S subunit for a stable and proper assembly of 48S pre-initiation complexes necessary for stringent AUG recognition on mRNAs.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Regulation of Gene Expression, Institute of Microbiology ASCR, v.v.i., Videnska 1083, Prague, 142 20, Czech Republic.

ABSTRACT
Translation initiation factor eIF3 acts as the key orchestrator of the canonical initiation pathway in eukaryotes, yet its structure is greatly unexplored. We report the 2.2 Å resolution crystal structure of the complex between the yeast seven-bladed β-propeller eIF3i/TIF34 and a C-terminal α-helix of eIF3b/PRT1, which reveals universally conserved interactions. Mutating these interactions displays severe growth defects and eliminates association of eIF3i/TIF34 and strikingly also eIF3g/TIF35 with eIF3 and 40S subunits in vivo. Unexpectedly, 40S-association of the remaining eIF3 subcomplex and eIF5 is likewise destabilized resulting in formation of aberrant pre-initiation complexes (PICs) containing eIF2 and eIF1, which critically compromises scanning arrest on mRNA at its AUG start codon suggesting that the contacts between mRNA and ribosomal decoding site are impaired. Remarkably, overexpression of eIF3g/TIF35 suppresses the leaky scanning and growth defects most probably by preventing these aberrant PICs to form. Leaky scanning is also partially suppressed by eIF1, one of the key regulators of AUG recognition, and its mutant sui1(G107R) but the mechanism differs. We conclude that the C-terminus of eIF3b/PRT1 orchestrates co-operative recruitment of eIF3i/TIF34 and eIF3g/TIF35 to the 40S subunit for a stable and proper assembly of 48S pre-initiation complexes necessary for stringent AUG recognition on mRNAs.

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Disrupting the b/PRT1–i/TIF34 interaction eliminates association of the i/TIF34-g/TIF35 mini-module from the MFC in vivo. (A and B) WCEs prepared from YAH06 (prt1Δ) bearing untagged b/PRT1 (lanes 1–3), 8xHis-tagged b/PRT1 (lanes 4–6), and two of its mutant derivatives (lanes 7–9 and 10–12) were incubated with Ni2+ agarose and the bound proteins were eluted and subjected to western blot analysis with the antibodies indicated in each row. (In) lanes contained 5% of the input WCEs; (E) lanes contained 100% of eluate from the resin; (FT) lanes contained 5% of the flow through. (B) The Western signals for indicated proteins in the E fractions of the wt PRT1-His and its mutants were quantified, normalized for the amounts of the wt b/PRT1 in these fractions and plotted in the histogram as percentages of the corresponding values calculated for the wt b/PRT1. (C and D) WCEs were prepared from YAH12 (prt1Δ tif34Δ) bearing untagged PRT1 and wt TIF34 (lanes 1–4) and from YAH11 (prt1Δ tif34Δ) bearing 8xHis-tagged PRT1 and either wt TIF34 plus empty vector (lanes 5–8) or mutant tif34-DD/KK plus empty vector (lanes 9–12) and analyzed analogously to (A and B).
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gkr765-F5: Disrupting the b/PRT1–i/TIF34 interaction eliminates association of the i/TIF34-g/TIF35 mini-module from the MFC in vivo. (A and B) WCEs prepared from YAH06 (prt1Δ) bearing untagged b/PRT1 (lanes 1–3), 8xHis-tagged b/PRT1 (lanes 4–6), and two of its mutant derivatives (lanes 7–9 and 10–12) were incubated with Ni2+ agarose and the bound proteins were eluted and subjected to western blot analysis with the antibodies indicated in each row. (In) lanes contained 5% of the input WCEs; (E) lanes contained 100% of eluate from the resin; (FT) lanes contained 5% of the flow through. (B) The Western signals for indicated proteins in the E fractions of the wt PRT1-His and its mutants were quantified, normalized for the amounts of the wt b/PRT1 in these fractions and plotted in the histogram as percentages of the corresponding values calculated for the wt b/PRT1. (C and D) WCEs were prepared from YAH12 (prt1Δ tif34Δ) bearing untagged PRT1 and wt TIF34 (lanes 1–4) and from YAH11 (prt1Δ tif34Δ) bearing 8xHis-tagged PRT1 and either wt TIF34 plus empty vector (lanes 5–8) or mutant tif34-DD/KK plus empty vector (lanes 9–12) and analyzed analogously to (A and B).

Mentions: Next we wished to demonstrate the effect of our mutations on the association of i/TIF34 with the rest of eIF3 in vivo. Towards this end we analyzed formation of the entire eIF3-containing MFC (see our model in Figure 4C) in yeast cells by Ni2+-chelation chromatography using His8-tagged PRT1 as bait. As reported previously (13), a fraction of a/TIF32, j/HCR1, eIF2, eIF5 and eIF1 co-purified specifically with wt b/PRT1-His but not with its untagged version (Figure 5A, lanes 4–6 versus 1–3). The prt1-W674A mutation of one of the two contacts between i/TIF34 and b/PRT1 severely diminishes (by >90%) association of i/TIF34, and in contrast to the above in vitro experiments, also that of g/TIF35 with the MFC, whereas prt1-W674F shows no effects (Figures 5A and B, lanes 7–9 versus 10–12). This concurs well with our genetic data (Figure 3A). In addition, the overall integrity of the MFC also seems to be modestly affected. Similarly, the tif34-DD/KK mutation of the other contact also severely reduces binding of i/TIF34 and g/TIF35 to the purified b/PRT1-His complex (Figures 5C and D, lanes 9–12 versus 5–8). Importantly, overexpressing His8-tagged TIF35 as bait in the background of tif34-DD/KK further supported these novel observations as g/TIF35-His practically failed to pull down any of the MFC components with the exception of i/TIF34 (Supplementary Figure S8A). The fact that the DD/KK mutation does not affect the mutual interaction between i/TIF34 and g/TIF35 in vivo is in perfect agreement with our in vitro binding data (Figure 4B). Together these results strongly suggest that binding of i/TIF34 with b/PRT1, in addition to the direct g/TIF35–b/PRT1 interaction, is a necessary prerequisite for stable eIF3-association of g/TIF35 in vivo indicating that the observed growth phenotypes are a direct consequence of the loss of the essential i/TIF34–g/TIF35 mini-module from the rest of eIF3. Hence the fact that the individual prt1-Y677A, prt1-R678A, tif34-D207K and tif34-D224K mutations diminish the b/PRT1–i/TIF34 interaction in vitro (Figure 4) yet produce no significant growth phenotypes (Figure 3) can be explained by proposing that their impact in vivo is largely overcome by a stabilization effect of simultaneous binding of g/TIF35 to i/TIF34 and b/PRT1 in the context of the entire eIF3 complex, as was observed (Supplementary Figure S8B and C). In other words, their aforementioned redundancy is dependent on the presence of g/TIF35 in vivo. Indeed, this effect is not powerful enough in the case of more deleterious double mutations. Since we had confirmation that genetic and molecular defects of both prt1-W674A and tif34-DD/KK mutations have the same nature, we decided to focus our further analysis on the latter.Figure 5.


Structural analysis of an eIF3 subcomplex reveals conserved interactions required for a stable and proper translation pre-initiation complex assembly.

Herrmannová A, Daujotyte D, Yang JC, Cuchalová L, Gorrec F, Wagner S, Dányi I, Lukavsky PJ, Valásek LS - Nucleic Acids Res. (2011)

Disrupting the b/PRT1–i/TIF34 interaction eliminates association of the i/TIF34-g/TIF35 mini-module from the MFC in vivo. (A and B) WCEs prepared from YAH06 (prt1Δ) bearing untagged b/PRT1 (lanes 1–3), 8xHis-tagged b/PRT1 (lanes 4–6), and two of its mutant derivatives (lanes 7–9 and 10–12) were incubated with Ni2+ agarose and the bound proteins were eluted and subjected to western blot analysis with the antibodies indicated in each row. (In) lanes contained 5% of the input WCEs; (E) lanes contained 100% of eluate from the resin; (FT) lanes contained 5% of the flow through. (B) The Western signals for indicated proteins in the E fractions of the wt PRT1-His and its mutants were quantified, normalized for the amounts of the wt b/PRT1 in these fractions and plotted in the histogram as percentages of the corresponding values calculated for the wt b/PRT1. (C and D) WCEs were prepared from YAH12 (prt1Δ tif34Δ) bearing untagged PRT1 and wt TIF34 (lanes 1–4) and from YAH11 (prt1Δ tif34Δ) bearing 8xHis-tagged PRT1 and either wt TIF34 plus empty vector (lanes 5–8) or mutant tif34-DD/KK plus empty vector (lanes 9–12) and analyzed analogously to (A and B).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3300007&req=5

gkr765-F5: Disrupting the b/PRT1–i/TIF34 interaction eliminates association of the i/TIF34-g/TIF35 mini-module from the MFC in vivo. (A and B) WCEs prepared from YAH06 (prt1Δ) bearing untagged b/PRT1 (lanes 1–3), 8xHis-tagged b/PRT1 (lanes 4–6), and two of its mutant derivatives (lanes 7–9 and 10–12) were incubated with Ni2+ agarose and the bound proteins were eluted and subjected to western blot analysis with the antibodies indicated in each row. (In) lanes contained 5% of the input WCEs; (E) lanes contained 100% of eluate from the resin; (FT) lanes contained 5% of the flow through. (B) The Western signals for indicated proteins in the E fractions of the wt PRT1-His and its mutants were quantified, normalized for the amounts of the wt b/PRT1 in these fractions and plotted in the histogram as percentages of the corresponding values calculated for the wt b/PRT1. (C and D) WCEs were prepared from YAH12 (prt1Δ tif34Δ) bearing untagged PRT1 and wt TIF34 (lanes 1–4) and from YAH11 (prt1Δ tif34Δ) bearing 8xHis-tagged PRT1 and either wt TIF34 plus empty vector (lanes 5–8) or mutant tif34-DD/KK plus empty vector (lanes 9–12) and analyzed analogously to (A and B).
Mentions: Next we wished to demonstrate the effect of our mutations on the association of i/TIF34 with the rest of eIF3 in vivo. Towards this end we analyzed formation of the entire eIF3-containing MFC (see our model in Figure 4C) in yeast cells by Ni2+-chelation chromatography using His8-tagged PRT1 as bait. As reported previously (13), a fraction of a/TIF32, j/HCR1, eIF2, eIF5 and eIF1 co-purified specifically with wt b/PRT1-His but not with its untagged version (Figure 5A, lanes 4–6 versus 1–3). The prt1-W674A mutation of one of the two contacts between i/TIF34 and b/PRT1 severely diminishes (by >90%) association of i/TIF34, and in contrast to the above in vitro experiments, also that of g/TIF35 with the MFC, whereas prt1-W674F shows no effects (Figures 5A and B, lanes 7–9 versus 10–12). This concurs well with our genetic data (Figure 3A). In addition, the overall integrity of the MFC also seems to be modestly affected. Similarly, the tif34-DD/KK mutation of the other contact also severely reduces binding of i/TIF34 and g/TIF35 to the purified b/PRT1-His complex (Figures 5C and D, lanes 9–12 versus 5–8). Importantly, overexpressing His8-tagged TIF35 as bait in the background of tif34-DD/KK further supported these novel observations as g/TIF35-His practically failed to pull down any of the MFC components with the exception of i/TIF34 (Supplementary Figure S8A). The fact that the DD/KK mutation does not affect the mutual interaction between i/TIF34 and g/TIF35 in vivo is in perfect agreement with our in vitro binding data (Figure 4B). Together these results strongly suggest that binding of i/TIF34 with b/PRT1, in addition to the direct g/TIF35–b/PRT1 interaction, is a necessary prerequisite for stable eIF3-association of g/TIF35 in vivo indicating that the observed growth phenotypes are a direct consequence of the loss of the essential i/TIF34–g/TIF35 mini-module from the rest of eIF3. Hence the fact that the individual prt1-Y677A, prt1-R678A, tif34-D207K and tif34-D224K mutations diminish the b/PRT1–i/TIF34 interaction in vitro (Figure 4) yet produce no significant growth phenotypes (Figure 3) can be explained by proposing that their impact in vivo is largely overcome by a stabilization effect of simultaneous binding of g/TIF35 to i/TIF34 and b/PRT1 in the context of the entire eIF3 complex, as was observed (Supplementary Figure S8B and C). In other words, their aforementioned redundancy is dependent on the presence of g/TIF35 in vivo. Indeed, this effect is not powerful enough in the case of more deleterious double mutations. Since we had confirmation that genetic and molecular defects of both prt1-W674A and tif34-DD/KK mutations have the same nature, we decided to focus our further analysis on the latter.Figure 5.

Bottom Line: Mutating these interactions displays severe growth defects and eliminates association of eIF3i/TIF34 and strikingly also eIF3g/TIF35 with eIF3 and 40S subunits in vivo.Leaky scanning is also partially suppressed by eIF1, one of the key regulators of AUG recognition, and its mutant sui1(G107R) but the mechanism differs.We conclude that the C-terminus of eIF3b/PRT1 orchestrates co-operative recruitment of eIF3i/TIF34 and eIF3g/TIF35 to the 40S subunit for a stable and proper assembly of 48S pre-initiation complexes necessary for stringent AUG recognition on mRNAs.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Regulation of Gene Expression, Institute of Microbiology ASCR, v.v.i., Videnska 1083, Prague, 142 20, Czech Republic.

ABSTRACT
Translation initiation factor eIF3 acts as the key orchestrator of the canonical initiation pathway in eukaryotes, yet its structure is greatly unexplored. We report the 2.2 Å resolution crystal structure of the complex between the yeast seven-bladed β-propeller eIF3i/TIF34 and a C-terminal α-helix of eIF3b/PRT1, which reveals universally conserved interactions. Mutating these interactions displays severe growth defects and eliminates association of eIF3i/TIF34 and strikingly also eIF3g/TIF35 with eIF3 and 40S subunits in vivo. Unexpectedly, 40S-association of the remaining eIF3 subcomplex and eIF5 is likewise destabilized resulting in formation of aberrant pre-initiation complexes (PICs) containing eIF2 and eIF1, which critically compromises scanning arrest on mRNA at its AUG start codon suggesting that the contacts between mRNA and ribosomal decoding site are impaired. Remarkably, overexpression of eIF3g/TIF35 suppresses the leaky scanning and growth defects most probably by preventing these aberrant PICs to form. Leaky scanning is also partially suppressed by eIF1, one of the key regulators of AUG recognition, and its mutant sui1(G107R) but the mechanism differs. We conclude that the C-terminus of eIF3b/PRT1 orchestrates co-operative recruitment of eIF3i/TIF34 and eIF3g/TIF35 to the 40S subunit for a stable and proper assembly of 48S pre-initiation complexes necessary for stringent AUG recognition on mRNAs.

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Related in: MedlinePlus