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. 2013 Nov 8;8(11):e78715.
doi: 10.1371/journal.pone.0078715. eCollection 2013.

Human-like eukaryotic translation initiation factor 3 from Neurospora crassa

M Duane Smith  1 , Yu Gu, Jordi Querol-Audí, Jacob M Vogan, Adam Nitido, Jamie H D Cate
Affiliations

Human-like eukaryotic translation initiation factor 3 from Neurospora crassa

M Duane Smith et al. PLoS One. .

Abstract

Eukaryotic translation initiation factor 3 (eIF3) is a key regulator of translation initiation, but its in vivo assembly and molecular functions remain unclear. Here we show that eIF3 from Neurospora crassa is structurally and compositionally similar to human eIF3. N. crassa eIF3 forms a stable 12-subunit complex linked genetically and biochemically to the 13(th) subunit, eIF3j, which in humans modulates mRNA start codon selection. Based on N. crassa genetic analysis, most subunits in eIF3 are essential. Subunits that can be deleted (e, h, k and l) map to the right side of the eIF3 complex, suggesting that they may coordinately regulate eIF3 function. Consistent with this model, subunits eIF3k and eIF3l are incorporated into the eIF3 complex as a pair, and their insertion depends on the presence of subunit eIF3h, a key regulator of vertebrate development. Comparisons to other eIF3 complexes suggest that eIF3 assembles around an eIF3a and eIF3c dimer, which may explain the coordinated regulation of human eIF3 levels. Taken together, these results show that Neurospora crassa eIF3 provides a tractable system for probing the structure and function of human-like eIF3 in the context of living cells.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The stoichiometric subunit composition of eIF3 varies across species.
Cladogram constructed using sequences of 18S rRNA from the listed organisms. The subunit composition of eIF3 from each organism is depicted using spherical models. Subunit count in the stoichiometric complex of the displayed organisms is as follows: H. sapiens, D. rerio, M. musculus, C. elegans, D. melanogaster, N. crassa, D. discoideum and A. niger (12); A. thaliana (11); S. pombe (Csn7b, left, or Int6, right, complexes) (8 each); T. brucei (8); S. cerevisiae (5), The size of the spheres used to depict eIF3 subunits are relative to their respective molecular weights. The tree was constructed using tools at www.phylogeny.fr .
Figure 2
Figure 2. Viable Nc eIF3 knock-out strains display defects in conidiation and linear growth.
(A) Growth of Nc eIF3 single and double knock-out (KO) strains on Vogel’s minimal media (MM) with 2% sucrose. Plates were spotted from frozen stocks of the indicated eIF3 subunit single or double knock-out strain, grown in the dark and photographed on the indicated days. (B) Linear growth of Nc eIF3 knock-out strains on Vogel’s MM with 2% sucrose (black bars) or water agar (white bars). In all graphs linear growth is plotted as a fraction of wild-type growth on comparable media. Linear growth of wild-type (WT) on water agar as a fraction of MM with 2% sucrose is 0.63 (Table S2 in File S1). Linear growth of eIF3 single knock-out strains is compared to WT, where the eIF3 subunit knock-out is indicated below the bars (graph eIF3 KO). Linear growth of eIF3 knock-out compared to wild-type and the knock-out strain with an N-terminally tagged (N) or C-terminally tagged (C) recombinant eIF3 subunit at the his-3 locus (graphs eIF3h, j, k, l). The recombinant eIF3 subunit and corresponding KO strain is indicated below each graph. (C) Linear growth of eIF3 double knock-out strains compared to the corresponding single knock-outs (white bars) on Vogel’s MM with 2% sucrose and 2% agar or Vogel’s MM with 2% agar (water agar). Black bars represent the calculated multiplicative effect from both single knock-outs. Each graph has a broken vertical axis to visually emphasize the difference in linear growth rates. Error bars indicate the standard error on the mean. Numerical values for linear growth and errors are in Table S2 in File S1.
Figure 3
Figure 3. Composition and structure of eIF3 assemblies.
(A) Coomassie stained purified N. crassa eIF3 with C-terminally tagged eIF3l. Subunits identified by tandem mass spectrometry are labelled. Subunits f and h co-migrate on the gel. The tagged subunit is indicated with an asterisk. (B) 2-D EM class averages of negatively stained N. crassa eIF3 dodecamer compared with the equivalent view of the human eIF3 octamer. The 3-D cryo-EM reconstruction of the human eIF3 octameric core is shown for reference, with labeled features and subunits (adapted from [11]). (C) Coomassie stained affinity purifications of FLAG tagged Neurospora eIF3j (C- or N-terminally tagged). The major bands are Nc eIF3j. Minor bands are interacting proteins (Table S3 in File S1).
Figure 4
Figure 4. Affinity purification of eIF3 knock-out strains reveal subunit interdependence during eIF3 assembly.
(A) Anti-FLAG affinity purifications from N. crassa extracts using N-terminally tagged eIF3l or eIF3k in the ΔeIF3kl double knock-out background (lanes 1 and 2). Affinity purification from N. crassa extract obtained from a heterokaryon strain (lane 3) made from strains used in lanes 1 and 2. N-terminally tagged k and l subunits expressed from the his-3 locus are indicated above each lane. All strains are ΔeIF3kl double knock-outs. Contaminant bands in lanes 1 and 2 were identified as phenylalanyl tRNA synthetase beta chain (73 kDa) and tRNA ligase (58 kDa). (B) Anti-FLAG Western blot of the affinity purifications from the gel in (A). Each lane has the exact amount of total protein loaded in (A). Anti-FLAG affinity purifications from N. crassa extracts using N-terminally tagged eIF3h in ΔeIF3h or ΔeIF3hl strains (C) or N-terminally tagged eIF3k in ΔeIF3k or ΔeIF3hk strains (D). The tagged h or k subunits are indicated with asterisks. Arrows indicate the missing k and l subunits in the ΔeIF3hl strain (C) or a degradation product of eIF3a (D). All gels in panels (A), (C) and (D) are stained with Coomassie blue.
Figure 5
Figure 5. Redefining the core of eIF3 and proposed models for assembly.
(A) Venn diagram highlighting common subunits among three definitions of the eIF3 core complex: 1) the phylogenetically conserved complex from S. cerevisiae, 2) the reconstituted human PCI-MPN core complex and 3) the biologically essential subunits from N. crassa eIF3. (B) Proposed models for human-like eIF3 assembly. Individual subunits or sub-complexes assemble onto an ac dimer. Solid and dashed arrows represent assembly and alternative assembly interactions and are drawn to reconcile the compositional breadth of eIF3 sub-complexes across species as well as biochemical data , .
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