Structural insights into the recruitment of viral type 2 IRES to ribosomal preinitiation complex for protein synthesis
- Department of Developmental Biology and Genetics, Indian Institute of Science, India
Figures
Figure 1 with 2 supplements
Features of EMCV IRES-48S PIC.
(A) Different views of encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES)-48S preinitiation complex (PIC) (Map B1) by 45° rotation along one axis. The map shows densities assigned to 40S ribosome, RNA (in channel), ternary complex, and IRES density at the inter-subunit region of head. (B) View of Map B and zoomed view of densities corresponding to IRES and initiator tRNA (tRNAi).
Figure 1—figure supplement 1
Sample preparation and data processing.
(A) Isolation of encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES)-48S preinitiation complex (PIC) from Talon affinity chromatography. Talon affinity chromatography profile and elution fractions were analyzed and pelleted using 30% cushion on 1.5% agarose gel, followed by uranyl acetate staining of elution pellet and observation under transmission electron microscope at ×57k magnification Figure 1—figure supplement 1—source data 1 and 2. (B) Processing of cryo-electron microscopy (cryo-EM) data and features of obtained maps using CryoSPARCv4.3.0 and GSFSC resolution estimates for different maps obtained.
-
Figure 1—figure supplement 1—source data 1
Original file of the full raw uncropped gels or blots.
Isolation of encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES)-48S preinitiation complex (PIC) from Talon affinity chromatography, including Talon affinity chromatography profile.
- https://cdn.elifesciences.org/articles/107788/elife-107788-fig1-figsupp1-data1-v1.zip
-
Figure 1—figure supplement 1—source data 2
Original file of the full raw uncropped gels or blots.
Analysis of elution fractions – pelleted using 30% cushion on 1.5% agarose gel.
- https://cdn.elifesciences.org/articles/107788/elife-107788-fig1-figsupp1-data2-v1.zip
Figure 1—figure supplement 2
Local resolution of map and unresolved extra densities.
(A) Local resolution estimation of Map B. (Right) Surface view; (right-top) zoomed view on internal ribosome entry site (IRES) density; (left-bottom) cross-section of Map B to show resolution at the core of the ribosome. (B) Local resolution estimation of Map B1. (Right) Surface view; (left-bottom) cross-section of Map B1 to show resolution at the core of ribosome. (C) Extra density at the mRNA entry site of Map B/B1. Ribosomal proteins and RNA in contact with the unassigned extra density. (D) Superimposition of encephalomyocarditis virus (EMCV) IRES-48S preinitiation complex (PIC) on human 48S PIC (PDB Id – 8OZ0) shows the extra density coincides with the location of eIF3g-RRM and downstream mRNA. (E) Extra density at the mRNA entry site of Map A. Ribosomal proteins and RNA in contact with the unassigned extra density.
Figure 2 with 1 supplement
Closed conformation of EMCV IRES-48S PIC.
(A) Fitting of initiator tRNA (tRNAi)-base paired to start codon AUG (left). Zoomed view of codon-anticodon interaction (middle), and B-factor for codon-anticodon interaction (right). (B) The tRNAi in encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES)-48S preinitiation complex (PIC) is in PIN state similar to that in human 48S PIC PIN state (PDB Id – 7QP7) and its anticodon shows ~7 Å shift as compared to human 48S PIC open state (PDB Id – 7QP6). (C) Comparison of ribosomal conformation (18S rRNA) of EMCV IRES 48S PIC with human open and closed state (PDB Id – 7QP6 and 7QP7, respectively). Focusing on the entry site and the helices governing latch conformation, helix 34 moves toward helix 18 by 9 Å. (D) Movement of eS17 in open and closed states of ribosome. Zoomed view of comparison between eS17 in EMCV IRES 48S PIC and human 48S PIC open state, showing an upward shift of the N-terminal domain by ~10 Å.
Figure 2—figure supplement 1
Fitting of model into map.
(A) From Map B to model (encephalomyocarditis virus [EMCV] internal ribosome entry sites [IRES]-48S preinitiation complex [PIC]). Obtained model showing 40S ribosome, RNA in channel, initiator tRNA, IRES domain. (Right) Fourier shell correlation (FSC) of map to model fit. (B) From Map B1 to model (EMCV IRES-48S PIC). Obtained model showing 40S ribosome, RNA in channel, initiator tRNA, IRES domain, eIF2αγ. (Right) FSC of map to model fit. (C) From Map A to model by fitting of 40S ribosome without any factors. (Right) FSC of map to model fit. (D) The sequence of EMCV IRES mRNA in the channel is provided. Residues in orange colour have no density in the obtained Map B. (E) Comparing the state of 18S rRNA in Map A and Map B. (F) eS17-NTD position in 40S ribosome. (Right) Fitting of eS17 in EMCV IRES-48S PIC map.
Figure 3 with 2 supplements
Deciphering the IRES density.
(A) Extra density connecting the head of 40S to initiator tRNA (tRNAi) elbow region is contributed by encephalomyocarditis virus (EMCV) internal ribosome entry sites (IRES) RNA in Map B1. Rotated views of RNA density showing its connection to the 40S head via uS13 and uS19 and to tRNAi via its elbow region. (B) Organisation of EMCV IRES domains from D to L, where H-L makes the functional IRES moiety (Hellen and Wimmer, 1995). (C) Deciphering the architecture of the obtained IRES density. The density could be interpreted as a long main stem (S1) extending away from the ribosome, where the base is anchored to the ribosome by two branches (B1 and B2) and to tRNAi by one branch (B3), further divided into two sub-branches (B3a and B3b). (D) Secondary structure of apical region of domain I (made using RNAfold) marking the stem and branches, along with imported loops. (E) Fitting of domain I apex in the density (rotated views). The sub-domains are coloured as proposed in C,D.
Figure 3—figure supplement 1
Model building of domain I apex.
(A) Process of fitting domain I apex from AlphaFold3 generated model. (B) Correlation coefficient per residue in domain I apex for fit in the density map. (C) B-factor for each residue in the final model. The range is depicted in the colour key where blue is low and red is high.
Figure 3—figure supplement 2
Fitting of AlphaFold3 predicted models of individual IRES domains in the extra density.
(A–C) AlphaFold3 predicted tertiary structures of individual domains in encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) and their corresponding predicted aligned error (PAE) plot: (A) domain D, (B) domain E, (C) domain F. (D) Fitting of predicted tertiary structure of domains D, E, F in the experimental model of domain D-F (Dorn et al., 2023), showing potential correlation. (E–G) AlphaFold3 predicted tertiary structures of domain G (E), domain H (F), and domain I, and zoomed view of domain I apex showing the architecture of the apical region (G) and its corresponding PAE plot. (H) AlphaFold3 predicted tertiary structures of domain J-K, its PAE plot, and fitting of predicted structure in the cryo-EM model of domain J-K (PDB Id – 8HUJ). (I) AlphaFold3 predicted tertiary structures of domain L and its PAE plot. (J) Colour scheme describing the expected position error of the above-mentioned predictions. (K) Possibility of fitting domain D-F model (as determined in Dorn et al., 2023) in the IRES density connecting the 40S head to tRNA in EMCV IRES-48S preinitiation complex (PIC). (L) Possibility of fitting domain H in the IRES density. (M) Possibility of fitting domain J-K (PDB Id – 8HUJ) in density. The domain J-K is way too long to fit in map and does not account for certain region. The extending loops would clash with 40S ribosome and initiator tRNA (tRNAi).
Figure 4 with 1 supplement
EMCV IRES domain I apex contacts uS13, uS19 and tRNAi.
(A) Model showing connections of domain I apex with uS13, uS19, and initiator tRNA (tRNAi). (B) uS13 interacts with B3 branch or sub-domain of internal ribosome entry site (IRES) via its alpha helix (100–117 residues). (C) Multiple points of contacts between uS19 and domain I motifs: RAAA and AAG. The electrostatic potential map of uS19 suggests that encephalomyocarditis virus (EMCV) IRES interacts via ionic interactions with its phosphate backbone. (D) Sequence alignment of h38 with domain I apex of EMCV IRES showing sequence identity. Overlapping of uS19 from 80S (PDB Id – 4UG0) and EMCV IRES-48S preinitiation complex (PIC) show that the interaction of uS19 with EMCV IRES is similar to interaction with that of h38. (E) The fit of GNRA or GCGA stem and its contact with tRNAi at the elbow region and acceptor stem.
Figure 4—figure supplement 1
Importance of EMCV IRES domain I apex motifs.
(A) Luciferase reporter assay showing the translation efficiency of wild-type (WT) encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) with IRES mutants – GNRAm and RAAAm. The error bars represent the standard deviations of the three biological replicates. The value for each biological replicate was determined as a mean of three technical replicates. Student’s unpaired two-tailed t-test was used for statistical analysis. (B) Interaction of uS19 and uS13 in 40S subunit with 60S subunit, involving helix 38 and uL5 to form inter-subunit bridges – B1a and B1b/c – respectively in 80S (PDB Id – 4UG0). (C) Estimation of clash with IRES domain I during 60S joining to form 80S-elongation competent complex (PDB Id – 4UG0). uL5 and helix 38 of 28S rRNA would clash with the IRES I domain. (Left) Superimposition of 80S ribosome on EMCV IRES-48S preinitiation complex (PIC). (D) Reported GNRA (GUGA) interaction with the minor groove formed by C-G stem in bacterial RNaseP (PDB Id – 3Q1Q).
Figure 5
State of ternary complex in EMCV IRES-48S PIC.
(A) Inter-subunit view of encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) 48S preinitiation complex (PIC) showing position for ternary complex on map. Fitting of eIF2α and eIF2γ in its corresponding density in Map B1. (B) Overlapping EMCV IRES 48S PIC on mammalian late-stage 48S PIC (PDB Id – 6YAN) indicates a shift in position of eIF2γ towards 40S head. (C) Zoomed view showing the positions of eIF2γ, initiator tRNA (tRNAi), and eIF2α in the EMCV IRES 48S PIC relative to those in the mammalian late-stage 48S PIC (PDB ID 6YAN). The black arrow indicates the shift in position. (D) Superimposition of the EMCV IRES-48S PIC on the human late-stage 48S PIC (before 60S joining; PDB Id – 8PJ5), showing the conformation of tRNAi in association with eIF2 and IRES and tRNAi with eIF5B in the canonical context, respectively. (Right) Zoomed view of tRNAi conformation in both complexes.
Figure 6 with 1 supplement
Summarising the known interactions of EMCV IRES domains in context to 48S PIC.
(A) Encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) secondary structure depicting the role of binding partners for each domain in context to translation initiation. The known tertiary structure of EMCV IRES and its binding partners are depicted. The domains responsible for binding PTB1 are boxed. (B) Conservation of domain I apex sequence and secondary structure across type 2 IRES family. (C) Comparison of secondary structure of EMCV and foot and mouth disease virus (FMDV) IRES to that of polioviral IRES (type 1).
Figure 6—video 1
The movie depicts the position of encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) (brown) on the 40S ribosomal subunit, in contact with initiator tRNA (tRNAi) (green) in a 48S preinitiation complex (PIC) state.
On zooming, the IRES domain I apical part is contacting the ribosomal proteins uS13 (golden yellow) and uS19 (violet), as well as the elbow and acceptor arm of tRNAi, which is base-paired to start codon.
Author response image 1
The selected 2D classes and the rejected 2D classes from initial round of classification, and the final selected 2D classes, which were subjected to Ab-initio reconstruction to get the good ribosome particles.
Author response image 2
Reprocessing of the entire dataset using Relion5 for polishing of selected particles, followed by 3D classification and refinements in cryoSPARC.
Author response image 3
Prediction of tertiary structure of EMCV IRES (280-905 nucleotides) and zoomed features for each domain present in the IRES.
The predicted aligned error plot for the RNA structure is shown.
Author response image 4
Data processing- Map B particles were 2D classified, and further junk was cleared as rejected particles.
The selected particles were refined using non-uniform refinement to get Map B11, and later, a focused mask circling the head-tRNA-IRES region was used for local refinement in the region to yield map B111.
Author response image 5
Comparison of local resolution across head-IRES-tRNA in map B1 (as reported in the manuscript) and Map B111.
Author response image 6
(left) Fitting of eIF2α model in the map. (right) Fitting of Cα backbone of eIF2α and mRNA in the map.
Author response image 7
(upper left) Location of eIF4G-eIF4A in canonical human 48S PIC (PDB Id- 8OZ0). (upper right) Superimposition of 18S rRNA from human 48S and EMCV IRES 48S. (lower left) Superimposition of Human Closed 48S PIC structure (PDB Id- 8OZ0) on EMCV IRES-48S PIC model and placement of EMCV IRES- J-K domain-HEAT1-eIF4A structure (PDB Id- 8HUJ) with respect to eIF4G-HEAT1 domain. (lower right) Predicting location of eIF3 and eIF4 proteins in EMCV IRES-48S PIC.
Author response image 8
(left) The shortest distance between the last fitted residue- 825th of EMCV IRES to 785th of J-K domain of IRES (keeping eIF4G position same as that of PDB Id- 8OZ0) is 173 Å. (right) Tracing the path of mRNA (red) upstream of AUG coming out of the exit site of 40S ribosome and the possible position of eIF4G on EMCV IRES-48S PIC. Addition of nucleotides between C-785 and G-825 would fill the gap. The route of predicted mRNA from the exit channel is based on the mRNA (green) exiting the channel (PDB Id- 8OZ0).
Author response image 9
Rotated views of EMCV IRES domains- I apical part in contact with 40S head and tRNAi and predicted location of J-K domain in contact with eIF4G, close to the left foot of 40S (predicted from PDB Id- 8OZ0).
The minimum distance connecting 601st nucleotide in I domain to 682nd nucleotide in J-K domain is 295.5 Å.
Tables
Table 1
Cryo-electron microscopy (cryo-EM) data and model statistics.
| Data collection | Map A – 40S without factors | Map B – 40S-tRNAi-EMCV IRES | Map B1 – 40S-tRNAi-EMCV IRES-eIF2α-eIF2γ |
|---|---|---|---|
| Microscope | Talos Arctica | ||
| Camera | Gatan K2 Summit Direct Detector | ||
| Magnification | ×36,000 | ||
| Voltage (kV) | 200 | ||
| Electron dose (e-/Å2) | 50–55 | ||
| Defocus range | –2 to –0.5 | ||
| Pixel size (Å) | 1.17 | ||
| Number of micrographs | 22549 | ||
| Cryo-EM reconstruction | |||
| Final number of particles | 125,503 | 55,231 | 28,439 |
| Point group symmetry | C1 | C1 | C1 |
| FSC threshold | 0.143 | 0.143 | 0.143 |
| Map overall resolution (Å) | 4.51 | 4.55 | 5.01 |
| Resolution metric | Gold-standard FSC | Gold-standard FSC | Gold-standard FSC |
| Sharpening B factor(Å2) | –153.5 | –117.9 | –122.3 |
| Atomic model refinement | |||
| Resolution (0.5) (Å) | 5.0 | 5.4 | 6.0 |
| D FSC model (0/0.143/0.5) | 4.4/4.5/5.0 | 4.4/4.5/5.4 | 4.9/4.9/6.0 |
| Initial models used | 6YAN | 6YAN; 8OZ0 | 6YAN; 8OZ0 |
| CC overall | |||
| CC(mask)/(box)/(peaks)/(volume) | 0.73/0.83/0.72/0.66 | 0.77/0.84/0.71/0.77 | 0.78/0.84/0.72/0.78 |
| Molprobity score | 2.3 | 2.28 | 2.23 |
| Clash score | 19.08 | 19.09 | 18.64 |
| No. of atoms/No. of residues | |||
| Chains | 39 | 41 | 39 |
| Total atoms (Hydrogens:0) | 76455 | 80057 | 83717 |
| No. of residues – proteins/nucleotides | 4916/1744 | 4918/1912 | 5660/1912 |
| Bond (RMSD) Lengths (Å) | 0.002 | 0.003 | 0.003 |
| Bond (RMSD) Angles (⁰) | 0.658 | 0.650 | 0.659 |
| Ramachandran plot (%) | |||
| Outliers | 0.21 | 0.27 | 0.20 |
| Allowed | 8.94 | 8.21 | 7.09 |
| Favoured | 90.86 | 91.52 | 92.72 |
| Rotamer outliers (%) | 0.02 | 0.02 | 0.00 |
| Cβ outliers (%) | 0.00 | 0.00 | 0.00 |
| CaBLAM outliers (%) | 6.09 | 5.82 | 5.47 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Homo sapiens) | PTB1 | Nucleotide database | NM_002819.5 | 3C protease site (LEVLFQGP) was inserted at the N-terminal and inserted into pET28a in between BamHI and HindIII restriction sites, retaining the N-terminal 6X Histidine tag |
| Gene (Human Rhinovirus) | HRV_3C protease | Protein database | 1CQQ_A | |
| Gene (Encephalomyocarditis virus) | EMCV IRES | Nucleotide database | NC_001479.1 | 280–905 nucleotides |
| Gene (Photinus pyralis) | LUCIFERASE | Snapgene | pGL3-basic | Firefly luciferase |
| Recombinant DNA reagent | pcDNA3.1 (Plasmid) | Snapgene | pcDNA3.1 | Vector |
| Recombinant DNA reagent | pET28a (Plasmid) | Snapgene | pET28a | Vector |
| Strain, strain background (Escherichia coli) | BL21 (DE3) | Recombinant protein expression strain | Maintained in the lab | |
| Commercial assay, kit | RiboMAX Large Scale RNA Production Systems | Promega | P1280 | In vitro transcription kit |
| Commercial assay, kit | Rabbit Reticulocyte Lysate, Nuclease-Treated | Promega | L4960 | Lysate used for pull-down |
| Commercial assay, kit | Luciferase substrate- Steady-Glo Luciferase Assay System | Promega | E2510 |
Additional files
-
Supplementary file 1
AlphaFold3 input sequences and Oligo sequences.
- https://cdn.elifesciences.org/articles/107788/elife-107788-supp1-v1.docx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/107788/elife-107788-mdarchecklist1-v1.docx
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Structural insights into the recruitment of viral type 2 IRES to ribosomal preinitiation complex for protein synthesis
eLife 14:RP107788.
https://doi.org/10.7554/eLife.107788.3
