Fig 1: Localization and involvement of translation-related proteins at invadosomes. a Dose response of the number of rosettes per nuclei after translation inhibitor treatment. NIH-3T3-Src cells were treated with anisomycin or cycloheximide (CHX) for 24 h. Representatives images of cells treated the minimum concentration. The bar graph represents the number of rosettes per nuclei. Error bars (SEM, n = 20 fields, three independent experiments; ***P < 0.001 as compared with the non-treated cells as control, one-way ANOVA followed by Bonferroni test as compared with the siRNA control). Scale bar: 50 µm. b Confocal microscopy images of lifeact-mRuby (red)-expressing NIH-3T3-Src cells immunostained for eEF1A1 (green) or transfected with eEF2-GFP (green) or Caprin 1-myc (green). Left panels show in each channel in black and white. Right panels show merge images with enlarged views of the boxed regions. Scale bars: 10 µm (EEF1A1 and Caprin 1), 20 µm (eEF2). c Lifeact-mRuby (gray)-expressing NIH-3T3-Src cells were transfected with a siRNA control (siCtrl) or two independent siRNA targeting eEF2, eEF1A1, or Caprin 1 involved in translation activity. As controls the cells were treated with the Src inhibitor PP2 (5 µM). The upper panel shows representative images of the rosette number determined by the mask applied by the software (red areas). Scale bar: 5 µm. In the lower panel, the bar graph shows the number of rosettes per nuclei. The black bars represent the control conditions of the experiment. Error bars (SEM, n = 75 fields, three independent experiments; ns, not significant; *P < 0.05; **P < 0.005; ***P < 0.001 by one-way ANOVA followed by Bonferroni test as compared with the siRNA control). d NIH-3T3-Src cells transfected with a siRNA control (siCtrl) or two independent siRNA targeting eEF2, eEF1A1, or Caprin 1 were seeded on a fluorescent gelatin matrix. As a control, cells were treated with PP2 (5 µM). The upper panel shows representative images of the degraded area (black), insets on the bottom show the nuclei of the same field. Scale bar: 50 µm. In the lower panel, the bar graph shows the gelatin area degraded per cell after 24 h. Error bars (SEM, n = 30 fields, three independent experiments; ns, not significant, ***P < 0.001 as compared with the control siRNA, one-way ANOVA followed by Bonferroni test as compared to the siRNA control)
Fig 2: Isolation of Translational Decoding Complexes for Cryo-EM, Related to Figure 1(A) Schematic of the mRNA constructs used for in vitro translation and isolation of ribosome-nascent chain complexes (RNCs). The start codon (AUG), stop codon (UAG or UGA), and coding regions for the 3X Flag tag (green), the autonomously-folding villin headpiece (VHP) domain (blue), the cytosolic portion of Sec61β (orange), and KRas (purple) are indicated.(B) Experimental strategies for isolating the indicated RNCs from in vitro translation (IVT) reactions.(C) SDS-PAGE and Coomassie staining of isolated RNCs representing the elongation complex (80S⋅aa-tRNA⋅eEF1A); pre-accommodated (80S⋅eRF1⋅eRF3) or accommodated (80S⋅eRF1) termination complexes; and rescue complex (80S⋅Pelota⋅Hbs1l) reconstituted with a truncated mRNA (see panel A). Copurified, exogenously-added, and ribosomal (ribo. prot.) proteins are indicated.(D) The long NC construct (see panel A) was translated in vitro in rabbit reticulocyte lysate (RRL) with the indicated translational inhibitors added at the following concentrations: 50 μg/mL cycloheximide (CHX), 10 μM anisomycin, 200 μM emetine, and 50 μM didemnin B. The translation reactions were affinity purified via the 3X Flag tag on the nascent chain. The elutions and inputs were analyzed by SDS-PAGE and immunoblotting for the indicated proteins, revealing that didemnin B specifically traps eEF1A on the isolated RNCs.(E) The NC-stop construct was translated in vitro in RRL in the presence of 35S-methionine and mutant eRF1(AAQ) to trap RNCs with the UGA stop codon in the A site. The RNCs were isolated under high salt conditions and subjected to affinity purification via the 3X Flag tag on the nascent chain. The isolated RNCs were incubated with 1 mM puromycin or recombinant wild-type eRF1, wild-type eRF3, and 0.5 mM GMPPCP or GTP as indicated, and then directly analyzed by SDS-PAGE and autoradiography. The bands corresponding to ribosome-associated nascent chain-tRNA (NC-tRNA) and released nascent chains (NC) are indicated. This demonstrates the functionality of the components of the reconstituted termination complex in mediating the release of the nascent chain, which is inhibited by the nonhydrolyzable GTP analog, GMPPCP.
Fig 3: GTPase Active Sites, Related to Figure 5(A) EM map density and model for GDP and GTP analogs in the indicated structures. eEF1A-bound GDP density is contoured at 7σ; Hbs1l-bound GMPPCP density is contoured at 6σ. Coordinating residues (pink) and magnesium ions (green) are indicated.(B) Interactions of the sarcin-ricin loop (SRL) with the catalytic histidine (teal) of the indicated GTPase. The residues of the hydrophobic gate are indicated in yellow.
Fig 4: Structure of the Mammalian Elongation Complex(A) Overview of the elongation complex comprising the large (60S) and small (40S) ribosomal subunits, P- (green) and E-site (gold) tRNAs, mRNA (slate), aminoacyl-tRNA in the A/T state (aa-tRNA; purple), and eEF1A (red).(B) Decoding center of the elongation complex. eS30 (teal) and the decoding nucleotides of 18S rRNA (yellow) are indicated.(C) EM map density and models of the interactions within the decoding center of the elongation complex. Decoding nucleotides of 18S rRNA (yellow), aa-tRNA (purple), the A-site codon (+1 to +3) of mRNA (slate), and uS12 (orange) are indicated.(D) Density and models of the interaction between His76 of the N terminus of eS30 (teal) within the decoding center of the elongation complex. In panels (C) and (D), density for mRNA, tRNA, and rRNA is contoured at 9σ; density for uS12 and eS30 is contoured at 5σ.(E) The C termini of uS19 (bronze) and uS13 (brown) of the mammalian (80S) elongation complex compared to the homologous proteins in a 70S bacterial elongation complex (gray, PDB: 4V51), showing the potential interactions of the C terminus of uS19 in mammals or uS13 in bacteria with the anticodon stem loops of A/T aa-tRNA (purple) and P-site tRNA (green).See also Figures S1, S2, S3, and S4.
Fig 5: Details of Pre-accommodation Architectures, Related to Figure 5(A) The acceptor stem of aa-tRNA (purple) binds in a cleft between the G domain (red) and domains 2 (orange) and 3 (yellow) of eEF1A.(B) Surface model of eEF1A colored by electrostatic potential (same view as panel A).(C) EM map density contoured at 7σ and models of the interactions between the 3′ end of aa-tRNA (purple) and domain 2 (orange) and G domain (red) of eEF1A.(D and E) The M domains of (D) eRF1 and (E) Pelota bind their respective GTPase partners in a cleft analogous to where aa-tRNA binds eEF1A. Structures are aligned as in panel (A).(F and G) Surface model colored by electrostatic potential of (F) eRF3, and (G) Hbs1l.(H and I) Superposition of (H) the crystal structure of aRF1⋅aEF1A⋅GTP (gray) on ribosome-bound eRF1⋅eRF3⋅GMPPCP or of (I) the crystal structure of aPelota⋅aEF1A⋅GTP (gray) on ribosome-bound Pelota⋅Hbs1l⋅GMPPCP via domains 2 and 3 of the GTPase. Upon ribosome binding, the N domain of the decoding factor is reoriented, while the M domain forms additional contacts with the G domain of the GTPase.(J and K) Interactions between the M domains of (J) eRF1 or of (K) Pelota with the G domain of the respective GTPase. The β7-α5 loop, which harbors the GGQ motif of eRF1, makes interactions with the Switch 1 (Sw1, red) motif, and additional interactions are formed with the Switch 2 (Sw2, teal) motif harboring the catalytic histidine.(L) The backbone and CCA end of A/T aa-tRNA also interacts with catalytically important motifs of the G domain of eEF1A.
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