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. 2023 Feb 28;12:e81302. doi: 10.7554/eLife.81302

Figure 3. The amino acid substitution in the Ophiocordyceps sp. BRM1 eIF4A confers translational resistance to rocaglates in fungi.

(A) RocA-mediated translational repression recapitulated by an in vitro reconstitution system with human factors. Recombinant proteins of H. sapiens eIF4A1 WT or Phe163Gly were added to the reaction with RocA. Reporter mRNA with CAA repeats or polypurine motifs was translated in the reaction. The data are presented as the mean and s.d. values (n = 3). (B) Translation of complex-preformed mRNAs to test the RocA gain of function. Recombinant proteins of Ophiocordyceps sp. BRM1 eIF4A1 WT or the Gly172His mutant were preincubated with the reporter mRNA possessing polypurine motifs in the presence or absence of RocA. After removal of free RocA by gel filtration, the protein-mRNA complex was added to RRL to monitor protein synthesis. The data are presented as the mean and s.d. values (n = 3). (C) MA (M, log ratio; A, mean average) plot of the translation efficiency changes caused by 3 µM aglafoline treatment in C. orbiculare eIF4AWT conidia. Resistant and sensitive mRNAs (FDR < 0.05) are highlighted. (D) Cumulative distribution of the translation efficiency changes in aglafoline-sensitive mRNAs (defined in C) in C. orbiculare eIF4AWT conidia treated with 0.3 or 3 µM aglafoline. (E) Cumulative distribution of the translation efficiency changes in aglafoline-sensitive mRNAs (defined in C) induced by 3 µM aglafoline treatment in C. orbiculare eIF4AWT and eIF4AHis153Gly conidia. (F) Cumulative distribution of the global translation alterations, which are footprint changes normalized to mitochondrial footprints, in aglafoline-sensitive mRNAs (defined in C) induced by 3 µM aglafoline treatment in C. orbiculare eIF4AWT and eIF4AHis153Gly conidia. (G) Box plot of the translation efficiency changes caused by 3 µM aglafoline treatment in conidia across mRNAs with or without an [A/G]6 motif in the 5′ UTR. The p values in (D–G) were calculated by the Mann–Whitney U test.

Figure 3—source data 1. Files for the primary data corresponding to Figure 3A.
Figure 3—source data 2. Files for the primary data corresponding to Figure 3B.

Figure 3.

Figure 3—figure supplement 1. Establishment of eIF4A-engineered C. orbiculare strains.

Figure 3—figure supplement 1.

(A) Schematics of eIF4A recombination in C. orbiculare. NPTII, neomycin phosphotransferase II. (B) PCR-based screening of the recombined strains. The primer sets used for screening are depicted in (A). (C, D) Colony formation of the indicated C. orbiculare strains cultured in PDA for 5 days (C). The measured colony diameters are shown in the box plot (n = 5) (D). (E–G) Fluorescence polarization assay for FAM-labeled RNA ([AG]10) (10 nM). WT and mutated Ophiocordyceps sp. BRM1 eIF4A proteins were used. To measure ATP-independent RNA clamping induced by aglafoline (50 µM), ADP and Pi were included in the reaction. The data are presented as the mean and s.d. values (n = 3). (H) Summary of the Kd values in (E–G) under treatment with aglafoline. The data are presented as the mean and s.d. values.
Figure 3—figure supplement 1—source data 1. Files for the full and unedited gel images corresponding to Figure 3—figure supplement 1B and C.
Figure 3—figure supplement 1—source data 2. Files for the primary data corresponding to Figure 3—figure supplement 1D.
Figure 3—figure supplement 1—source data 3. Files for the primary data corresponding to Figure 3—figure supplement 1E–G.
Figure 3—figure supplement 2. Characterization of ribosome footprints in C. orbiculare.

Figure 3—figure supplement 2.

(A) Distribution of ribosome footprint length in conidia and mycelia. (B) Tile plot of reading frames at each ribosome footprint length in conidia and mycelia. The 5′ end positions of the ribosome footprints are depicted. The footprint count scales are shown in the color bars. (C) Metagene plot of 29-nt ribosome footprints around start (left) and stop (right) codons in conidia and mycelia. The 5′ end positions of the ribosome footprints are depicted. RPM: reads per million mapped reads.
Figure 3—figure supplement 3. Translation changes by aglafoline treatment in recombined C. orbiculare.

Figure 3—figure supplement 3.

(A) GO term analysis of aglafoline-sensitive mRNAs (defined in Figure 3C). GO terms associated with yeast homologs were analyzed by DAVID (Huang et al., 2009a; Huang et al., 2009b). (B) MA plot of the translation efficiency changes induced by 3 µM aglafoline treatment in C. orbiculare eIF4AWT mycelia. Resistant and sensitive mRNAs (false discovery rate [FDR] < 0.05) are highlighted. (C) Cumulative distribution of the translation efficiency changes in aglafoline-sensitive mRNAs (defined in B) in C. orbiculare eIF4AWT mycelia treated with 0.3 or 3 µM aglafoline. (D) Cumulative distribution of the translation efficiency changes in aglafoline-sensitive mRNAs (defined in B) induced by 3 µM aglafoline treatment in C. orbiculare eIF4AWT and eIF4AHis153Gly mycelia. (E) Cumulative distribution of the global translation alterations, which are footprint changes normalized to mitochondrial footprints, in aglafoline-sensitive mRNAs (defined in B) induced by 3 µM aglafoline treatment in C. orbiculare eIF4AWT and eIF4AHis153Gly conidia. (F) Box plot of translation efficiency changes caused by 3 µM aglafoline treatment in mycelia across mRNAs with or without an [A/G]6 motif in the 5′ UTR. (G) Venn diagram of the overlap between aglafoline-sensitive mRNAs in conidia (defined in Figure 3C) and mycelia (defined in B). The p values in (C–F) were calculated by the Mann–Whitney U test.