Evidence for a functionally relevant rocaglamide binding site on the eIF4A-RNA complex

Abstract

Translation initiation is an emerging target in oncology and neurobiology indications. Naturally derived and synthetic rocaglamide scaffolds have been used to interrogate this pathway; however, there is uncertainty regarding their precise mechanism(s) of action. We exploited the genetic tractability of yeast to define the primary effect of both a natural and a synthetic rocaglamide in a cellular context and characterized the molecular target using biochemical studies and in silico modeling. Chemogenomic profiling and mutagenesis in yeast identified the eIF (eukaryotic Initiation Factor) 4A helicase homologue as the primary molecular target of rocaglamides and defined a discrete set of residues near the RNA binding motif that confer resistance to both compounds. Three of the eIF4A mutations were characterized regarding their functional consequences on activity and response to rocaglamide inhibition. These data support a model whereby rocaglamides stabilize an eIF4A-RNA interaction to either alter the level and/or impair the activity of the eIF4F complex. Furthermore, in silico modeling supports the annotation of a binding pocket delineated by the RNA substrate and the residues identified from our mutagenesis screen. As expected from the high degree of conservation of the eukaryotic translation pathway, these observations are consistent with previous observations in mammalian model systems. Importantly, we demonstrate that the chemically distinct silvestrol and synthetic rocaglamides share a common mechanism of action, which will be critical for optimization of physiologically stable derivatives. Finally, these data confirm the value of the rocaglamide scaffold for exploring the impact of translational modulation on disease.

Figure 1

Components of the translation initiation pathway alter the cellular response to rocaglamides. A. Chemical scaffolds examined in this study. B. Normalized growth (measured by OD600) of wild-type (gray) and Δ7 (black) yeast strains in the presence of ROC-N (squares) and silvestrol (circles). Shown are biological and technical replicates. n = 2. C. ROC-N inhibits expression of a luciferase reporter. Luciferase levels relative to a DMSO control are plotted versus compound concentration (log). n=3 ± SEM D. Haploinsufficiency profile of ROC-N (6 µM) with relative strain sensitivity plotted as a function of statistical significance (z-score). Strains essential for viability (black) and non-essential (gray) are indicated. Deletion strains involved in key translation initiation complexes are highlighted, together with the corresponding mammalian nomenclature annotated. E. Haploinsufficiency profile (z-score) of ROC-N (6 µM) is plotted as a function of silvestrol (200 µM). Commonly affected strains are highlighted.

Figure 2

Mutagenesis screening identifies three complementation groups showing resistance to ROC-N. A. Annotation of gene targets found mutated for individual clones within each complementation group. B. Sensitivity of strains from the indicated complementation classes to growth inhibition by ROC-N. Serial dilutions of individual strains were spotted onto minimal media with or without ROC-N (0.7 µM). Note that the parental strain (Δ7) lacks key drug resistance mechanisms, resulting in increased sensitivity to the compound (see Methods). Selected haploid resistant clones demonstrated increased ability to form colonies in the presence of compound as compared to the parental strain. Complementation of the mutant strains with a plasmid-borne (pRS416) wild-type ORF restores compound sensitivity. Replacement of the TIF4631 and KEM1 ORFs in the Δ7 background also leads to increased survival to ROC-N.

Figure 3

Mutation at a discrete set of amino acids provides resistance to silvestrol and ROC-N. Haploid yeast strains carrying a single mutated TIF1 allele in a Δ7Δtif2 background were grown in liquid culture supplemented with DMSO (left panels), ROC-N (0.7 µM, middle panels), or silvestrol (65 µM right panels). Growth was monitored by the Absorbance at 600 nM and measured over time.

Figure 4

Effect of TIF mutations and rocaglamides on RNA binding activity. A. SDS-PAGE analysis of purified recombinant TIF1 proteins. One microgram of each wild-type and mutant proteins were separated by SDS-PAGE and stained with Coomassie blue. B. RNA binding activity of TIF1 mutants. Asterisk denotes RNA control in the absence of protein. Assays were performed as described in the Methods in the presence of 0.5 %DMSO, 10 µM ROC-N, or 50 µM silvestrol (concentrations chosen based on the ability to block translation (Figure 1C and data not shown). n=3 ± SEM.

Figure 5

Effect of TIF1 mutations and rocaglamides on ATP hydrolysis. Assays were performed as described in the Methods in the presence of 0.5% DMSO (black), 10 µM ROC-N (white), or 50 µM silvestrol (gray). The % ATP hydrolysis is plotted as a ratio of ³²P-Pi/(³²P-ATP + ³²P-Pi). A background value of 5 % Pi obtained in the absence of protein and RNA was subtracted from each value. n=3 ± SEM.

Figure 6

Characterization of rocaglamides on RNA helicase activity of TIF1 mutants. A. Schematic representation of RNA substrate used in the helicase assay. B. Assays were performed as described in the Methods in the presence of 0.5% DMSO, 10 µM ROC-N, or 50 µM silvestrol. The position of migration of double-stranded and single-stranded RNA molecules is indicated to the left with an asterisk denoting the radiolabel. Lane 1, duplex RNA incubated under unwinding conditions without protein for 15 min at 35°C. Lane 2, duplex RNA incubated for 5 min without protein at 95°C. C. Quantification of TIF helicase activity. The percentage of unwinding was determined as the ratio of (monomer RNA + duplex RNA)/monomer RNA. The percentage of monomer present in the duplex input samples (Panel B; lane 1) was set at 0% and subtracted from the values obtained in the sample lanes. n=4 ± SEM, (*p<0.05, **p<0.005).

Figure 7

In silico modeling of eIF4A homologues and putative rocaglamide binding site. A. Aligned ribbon diagrams of D1 domains for human eIF4AIII (PDB 2HYI, green) and yeast TIF1/2 helicases (PDB 2VSO, blue). The two domains were aligned via backbone atoms to an RMSD of 0.99A; alignment was somewhat better in the locality of helices alpha-3 and alpha-4. The RNA substrate from the eIF4AIII structure is shown in orange. TIF1/2 mutants identified in this work are highlighted in stick format and labeled, yellow for those most frequently mutated (P147, F151 and Q183) and orange for those less frequently mutated (T146, F180 and I187). Five of these residues are strictly conserved in eIF4AIII (T163, P164, F168, F197 and Q200) whereas I187 is partially conserved as V204. B. The same region of the eIF4AIII helicase D1 domain visualized in A., RNA substrate visualized as colored sticks. The described SiteMap sites are highlighted in grey surface rendering, with individual hydrophobic (yellow) and hydrophilic (red and blue) features outlined with colored grids; white dots represent site points used by SiteMap software to identify and merge adjacent sub-pockets (DScore of 1.02).