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Dual targeting of DDX3 and eIF4A by the translation inhibitor rocaglamide A

ABSTRACT: Small molecule compounds that sense the nucleic acid sequences, promise the attractive venue for drug development. Such an unusual effect has been observed in the natural product Rocaglamide A (RocA) from Aglaia plant, proving to exhibit anti-tumor effects by clamping eukaryotic initiation factor (eIF) 4A onto mRNA polypurine sequences. Although eIF4A has been speculated the unique target of RocA, the insensitization of eIF4A in human cells only partially rescued the translation repression from RocA, suggesting another alternative target of this compound. Here, we revealed that DDX3 is an alternative target of RocA. Developing a RocA derivative with an O-nitrobenzoxadiazole unit (RocA-O-NBD), which can covalently bind to proximate proteins and provide fluorescence to them, we identified DDX3 bound to RocA. As observed in eIF4A, RocA locked the DDX3 protein onto polypurine sequences of RNA in an ATP-independent manner. De novo assembled Aglaia plant transcriptome uncovered the natural amino acid substitutions in Aglaia DDX3 to protect itself from RocA toxicity. Because of the dominant negative effect of RocA, we also proved the protein abundance of eIF4A and DDX3 in cancer cells determines their sensitivity to RocA. Overall, this study discovered DDX3 as another target of RocA and suggests the probability to predict tumor toxicity of RocA by the target abundance.

ORGANISM(S): Homo sapiens

PROVIDER: GSE148636 | GEO | 2020/12/07

REPOSITORIES: GEO

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Dual targeting of DDX3 and eIF4A by the translation inhibitor rocaglamide A [Bind-n-Seq]

Project description:Small molecule compounds that sense the nucleic acid sequences, promise the attractive venue for drug development. Such an unusual effect has been observed in the natural product Rocaglamide A (RocA) from Aglaia plant, proving to exhibit anti-tumor effects by clamping eukaryotic initiation factor (eIF) 4A onto mRNA polypurine sequences. Although eIF4A has been speculated the unique target of RocA, the insensitization of eIF4A in human cells only partially rescued the translation repression from RocA, suggesting another alternative target of this compound. Here, we revealed that DDX3 is an alternative target of RocA. Developing a RocA derivative with an O-nitrobenzoxadiazole unit (RocA-O-NBD), which can covalently bind to proximate proteins and provide fluorescence to them, we identified DDX3 bound to RocA. As observed in eIF4A, RocA locked the DDX3 protein onto polypurine sequences of RNA in an ATP-independent manner. De novo assembled Aglaia plant transcriptome uncovered the natural amino acid substitutions in Aglaia DDX3 to protect itself from RocA toxicity. Because of the dominant negative effect of RocA, we also proved the protein abundance of eIF4A and DDX3 in cancer cells determines their sensitivity to RocA. Overall, this study discovered DDX3 as another target of RocA and suggests the probability to predict tumor toxicity of RocA by the target abundance.

2020-12-07 | GSE150111 | GEO

Ternary protein•drug•RNA interface explains RocA sequence selectivity and Aglaia resistance

Project description:A novel class of translation inhibitors isolated from Aglaia plants, exemplified by Rocaglamide A (RocA), shows promise as an anti-cancer therapy. RocA converts its molecular target, translation initiation factor 4A (eIF4A), a DEAD-box protein, into a purine-selective RNA-binding protein that represses protein synthesis from a subset of mRNAs. This unusual mechanism of action raises the question of how the drug induces selectivity for polypurine sequences. Furthermore, as eIF4A is found in all eukaryotes, it is unclear how Aglaia resists the effects of RocA. Here, we assembled the Aglaia transcriptome de novo and identified highly specific mutations in eIF4A that enable it to evade RocA-dependent translation inhibition. Furthermore, we determined the crystal structure of the human eIF4A1•ATP analog•RocA•polypurine RNA complex. RocA binds residues mutated in Aglaia and intercalates between consecutive purines at a sharp RNA bend, revealing the structural basis of polypurine-selective translational inhibition by RocA in human and resistance in Aglaia.

2018-12-30 | GSE102720 | GEO

Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor

Project description:Rocaglamide A (RocA) typifies a class of protein synthesis inhibitors that selectively kill aneuploid tumor cells and repress translation of specific mRNAs. RocA targets eukaryotic initiation factor 4A (eIF4A), an ATP-dependent DEAD-box RNA helicase; its mRNA selectivity is proposed to reflect highly structured 5â² UTRs that depend strongly on eIF4A-mediated unwinding. However, rocaglate treatment may not phenocopy the loss of eIF4A activity, as these drugs actually increase the affinity between eIF4A and RNA. Here, we show that secondary structure in 5â² UTRs is only a minor determinant for RocA selectivity and RocA does not repress translation by reducing eIF4A availability. Rather, in vitro and in cells, RocA specifically clamps eIF4A onto polypurine sequences in an ATP-independent manner. This artificially clamped eIF4A blocks 43S scanning, leading to premature, upstream translation initiation and reducing protein expression from transcripts bearing the RocA-eIF4A target sequence. In elucidating the mechanism of selective translation repression by this lead anti-cancer compound, we provide an example of a drug stabilizing sequence-selective RNA-protein interactions. Bind-n-Seq and iCLIP-Seq

2016-06-15 | E-GEOD-79392 | biostudies-arrayexpress

Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor

Project description:Rocaglamide A (RocA) typifies a class of protein synthesis inhibitors that selectively kill aneuploid tumor cells and repress translation of specific mRNAs. RocA targets eukaryotic initiation factor 4A (eIF4A), an ATP-dependent DEAD-box RNA helicase; its mRNA selectivity is proposed to reflect highly structured 5′ UTRs that depend strongly on eIF4A-mediated unwinding. However, rocaglate treatment may not phenocopy the loss of eIF4A activity, as these drugs actually increase the affinity between eIF4A and RNA. Here, we show that secondary structure in 5′ UTRs is only a minor determinant for RocA selectivity and RocA does not repress translation by reducing eIF4A availability. Rather, in vitro and in cells, RocA specifically clamps eIF4A onto polypurine sequences in an ATP-independent manner. This artificially clamped eIF4A blocks 43S scanning, leading to premature, upstream translation initiation and reducing protein expression from transcripts bearing the RocA-eIF4A target sequence. In elucidating the mechanism of selective translation repression by this lead anti-cancer compound, we provide an example of a drug stabilizing sequence-selective RNA-protein interactions.

2016-06-15 | GSE79392 | GEO

Rocaglamide A converts RNA helicase eIF4A into a sequence-specific translational repressor

2016-06-15 | GSE70211 | GEO

Rocaglamide A converts RNA helicase eIF4A into a sequence-specific translational repressor

Project description:Rocaglamide A (RocA) typifies a novel class of protein synthesis inhibitors that selectively kill aneuploid tumor cells and repress translation of specific mRNAs. RocA targets eukaryotic initiation factor 4A (eIF4A), the prototypical DEAD-box RNA helicase, and its mRNA selectivity is proposed to reflect highly structured 5â² UTRs that are very dependent on eIF4A-mediated unwinding. Here, we show that secondary structure in 5â² UTRs is only a minor determinant for RocA selectivity and RocA does not repress translation by reducing eIF4A activity. Rather, in vitro and in vivo, RocA clamps eIF4A onto a specific sequence motif even after ATP hydrolysis. This artificially clamped eIF4A blocks 43S scanning, leading to premature, upstream translation initiation and reducing gene expression on transcripts bearing the RocA-eIF4A target sequence. In elucidating the mechanism of this lead anti-cancer compound and explaining its mRNA selectivity, we provide the first example of a drug stabilizing sequence-specific RNA-protein interactions. Ribosome profiling, mRNA-Seq, RIP-Seq, and Bind-n-Seq Ribosome profiling for sample 1-5, and 11-15. Sample1 and 2 are replicates of control of DMSO treatment for sample 3-5, and 11, with RocA and PP242 treatments. Sample 12 and 13 are replicates of control of DMSO treatment for sample 14 and 15 with Hipp treatments. mRNA-Seq for sample 6-10. Sample 6 and 7 are replicates of control of DMSO treatment for sample 8-10 with RocA treatments. RIP-Seq for 16-19. Sample 16 and 17 are replicates of control of DMSO treatment for sample 18-19 with RocA treatments. Bind-n-Seq for 20-23. Sample 21 is control of DMSO treatment for sample 22-23 with RocA treatments. Sample 20 is a input contol for protein-bound fraction of sample 21. We stably expressed SBP (streptavidin binding peptide)-tagged eIF4A in HEK 293T-REx cells and purified it via M270 streptavidin beads (life techonologies).

2016-06-15 | E-GEOD-70211 | biostudies-arrayexpress

Disome profiling of rocaglamide A treatment in HEK293FT cells

Project description:For the past decade, extensive studies of translation have produced a vast amount of ribosome profiling data, an insightful resource for mining of critical details about the dynamics of translation regulation under various biological contexts. Previously, Rocaglamide A (RocA), an anti-tumor heterotricyclic natural compound, has been shown to selectively inhibit translation initiation of a large group of mRNA species by clamping eukaryotic translation initiation factor 4A (eIF4A) onto poly-purine motifs in the 5’ un-translational regions (5’UTRs). However, re-analysis of the previous ribosome profiling datasets revealed an unexpected shift of the ribosome occupancy pattern during early translation elongation upon RocA treatment in various types of cells, for a specific group of mRNA transcripts without poly-purine motifs over-presented in their 5’UTRs. Such perturbation of the translation elongation dynamics can be attributed to the blockage of translating ribosomes due to the binding of eIF4A to the poly-purine sequence in coding sequences. This new mode of action of RocA, which is complementary to the canonical function inhibiting translation initiation, selectively interfere with the translation of the genes involved in fundamental processes such as ATP synthase, mitochondrial respiratory chain complex, et al. In summary, re-analyses of previous ribosome profiling data has revealed the complete dual modes of RocA in blocking translation and thus underlying the potent anti-tumor effect

2022-12-17 | GSE201364 | GEO

A parasitic fungus employs mutated eIF4A to survive on rocaglate-synthesizing Aglaia plants

Project description:Plants often generate secondary metabolites with antifungal properties as defense mechanisms against parasites. Although some fungi may potentially overcome the barrier of antimicrobial compounds, only a limited number of examples and molecular mechanisms of resistance have been reported. Here, we found an Aglaia plant-parasitizing fungus that overcomes the toxicity of rocalgates, which are translation inhibitors synthesized by the plant, through an amino acid substitution in a translation initiation factor (eIF). De novo transcriptome assembly of the fungus revealed that eIF4A, a molecular target of rocaglates, replaces a critical amino acid in the rocaglate binding site. Moreover, genome-wide ribosome profiling harnessing a cucumber-infecting fungus, Colletotrichum orbiculare, demonstrated that the translational inhibitory effects of rocaglates were largely attenuated by the mutation found in the Aglaia parasite. The engineered Colletotrichum orbiculare showed a survival advantage on cucumber plants with rocaglates. Our study exemplifies a plant-fungus tug-of-war centered on secondary metabolites produced by host plants.

2023-02-20 | GSE200060 | GEO

Profile of RNAs bound by wild-type (WT) or R534H variant of DDX3 with or without Sodium Arsenite treatment [iCLIP-Seq]

Project description:By iCLIP-seq, we demonstrated that DDX3 binds to specific sites on 18S ribosomal RNA and to messenger RNAs at 5’ leader and guanidine-rich sequences immediately upstream of ribosome-dense regions and that arsenite treatment redistributes DDX3 binding from 5'UTR to CDS, which is attenuated by DDX3 R534H.

2016-04-21 | GSE70801 | GEO

eIF4A supports an oncogenic translation program in pancreatic ductal adenocarcinoma

Project description:Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy with limited treatment options. Although metabolic reprogramming is a hallmark of many cancers, including PDA, previous attempts to target metabolic changes therapeutically have been stymied by drug toxicity and tumour cell plasticity. Here, we show that PDA cells engage an eIF4F-dependent translation program that supports redox and central carbon metabolism. Inhibition of the eIF4F subunit, eIF4A, using the synthetic rocaglate CR-1-31-B (CR-31) reduced the viability of PDA organoids relative to their normal counterparts. In vivo, CR-31 suppresses tumour growth and extends survival of genetically-engineered murine models of PDA. Surprisingly, inhibition of eIF4A also induces glutamine reductive carboxylation. As a consequence, combined targeting of eIF4A and glutaminase activity more effectively inhibits PDA cell growth both in vitro and in vivo. Overall, our work demonstrates the importance of eIF4A in translational control of pancreatic tumour metabolism and as a therapeutic target against PDA.

2019-10-03 | GSE125380 | GEO

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