Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor
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ABSTRACT:
ORGANISM(S): synthetic construct Homo sapiens
PROVIDER: GSE79392 | GEO | 2016/06/15
SECONDARY ACCESSION(S): PRJNA315688
REPOSITORIES: GEO
Similar Datasets
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
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 5′ UTRs that are very dependent on eIF4A-mediated unwinding. 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 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.
2016-06-15 | GSE70211 | GEO
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
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
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
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 | GSE148636 | GEO
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
Project description:Protein synthesis-targeting agents against eIF4A activity, including rocaglates, are actively pursued as anticancer and antiviral therapies. Yet, their full effect on the translational landscape is unknown, especially with regards to up-regulated proteins and drug-activated translation factors that mediate rocaglates’ remarkable anticancer potency. Here, we investigated rocaglate-driven global translational remodeling in cancer cells. Proteomic translatome analysis by TMT-pulse-SILAC revealed an extensive repertoire of rocaglate-inducible proteins that regulate hitherto unrecognized mechanisms of cytotoxicity. As proof-of-concept, we show that GEF-H1 induction activates anti-survival RHOA/JNK signaling. Intriguingly, rocaglate responses persist in eIF4A-depleted cells. Global MATRIX survey of translation machinery adaptations to rocaglates revealed augmented translational activities of the general translation factor eEF1ε1, and the DEAD-box RNA helicase DDX17, which drive rocaglate-specific protein induction and drug response. This original unbiased proteomic interrogation of rocaglate-driven translational reprogramming transforms the current definition of rocaglates as one-dimensional eIF4A inhibitors to comprehensive remodelers of the protein synthesis landscape.
2021-11-23 | PXD022556 | Pride
Project description:DEAD-box RNA helicases eIF4A and Ded1 promote translation by resolving mRNA secondary structures that impede preinitiation complex (PIC) attachment to mRNA or scanning. eIF4B is a cofactor for eIF4A but might also function independently of eIF4A. Ribosome profiling of mutants lacking eIF4B or with impaired eIF4A or Ded1 activity revealed that eliminating eIF4B reduces the relative translational efficiencies of many more genes than does inactivation of eIF4A, despite comparable reductions in bulk translation, and few genes display unusually strong requirements for both factors. However, either eliminating eIF4B or inactivating eIF4A preferentially impacts mRNAs with longer, more structured 5’UTRs. These findings reveal an eIF4A-independent role for eIF4B in addition to its function as eIF4A cofactor in promoting PIC attachment or scanning on structured mRNAs. eIF4B, eIF4A, and Ded1 mutations also preferentially impair translation of longer mRNAs in a fashion mitigated by the ability to form closed-loop mRNPs via eIF4F-Pab1 association, suggesting cooperation between closed-loop assembly and eIF4B/helicase functions. Remarkably, depleting eIF4G, the scaffold subunit of eIF4F, preferentially impacts short mRNAs with strong closed-loop potential and unstructured 5’UTRs, exactly the opposite features associated with hyperdependence on the eIF4B/helicases. We propose that short, highly efficient mRNAs preferentially depend on the stimulatory effects of eIF4G-dependent closed-loop assembly.
2016-05-27 | GSE81966 | GEO
Project description:Translational dysregulation is an emerging hallmark of cancer, and increased activity of the mRNA helicase eIF4A is associated with poor survival in malignancies. This is believed to be due to the unwinding of secondary structures within the 5’UTRs of oncogenic mRNAs, with studies showing that in general eIF4A-dependent mRNAs have longer 5’UTRs with more stable secondary structures, yet our ability to predict eIF4A-dependency from 5’UTR properties alone remains poor. We therefore used Structure-seq 2 to measure transcriptome-wide changes in RNA structure in MCF7 cells, following eIF4A inhibition with hippuristanol. This technique measures the single-strandedness of RNA by specific and rapid methylation of single-stranded adenosines and cytosines with Dimethyl Sulphate (DMS). When paired with polysome profiling data to identify which mRNAs are most translationally repressed, we can identify the structural determinants of eIF4A-dependency. Upon eIF4A inhibition, both 5’UTRs and CDSs become generally more structured, while overall this was not observed for 3’UTRs. This was most pronounced in CDSs, supporting recent findings that the ribosome sculpts RNA structure in this region. 5’UTRs are generally more structured at their 5’ ends and highly translated mRNAs are less structured just upstream of the CDS. Following eIF4A inhibition, the 5’UTR is remodelled. eIF4A-dependent mRNAs have greater localised gains of structure. The degree of these structural changes is strongly correlated with 5’UTR length, explaining why eIF4A-dependent mRNAs have longer 5’UTRs. Crucially, in eIF4A-dependent mRNAs these highly-structured elements are located predominantly at the 3’ end of the 5’UTR, suggesting that increased structure just upstream of the CDS is most inhibitory to translation following eIF4A inhibition and is a key determinant of eIF4A-dependency.
2019-11-26 | GSE134865 | GEO