Rocaglamide A converts RNA helicase eIF4A into a sequence-specific translational repressor
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ABSTRACT:
PROVIDER: PRJNA287879 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR2075930.fastq.gz | Fastqsanger.gz | |||
| SRR2075940.fastq.gz | Fastqsanger.gz | |||
| SRR2075931.fastq.gz | Fastqsanger.gz | |||
| SRR2075941.fastq.gz | Fastqsanger.gz | |||
| SRR2075932.fastq.gz | Fastqsanger.gz |
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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: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 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
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: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:Dual targeting of DDX3 and eIF4A by the translation inhibitor rocaglamide A
| PRJNA625261 | ENA
Project description:The translational control of oncoprotein expression is implicated in many cancers. Here we report an eIF4A/DDX2 RNA helicase-dependent mechanism of translational control that contributes to oncogenesis and underlies the anticancer effects of Silvestrol and related compounds. For example, eIF4A promotes T-ALL development in vivo and is required for leukaemia maintenance. Accordingly, inhibition of eIF4A with Silvestrol has powerful therapeutic effects in vitro and in vivo. We use transcriptome-scale ribosome footprinting to identify the hallmarks of eIF4A-dependent transcripts. These include 5'UTR sequences such as the 12-mer guanine quartet (CGG)4 motif that can form RNA G-quadruplex structures. Notably, among the most eIF4A-dependent and Silvestrol-sensitive transcripts are a number of oncogenes, super-enhancer associated transcription factors, and epigenetic regulators. Hence, the 5'UTRs of selected cancer genes harbour a targetable requirement for the eIF4A RNA helicase. Comparison of ribosome-protected RNA for drug treated and DMSO treated KOPT-K1 cell, two replicates of ribosome-protected RNA sequencing and three replicates of RNA-seq.
2014-07-27 | E-GEOD-56887 | biostudies-arrayexpress
Project description:The translational control of oncoprotein expression is implicated in many cancers. Here we report an eIF4A/DDX2 RNA helicase-dependent mechanism of translational control that contributes to oncogenesis and underlies the anticancer effects of Silvestrol and related compounds. For example, eIF4A promotes T-ALL development in vivo and is required for leukaemia maintenance. Accordingly, inhibition of eIF4A with Silvestrol has powerful therapeutic effects in vitro and in vivo. We use transcriptome-scale ribosome footprinting to identify the hallmarks of eIF4A-dependent transcripts. These include 5'UTR sequences such as the 12-mer guanine quartet (CGG)4 motif that can form RNA G-quadruplex structures. Notably, among the most eIF4A-dependent and Silvestrol-sensitive transcripts are a number of oncogenes, super-enhancer associated transcription factors, and epigenetic regulators. Hence, the 5'UTRs of selected cancer genes harbour a targetable requirement for the eIF4A RNA helicase.
2014-07-27 | GSE56887 | GEO
Project description:Dual targeting of DDX3 and eIF4A by the translation inhibitor rocaglamide A [Bind-n-Seq]
| PRJNA631213 | ENA