Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor
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ABSTRACT:
PROVIDER: PRJNA315688 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR3238820.fastq.gz | Fastqsanger.gz | |||
| SRR3238821.fastq.gz | Fastqsanger.gz | |||
| SRR3238814.fastq.gz | Fastqsanger.gz | |||
| SRR3238815.fastq.gz | Fastqsanger.gz | |||
| SRR3238816.fastq.gz | Fastqsanger.gz |
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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 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:Rocaglamide A (RocA) typifies a class of protein synthesis inhibitors that selectively kill aneuploid tumour cells and repress translation of specific messenger RNAs. RocA targets eukaryotic initiation factor 4A (eIF4A), an ATP-dependent DEAD-box RNA helicase; its messenger RNA selectivity is proposed to reflect highly structured 5' untranslated regions 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' untranslated regions is only a minor determinant for RocA selectivity and that 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.
| S-EPMC4946961 | biostudies-literature
Project description:DEAD-box RNA helicases eIF4A and Ded1 are believed to promote translation initiation by resolving mRNA secondary structures that impede ribosome attachment at the mRNA 5’ end or subsequent scanning of the 5’UTR, but whether they perform distinct functions or act redundantly in vivo is poorly understood. We compared the effects of mutations in Ded1 or eIF4A on global translational efficiencies (TEs) in yeast by ribosome footprint profiling. Despite similar reductions in bulk translation, inactivation of a cold-sensitive Ded1 mutant substantially reduced the TEs of >600 mRNAs, whereas inactivation of a temperature-sensitive eIF4A mutant yielded <40 similarly impaired mRNAs. The broader requirement for Ded1 did not reflect more pervasive secondary structures at low temperature, as inactivation of temperature-sensitive and cold-sensitive ded1 mutants gave highly correlated results. Interestingly, Ded1-dependent mRNAs exhibit greater than average 5’UTR length and propensity for secondary structure, implicating Ded1 in scanning though structured 5' UTRs. Reporter assays confirmed that cap- distal stem-loop insertions increase dependence on Ded1 but not eIF4A for efficient translation. While only a small fraction of mRNAs is strongly dependent on eIF4A, this dependence is significantly correlated with requirements for Ded1 and 5’UTR features characteristic of Ded1- dependent mRNAs. Our findings suggest that Ded1 is critically required to promote scanning through secondary structures within 5’UTRs; and while eIF4A cooperates with Ded1 in this function, it also promotes a step of initiation common to virtually all yeast mRNAs. We compared the effects of mutations in Ded1 or eIF4A on global translational efficiencies (TEs) in yeast by ribosome footprint profiling.The study includes 32 samples, comprised of 16 mRNA-Seq samples and 16 ribosome footprint profiling samples, derived from biological replicates of 3 mutant strains, ded1-cs, ded1-ts and tif1-ts, and the corresponding wild-type strains. The tif1-ts mutant and its wild-type counterpart were analyzed at 30°C and 37°C.
2015-03-02 | E-GEOD-66411 | biostudies-arrayexpress
Project description:DEAD-box RNA helicases eIF4A and Ded1 are believed to promote translation initiation by resolving mRNA secondary structures that impede ribosome attachment at the mRNA 5’ end or subsequent scanning of the 5’UTR, but whether they perform distinct functions or act redundantly in vivo is poorly understood. We compared the effects of mutations in Ded1 or eIF4A on global translational efficiencies (TEs) in yeast by ribosome footprint profiling. Despite similar reductions in bulk translation, inactivation of a cold-sensitive Ded1 mutant substantially reduced the TEs of >600 mRNAs, whereas inactivation of a temperature-sensitive eIF4A mutant yielded <40 similarly impaired mRNAs. The broader requirement for Ded1 did not reflect more pervasive secondary structures at low temperature, as inactivation of temperature-sensitive and cold-sensitive ded1 mutants gave highly correlated results. Interestingly, Ded1-dependent mRNAs exhibit greater than average 5’UTR length and propensity for secondary structure, implicating Ded1 in scanning though structured 5' UTRs. Reporter assays confirmed that cap- distal stem-loop insertions increase dependence on Ded1 but not eIF4A for efficient translation. While only a small fraction of mRNAs is strongly dependent on eIF4A, this dependence is significantly correlated with requirements for Ded1 and 5’UTR features characteristic of Ded1- dependent mRNAs. Our findings suggest that Ded1 is critically required to promote scanning through secondary structures within 5’UTRs; and while eIF4A cooperates with Ded1 in this function, it also promotes a step of initiation common to virtually all yeast mRNAs.
2015-03-02 | GSE66411 | 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 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:DEAD-box ATPases belong to an abundant class of proteins that are involved in virtually all aspects of RNA metabolism and are found in all kingdoms of life. When bound to a DEAD-box ATPase, the RNA substrate is forced into a kinked conformation that is incompatible with helical structures. Distortion of the RNA can result in unwinding of short RNA duplexes (helicase activity) or destabilize RNA-protein interactions, allowing DEAD-box ATPases to remodel mRNPs (RNPase activity). The RNPase activity makes DEAD-box ATPases suitable molecular building blocks for the implementation of checkpoints that confer directionality to the process of RNA biogenesis. Here, we provide data that characterizes the DEAD-box ATPase Dbp2 (SPBP8B7.16c) of the fission yeast Schizosaccharomyces pombe. Using ChIP-seq, we determined the sites where HTP-tagged Dbp2 associates with chromatin. ChIP-seq of Srp2-HTP is included as a reference protein that is known to associate with transcribing RNA polymerase II (RNAPII).
2024-06-30 | E-MTAB-13712 | biostudies-arrayexpress
Project description:DEAD-box ATPases belong to an abundant class of proteins that are involved in virtually all aspects of RNA metabolism and are found in all kingdoms of life. When bound to a DEAD-box ATPase, the RNA substrate is forced into a kinked conformation that is incompatible with helical structures. Distortion of the RNA can result in unwinding of short RNA duplexes (helicase activity) or destabilize RNA-protein interactions, allowing DEAD-box ATPases to remodel mRNPs (RNPase activity). The RNPase activity makes DEAD-box ATPases suitable molecular building blocks for the implementation of checkpoints that confer directionality to the process of RNA biogenesis. Here, we provide data that characterizes the DEAD-box ATPase Dbp2 (SPBP8B7.16c) of the fission yeast Schizosaccharomyces pombe. Using RNA-seq, we determined RNA expression profiles of a conditional depletion strain of Dbp2 and the corresponding wild type. For this, we placed the endogenous dbp2 gene under the control of the P.nmt1 promoter, which is repressed in the presence of thiamine. Cells were harvested at the beginning (t0) or the end (t5 or t9) of shift to thiamine-containing YES medium. S. cerevisiae spike-in cells were added in a 1:5 OD600 ratio immediately before harvesting.
2024-06-30 | E-MTAB-13717 | biostudies-arrayexpress
Project description:DEAD-box ATPases belong to an abundant class of proteins that are involved in virtually all aspects of RNA metabolism and are found in all kingdoms of life. When bound to a DEAD-box ATPase, the RNA substrate is forced into a kinked conformation that is incompatible with helical structures. Distortion of the RNA can result in unwinding of short RNA duplexes (helicase activity) or destabilize RNA-protein interactions, allowing DEAD-box ATPases to remodel mRNPs (RNPase activity). The RNPase activity makes DEAD-box ATPases suitable molecular building blocks for the implementation of checkpoints that confer directionality to the process of RNA biogenesis. Here, we provide data that characterizes the DEAD-box ATPase Dbp2 (SPBP8B7.16c) of the fission yeast Schizosaccharomyces pombe. Using calibrated RNAPII-ChIP-seq, we determined the transcription profiles of a conditional depletion strain of Dbp2 and the corresponding wild type. For this, we placed the endogenous dbp2 gene under the control of the P.nmt1 promoter, which is repressed in the presence of thiamine. Cells were crosslinked at the beginning (t0) or the end (t9) of a 9h shift to thiamine-containing YES medium. S. cerevisiae spike-in cells were added in a 1:5 OD600 ratio immediately before crosslinking.
2024-06-30 | E-MTAB-13714 | biostudies-arrayexpress