Transcriptome wide analysis of translation efficiency in MCF7 cells using polysome profiling with and without eIF4A inhibition by hippuristanol treatment
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE134888 | GEO | 2019/11/21
REPOSITORIES: GEO
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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 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: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
Project description:Hippuristanol (Hipp) is a natural product that selectively inhibits protein synthesis by targeting eukaryotic initiation factor (eIF) 4A, a DEAD-box RNA helicase required for ribosome recruitmentt o mRNA templates. Using a CRISPR/Cas9-based variomics screen, we identify functional eIF4A1 Hipp-resistant alleles, which in turn allow us to link the translation-inhibitory and cytotoxic properties of Hipp to eIF4A1 target-engagement. Genome-wide translational profiling in the absence or presence of Hipp (~EC50) were undertaken and our validation studies provided insight into structure-activity relationships of eIF4A-dependent mRNAs.
2020-08-05 | GSE151687 | GEO
Project description:Ribosome profiling of MDA-MB-231 cells treated with Silvestrol to monitor transcriptome wide, eIF4A-dependent changes in translation efficiency Translation efficiency (TE) of mRNAs dervied from ribosome footprints was monitored in the presence or absence of 25 nM Silvestrol, an inhibitor of eukaryotic translation initiation factor 4A (eIF4A). Transcripts with reduced TE in the presence of Silvestrol were compare to transcripts with reduced TE in the presence of INK128, a catalytic mTOR inhbitor.
2014-09-13 | E-GEOD-61375 | 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: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: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: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:New and effective therapeutics are urgently needed for the treatment of pancreatic ductal adenocarcinoma (PDAC). The eIF4A/DDX2 RNA helicase drives translation of mRNAs with highly structured 5’UTRs. The natural compound silvestrol and synthetic analogues are potent and selective inhibitors of eIF4A1/2 that show promising activity in models of hematological malignancies. Here, we show silvestrol analogues have nanomolar activity against PDAC cell lines and organoids in vitro. Moreover, we see single agent activity in the KRAS/p53 mouse PDAC model and also against PDAC xenografts and primary, patient derived PDAC tumors. These therapeutic effects occur at non-toxic dose levels. Transcriptome-wide ribosome profiling, analyses of protein and gene expression, and translation reporter studies reveal that eIF4A inhibitors block an oncogenic translation program in PDAC cells that includes G-quadruplex containing mRNAs such as KRAS, MYC, YAP1, MET, SMAD3, TGFβ and others. Together, our data indicate that pharmacological inhibition of eIF4A disrupts oncoprotein production and shows efficacy across several PDAC models.
2021-03-03 | GSE120159 | GEO