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| Status |
Public on Jun 15, 2016 |
| Title |
Rocaglamide A converts RNA helicase eIF4A into a sequence-specific translational repressor |
| Organisms |
Homo sapiens; synthetic construct |
| Experiment type |
Expression profiling by high throughput sequencing
Other
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| Summary |
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.
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| Overall design |
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).
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| |
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| Contributor(s) |
Iwasaki S, Floor SN, Ingolia NT |
| Citation(s) |
27309803 |
| |
| Submission date |
Jun 24, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Shintaro Iwasaki |
| E-mail(s) |
[email protected]
|
| Organization name |
RIKEN
|
| Department |
Cluster for Pioneering Research
|
| Street address |
S205 Bioscience Bldg. 2-1 Hirosawa
|
| City |
Wako |
| State/province |
Saitama |
| ZIP/Postal code |
351-0198 |
| Country |
Japan |
| |
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| Platforms (2) |
| GPL11154 |
Illumina HiSeq 2000 (Homo sapiens) |
| GPL15228 |
Illumina HiSeq 2000 (synthetic construct) |
|
| Samples (23)
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| Relations |
| BioProject |
PRJNA287879 |
| SRA |
SRP059825 |
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