Rocaglamide A (RocA) typifies a class of protein synthesis inhibitors that selectively kill aneuploid tumour cells and repress translation of specific messenger RNAs1,2,3,4. 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 unwinding5. However, rocaglate treatment may not phenocopy the loss of eIF4A activity, as these drugs actually increase the affinity between eIF4A and RNA1,2,6. 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.
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We are grateful to J. Tanaka for providing hippuristanol, to Y. Tomari for sharing DNA constructs, to H. Asahara and University of California, Berkeley DNA sequencing facility for help with the toeprinting assay, and to A. Pinder and F. Tan for support with deep sequencing analysis. We also thank the members of Ingolia, Lareau, and Tomari laboratories for discussion and technical support. N.T.I. is a Damon-Runyon-Rachleff Innovator supported in part by the Damon Runyon Cancer Research Foundation (DRR-37-15), the Searle Scholars Program (11-SSP-229), and the National Institute of General Medical Sciences of the National Institutes of Health (P50GM102706). This work used the Vincent J. Coates Genomics Sequencing Laboratory at University of California, Berkeley, supported by National Institutes of Health S10 Instrumentation Grants S10RR029668, S10RR027303, and OD018174. S.I. is a recipient of Human Frontier Science Program long-term fellowship. S.N.F. is a Howard Hughes Medical Institute Fellow of the Helen Hay Whitney Foundation.
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This file contains the raw data for Figure 4e.
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Nature Reviews Molecular Cell Biology (2017)