Letter | Published:

Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor

Nature volume 534, pages 558561 (23 June 2016) | Download Citation

Abstract

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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Data deposits

Sequences generated in this study have been deposited in the Gene Expression Omnibus under accession numbers GSE70211 and GSE79392.

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Acknowledgements

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.

Author information

Affiliations

  1. Department of Molecular and Cell Biology, Center for RNA Systems Biology, University of California, Berkeley, California 94720, USA

    • Shintaro Iwasaki
    • , Stephen N. Floor
    •  & Nicholas T. Ingolia

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Contributions

S.I. performed all experiments and analysed the data. Recombinant protein purification and the fluorescence polarization assay were performed with the help of S.N.F. S.I. and N.T.I. designed the experiments and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Nicholas T. Ingolia.

Extended data

Supplementary information

Excel files

  1. 1.

    Supplementary Table 1

    High-sensitivity (a) and low-sensitivity (b) mRNAs to 3 µM RocA. Each transcript is listed with its UCSC identifier, translation -fold change to mean [log2], q value, translation -fold change normalized to mitochondria footprints [log2], length of 5′ UTR, minimum ΔG calculated along 5′ UTR with 30-mer window, gene name, and gene description. (c) High-sensitivity mRNAs to 0.03 µM RocA are listed the same as (a).

  2. 2.

    Supplementary Table 2

    High-sensitivity (a) and low-sensitivity (b) mRNAs to 1 µM Hipp are listed the same as Supplemental Table 1.

  3. 3.

    Supplementary Table 3

    (a) PP242 target mRNAs. Each transcript is listed with its UCSC identifier, translation -fold change to mean [log2], q value, translation -fold change normalized to mitochondria footprints [log2], gene name, and gene description.

PDF files

  1. 1.

    Supplementary Figure 1

    This file contains the raw data for Figure 4e.

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DOI

https://doi.org/10.1038/nature17978

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