Functional characterization of IRESes by an inhibitor of the RNA helicase eIF4A

Abstract

RNA helicases are molecular motors that are involved in virtually all aspects of RNA metabolism. Eukaryotic initiation factor (eIF) 4A is the prototypical member of the DEAD-box family of RNA helicases. It is thought to use energy from ATP hydrolysis to unwind mRNA structure and, in conjunction with other translation factors, it prepares mRNA templates for ribosome recruitment during translation initiation. In screening marine extracts for new eukaryotic translation initiation inhibitors, we identified the natural product hippuristanol. We show here that this compound is a selective and potent inhibitor of eIF4A RNA-binding activity that can be used to distinguish between eIF4A-dependent and -independent modes of translation initiation in vitro and in vivo. We also show that poliovirus replication is delayed when infected cells are exposed to hippuristanol. Our study demonstrates the feasibility of selectively targeting members of the DEAD-box helicase family with small-molecule inhibitors.

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Figure 1: Hippuristanol inhibits cap-dependent translation initiation.
Figure 2: Hippuristanol inhibits translation initiation.
Figure 3: Effect of hippuristanol on ATP hydrolysis, ATP-binding and RNA-binding activities of eIF4A.
Figure 4: Selective inhibition of eIF4A activity by hippuristanol.
Figure 5: Translation driven by the PTV-1 IRES is independent of eIF4A in vitro and in vivo.
Figure 6: Poliovirus replication is delayed by inhibition of eIF4A in vivo.

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Acknowledgements

We thank R. Cencic and F. Robert for their comments on the manuscript; I. Harvey and I. Ko for technical assistance; E. Jankowsky (Case Western Reserve University) for his gift of Ded1p; W.C. Merrick (Case Western Reserve University) for his gift of eIF4F; N. Sonenberg (McGill University) for his gift of anti-eIF4GII antibody; and M. Moore (Brandeis University) for the generous supply of HeLa nuclear extracts. Plasmids pcDNA3-RLUC-POLIOIRES-FLUC and pGL3/Ren/CrPV/FF were kindly provided by N. Sonenberg and P. Sarnow (Stanford University), respectively. We appreciate the expertise provided to us by Y. Svitkin (McGill University) with the poliovirus infections. M.-E.B. was supported by a Canadian Institutes of Health Research (CIHR) Cancer Consortium Training Grant Award and a Fonds de Recherche en Santé du Québec studentship award; M.O. by the Max Kade Foundation (New York); L.L. by a CIHR Chemical Biology Fellowship and a Maysie MacSporran studentship; and L.S.C. by a UK Biotechnology and Biological Sciences Research Council studentship. This work was supported by a National Cancer Institute of Canada (#014313) and a US National Institutes of Health (NIH) grant (CA114475) to J.P., a Japan Ministry of Education, Culture, Sports, Science and Technology (MEXT) grant (17035067) to J.T. and an NIH grant (CA068262) to G.W. J.P. is a CIHR Senior Investigator.

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Correspondence to Jerry Pelletier.

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Supplementary information

Supplementary Fig. 1

Hippuristanol inhibits translation in rabbit reticulocyte lysates and wheat germ extracts. (PDF 50 kb)

Supplementary Fig. 2

Effect of hippuristanol on protein, RNA and DNA synthesis in vivo. (PDF 75 kb)

Supplementary Fig. 3

Hippuristanol does not inhibit crosslinking of eIF4B to RNA. (PDF 102 kb)

Supplementary Fig. 4

Hippuristanol can distinguish between eIF4A–dependent and eIF4A–independent translation in vitro. (PDF 205 kb)

Supplementary Fig. 5

Determination of the IC50 for hippuristanol on HeLa cell proliferation. (PDF 64 kb)

Supplementary Fig. 6

Structure–activity relationship of hippuristanol congeners. (PDF 89 kb)

Supplementary Fig. 7

Effect of hippuristanol congeners on eIF4AIf mediated helicase activity. (PDF 159 kb)

Supplementary Methods (PDF 53 kb)

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Bordeleau, M., Mori, A., Oberer, M. et al. Functional characterization of IRESes by an inhibitor of the RNA helicase eIF4A. Nat Chem Biol 2, 213–220 (2006). https://doi.org/10.1038/nchembio776

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