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eIF4G is required for the pioneer round of translation in mammalian cells

Nature Structural & Molecular Biology volume 11, pages 992–1000 (2004)Cite this article

Abstract

Nonsense-mediated mRNA decay (NMD) in mammalian cells targets cap-binding protein 80 (CBP80)-bound mRNA during or after a pioneer round of translation. It is unknown whether eukaryotic translation initiation factor 4G (eIF4G) functions in the pioneer round. We show that baculovirus-produced CBP80 and CBP20 independently interact with eIF4GI. The interactions between eIF4G and the heterodimer CBP80/20 suggest that eIF4G has a function in the pioneer initiation complex rather than merely a presence during remodeling to the steady-state complex. First, NMD is inhibited upon eIF4G cleavage by HIV-2 or poliovirus 2A protease. Second, eIF4GI coimmunopurifies with pre-mRNA, indicating that it associates with transcripts before the pioneer round. Third, eIF4G immunopurifies with Upf NMD factors and eIF4AIII, which are constituents of the pioneer translation initiation complex. We propose a model in which eIF4G serves to connect CBP80/20 with other initiation factors during the pioneer round of translation.

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Figure 1: Baculovirus-produced and purified CBP20-His and CBP80-His interact with cellular eIF4G and exogenously produced myc-eIF4GI using far-western analysis.
Figure 2: Evidence that three regions of eIF4GI independently interact with CBP80 based on coimmunopurifications using Cos cells.
Figure 3: CBP80 coimmunopurifies with HA-eIF4GII.
Figure 4: eIF4G associates with intron-containing pre-mRNA, the Upf2 and Upf3X NMD factors in an RNA-dependent manner, and the EJC component eIF4AIII.
Figure 5: Evidence that eIF4G functions in NMD: the kinetics of eIF4G cleavage by HIV-2 protease correlates with an inhibition of NMD.
Figure 6: Additional evidence that eIF4G functions in NMD: the kinetics of eIF4G cleavage by poliovirus 2A (PV 2A) protease correlates with an inhibition of NMD.
Figure 7: Model for the pioneer translation initiation complex in comparison to its product, the steady-state translation initiation complex.

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Acknowledgements

We thank M. Coldwell, L. McKendrick and S. Morley for pmyc-eIF4GI (1–1600) and derivatives, pBS-His-CBP80, anti-eIF4G and anti-PAPB; N. Sonenberg for anti-eIF4GII, pCMV-HA-eIF4GII and pcDNA3-HA-eIF4AIII; P. Mitchell and D. Tollervey for anti-Rrp4; K. Borden for the GST-eIF4E expression vector; J. Lewis for pRSET-CBP20; H. Baumann and B. Held for anti-MUP; T. Ohlmann for HIV-1 and HIV-2 protease expression vectors and R. Lloyd for the polio 2A protease expression vector. We are grateful to R. Lloyd, T. Ohlmann and S. Cusack for helpful conversations, Y.K. Kim for comments on the manuscript, and M. Kmieciak for help generating figures. This work was supported by US National Institutes of Health grants to L.E.M.

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  1. Fabrice Lejeune and Aparna C Ranganathan: These authors contributed equally to this work.

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  1. Department of Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester, 601 Elmwood Avenue, Box 712, Rochester, 14642, New York, USA

    Fabrice Lejeune, Aparna C Ranganathan & Lynne E Maquat

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Lejeune, F., Ranganathan, A. & Maquat, L. eIF4G is required for the pioneer round of translation in mammalian cells. Nat Struct Mol Biol 11, 992–1000 (2004). https://doi.org/10.1038/nsmb824

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