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Dynamic refolding of IFN-γ mRNA enables it to function as PKR activator and translation template

Nature Chemical Biology volume 5, pages 896–903 (2009)Cite this article

Abstract

Interferon-γ mRNA activates the RNA-dependent protein kinase PKR, which in turn strongly attenuates translation of interferon-γ mRNA. Unlike riboswitches restricted to noncoding regions, the interferon-γ RNA domain that activates PKR comprises the 5′ UTR and 26 translated codons. Extensive interferon-γ coding sequence is thus dedicated to activating PKR and blocking interferon-γ synthesis. This implies that the PKR activator is disrupted by ribosomes during translation initiation and must refold promptly to restore PKR activation. The activator structure harbors an essential kink-turn, probably to allow formation of a pseudoknot that is critical for PKR activation. Three indispensable short helices, bordered by orientation-sensitive base pairs, align with the pseudoknot stem, generating RNA helix of sufficient length to activate PKR. Through gain-of-function mutations, we show that the RNA activator can adopt alternative conformations that activate PKR. This flexibility promotes efficient refolding of interferon-γ mRNA, which is necessary for its dual function as translation template and activator of PKR, and which thus prevents overexpression of this inflammatory cytokine.

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Figure 1: Structure of the PKR activator element in human IFN-γ mRNA.
Figure 2: A-U pairs at the base of helices S1 and S3 are critical for PKR activation.
Figure 3: A K-turn motif is essential for PKR activation.
Figure 4: Alternative RNA conformations in the loop mediate PKR activation.
Figure 5: Alternative RNA conformations in helix S2 mediate PKR activation.
Figure 6: Phylogenetic conservation of the RNA activator of PKR in IFN-γ mRNA.

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Acknowledgements

We thank E. Westhof for valuable suggestions and V. Bafna for bioinformatic analysis of the rodent IFN-γ genes. We thank A. Billiau (Catholic University of Leuven) for porcine IFN-γ cDNA. This work was supported by grants from the Israel Science Foundation and the German Research Foundation (DFG).

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, The Hebrew University-Hadassah Medical School, Jerusalem, Israel

    Smadar Cohen-Chalamish, Anat Hasson, Dahlia Weinberg, Lise Sarah Namer, Yona Banai, Farhat Osman & Raymond Kaempfer

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  1. Smadar Cohen-Chalamish
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Contributions

S.C.-C. performed in-line structure probing and cloned mouse IFN-γ DNA; S.C.-C., A.H., D.W. and F.O. generated mutant RNA transcripts; L.S.N. and F.O. expressed recombinant PKR; R.K. and Y.B. prepared rabbit reticulocyte ribosomes; A.H., Y.B., D.W. and S.C.-C. assayed PKR activation; A.H. and Y.B. measured translation efficiency in cells; R.K. and S.C.-C. designed the study.

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Correspondence to Raymond Kaempfer.

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Cohen-Chalamish, S., Hasson, A., Weinberg, D. et al. Dynamic refolding of IFN-γ mRNA enables it to function as PKR activator and translation template. Nat Chem Biol 5, 896–903 (2009). https://doi.org/10.1038/nchembio.234

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