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A conserved PUF–Ago–eEF1A complex attenuates translation elongation

Nature Structural & Molecular Biology volume 19, pages 176–183 (2012)Cite this article

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Abstract

PUF (Pumilio/FBF) RNA-binding proteins and Argonaute (Ago) miRNA-binding proteins regulate mRNAs post-transcriptionally, each acting through similar, yet distinct, mechanisms. Here, we report that PUF and Ago proteins can also function together in a complex with a core translation elongation factor, eEF1A, to repress translation elongation. Both nematode (Caenorhabditis elegans) and mammalian PUF–Ago–eEF1A complexes were identified, using coimmunoprecipitation and recombinant protein assays. Nematode CSR-1 (Ago) promoted repression of FBF (PUF) target mRNAs in in vivo assays, and the FBF-1–CSR-1 heterodimer inhibited EFT-3 (eEF1A) GTPase activity in vitro. Mammalian PUM2–Ago–eEF1A inhibited translation of nonadenylated and polyadenylated reporter mRNAs in vitro. This repression occurred after translation initiation and led to ribosome accumulation within the open reading frame, roughly at the site where the nascent polypeptide emerged from the ribosomal exit tunnel. Together, these data suggest that a conserved PUF–Ago–eEF1A complex attenuates translation elongation.

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Figure 1: FBF-1 binds CSR-1 to repress target mRNA.
Figure 2: FBF-1–CSR-1–EFT-3 ternary complex formation reduces EFT-3 GTPase activity.
Figure 3: PUM2–Ago–eEF1A complex inhibits protein production.
Figure 4: Kinetics of PUM2 translational repression.
Figure 5: Human PUM2 attenuates translation elongation.

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Acknowledgements

We thank H. Tabara (Kyoto University) for providing CSR-1 and DRH-3 antibodies and E. Kipreos (University of Georgia) for the CYE-1 antibody. We thank Kimble and Wickens lab members for discussion; we also thank E. Lund and S. Kennedy for critical reading of the manuscript and A. Helsley-Marchbanks and L. Vanderploeg for help with the manuscript and figure preparation. Mass spectrometry was carried out with support from the Human Proteomics Program at the University of Wisconsin-Madison.

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Authors and Affiliations

  1. Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA

    Kyle Friend, Zachary T Campbell, Amy Cooke, Marvin P Wickens & Judith Kimble

  2. Graduate Program in Cellular and Molecular Biology, University of Wisconsin-Madison, Madison, Wisconsin, USA

    Amy Cooke, Marvin P Wickens & Judith Kimble

  3. Howard Hughes Medical Institute, University of Wisconsin-Madison, Madison, Wisconsin, USA

    Peggy Kroll-Conner & Judith Kimble

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Contributions

K.F. conducted the experiments with the exception of phylogenetic analysis (Z.T.C.), luciferase reporter mRNA production (A.C.) and csr-1 mutant generation (P.K.-C.). K.F., M.P.W. and J.K. prepared the manuscript. K.F. is supported by PF-10-127-01-DDC from the American Cancer Society, Z.T.C. by US National Institutes of Health (NIH) postdoctoral fellowship F32 GM095169, A.C. by NIH training grant T32 GM07215 and an Advanced Opportunity Fellowship from the University of Wisconsin-Madison, M.P.W. by NIH grants GM031892 and GM050942 and J.K. by NIH grant GM069454. J.K. is an investigator of the Howard Hughes Medical Institute.

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Correspondence to Judith Kimble.

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Friend, K., Campbell, Z., Cooke, A. et al. A conserved PUF–Ago–eEF1A complex attenuates translation elongation. Nat Struct Mol Biol 19, 176–183 (2012). https://doi.org/10.1038/nsmb.2214

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