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Prolyl 4-hydroxylation regulates Argonaute 2 stability

Nature volume 455, pages 421–424 (2008)Cite this article

Abstract

Human Argonaute (Ago) proteins are essential components of the RNA-induced silencing complexes (RISCs). Argonaute 2 (Ago2) has a P-element-induced wimpy testis (PIWI) domain, which folds like RNase H and is responsible for target RNA cleavage in RNA interference1. Proteins such as Dicer, TRBP, MOV10, RHA, RCK/p54 and KIAA1093 associate with Ago proteins and participate in small RNA processing, RISC loading and localization of Ago proteins in the cytoplasmic messenger RNA processing bodies1,2. However, mechanisms that regulate RNA interference remain obscure. Here we report physical interactions between Ago2 and the α-(P4H-α(I)) and β-(P4H-β) subunits of the type I collagen prolyl-4-hydroxylase (C-P4H(I)). Mass spectrometric analysis identified hydroxylation of the endogenous Ago2 at proline 700. In vitro, both Ago2 and Ago4 seem to be more efficiently hydroxylated than Ago1 and Ago3 by recombinant human C-P4H(I). Importantly, human cells depleted of P4H-α(I) or P4H-β by short hairpin RNA and P4H-α(I) null mouse embryonic fibroblast cells showed reduced stability of Ago2 and impaired short interfering RNA programmed RISC activity. Furthermore, mutation of proline 700 to alanine also resulted in destabilization of Ago2, thus linking Ago2 P700 and hydroxylation at this residue to its stability regulation. These findings identify hydroxylation as a post-translational modification important for Ago2 stability and effective RNA interference.

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Figure 1: Human Ago2 associates with C-P4H(I) subunits.
Figure 2: In vivo and in vitro hydroxylation of Ago2.
Figure 3: Impaired hydroxylation downregulates Ago2 stability.
Figure 4: Impaired C-P4H(I) reduced let-7 guided siRISC activity.

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Acknowledgements

We thank C. C. Mello, A. Grishok and P. A. Sharp for reading this manuscript and discussion. We thank E. Lehtimäki and R. Juntunen for in vitro hydroxylation assays, J. Lykke-Andersen and N. Kedersha for polyclonal anti-Dcp1a antibody and anti-Tia1 antibody, and the personnel of the Biocenter Oulu Transgenic Animal Core Facility and the University of Oulu Laboratory Animal Centre for technical assistance. We also thank the Kazusa DNA Research Institute (Japan) for providing the KIAA clones with numbers 1567, 1093, 1582 and 1460 and N. R. Wall for pOZ-FH-Ago1 plasmid. H.H.Q. was supported by a Canadian Institute of Health Research postdoctoral fellowship. This work was supported by grants from the Health Science Council of the Academy of Finland (202469) and the S. Jusélius foundation to J.M. and National Institutes of Health grants to J.P. (AG025688) and Y.S. (GM53874).

Author Contributions H.H.Q. performed tandem affinity purification, Co-IP, Ago2 stability, si/miRISC activity, P-body and stress-granule experiments; P.P.O. established GFP reporter stable cell lines and performed MG132 experiments in the Lee laboratory; D.C. and J.P. performed mass-spectrometric analysis for the tandem affinity purified Ago complexes and hydroxylation; J.M. and O.P. conducted in vitro hydroxylation and provided P4H-α(I) null MEF cells; Yujiang Shi helped in tandem affinity purification. H.H.Q. and Yang Shi conceived and designed the study, and wrote the manuscript.

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Authors and Affiliations

  1. Department of Pathology, Harvard Medical School, New Research Building 854, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA,

    Hank H. Qi & Yang Shi

  2. Cutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts 02129, USA ,

    Pat P. Ongusaha & Sam W. Lee

  3. Biocenter Oulu and Department of Medical Biochemistry and Molecular Biology, Oulu Centre for Cell-Matrix Research, University of Oulu, FIN-90014 Oulu, Finland

    Johanna Myllyharju & Outi Pakkanen

  4. Department of Human Genetics, Center for Neurodegenerative Disease, School of Medicine, Emory University, Atlanta, Georgia 30322, USA,

    Dongmei Cheng & Junmin Peng

  5. Division of Endocrinology, Department of Medicine and BCMP, Diabetes, and Hypertension, Brigham and Women’s Hospital and Harvard Medical School, 221 Longwood Avenue, Boston, Massachusetts 02115, USA,

    Yujiang Shi

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  1. Hank H. Qi
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Correspondence to Yang Shi.

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Qi, H., Ongusaha, P., Myllyharju, J. et al. Prolyl 4-hydroxylation regulates Argonaute 2 stability. Nature 455, 421–424 (2008). https://doi.org/10.1038/nature07186

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