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Turning catalytically inactive human Argonaute proteins into active slicer enzymes

Nature Structural & Molecular Biology volume 20, pages 814–817 (2013)Cite this article

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Abstract

Argonaute proteins interact with small RNAs that guide them to complementary target RNAs, thus leading to inhibition of gene expression. Some but not all Argonaute proteins are endonucleases and can cleave the complementary target RNA. Here, we have mutated inactive human Ago1 and Ago3 and generated catalytic Argonaute proteins. We find that two short sequence elements at the N terminus are important for activity. In addition, PIWI-domain mutations in Ago1 may misarrange the catalytic center. Our work helps in understanding of the structural requirements that make an Argonaute protein an active endonucleolytic enzyme.

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Figure 1: Passenger-strand cleavage activity.
Figure 2: The N-terminal domain of Ago3 inhibits its cleavage activity.
Figure 3: Two short sequence elements in the N-terminal domain of Ago3 are required for cleavage activity.
Figure 4: Alterations in the PIWI domain and the N terminus restore Ago1 slicing activity.

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Acknowledgements

We thank S. Ammon and C. Friederich for technical assistance. Our research is supported by grants from the Deutsche Forschungsgemeinschaft (SFB 960 projects B3 and B4 to G.M. and FOR855 to G.M.), the European Research Council (grant 242792 'sRNAs' to G.M.), the Bavarian Genome Research Network (BayGene to G.M.), the Bavarian Systems-Biology Network (BioSysNet to G.M.) and the European Union (grant 201102 'ONCOMIRs' to G.M.).

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Authors and Affiliations

  1. Biochemistry Center Regensburg, Laboratory for RNA Biology, University of Regensburg, Regensburg, Germany

    Judith Hauptmann, Anne Dueck, Simone Harlander, Janina Pfaff & Gunter Meister

  2. Institute of Biophysics and Physical Biochemistry, University of Regensburg, Regensburg, Germany

    Rainer Merkl

Authors
  1. Judith Hauptmann
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  2. Anne Dueck
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  3. Simone Harlander
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  4. Janina Pfaff
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  6. Gunter Meister
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Contributions

J.H., A.D. and G.M. designed the experiments. J.H. and A.D. performed the experiments. S.H. and J.P. prepared several Ago mutants and recombinant Ago proteins. R.M. did bioinformatical analyses and structural modeling. G.M. and J.H. wrote the manuscript.

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Correspondence to Gunter Meister.

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The authors declare no competing financial interests.

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Hauptmann, J., Dueck, A., Harlander, S. et al. Turning catalytically inactive human Argonaute proteins into active slicer enzymes. Nat Struct Mol Biol 20, 814–817 (2013). https://doi.org/10.1038/nsmb.2577

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