Abstract
A paramount task in RNA interference research is to decipher the complex biology of cellular effectors, exemplified in humans by four pleiotropic Argonaute proteins (Ago1–Ago4). Here, we exploited DNA family shuffling, a molecular evolution technology, to generate chimeric Ago protein libraries for dissection of intricate phenotypes independently of prior structural knowledge. Through shuffling of human Ago2 and Ago3, we discovered two N-terminal motifs that govern RNA cleavage in concert with the PIWI domain. Structural modeling predicts an impact on protein rigidity and/or RNA-PIWI alignment, suggesting new mechanistic explanations for Ago3's slicing deficiency. Characterization of hybrids including Ago1 and Ago4 solidifies that slicing requires the juxtaposition and combined action of multiple disseminated modules. We also present a Gateway library of codon-optimized chimeras of human Ago1–Ago4 and molecular evolution analysis software as resources for future investigations into RNA interference sequence-structure-function relationships.
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Acknowledgements
The authors greatly appreciate funding of their group and work by the Heidelberg University Cluster of Excellence CellNetworks (grant number EXC 81) as well as by the Chica and Heinz Schaller Foundation. L.G.T. was supported by an European Molecular Biology Organization fellowship (EMBO ALTF 676-2010). We moreover thank H.-G. Kräusslich, R. Bartenschlager, G. Stöcklin and all lab members for helpful discussions and suggestions. In addition, we are indebted to S. Ruedel (University of Regensburg, Regensburg, Germany) for kindly providing her protocol for slicer assays as well as to W. Filipowicz (Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland) for his much appreciated gifts of Ago2-knockdown cells and of various plasmids for the tethering assays. Finally, we are grateful to E. Wiedtke for technical support as well as to S. Grosse for her extensive critical testing of our Salanto software and for providing sundry useful ideas for improvements.
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N.S. and D.G. designed the experiments in this work and wrote the manuscript. N.S. conducted all experiments. L.G.T. and R.B.R. contributed the Ago2 and Ago3 Rosetta models and assisted in their interpretation. C.B. performed the bioinformatical analyses of the DFS reactions and products, using the Salanto software tool that he, together with N.S., developed for this work.
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Schürmann, N., Trabuco, L., Bender, C. et al. Molecular dissection of human Argonaute proteins by DNA shuffling. Nat Struct Mol Biol 20, 818–826 (2013). https://doi.org/10.1038/nsmb.2607
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DOI: https://doi.org/10.1038/nsmb.2607
