A conserved motif in Argonaute-interacting proteins mediates functional interactions through the Argonaute PIWI domain

Abstract

Argonaute (Ago) proteins mediate silencing of nucleic acid targets by small RNAs. In fission yeast, Ago1, Tas3 and Chp1 assemble into a RITS complex, which silences transcription near centromeres. Here we describe a repetitive motif within Tas3, termed the 'Argonaute hook', that is conserved from yeast to humans and binds Ago proteins through their PIWI domains in vitro and in vivo. Site-directed mutation of key residues in the motif disrupts Ago binding and heterochromatic silencing in vivo. Unexpectedly, a PIWI domain pocket that binds the 5′ end of the short interfering RNA guide strand is required for direct binding of the Ago hook. Moreover, wild-type but not mutant Ago hook peptides derepress microRNA-mediated translational silencing of a target messenger RNA. Proteins containing the conserved Ago hook may thus be important regulatory components of effector complexes in RNA interference.

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Figure 1: A conserved protein motif mediates interaction with the S. pombe and human Argonaute proteins of the Ago subfamily34.
Figure 2: A bipartite linear motif in TNRC6B acts as an Ago hook by binding human AGO2 in vitro and in vivo.
Figure 3: The human, Ago-binding TNRC6B ortholog actively shuttles between cytoplasm and nucleus.
Figure 4: The Ago hook binds the AGO2 PIWI domain in a pocket crucial for 5′-end siRNA guide-strand recognition.
Figure 5: The Ago hook is essential for establishing TGS in S. pombe and specifically interferes with miRNA-mediated translational repression in D. melanogaster.

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Acknowledgements

We thank N. Daigle and J. Ellenberg for advice on imaging; D. Brunner (EMBL) and R. Allshire (Wellcome Trust Centre for Cell Biology) for help with S. pombe and strains; R. Pillai (EMBL) and W. Filipowicz (Friedich Miescher Institute) for Hiwi plasmid, advice and discussion; T. Gibson for linear-motif discussions; S. Narumiya (Kyoto University) for HeLa Kyoto cells; J. Parker and D. Barford for the A. fulgidus PIWI purification protocol; and members of A.G.L.'s laboratory, as well as A. Akhtar, J. Ellenberg, D. Brunner, J. Müller, K. Rippe, I. Mattaj and C. Margulies, for discussion. This work was supported by the EMBL and by grants from the EU Sixth Framework Programme, Marie Curie Early-Stage Training in Advanced Life Science Research (E.L.), the Peter and Traudl Engelhorn Foundation (M.H.), the Deutsche Forschungsgemeinschaft (M.W.H.), the Marie Curie Research Training Network 'Chromatin Plasticity' (A.G.L.) and the Network of Excellence 'The Epigenome' (A.G.L.).

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S.T., E.L., R.T., M.B., D.E., C.H. and A.G.L. performed the experiments. S.T., E.L., R.T., M.B., M.H., D.E., M.W.H. and A.G.L. designed, analyzed and interpreted the experiments. S.T. and A.G.L. wrote the paper with input from all other authors.

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Correspondence to Andreas G Ladurner.

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Till, S., Lejeune, E., Thermann, R. et al. A conserved motif in Argonaute-interacting proteins mediates functional interactions through the Argonaute PIWI domain. Nat Struct Mol Biol 14, 897–903 (2007). https://doi.org/10.1038/nsmb1302

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