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


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.


  1. 1

    Motamedi, M.R. et al. Two RNAi complexes, RITS and RDRC, physically interact and localize to noncoding centromeric RNAs. Cell 119, 789–802 (2004).

    CAS  Article  Google Scholar 

  2. 2

    Noma, K. et al. RITS acts in cis to promote RNA interference-mediated transcriptional and post-transcriptional silencing. Nat. Genet. 36, 1174–1180 (2004).

    CAS  Article  Google Scholar 

  3. 3

    Sugiyama, T., Cam, H., Verdel, A., Moazed, D. & Grewal, S.I. RNA-dependent RNA polymerase is an essential component of a self-enforcing loop coupling heterochromatin assembly to siRNA production. Proc. Natl. Acad. Sci. USA 102, 152–157 (2005).

    CAS  Article  Google Scholar 

  4. 4

    Verdel, A. et al. RNAi-mediated targeting of heterochromatin by the RITS complex. Science 303, 672–676 (2004).

    CAS  Article  Google Scholar 

  5. 5

    Buhler, M., Verdel, A. & Moazed, D. Tethering RITS to a nascent transcript initiates RNAi- and heterochromatin-dependent gene silencing. Cell 125, 873–886 (2006).

    CAS  Article  Google Scholar 

  6. 6

    Kim, D.H., Villeneuve, L.M., Morris, K.V. & Rossi, J.J. Argonaute-1 directs siRNA-mediated transcriptional gene silencing in human cells. Nat. Struct. Mol. Biol. 13, 793–797 (2006).

    CAS  Article  Google Scholar 

  7. 7

    Ting, A.H., Schuebel, K.E., Herman, J.G. & Baylin, S.B. Short double-stranded RNA induces transcriptional gene silencing in human cancer cells in the absence of DNA methylation. Nat. Genet. 37, 906–910 (2005).

    CAS  Article  Google Scholar 

  8. 8

    Janowski, B.A. et al. Involvement of AGO1 and AGO2 in mammalian transcriptional silencing. Nat. Struct. Mol. Biol. 13, 787–792 (2006).

    CAS  Article  Google Scholar 

  9. 9

    Herr, A.J., Jensen, M.B., Dalmay, T. & Baulcombe, D.C. RNA polymerase IV directs silencing of endogenous DNA. Science 308, 118–120 (2005).

    CAS  Article  Google Scholar 

  10. 10

    Lippman, Z. & Martienssen, R. The role of RNA interference in heterochromatic silencing. Nature 431, 364–370 (2004).

    CAS  Article  Google Scholar 

  11. 11

    Matzke, M.A. & Birchler, J.A. RNAi-mediated pathways in the nucleus. Nat. Rev. Genet. 6, 24–35 (2005).

    CAS  Article  Google Scholar 

  12. 12

    Verdel, A. & Moazed, D. RNAi-directed assembly of heterochromatin in fission yeast. FEBS Lett. 579, 5872–5878 (2005).

    CAS  Article  Google Scholar 

  13. 13

    Eulalio, A., Behm-Ansmant, I. & Izaurralde, E. P bodies: at the crossroads of post-transcriptional pathways. Nat. Rev. Mol. Cell Biol. 8, 9–22 (2007).

    CAS  Article  Google Scholar 

  14. 14

    Bruno, I. & Wilkinson, M.F. P-bodies react to stress and nonsense. Cell 125, 1036–1038 (2006).

    CAS  Article  Google Scholar 

  15. 15

    Marx, J. Molecular biology. P-bodies mark the spot for controlling protein production. Science 310, 764–765 (2005).

    CAS  Article  Google Scholar 

  16. 16

    Sheth, U. & Parker, R. Decapping and decay of messenger RNA occur in cytoplasmic processing bodies. Science 300, 805–808 (2003).

    CAS  Article  Google Scholar 

  17. 17

    Meister, G. et al. Identification of novel argonaute-associated proteins. Curr. Biol. 15, 2149–2155 (2005).

    CAS  Article  Google Scholar 

  18. 18

    Behm-Ansmant, I. et al. mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes. Genes Dev. 20, 1885–1898 (2006).

    CAS  Article  Google Scholar 

  19. 19

    Liu, J. et al. A role for the P-body component GW182 in microRNA function. Nat. Cell Biol. 7, 1261–1266 (2005).

    Article  Google Scholar 

  20. 20

    Jakymiw, A. et al. Disruption of GW bodies impairs mammalian RNA interference. Nat. Cell Biol. 7, 1267–1274 (2005).

    Article  Google Scholar 

  21. 21

    Ding, L., Spencer, A., Morita, K. & Han, M. The developmental timing regulator AIN-1 interacts with miRISCs and may target the argonaute protein ALG-1 to cytoplasmic P bodies in C. elegans. Mol. Cell 19, 437–447 (2005).

    CAS  Article  Google Scholar 

  22. 22

    Lingel, A., Simon, B., Izaurralde, E. & Sattler, M. Nucleic acid 3′-end recognition by the Argonaute2 PAZ domain. Nat. Struct. Mol. Biol. 11, 576–577 (2004).

    CAS  Article  Google Scholar 

  23. 23

    Lingel, A., Simon, B., Izaurralde, E. & Sattler, M. Structure and nucleic-acid binding of the Drosophila Argonaute 2 PAZ domain. Nature 426, 465–469 (2003).

    CAS  Article  Google Scholar 

  24. 24

    Song, J.J. et al. The crystal structure of the Argonaute2 PAZ domain reveals an RNA binding motif in RNAi effector complexes. Nat. Struct. Biol. 10, 1026–1032 (2003).

    CAS  Article  Google Scholar 

  25. 25

    Ma, J.B., Ye, K. & Patel, D.J. Structural basis for overhang-specific small interfering RNA recognition by the PAZ domain. Nature 429, 318–322 (2004).

    CAS  Article  Google Scholar 

  26. 26

    Yan, K.S. et al. Structure and conserved RNA binding of the PAZ domain. Nature 426, 468–474 (2003).

    Article  Google Scholar 

  27. 27

    Parker, J.S., Roe, S.M. & Barford, D. Structural insights into mRNA recognition from a PIWI domain-siRNA guide complex. Nature 434, 663–666 (2005).

    CAS  Article  Google Scholar 

  28. 28

    Ma, J.B. et al. Structural basis for 5′-end-specific recognition of guide RNA by the A. fulgidus Piwi protein. Nature 434, 666–670 (2005).

    CAS  Article  Google Scholar 

  29. 29

    Yuan, Y.R. et al. Crystal structure of A. aeolicus argonaute, a site-specific DNA-guided endoribonuclease, provides insights into RISC-mediated mRNA cleavage. Mol. Cell 19, 405–419 (2005).

    CAS  Article  Google Scholar 

  30. 30

    Rivas, F.V. et al. Purified Argonaute2 and an siRNA form recombinant human RISC. Nat. Struct. Mol. Biol. 12, 340–349 (2005).

    CAS  Article  Google Scholar 

  31. 31

    Liu, J. et al. Argonaute2 is the catalytic engine of mammalian RNAi. Science 305, 1437–1441 (2004).

    CAS  Article  Google Scholar 

  32. 32

    Meister, G. et al. Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. Mol. Cell 15, 185–197 (2004).

    CAS  Article  Google Scholar 

  33. 33

    Miyoshi, K., Tsukumo, H., Nagami, T., Siomi, H. & Siomi, M.C. Slicer function of Drosophila Argonautes and its involvement in RISC formation. Genes Dev. 19, 2837–2848 (2005).

    CAS  Article  Google Scholar 

  34. 34

    Tolia, N.H. & Joshua-Tor, L. Slicer and the Argonautes. Nat. Chem. Biol. 3, 36–43 (2007).

    CAS  Article  Google Scholar 

  35. 35

    Girard, A., Sachidanandam, R., Hannon, G.J. & Carmell, M.A. A germline-specific class of small RNAs binds mammalian Piwi proteins. Nature 442, 199–202 (2006).

    Article  Google Scholar 

  36. 36

    Aravin, A. et al. A novel class of small RNAs bind to MILI protein in mouse testes. Nature 442, 203–207 (2006).

    CAS  Article  Google Scholar 

  37. 37

    Grivna, S.T., Beyret, E., Wang, Z. & Lin, H. A novel class of small RNAs in mouse spermatogenic cells. Genes Dev. 20, 1709–1714 (2006).

    CAS  Article  Google Scholar 

  38. 38

    Okamura, K., Ishizuka, A., Siomi, H. & Siomi, M.C. Distinct roles for Argonaute proteins in small RNA-directed RNA cleavage pathways. Genes Dev. 18, 1655–1666 (2004).

    CAS  Article  Google Scholar 

  39. 39

    Rand, T.A., Ginalski, K., Grishin, N.V. & Wang, X. Biochemical identification of Argonaute 2 as the sole protein required for RNA-induced silencing complex activity. Proc. Natl. Acad. Sci. USA 101, 14385–14389 (2004).

    CAS  Article  Google Scholar 

  40. 40

    Rehwinkel, J., Behm-Ansmant, I., Gatfield, D. & Izaurralde, E. A crucial role for GW182 and the DCP1:DCP2 decapping complex in miRNA-mediated gene silencing. RNA 11, 1640–1647 (2005).

    CAS  Article  Google Scholar 

  41. 41

    Eystathioy, T. et al. A phosphorylated cytoplasmic autoantigen, GW182, associates with a unique population of human mRNAs within novel cytoplasmic speckles. Mol. Biol. Cell 13, 1338–1351 (2002).

    CAS  Article  Google Scholar 

  42. 42

    Petrie, V.J., Wuitschick, J.D., Givens, C.D., Kosinski, A.M. & Partridge, J.F. RNA interference (RNAi)-dependent and RNAi-independent association of the Chp1 chromodomain protein with distinct heterochromatic loci in fission yeast. Mol. Cell. Biol. 25, 2331–2346 (2005).

    CAS  Article  Google Scholar 

  43. 43

    Carmichael, J.B. et al. RNA interference effector proteins localize to mobile cytoplasmic puncta in Schizosaccharomyces pombe. Traffic 7, 1032–1044 (2006).

    CAS  Article  Google Scholar 

  44. 44

    Sen, G.L. & Blau, H.M. Argonaute 2/RISC resides in sites of mammalian mRNA decay known as cytoplasmic bodies. Nat. Cell Biol. 7, 633–636 (2005).

    CAS  Article  Google Scholar 

  45. 45

    Liu, J., Valencia-Sanchez, M.A., Hannon, G.J. & Parker, R. MicroRNA-dependent localization of targeted mRNAs to mammalian P-bodies. Nat. Cell Biol. 7, 719–723 (2005).

    CAS  Article  Google Scholar 

  46. 46

    Parker, J.S., Roe, S.M. & Barford, D. Crystal structure of a PIWI protein suggests mechanisms for siRNA recognition and slicer activity. EMBO J. 23, 4727–4737 (2004).

    CAS  Article  Google Scholar 

  47. 47

    Song, J.J., Smith, S.K., Hannon, G.J. & Joshua-Tor, L. Crystal structure of Argonaute and its implications for RISC slicer activity. Science 305, 1434–1437 (2004).

    CAS  Article  Google Scholar 

  48. 48

    Ehrenberg, M. & Tenson, T. A new beginning of the end of translation. Nat. Struct. Biol. 9, 85–87 (2002).

    CAS  Article  Google Scholar 

  49. 49

    Nakamura, Y. & Ito, K. Making sense of mimic in translation termination. Trends Biochem. Sci. 28, 99–105 (2003).

    CAS  Article  Google Scholar 

  50. 50

    Lewis, B.P., Shih, I.H., Jones-Rhoades, M.W., Bartel, D.P. & Burge, C.B. Prediction of mammalian microRNA targets. Cell 115, 787–798 (2003).

    CAS  Article  Google Scholar 

  51. 51

    Stark, A., Brennecke, J., Russell, R.B. & Cohen, S.M. Identification of Drosophila MicroRNA targets. PLoS Biol. 1, E60 (2003).

    Article  Google Scholar 

  52. 52

    Doench, J.G. & Sharp, P.A. Specificity of microRNA target selection in translational repression. Genes Dev. 18, 504–511 (2004).

    CAS  Article  Google Scholar 

  53. 53

    Lewis, B.P., Burge, C.B. & Bartel, D.P. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120, 15–20 (2005).

    CAS  Article  Google Scholar 

  54. 54

    Thermann, R. & Hentze, M.W. Drosophila miR2 induces pseudo-polysomes and inhibits translation initiation. Nature 447, 875–878 (2007).

    CAS  Article  Google Scholar 

  55. 55

    Partridge, J.F. et al. Functional separation of the requirements for establishment and maintenance of centromeric heterochromatin. Mol. Cell 26, 593–602 (2007).

    CAS  Article  Google Scholar 

  56. 56

    Golemis, E.A., Serebriiskii, I. & Law, S.F. The yeast two-hybrid system: criteria for detecting physiologically significant protein-protein interactions. Curr. Issues Mol. Biol. 1, 31–45 (1999).

    CAS  PubMed  Google Scholar 

  57. 57

    Kustatscher, G., Hothorn, M., Pugieux, C., Scheffzek, K. & Ladurner, A.G. Splicing regulates NAD metabolite binding to histone macroH2A. Nat. Struct. Mol. Biol. 12, 624–625 (2005).

    CAS  Article  Google Scholar 

  58. 58

    Moreno, S., Klar, A. & Nurse, P. Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol. 194, 795–823 (1991).

    CAS  Article  Google Scholar 

  59. 59

    Kelly, T.J. et al. The fission yeast cdc18+ gene product couples S phase to START and mitosis. Cell 74, 371–382 (1993).

    CAS  Article  Google Scholar 

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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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