Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Argonaute proteins: functional insights and emerging roles

Subjects

Key Points

  • There have been recent insights from structural studies of Argonaute proteins, including human Argonautes.

  • Mechanisms of small RNA loading and sorting into distinct Argonaute proteins are emerging.

  • Distinct members of the AGO clade have different functions, and there are different repertoires of AGO proteins across species.

  • This Review discusses important and experimentally validated Argonaute protein binding partners.

  • Argonaute proteins can be regulated by post-translational modifications

  • Recently identified novel functions of AGO clade proteins include transcriptional silencing processes, alternative splicing and DNA double-strand break repair.

Abstract

Small-RNA-guided gene regulation has emerged as one of the fundamental principles in cell function, and the major protein players in this process are members of the Argonaute protein family. Argonaute proteins are highly specialized binding modules that accommodate the small RNA component — such as microRNAs (miRNAs), short interfering RNAs (siRNAs) or PIWI-associated RNAs (piRNAs) — and coordinate downstream gene-silencing events by interacting with other protein factors. Recent work has made progress in our understanding of classical Argonaute-mediated gene-silencing principles, such as the effects on mRNA translation and decay, but has also implicated Argonaute proteins in several other cellular processes, such as transcriptional regulation and splicing.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: A model for loading of small RNAs into AGO clade proteins.
Figure 2: A model for the recruitment of AGO proteins to target RNAs.
Figure 3: Post-translational AGO protein modifications.
Figure 4: Potential nuclear functions of AGO clade proteins.

References

  1. 1

    Drinnenberg, I. A. et al. RNAi in budding yeast. Science 326, 544–550 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. 2

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

    Article  CAS  Google Scholar 

  3. 3

    Peters, L. & Meister, G. Argonaute proteins: mediators of RNA silencing. Mol. Cell 26, 611–623 (2007).

    Article  CAS  PubMed  Google Scholar 

  4. 4

    Hutvagner, G. & Simard, M. J. Argonaute proteins: key players in RNA silencing. Nature Rev. Mol. Cell Biol. 9, 22–32 (2008).

    Article  CAS  Google Scholar 

  5. 5

    Siomi, M. C., Sato, K., Pezic, D. & Aravin, A. A. PIWI-interacting small RNAs: the vanguard of genome defence. Nature Rev. Mol. Cell Biol. 12, 246–258 (2011).

    Article  CAS  Google Scholar 

  6. 6

    Yigit, E. et al. Analysis of the C. elegans Argonaute family reveals that distinct Argonautes act sequentially during RNAi. Cell 127, 747–757 (2006).

    Article  CAS  PubMed  Google Scholar 

  7. 7

    Van Ex, F., Jacob, Y. & Martienssen, R. A. Multiple roles for small RNAs during plant reproduction. Curr. Opin. Plant Biol. 14, 588–593 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. 8

    Jinek, M. & Doudna, J. A. A three-dimensional view of the molecular machinery of RNA interference. Nature 457, 405–412 (2009).

    Article  CAS  PubMed  Google Scholar 

  9. 9

    Simon, B. et al. Recognition of 2′-O-methylated 3′-end of piRNA by the PAZ domain of a Piwi protein. Structure 19, 172–180 (2011).

    Article  CAS  PubMed  Google Scholar 

  10. 10

    Kwak, P. B. & Tomari, Y. The N domain of Argonaute drives duplex unwinding during RISC assembly. Nature Struct. Mol. Biol. 19, 145–151 (2012).

    Article  CAS  Google Scholar 

  11. 11

    Nakanishi, K., Weinberg, D. E., Bartel, D. P. & Patel, D. J. Structure of yeast Argonaute with guide RNA. Nature 486, 368–374 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. 12

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

    Article  CAS  PubMed  Google Scholar 

  13. 13

    Meister, G. et al. Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. Mol. Cell 15, 185–197 (2004). References 12 and 13 show that AGO2 is the catalytic subunit of human RISC that cleaves target RNAs complementary to the bound siRNA or miRNA.

    Article  CAS  PubMed  Google Scholar 

  14. 14

    Hauptmann, J. et al. Turning catalytically inactive human Argonaute proteins into active slicer enzymes. Nature Struct. Mol. Biol. 12 May 2013 (10.1038/nsmb.2577).

  15. 15

    Elkayam, E. et al. The structure of human Argonaute-2 in complex with miR-20a. Cell 150, 100–110 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. 16

    Schirle, N. T. & MacRae, I. J. The crystal structure of human Argonaute2. Science 336, 1037–1040 (2012). References 11, 15 and 16 solve the crystal structures of full-length eukaryotic AGO proteins. Reference 15 solves the structure of human AGO2 together with a bound miRNA. Reference 16 solves the structure in the presence of tryptophan and provides a model for TRP binding on the surface of the PIWI domain, which is used by G182 proteins to interact with AGO proteins. Reference 11 identifies a fourth catalytic residue defining a catalytic tetrad.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. 17

    Khvorova, A., Reynolds, A. & Jayasena, S. D. Functional siRNAs and miRNAs exhibit strand bias. Cell 115, 209–216 (2003).

    Article  CAS  PubMed  Google Scholar 

  18. 18

    Schwarz, D. S. et al. Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199–208 (2003).

    Article  CAS  PubMed  Google Scholar 

  19. 19

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

    Article  CAS  PubMed  Google Scholar 

  20. 20

    Maniataki, E. & Mourelatos, Z. A human, ATP-independent, RISC assembly machine fueled by pre-miRNA. Genes Dev. 19, 2979–2990 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. 21

    Gregory, R. I., Chendrimada, T. P., Cooch, N. & Shiekhattar, R. Human RISC couples microRNA biogenesis and posttranscriptional gene silencing. Cell 123, 631–640 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. 22

    Forstemann, K. et al. Normal microRNA maturation and germ-line stem cell maintenance requires loquacious, a double-stranded RNA-binding domain protein. PLoS Biol. 3, e236 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

    Liu, Q. et al. R2D2, a bridge between the initiation and effector steps of the Drosophila RNAi pathway. Science 301, 1921–1925 (2003).

    Article  PubMed  Google Scholar 

  24. 24

    Tomari, Y., Matranga, C., Haley, B., Martinez, N. & Zamore, P. D. A protein sensor for siRNA asymmetry. Science 306, 1377–1380 (2004).

    Article  CAS  PubMed  Google Scholar 

  25. 25

    Chendrimada, T. P. et al. TRBP recruits the Dicer complex to AGO2 for microRNA processing and gene silencing. Nature 436, 740–744 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. 26

    Haase, A. D. et al. TRBP, a regulator of cellular PKR and HIV-1 virus expression, interacts with Dicer and functions in RNA silencing. EMBO Rep. 6, 961–967 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

    Lee, Y. et al. The role of PACT in the RNA silencing pathway. EMBO J. 25, 522–532 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. 28

    Noland, C. L. Ma, E. & Doudna, J. A. siRNA repositioning for guide strand selection by human Dicer complexes. Mol. Cell 43, 110–121 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. 29

    Johnston, M., Geoffroy, M. C., Sobala, A., Hay, R. & Hutvagner, G. HSP90 protein stabilizes unloaded argonaute complexes and microscopic P-bodies in human cells. Mol. Biol. Cell 21, 1462–1469 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. 30

    Iki, T. et al. In vitro assembly of plant RNA-induced silencing complexes facilitated by molecular chaperone HSP90. Mol. Cell 39, 282–291 (2010).

    Article  CAS  PubMed  Google Scholar 

  31. 31

    Iwasaki, S. et al. Hsc70/Hsp90 chaperone machinery mediates ATP-dependent RISC loading of small RNA duplexes. Mol. Cell 39, 292–299 (2010).

    Article  CAS  PubMed  Google Scholar 

  32. 32

    Frohn, A. et al. Dicer-dependent and -independent Argonaute2 protein interaction networks in mammalian cells. Mol. Cell Proteom. 11, 1442–1456 (2012).

    Article  CAS  Google Scholar 

  33. 33

    Wang, B. et al. Distinct passenger strand and mRNA cleavage activities of human Argonaute proteins. Nature Struct. Mol. Biol. 16, 1259–1266 (2009).

    Article  CAS  Google Scholar 

  34. 34

    Robb, G. B. & Rana, T. M. RNA helicase A interacts with RISC in human cells and functions in RISC loading. Mol. Cell 26, 523–537 (2007).

    Article  CAS  PubMed  Google Scholar 

  35. 35

    Gu, S. et al. Thermodynamic stability of small hairpin RNAs highly influences the loading process of different mammalian Argonautes. Proc. Natl Acad. Sci. USA 108, 9208–9213 (2011).

    Article  PubMed  Google Scholar 

  36. 36

    Tomari, Y. et al. RISC assembly defects in the Drosophila RNAi mutant armitage. Cell 116, 831–841 (2004).

    Article  CAS  PubMed  Google Scholar 

  37. 37

    Petri, S. et al. Increased siRNA duplex stability correlates with reduced off-target and elevated on-target effects. RNA 17, 737–749 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. 38

    Rand, T. A., Petersen, S., Du, F. & Wang, X. Argonaute2 cleaves the anti-guide strand of siRNA during RISC activation. Cell 123, 621–629 (2005).

    Article  CAS  PubMed  Google Scholar 

  39. 39

    Matranga, C., Tomari, Y., Shin, C., Bartel, D. P. & Zamore, P. D. Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes. Cell 123, 607–620 (2005).

    Article  CAS  PubMed  Google Scholar 

  40. 40

    Leuschner, P. J., Ameres, S. L., Kueng, S. & Martinez, J. Cleavage of the siRNA passenger strand during RISC assembly in human cells. EMBO Rep. 7, 314–320 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. 41

    Ye, X. et al. Structure of C3PO and mechanism of human RISC activation. Nature Struct. Mol. Biol. 18, 650–657 (2011).

    Article  CAS  Google Scholar 

  42. 42

    Liu, Y. et al. C3PO, an endoribonuclease that promotes RNAi by facilitating RISC activation. Science 325, 750–753 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. 43

    Haase, A. D. et al. Probing the initiation and effector phases of the somatic piRNA pathway in Drosophila. Genes Dev. 24, 2499–2504 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. 44

    Zheng, K. et al. Mouse MOV10L1 associates with Piwi proteins and is an essential component of the Piwi-interacting RNA (piRNA) pathway. Proc. Natl Acad. Sci. USA 107, 11841–11846 (2010).

    Article  Google Scholar 

  45. 45

    Frost, R. J. et al. MOV10L1 is necessary for protection of spermatocytes against retrotransposons by Piwi-interacting RNAs. Proc. Natl Acad. Sci. USA 107, 11847–11852 (2010).

    Article  Google Scholar 

  46. 46

    Olivieri, D., Senti, K. A., Subramanian, S., Sachidanandam, R. & Brennecke, J. The cochaperone shutdown defines a group of biogenesis factors essential for all piRNA populations in Drosophila. Mol. Cell 47, 954–969 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. 47

    Preall, J. B. Czech, B., Guzzardo, P. M., Muerdter, F. & Hannon, G. J. Shutdown is a component of the Drosophila piRNA biogenesis machinery. RNA 18, 1446–1457 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. 48

    Xiol, J. et al. A role for FKBP6 and the chaperone machinery in piRNA amplification and transposon silencing. Mol. Cell 47, 970–979 (2012).

    Article  CAS  PubMed  Google Scholar 

  49. 49

    Czech, B. et al. Hierarchical rules for Argonaute loading in Drosophila. Mol. Cell 36, 445–456 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. 50

    Forstemann, K., Horwich, M. D., Wee, L., Tomari, Y. & Zamore, P. D. Drosophila microRNAs are sorted into functionally distinct Argonaute complexes after production by Dicer-1. Cell 130, 287–297 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. 51

    Tomari, Y., Du, T. & Zamore, P. D. Sorting of Drosophila small silencing RNAs. Cell 130, 299–308 (2007). References 50 and 51 show that D. melanogaster miRNAs are sorted into Ago1, and siRNAs are sorted into Ago2. It is also showed that the double-stranded nature of the precursor is important for sorting.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. 52

    Okamura, K., Liu, N. & Lai, E. C. Distinct mechanisms for microRNA strand selection by Drosophila Argonautes. Mol. Cell 36, 431–444 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. 53

    Ameres, S. L., Hung, J. H., Xu, J., Weng, Z. & Zamore, P. D. Target RNA-directed tailing and trimming purifies the sorting of endo-siRNAs between the two Drosophila Argonaute proteins. RNA 17, 54–63 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. 54

    Czech, B. & Hannon, G. J. Small RNA sorting: matchmaking for Argonautes. Nature Rev. Genet. 12, 19–31 (2011).

    Article  CAS  PubMed  Google Scholar 

  55. 55

    Voinnet, O. Origin, biogenesis, and activity of plant microRNAs. Cell 136, 669–687 (2009).

    Article  CAS  Google Scholar 

  56. 56

    Eun, C. et al. AGO6 functions in RNA-mediated transcriptional gene silencing in shoot and root meristems in Arabidopsis thaliana. PLoS ONE 6, e25730 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. 57

    Jouannet, V. et al. Cytoplasmic Arabidopsis AGO7 accumulates in membrane-associated siRNA bodies and is required for ta-siRNA biogenesis. EMBO J. 31, 1704–1713 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. 58

    Montgomery, T. A. et al. Specificity of ARGONAUTE7–miR390 interaction and dual functionality in TAS3 trans-acting siRNA formation. Cell 133, 128–141 (2008).

    Article  CAS  Google Scholar 

  59. 59

    Mi, S. et al. Sorting of small RNAs into Arabidopsis Argonaute complexes is directed by the 5′ terminal nucleotide. Cell 133, 116–127 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. 60

    Olmedo-Monfil, V. et al. Control of female gamete formation by a small RNA pathway in Arabidopsis. Nature 464, 628–632 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. 61

    Zhu, H. et al. Arabidopsis Argonaute10 specifically sequesters miR166/165 to regulate shoot apical meristem development. Cell 145, 242–256 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. 62

    Burroughs, A. M. et al. Deep-sequencing of human Argonaute-associated small RNAs provides insight into miRNA sorting and reveals Argonaute association with RNA fragments of diverse origin. RNA Biol. 8, 158–177 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. 63

    Dueck, A. Ziegler, C., Eichner, A., Berezikov, E. & Meister, G. microRNAs associated with the different human Argonaute proteins. Nucleic Acids Res. 40, 9850–9862 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. 64

    Wang, D. et al. Quantitative functions of Argonaute proteins in mammalian development. Genes Dev. 26, 693–704 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. 65

    Modzelewski, A. J., Holmes, R. J., Hilz, S., Grimson, A. & Cohen, P. E. AGO4 regulates entry into meiosis and influences silencing of sex chromosomes in the male mouse germline. Dev. Cell 23, 251–264 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. 66

    Hu, Q. et al. DICER- and AGO3-dependent generation of retinoic acid-induced DR2 Alu RNAs regulates human stem cell proliferation. Nature Struct. Mol. Biol. 19, 1168–1175 (2012).

    Article  CAS  Google Scholar 

  67. 67

    Cheloufi, S., Dos Santos, C. O., Chong, M. M. & Hannon, G. J. A Dicer-independent miRNA biogenesis pathway that requires AGO catalysis. Nature 465, 584–589 (2010). Reference 67 found that miR-451 is processed by AGO2 independently of Dicer. It also provides a model for the conservation of AGO2 cleavage activity. Reference 156 reported similar results in zebrafish.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. 68

    Frank, F., Sonenberg, N. & Nagar, B. Structural basis for 5′-nucleotide base-specific recognition of guide RNA by human AGO2. Nature 465, 818–822 (2010).

    Article  CAS  Google Scholar 

  69. 69

    Frank, F., Hauver, J., Sonenberg, N. & Nagar, B. Arabidopsis Argonaute MID domains use their nucleotide specificity loop to sort small RNAs. EMBO J. 31, 3588–3595 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. 70

    Bartel, D. P. MicroRNAs: target recognition and regulatory functions. Cell 136, 215–233 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. 71

    Huntzinger, E. & Izaurralde, E. Gene silencing by microRNAs: contributions of translational repression and mRNA decay. Nature Rev. Genet. 12, 99–110 (2011).

    Article  CAS  PubMed  Google Scholar 

  72. 72

    Krol, J., Loedige, I. & Filipowicz, W. The widespread regulation of microRNA biogenesis, function and decay. Nature Rev. Genet. 11, 597–610 (2010).

    Article  CAS  PubMed  Google Scholar 

  73. 73

    Meister, G. & Tuschl, T. Mechanisms of gene silencing by double-stranded RNA. Nature 431, 343–349 (2004).

    Article  CAS  Google Scholar 

  74. 74

    Bazzini, A. A., Lee, M. T. & Giraldez, A. J. Ribosome profiling shows that miR-430 reduces translation before causing mRNA decay in zebrafish. Science 336, 233–237 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. 75

    Bethune, J., Artus-Revel, C. G. & Filipowicz, W. Kinetic analysis reveals successive steps leading to miRNA-mediated silencing in mammalian cells. EMBO Rep. 13, 716–723 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. 76

    Djuranovic, S. Nahvi, A. & Green, R. miRNA-mediated gene silencing by translational repression followed by mRNA deadenylation and decay. Science 336, 237–240 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. 77

    Guo, H., Ingolia, N. T., Weissman, J. S. & Bartel, D. P. Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature 466, 835–840 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. 78

    Liu, J. et al. A role for the P-body component GW182 in microRNA function. Nature Cell Biol. 7, 1161–1166 (2005).

    Article  CAS  Google Scholar 

  79. 79

    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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. 80

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

    Article  CAS  PubMed  Google Scholar 

  81. 81

    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). References 78–81 together with reference 19 identify GW proteins as important interactors of AGO proteins. It was also shown that GW proteins are essential for miRNA-guided gene silencing in various organisms.

    Article  CAS  PubMed  Google Scholar 

  82. 82

    Fabian, M. R. et al. Mammalian miRNA RISC recruits CAF1 and PABP to affect PABP-dependent deadenylation. Mol. Cell 35, 868–880 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. 83

    Zekri, L., Huntzinger, E., Heimstadt, S. & Izaurralde, E. The silencing domain of GW182 interacts with PABPC1 to promote translational repression and degradation of microRNA targets and is required for target release. Mol. Cell. Biol. 29, 6220–6231 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. 84

    Moretti, F., Kaiser, C., Zdanowicz-Specht, A. & Hentze, M. W. PABP and the poly(A) tail augment microRNA repression by facilitated miRISC binding. Nature Struct. Mol. Biol. 19, 603–608 (2012).

    Article  CAS  Google Scholar 

  85. 85

    Fabian, M. R. et al. miRNA-mediated deadenylation is orchestrated by GW182 through two conserved motifs that interact with CCR4-NOT. Nature Struct. Mol. Biol. 18, 1211–1217 (2011).

    Article  CAS  Google Scholar 

  86. 86

    Chekulaeva, M. et al. miRNA repression involves GW182-mediated recruitment of CCR4-NOT through conserved W-containing motifs. Nature Struct. Mol. Biol. 18, 1218–1226 (2011).

    Article  CAS  Google Scholar 

  87. 87

    Braun, J. E., Huntzinger, E., Fauser, M. & Izaurralde, E. GW182 proteins directly recruit cytoplasmic deadenylase complexes to miRNA targets. Mol. Cell 44, 120–133 (2011).

    Article  CAS  Google Scholar 

  88. 88

    Huntzinger, E. et al. The interactions of GW182 proteins with PABP and deadenylases are required for both translational repression and degradation of miRNA targets. Nucleic Acids Res. 41, 978–994 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. 89

    Hock, J. et al. Proteomic and functional analysis of Argonaute-containing mRNA-protein complexes in human cells. EMBO Rep. 8, 1052–1060 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. 90

    Weinmann, L. et al. Importin 8 is a gene silencing factor that targets Argonaute proteins to distinct mRNAs. Cell 136, 496–507 (2009).

    Article  CAS  PubMed  Google Scholar 

  91. 91

    Landthaler, M. et al. Molecular characterization of human Argonaute-containing ribonucleoprotein complexes and their bound target mRNAs. RNA 14, 2580–2596 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. 92

    van Kouwenhove, M., Kedde, M. & Agami, R. MicroRNA regulation by RNA-binding proteins and its implications for cancer. Nature Rev. Cancer 11, 644–656 (2011).

    Article  CAS  Google Scholar 

  93. 93

    Miles, W. O., Tschop, K., Herr, A., Ji, J. Y. & Dyson, N. J. Pumilio facilitates miRNA regulation of the E2F3 oncogene. Genes Dev. 26, 356–368 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. 94

    Jin, H. et al. Human UPF1 participates in small RNA-induced mRNA downregulation. Mol. Cell. Biol. 29, 5789–5799 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. 95

    James, V. et al. LIM-domain proteins, LIMD1, Ajuba, and WTIP are required for microRNA-mediated gene silencing. Proc. Natl Acad. Sci. USA 107, 12499–12504 (2010).

    Article  PubMed  Google Scholar 

  96. 96

    Su, H. et al. Mammalian hyperplastic discs homolog EDD regulates miRNA-mediated gene silencing. Mol. Cell 43, 97–109 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  97. 97

    Jannot, G. et al. The ribosomal protein RACK1 is required for microRNA function in both C. elegans and humans. EMBO Rep. 12, 581–586 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  98. 98

    Leung, A. K. et al. Genome-wide identification of AGO2 binding sites from mouse embryonic stem cells with and without mature microRNAs. Nature Struct. Mol. Biol. 18, 237–244 (2011).

    Article  CAS  Google Scholar 

  99. 99

    Friend, K. et al. A conserved PUF–Ago–eEF1A complex attenuates translation elongation. Nature Struct. Mol. Biol. 19, 176–183 (2012).

    Article  CAS  Google Scholar 

  100. 100

    Pinder, B. D. & Smibert, C. A. microRNA-independent recruitment of Argonaute 1 to nanos mRNA through the Smaug RNA-binding protein. EMBO Rep. 14, 80–86 (2013).

    Article  CAS  PubMed  Google Scholar 

  101. 101

    Kirino, Y. et al. Arginine methylation of Piwi proteins catalysed by dPRMT5 is required for Ago3 and Aub stability. Nature Cell Biol. 11, 652–658 (2009).

    Article  CAS  PubMed  Google Scholar 

  102. 102

    Meister, G., Eggert, C. & Fischer, U. SMN-mediated assembly of RNPs: a complex story. Trends Cell Biol. 12, 472–478 (2002).

    Article  CAS  PubMed  Google Scholar 

  103. 103

    Siomi, M. C., Mannen, T. & Siomi, H. How does the royal family of Tudor rule the PIWI-interacting RNA pathway? Genes Dev. 24, 636–646 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  104. 104

    Tanaka, T. et al. Tudor domain containing 7 (Tdrd7) is essential for dynamic ribonucleoprotein (RNP) remodeling of chromatoid bodies during spermatogenesis. Proc. Natl Acad. Sci. USA 108, 10579–10584 (2011).

    Article  PubMed  Google Scholar 

  105. 105

    Zamparini, A. L. et al. Vreteno, a gonad-specific protein, is essential for germline development and primary piRNA biogenesis in Drosophila. Development 138, 4039–4050 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  106. 106

    Qi, H. H. et al. Prolyl 4-hydroxylation regulates Argonaute 2 stability. Nature 455, 421–424 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  107. 107

    Leung, A. K. et al. Poly(ADP-ribose) regulates stress responses and microRNA activity in the cytoplasm. Mol. Cell 42, 489–499 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  108. 108

    Rybak, A. et al. The let-7 target gene mouse lin-41 is a stem cell specific E3 ubiquitin ligase for the miRNA pathway protein Ago2. Nature Cell Biol. 11, 1411–1420 (2009).

    Article  CAS  PubMed  Google Scholar 

  109. 109

    Loedige, I., Gaidatzis, D., Sack, R., Meister, G. & Filipowicz, W. The mammalian TRIM-NHL protein TRIM71/LIN-41 is a repressor of mRNA function. Nucleic Acids Res. 41, 518–532 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  110. 110

    Hammell, C. M. Lubin, I., Boag, P. R., Blackwell, T. K. & Ambros, V. nhl-2 modulates microRNA activity in Caenorhabditis elegans. Cell 136, 926–938 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  111. 111

    Schwamborn, J. C., Berezikov, E. & Knoblich, J. A. The TRIM-NHL protein TRIM32 activates microRNAs and prevents self-renewal in mouse neural progenitors. Cell 136, 913–925 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  112. 112

    Gibbings, D. et al. Selective autophagy degrades DICER and AGO2 and regulates miRNA activity. Nature Cell Biol. 14, 1314–1321 (2012).

    Article  CAS  Google Scholar 

  113. 113

    Mourelatos, Z. et al. miRNPs: a novel class of ribonucleoproteins containing numerous microRNAs. Genes Dev. 16, 720–728 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  114. 114

    Hutvágner, G. & Zamore, P. D. A microRNA in a multiple-turnover RNAi enzyme complex. Science 297, 2056–2060 (2002).

    Article  CAS  PubMed  Google Scholar 

  115. 115

    Zeng, Y., Sankala, H., Zhang, X. & Graves, P. R. Phosphorylation of Argonaute 2 at serine-387 facilitates its localization to processing bodies. Biochem. J. 413, 429–436 (2008).

    Article  CAS  PubMed  Google Scholar 

  116. 116

    Rudel, S. et al. Phosphorylation of human Argonaute proteins affects small RNA binding. Nucleic Acids Res. 39, 2330–2343 (2011).

    Article  CAS  PubMed  Google Scholar 

  117. 117

    Adams, B. D., Claffey, K. P. & White, B. A. Argonaute-2 expression is regulated by epidermal growth factor receptor and mitogen-activated protein kinase signaling and correlates with a transformed phenotype in breast cancer cells. Endocrinology 150, 14–23 (2009). References 115–117 show that AGO proteins can be phosphorylated at specific sites, and specific signalling pathways are implicated in AGO function.

    Article  CAS  PubMed  Google Scholar 

  118. 118

    Rudel, S., Flatley, A., Weinmann, L., Kremmer, E. & Meister, G. A multifunctional human Argonaute2-specific monoclonal antibody. RNA 14, 1244–1253 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  119. 119

    Robb, G. B., Brown, K. M., Khurana, J. & Rana, T. M. Specific and potent RNAi in the nucleus of human cells. Nature Struct. Mol. Biol. 12, 133–137 (2005).

    Article  CAS  Google Scholar 

  120. 120

    Cernilogar, F. M. et al. Chromatin-associated RNA interference components contribute to transcriptional regulation in Drosophila. Nature 480, 391–395 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  121. 121

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

    Article  CAS  Google Scholar 

  122. 122

    Verdel, A. et al. RNAi-mediated targeting of heterochromatin by the RITS complex. Science 303, 672–676 (2004). This paper identifies the Ago-containing RNA-induced initiation of transcriptional gene silencing (RITS) complex, which is the mediator of small-RNA-guided transcriptional gene-silencing processes in Schizosaccharomyces pombe.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  123. 123

    Grewal, S. I. RNAi-dependent formation of heterochromatin and its diverse functions. Curr. Opin. Genet. Dev. 20, 134–141 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  124. 124

    Qi, Y. et al. Distinct catalytic and non-catalytic roles of ARGONAUTE4 in RNA-directed DNA methylation. Nature 443, 1008–1012 (2006).

    Article  PubMed  Google Scholar 

  125. 125

    Zilberman, D., Cao, X. & Jacobsen, S. E. ARGONAUTE4 control of locus-specific siRNA accumulation and DNA and histone methylation. Science 299, 716–719 (2003).

    Article  CAS  Google Scholar 

  126. 126

    Zilberman, D. et al. Role of Arabidopsis ARGONAUTE4 in RNA-directed DNA methylation triggered by inverted repeats. Curr. Biol. 14, 1214–1220 (2004).

    Article  CAS  PubMed  Google Scholar 

  127. 127

    Li, C. F. et al. An ARGONAUTE4-containing nuclear processing center colocalized with Cajal bodies in Arabidopsis thaliana. Cell 126, 93–106 (2006).

    Article  CAS  Google Scholar 

  128. 128

    Pontes, O. et al. The Arabidopsis chromatin-modifying nuclear siRNA pathway involves a nucleolar RNA processing center. Cell 126, 79–92 (2006).

    Article  CAS  PubMed  Google Scholar 

  129. 129

    Matzke, M., Kanno, T., Daxinger, L., Huettel, B. & Matzke, A. J. RNA-mediated chromatin-based silencing in plants. Curr. Opin. Cell Biol. 21, 367–376 (2009).

    Article  CAS  Google Scholar 

  130. 130

    Vastenhouw, N. L. & Plasterk, R. H. RNAi protects the Caenorhabditis elegans germline against transposition. Trends Genet. 20, 314–319 (2004).

    Article  CAS  PubMed  Google Scholar 

  131. 131

    Vastenhouw, N. L. et al. A genome-wide screen identifies 27 genes involved in transposon silencing in C. elegans. Curr. Biol. 13, 1311–1316 (2003).

    Article  CAS  PubMed  Google Scholar 

  132. 132

    Sijen, T. & Plasterk, R. H. Transposon silencing in the Caenorhabditis elegans germ line by natural RNAi. Nature 426, 310–314 (2003).

    Article  CAS  PubMed  Google Scholar 

  133. 133

    Guang, S. et al. An Argonaute transports siRNAs from the cytoplasm to the nucleus. Science 321, 537–541 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  134. 134

    Guang, S. et al. Small regulatory RNAs inhibit RNA polymerase II during the elongation phase of transcription. Nature 465, 1097–1101 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  135. 135

    Buckley, B. A. et al. A nuclear Argonaute promotes multigenerational epigenetic inheritance and germline immortality. Nature 489, 447–451 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  136. 136

    Pal-Bhadra, M., Bhadra, U. & Birchler, J. A. RNAi related mechanism affect both transcriptional and posttranscriptional transgene silencing in Drosophila. Mol. Cell 9, 315–327 (2002).

    Article  CAS  PubMed  Google Scholar 

  137. 137

    Pal-Bhadra, M. et al. Heterochromatic silencing and HP1 localization in Drosophila are dependent on the RNAi machinery. Science 303, 669–672 (2004).

    Article  CAS  PubMed  Google Scholar 

  138. 138

    Moshkovich, N. et al. RNAi-independent role for Argonaute2 in CTCF/CP190 chromatin insulator function. Genes Dev. 25, 1686–1701 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  139. 139

    Nishi, K., Nishi, A., Nagasawa, T. & Ui-Tei, K. Human TNRC6A is an Argonaute-navigator protein for microRNA-mediated gene silencing in the nucleus. RNA 19, 17–35 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  140. 140

    Janowski, B. A. et al. Activating gene expression in mammalian cells with promoter-targeted duplex RNAs. Nature Chem. Biol. 3, 166–173 (2007).

    Article  CAS  Google Scholar 

  141. 141

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

    Article  CAS  Google Scholar 

  142. 142

    Schwartz, J. C. et al. Antisense transcripts are targets for activating small RNAs. Nature Struct. Mol. Biol. 15, 842–848 (2008).

    Article  CAS  Google Scholar 

  143. 143

    Chu, Y., Yue, X., Younger, S. T., Janowski, B. A. & Corey, D. R. Involvement of Argonaute proteins in gene silencing and activation by RNAs complementary to a non-coding transcript at the progesterone receptor promoter. Nucleic Acids Res. 38, 7736–7748 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  144. 144

    Benhamed, M., Herbig, U., Ye, T., Dejean, A. & Bischof, O. Senescence is an endogenous trigger for microRNA-directed transcriptional gene silencing in human cells. Nature Cell Biol. 14, 266–275 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  145. 145

    Allo, M. et al. Control of alternative splicing through siRNA-mediated transcriptional gene silencing. Nature Struct. Mol. Biol. 16, 717–724 (2009).

    Article  CAS  Google Scholar 

  146. 146

    Ameyar-Zazoua, M. et al. Argonaute proteins couple chromatin silencing to alternative splicing. Nature Struct. Mol. Biol. 19, 998–1004 (2012).

    Article  CAS  Google Scholar 

  147. 147

    Taliaferro, J. M. et al. Two new and distinct roles for Drosophila Argonaute-2 in the nucleus: alternative pre-mRNA splicing and transcriptional repression. Genes Dev. 27, 378–389 (2013). References 145–147 implicate AGO proteins in alternative splicing processes in human and fly cells.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  148. 148

    Wahl, M. C., Will, C. L. & Luhrmann, R. The spliceosome: design principles of a dynamic RNP machine. Cell 136, 701–718 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  149. 149

    Wei, W. et al. A role for small RNAs in DNA double-strand break repair. Cell 149, 101–112 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  150. 150

    Michalik, K. M., Bottcher, R. & Forstemann, K. A small RNA response at DNA ends in Drosophila. Nucleic Acids Res. 40, 9596–9603 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  151. 151

    Francia, S. et al. Site-specific DICER and DROSHA RNA products control the DNA-damage response. Nature 488, 231–235 (2012). References 149–151 showed that small RNAs are produced from DNA double-strand breaks. References 149 and 151 also show that gene-silencing factors, such as AGO proteins or Dicer, are important for DNA double-strand break repair mechanisms.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  152. 152

    Chatterjee, S. & Grosshans, H. Active turnover modulates mature microRNA activity in Caenorhabditis elegans. Nature 461, 546–549 (2009).

    Article  CAS  PubMed  Google Scholar 

  153. 153

    Salmena, L., Poliseno, L., Tay, Y., Kats, L. & Pandolfi, P. P. A ceRNA hypothesis: the Rosetta Stone of a hidden RNA language? Cell 146, 353–358 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  154. 154

    Westholm, J. O. & Lai, E. C. Mirtrons: microRNA biogenesis via splicing. Biochimie 93, 1897–1904 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  155. 155

    Ender, C. et al. A human snoRNA with microRNA-like functions. Mol. Cell 32, 519–528 (2008).

    Article  CAS  PubMed  Google Scholar 

  156. 156

    Cifuentes, D. et al. A novel miRNA processing pathway independent of Dicer requires Argonaute2 catalytic activity. Science 328, 1694–1698 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  157. 157

    Diederichs, S. & Haber, D. A. Dual role for Argonautes in microRNA processing and posttranscriptional regulation of microRNA expression. Cell 131, 1097–1108 (2007).

    Article  CAS  PubMed  Google Scholar 

  158. 158

    Liu, X., Jin, D. Y., McManus, M. T. & Mourelatos, Z. Precursor microRNA-programmed silencing complex assembly pathways in mammals. Mol. Cell 46, 507–517 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  159. 159

    Bouasker, S. & Simard, M. J. The slicing activity of miRNA-specific Argonautes is essential for the miRNA pathway in C. elegans. Nucleic Acids Res. 40, 10452–10462 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  160. 160

    Czech, B. et al. An endogenous small interfering RNA pathway in Drosophila. Nature 453, 798–802 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  161. 161

    Kawamura, Y. et al. Drosophila endogenous small RNAs bind to Argonaute 2 in somatic cells. Nature 453, 793–797 (2008).

    Article  CAS  PubMed  Google Scholar 

  162. 162

    Okamura, K. et al. The Drosophila hairpin RNA pathway generates endogenous short interfering RNAs. Nature 453, 803–806 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  163. 163

    Tam, O. H. et al. Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes. Nature 453, 534–538 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  164. 164

    Watanabe, T. et al. Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes. Nature 453, 539–543 (2008).

    Article  CAS  PubMed  Google Scholar 

  165. 165

    Vagin, V. V. et al. A distinct small RNA pathway silences selfish genetic elements in the germline. Science 313, 320–324 (2006).

    Article  CAS  PubMed  Google Scholar 

  166. 166

    Nishimasu, H. et al. Structure and function of Zucchini endoribonuclease in piRNA biogenesis. Nature 491, 284–287 (2012).

    Article  CAS  PubMed  Google Scholar 

  167. 167

    Voigt, F. et al. Crystal structure of the primary piRNA biogenesis factor Zucchini reveals similarity to the bacterial PLD endonuclease Nuc. RNA 18, 2128–2134 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  168. 168

    Ipsaro, J. J., Haase, A. D., Knott, S. R., Joshua-Tor, L. & Hannon, G. J. The structural biochemistry of Zucchini implicates it as a nuclease in piRNA biogenesis. Nature 491, 279–283 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  169. 169

    Zhang, Z. et al. Heterotypic piRNA ping-pong requires Qin, a protein with both E3 ligase and Tudor domains. Mol. Cell 44, 572–584 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  170. 170

    Gu, W. et al. CapSeq and CIP-TAP identify Pol II start sites and reveal capped small RNAs as C. elegans piRNA precursors. Cell 151, 1488–1500 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  171. 171

    Hartig, J. V. Tomari, Y. & Forstemann, K. piRNAs—the ancient hunters of genome invaders. Genes Dev. 21, 1707–1713 (2007).

    Article  CAS  PubMed  Google Scholar 

  172. 172

    Malone, C. D. & Hannon, G. J. Small RNAs as guardians of the genome. Cell 136, 656–668 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

I apologize to those whose work has not been included owing to space constraints. I am grateful to A. Dueck, D. Schraivogel and J. Hauptmann for critically reading the manuscript. Our research is supported by grants from the Deutsche Forschungsgemeinschaft (DFG, SFB 960 and FOR855), the European Research Council (ERC grant 'sRNAs'), the Bavarian Genome Research Network (BayGene), the Bavarian Systems-Biology Network (BioSysNet), the European Union (grant 'ONCOMIRs') and the German Cancer Aid.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Gunter Meister.

Ethics declarations

Competing interests

The author declares no competing financial interests.

Related links

PowerPoint slides

Glossary

Dicer

An RNase III family endonuclease that processes double-stranded RNA and precursor microRNAs into small interfering RNAs and microRNAs, respectively.

RNA-induced silencing complex-loading complex

(RISC-loading complex). A protein complex containing Dicer, a double-stranded RNA-binding protein, Argonaute, heat shock protein 90 (HSP90) and potentially other proteins that is required for loading small RNAs onto the Argonaute protein.

miRNA* strand

The microRNA (miRNA) strand within the miRNA precursor that is not selected as the mature miRNA. miRNA* strands are typically degraded and are therefore less abundant in cells.

Endogenous short interfering RNAs

(endo-siRNAs). Short interfering RNAs (siRNAs) that originate from endogenous double-stranded RNA sources, such as long hairpin structures or sense or antisense transcripts from specific genomic loci that form double-stranded RNAs.

Trans-acting siRNAs

(ta-siRNAs). Small RNAs derived from specific genes; the transcripts are cleaved by specialized miRNAs and the cleavage products can be converted into double-stranded RNAs by RNA-dependent RNA polymerases. A Dicer enzyme cleaves the double stranded RNA to ta-siRNAs. ta-siRNAs are involved in DNA methylation.

Repeat-associated siRNAs

Small RNAs that originate from repetitive regions such as transposable elements. These RNAs are involved in silencing transcripts that are produced from repetitive elements.

Shoot apical meristem

A plant tissue that is located at the tip of the shoot axis. It contains undifferentiated cells and gives rise to lateral organs as well as the stem. It can regenerate.

Poly(A)-binding protein-interacting motif 2

(PAM2). This motif is present in several proteins and interacts with the poly(A)-binding protein domain, which is found in several proteins, including PABPC1.

LIM domain

Named after the proteins LIN1L, ISL1 and MEC3, LIM domain proteins have been linked to the cytoskeleton, and they have also been implicated in regulating cell growth, migration, cell–cell adhesion and signalling.

Crosslinking immunoprecipitation experiments

(CLIP experiments). CLIP technology facilitates the identification and sequencing of short RNA regions that associate with RNA-binding proteins or with microRNA-induced silencing complexes in intact cells.

Tudor domains

Conserved protein motifs that are able to recognize symmetrically dimethylated arginines.

Spliceosome

A ribonucleoprotein complex that is involved in splicing nuclear precursor mRNA (pre-mRNA). It is composed of five small nuclear ribonucleoproteins (snRNPs) and more than 50 non-snRNPs that recognize and assemble on exon–intron boundaries to catalyse intron processing of the pre-mRNA.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Meister, G. Argonaute proteins: functional insights and emerging roles. Nat Rev Genet 14, 447–459 (2013). https://doi.org/10.1038/nrg3462

Download citation

Further reading

Search

Quick links