Home LiteratureArticle Details
PMID: 18978028 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

Molecular characterization of human Argonaute-containing ribonucleoprotein complexes and their bound target mRNAs.

RNA (New York, N.Y.) ·Vol. 14 ·No. 12 ·2008-12-00 ·Pages 2580-96

Landthaler M, Gaidatzis D, Rothballer A, Chen PY, Soll SJ, Dinic L, Ojo T, Hafner M, Zavolan M, Tuschl T

Abstract

microRNAs (miRNAs) regulate the expression of mRNAs in animals and plants through miRNA-containing ribonucleoprotein particles (RNPs). At the core of these miRNA silencing effector complexes are the Argonaute (AGO) proteins that bind miRNAs and mediate target mRNA recognition. We generated HEK293 cell lines stably expressing epitope-tagged human AGO proteins and other RNA silencing-related proteins and used these cells to purify miRNA-containing RNPs. Mass spectrometric analyses of the proteins associated with different AGO proteins revealed a common set of helicases and mRNA-binding proteins, among them the three trinucleotide repeat containing proteins 6 (TNRC6A,-B,-C). mRNA microarray analyses of these miRNA-associated RNPs revealed that AGO and TNRC6 proteins bind highly similar sets of transcripts enriched in binding sites for highly expressed endogenous miRNAs, indicating that the TNRC6 proteins are a component of the mRNA-targeting miRNA silencing complex. Together with the very similar proteomic composition of each AGO complex, this result suggests substantial functional redundancy within families of human AGO and TNRC6 proteins. Our results further demonstrate that we have developed an effective biochemical approach to identify physiologically relevant human miRNA targets.

MeSH Terms
Cell Line Eukaryotic Initiation Factors/metabolism Humans Immunoprecipitation MicroRNAs/metabolism Proteome RNA, Messenger/metabolism Ribonucleoproteins/metabolism Ribosomes/metabolism Transfection
Chemicals
Eukaryotic Initiation Factors MIRN122 microRNA, human MicroRNAs Proteome RNA, Messenger Ribonucleoproteins
Authors & Affiliations
10 authors, click to expand affiliations / ORCID
Landthaler Markus
Laboratory of RNA Molecular Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10065, USA.
Gaidatzis Dimos
Rothballer Andrea
Chen Po Yu
Soll Steven Joseph
Dinic Lana
Ojo Tolulope
Hafner Markus
Zavolan Mihaela
Tuschl Thomas
References (103)
103 references, click to expand
  1. Short RNAs repress translation after initiation in mammalian cells.
    Mol Cell. 2006 Feb 17;21(4):533-42 PMID: 16483934
  2. microRNA target predictions across seven Drosophila species and comparison to mammalian targets.
    PLoS Comput Biol. 2005 Jun;1(1):e13 PMID: 16103902
  3. Human let-7a miRNA blocks protein production on actively translating polyribosomes.
    Nat Struct Mol Biol. 2006 Dec;13(12):1108-14 PMID: 17128272
  4. Systematic identification of mRNAs recruited to argonaute 2 by specific microRNAs and corresponding changes in transcript abundance.
    PLoS One. 2008;3(5):e2126 PMID: 18461144
  5. A mammalian microRNA expression atlas based on small RNA library sequencing.
    Cell. 2007 Jun 29;129(7):1401-14 PMID: 17604727
  6. VICKZ proteins: a multi-talented family of regulatory RNA-binding proteins.
    Biol Cell. 2005 Jan;97(1):87-96 PMID: 15601260
  7. A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA.
    Science. 2001 Aug 3;293(5531):834-8 PMID: 11452083
  8. Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans.
    Genes Dev. 2001 Oct 15;15(20):2654-9 PMID: 11641272
  9. TRBP, a regulator of cellular PKR and HIV-1 virus expression, interacts with Dicer and functions in RNA silencing.
    EMBO Rep. 2005 Oct;6(10):961-7 PMID: 16142218
  10. MicroRNAs direct rapid deadenylation of mRNA.
    Proc Natl Acad Sci U S A. 2006 Mar 14;103(11):4034-9 PMID: 16495412
  11. Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs.
    Mol Cell. 2004 Jul 23;15(2):185-97 PMID: 15260970
  12. Structural biology of RNA silencing and its functional implications.
    Cold Spring Harb Symp Quant Biol. 2006;71:81-93 PMID: 17381284
  13. Structure and conserved RNA binding of the PAZ domain.
    Nature. 2003 Nov 27;426(6965):468-74 PMID: 14615802
  14. Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing.
    Cell. 2001 Jul 13;106(1):23-34 PMID: 11461699
  15. microRNA functions.
    Annu Rev Cell Dev Biol. 2007;23:175-205 PMID: 17506695
  16. Isolation of microRNA targets by miRNP immunopurification.
    RNA. 2007 Aug;13(8):1198-204 PMID: 17592038
  17. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets.
    Cell. 2005 Jan 14;120(1):15-20 PMID: 15652477
  18. Structural basis for 5'-end-specific recognition of guide RNA by the A. fulgidus Piwi protein.
    Nature. 2005 Mar 31;434(7033):666-70 PMID: 15800629
  19. Identification of many microRNAs that copurify with polyribosomes in mammalian neurons.
    Proc Natl Acad Sci U S A. 2004 Jan 6;101(1):360-5 PMID: 14691248
  20. MicroRNA targeting specificity in mammals: determinants beyond seed pairing.
    Mol Cell. 2007 Jul 6;27(1):91-105 PMID: 17612493
  21. Nuclear export of microRNA precursors.
    Science. 2004 Jan 2;303(5654):95-8 PMID: 14631048
  22. Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs.
    Nature. 2005 Feb 17;433(7027):769-73 PMID: 15685193
  23. The role of PACT in the RNA silencing pathway.
    EMBO J. 2006 Feb 8;25(3):522-32 PMID: 16424907
  24. GW182 interaction with Argonaute is essential for miRNA-mediated translational repression and mRNA decay.
    Nat Struct Mol Biol. 2008 Apr;15(4):346-53 PMID: 18345015
  25. Argonaute2, a link between genetic and biochemical analyses of RNAi.
    Science. 2001 Aug 10;293(5532):1146-50 PMID: 11498593
  26. MicroRNAs control translation initiation by inhibiting eukaryotic initiation factor 4E/cap and poly(A) tail function.
    Proc Natl Acad Sci U S A. 2005 Nov 22;102(47):16961-6 PMID: 16287976
  27. Identification of human microRNA targets from isolated argonaute protein complexes.
    RNA Biol. 2007 Jun;4(2):76-84 PMID: 17637574
  28. The functions of animal microRNAs.
    Nature. 2004 Sep 16;431(7006):350-5 PMID: 15372042
  29. Mechanisms of gene silencing by double-stranded RNA.
    Nature. 2004 Sep 16;431(7006):343-9 PMID: 15372041
  30. Slicer and the argonautes.
    Nat Chem Biol. 2007 Jan;3(1):36-43 PMID: 17173028
  31. Inhibition of translational initiation by Let-7 MicroRNA in human cells.
    Science. 2005 Sep 2;309(5740):1573-6 PMID: 16081698
  32. Specificity of microRNA target selection in translational repression.
    Genes Dev. 2004 Mar 1;18(5):504-11 PMID: 15014042
  33. Crystal structure of Argonaute and its implications for RISC slicer activity.
    Science. 2004 Sep 3;305(5689):1434-7 PMID: 15284453
  34. Systematic identification of C. elegans miRISC proteins, miRNAs, and mRNA targets by their interactions with GW182 proteins AIN-1 and AIN-2.
    Mol Cell. 2007 Nov 30;28(4):598-613 PMID: 18042455
  35. RNA-binding protein Dnd1 inhibits microRNA access to target mRNA.
    Cell. 2007 Dec 28;131(7):1273-86 PMID: 18155131
  36. Translation repression in human cells by microRNA-induced gene silencing requires RCK/p54.
    PLoS Biol. 2006 Jul;4(7):e210 PMID: 16756390
  37. Transcripts targeted by the microRNA-16 family cooperatively regulate cell cycle progression.
    Mol Cell Biol. 2007 Mar;27(6):2240-52 PMID: 17242205
  38. Proteomic and functional analysis of Argonaute-containing mRNA-protein complexes in human cells.
    EMBO Rep. 2007 Nov;8(11):1052-60 PMID: 17932509
  39. Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells.
    Mol Cell. 2002 Jun;9(6):1327-33 PMID: 12086629
  40. Structural insights into mRNA recognition from a PIWI domain-siRNA guide complex.
    Nature. 2005 Mar 31;434(7033):663-6 PMID: 15800628
  41. Bioconductor: open software development for computational biology and bioinformatics.
    Genome Biol. 2004;5(10):R80 PMID: 15461798
  42. Combinatorial microRNA target predictions.
    Nat Genet. 2005 May;37(5):495-500 PMID: 15806104
  43. Widespread changes in protein synthesis induced by microRNAs.
    Nature. 2008 Sep 4;455(7209):58-63 PMID: 18668040
  44. MicroRNAS and their regulatory roles in plants.
    Annu Rev Plant Biol. 2006;57:19-53 PMID: 16669754
  45. A microRNA in a multiple-turnover RNAi enzyme complex.
    Science. 2002 Sep 20;297(5589):2056-60 PMID: 12154197
  46. A biochemical approach to identifying microRNA targets.
    Proc Natl Acad Sci U S A. 2007 Dec 4;104(49):19291-6 PMID: 18042700
  47. The impact of microRNAs on protein output.
    Nature. 2008 Sep 4;455(7209):64-71 PMID: 18668037
  48. Involvement of microRNA in AU-rich element-mediated mRNA instability.
    Cell. 2005 Mar 11;120(5):623-34 PMID: 15766526
  49. Epstein-Barr virus-encoded microRNA miR-BART2 down-regulates the viral DNA polymerase BALF5.
    Nucleic Acids Res. 2008 Feb;36(2):666-75 PMID: 18073197
  50. RNA helicase A interacts with RISC in human cells and functions in RISC loading.
    Mol Cell. 2007 May 25;26(4):523-37 PMID: 17531811
  51. Regulation by let-7 and lin-4 miRNAs results in target mRNA degradation.
    Cell. 2005 Aug 26;122(4):553-63 PMID: 16122423
  52. AU-rich-element-mediated upregulation of translation by FXR1 and Argonaute 2.
    Cell. 2007 Mar 23;128(6):1105-18 PMID: 17382880
  53. microRNAs: a safeguard against turmoil?
    Cell. 2007 Aug 24;130(4):581-5 PMID: 17719533
  54. mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes.
    Genes Dev. 2006 Jul 15;20(14):1885-98 PMID: 16815998
  55. MicroRNA-directed cleavage of HOXB8 mRNA.
    Science. 2004 Apr 23;304(5670):594-6 PMID: 15105502
  56. Let-7 microRNA-mediated mRNA deadenylation and translational repression in a mammalian cell-free system.
    Genes Dev. 2007 Aug 1;21(15):1857-62 PMID: 17671087
  57. Argonaute proteins: mediators of RNA silencing.
    Mol Cell. 2007 Jun 8;26(5):611-23 PMID: 17560368
  58. Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs.
    Genes Dev. 2003 Dec 15;17(24):3011-6 PMID: 14681208
  59. The crystal structure of the Argonaute2 PAZ domain reveals an RNA binding motif in RNAi effector complexes.
    Nat Struct Biol. 2003 Dec;10(12):1026-32 PMID: 14625589
  60. Two genetic circuits repress the Caenorhabditis elegans heterochronic gene lin-28 after translation initiation.
    Dev Biol. 2002 Mar 15;243(2):215-25 PMID: 11884032
  61. Silencing of microRNAs in vivo with 'antagomirs'.
    Nature. 2005 Dec 1;438(7068):685-9 PMID: 16258535
  62. Dysregulation of cardiogenesis, cardiac conduction, and cell cycle in mice lacking miRNA-1-2.
    Cell. 2007 Apr 20;129(2):303-17 PMID: 17397913
  63. miRNPs: a novel class of ribonucleoproteins containing numerous microRNAs.
    Genes Dev. 2002 Mar 15;16(6):720-8 PMID: 11914277
  64. miRNP:mRNA association in polyribosomes in a human neuronal cell line.
    RNA. 2004 Mar;10(3):387-94 PMID: 14970384
  65. Single-stranded antisense siRNAs guide target RNA cleavage in RNAi.
    Cell. 2002 Sep 6;110(5):563-74 PMID: 12230974
  66. A Dicer-2-dependent 80s complex cleaves targeted mRNAs during RNAi in Drosophila.
    Cell. 2004 Apr 2;117(1):83-94 PMID: 15066284
  67. microRNA target predictions in animals.
    Nat Genet. 2006 Jun;38 Suppl:S8-13 PMID: 16736023
  68. A role for the P-body component GW182 in microRNA function.
    Nat Cell Biol. 2005 Dec;7(12):1261-6 PMID: 16284623
  69. Perfect seed pairing is not a generally reliable predictor for miRNA-target interactions.
    Nat Struct Mol Biol. 2006 Sep;13(9):849-51 PMID: 16921378
  70. Human MicroRNA targets.
    PLoS Biol. 2004 Nov;2(11):e363 PMID: 15502875
  71. Inference of miRNA targets using evolutionary conservation and pathway analysis.
    BMC Bioinformatics. 2007;8:69 PMID: 17331257
  72. Asymmetry in the assembly of the RNAi enzyme complex.
    Cell. 2003 Oct 17;115(2):199-208 PMID: 14567917
  73. Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans.
    Cell. 1993 Dec 3;75(5):855-62 PMID: 8252622
  74. The lin-4 regulatory RNA controls developmental timing in Caenorhabditis elegans by blocking LIN-14 protein synthesis after the initiation of translation.
    Dev Biol. 1999 Dec 15;216(2):671-80 PMID: 10642801
  75. Structural basis for overhang-specific small interfering RNA recognition by the PAZ domain.
    Nature. 2004 May 20;429(6989):318-22 PMID: 15152257
  76. Nucleic acid 3'-end recognition by the Argonaute2 PAZ domain.
    Nat Struct Mol Biol. 2004 Jun;11(6):576-7 PMID: 15156196
  77. Drosophila miR2 induces pseudo-polysomes and inhibits translation initiation.
    Nature. 2007 Jun 14;447(7146):875-8 PMID: 17507927
  78. Requirement of bic/microRNA-155 for normal immune function.
    Science. 2007 Apr 27;316(5824):608-11 PMID: 17463290
  79. Functional siRNAs and miRNAs exhibit strand bias.
    Cell. 2003 Oct 17;115(2):209-16 PMID: 14567918
  80. Switching from repression to activation: microRNAs can up-regulate translation.
    Science. 2007 Dec 21;318(5858):1931-4 PMID: 18048652
  81. The Drosha-DGCR8 complex in primary microRNA processing.
    Genes Dev. 2004 Dec 15;18(24):3016-27 PMID: 15574589
  82. In vitro reconstitution of the human RISC-loading complex.
    Proc Natl Acad Sci U S A. 2008 Jan 15;105(2):512-7 PMID: 18178619
  83. The human DiGeorge syndrome critical region gene 8 and Its D. melanogaster homolog are required for miRNA biogenesis.
    Curr Biol. 2004 Dec 14;14(23):2162-7 PMID: 15589161
  84. Zebrafish MiR-430 promotes deadenylation and clearance of maternal mRNAs.
    Science. 2006 Apr 7;312(5770):75-9 PMID: 16484454
  85. Isolation of microRNA targets using biotinylated synthetic microRNAs.
    Methods. 2007 Oct;43(2):162-5 PMID: 17889804
  86. Identification of novel argonaute-associated proteins.
    Curr Biol. 2005 Dec 6;15(23):2149-55 PMID: 16289642
  87. ATP modulates siRNA interactions with an endogenous human Dicer complex.
    RNA. 2005 Nov;11(11):1719-24 PMID: 16177131
  88. Drosha in primary microRNA processing.
    Cold Spring Harb Symp Quant Biol. 2006;71:51-7 PMID: 17381280
  89. Molecular basis for the recognition of primary microRNAs by the Drosha-DGCR8 complex.
    Cell. 2006 Jun 2;125(5):887-901 PMID: 16751099
  90. Evidence that microRNAs are associated with translating messenger RNAs in human cells.
    Nat Struct Mol Biol. 2006 Dec;13(12):1102-7 PMID: 17128271
  91. Identification of miRNA targets with stable isotope labeling by amino acids in cell culture.
    Nucleic Acids Res. 2006;34(16):e107 PMID: 16945957
  92. A crucial role for GW182 and the DCP1:DCP2 decapping complex in miRNA-mediated gene silencing.
    RNA. 2005 Nov;11(11):1640-7 PMID: 16177138
  93. Localization of the developmental timing regulator Lin28 to mRNP complexes, P-bodies and stress granules.
    RNA Biol. 2007 Jan-Mar;4(1):16-25 PMID: 17617744
  94. Genome-wide analysis of mRNAs regulated by Drosha and Argonaute proteins in Drosophila melanogaster.
    Mol Cell Biol. 2006 Apr;26(8):2965-75 PMID: 16581772
  95. TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing.
    Nature. 2005 Aug 4;436(7051):740-4 PMID: 15973356
  96. Specialization and evolution of endogenous small RNA pathways.
    Nat Rev Genet. 2007 Nov;8(11):884-96 PMID: 17943195
  97. Processing of primary microRNAs by the Microprocessor complex.
    Nature. 2004 Nov 11;432(7014):231-5 PMID: 15531879
  98. Disruption of GW bodies impairs mammalian RNA interference.
    Nat Cell Biol. 2005 Dec;7(12):1267-74 PMID: 16284622
  99. Relief of microRNA-mediated translational repression in human cells subjected to stress.
    Cell. 2006 Jun 16;125(6):1111-24 PMID: 16777601
  100. Argonaute2 is the catalytic engine of mammalian RNAi.
    Science. 2004 Sep 3;305(5689):1437-41 PMID: 15284456
  101. Role for a bidentate ribonuclease in the initiation step of RNA interference.
    Nature. 2001 Jan 18;409(6818):363-6 PMID: 11201747
  102. Effects of Dicer and Argonaute down-regulation on mRNA levels in human HEK293 cells.
    Nucleic Acids Res. 2006;34(17):4801-15 PMID: 16971455
  103. The Microprocessor complex mediates the genesis of microRNAs.
    Nature. 2004 Nov 11;432(7014):235-40 PMID: 15531877
Article Info
Journal
RNA (New York, N.Y.)
Abbr.
RNA
ISSN
1469-9001
DOI
10.1261/rna.1351608
Published
2008-12-00
Epub
2008-00-31
Pages
2580-96
Language
English
Region
United States
NLM ID
9509184
PMCID
PMC2590962
Subset
IM
Grants
NIGMS NIH HHS · R01 GM068476 · United States
Howard Hughes Medical Institute · United States
NIGMS NIH HHS · GM068476 · United States
NIGMS NIH HHS · GM073047 · United States
NIGMS NIH HHS · P01 GM073047 · United States
External Links
PubMed source DOI: 10.1261/rna.1351608 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: product@genelibs.com

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2