Structural determinants of miRNAs for RISC loading and slicer-independent unwinding.
MicroRNAs (miRNAs) regulate expression of their target mRNAs through the RNA-induced silencing complex (RISC), which contains an Argonaute (Ago) family protein as a core component. In Drosophila melanogaster, miRNAs are generally sorted into Ago1-containing RISC (Ago1-RISC). We established a native gel system that can biochemically dissect the Ago1-RISC assembly pathway. We found that miRNA-miRNA* duplexes are loaded into Ago1 as double-stranded RNAs in an ATP-dependent fashion. In contrast, unexpectedly, unwinding of miRNA-miRNA* duplexes is a passive process that does not require ATP or slicer activity of Ago1. Central mismatches direct miRNA-miRNA* duplexes into pre-Ago1-RISC, whereas mismatches in the seed or guide strand positions 12-15 promote conversion of pre-Ago1-RISC into mature Ago1-RISC. Our findings show that unwinding of miRNAs is a precise mirror-image process of target recognition, and both processes reflect the unique geometry of RNAs in Ago proteins.
Kawamata
Tomoko
T
Institute of Molecular and Cellular Biosciences, The University of Tokyo, Japan.
Seitz
Hervé
H
Tomari
Yukihide
Y
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D060565:Argonaute Proteins; D020137:Base Pair Mismatch; D001483:Base Sequence; D019281:Dimerization; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism; D039642:Eukaryotic Initiation Factors; D035683:MicroRNAs / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009690:Nucleic Acid Conformation / Q000187:drug effects; D011485:Protein Binding; D020365:RNA Helicases / Q000378:metabolism; D012326:RNA Splicing; D034743:RNA-Induced Silencing Complex / Q000378:metabolism*; D043244:Ribonuclease III / Q000378:metabolism
C489654:AGO1 protein, Drosophila; D060565:Argonaute Proteins; D029721:Drosophila Proteins; D039642:Eukaryotic Initiation Factors; D035683:MicroRNAs; D034743:RNA-Induced Silencing Complex; D000255:Adenosine Triphosphate; C515033:Dcr-1 protein, Drosophila; D043244:Ribonuclease III; D020365:RNA Helicases
Science. 2003 Sep 26;301(5641):1921-5
14512631
Nat Struct Mol Biol. 2008 Apr;15(4):346-53
18345015
Genes Dev. 2002 Oct 1;16(19):2497-508
12368261
Cell. 2005 Nov 18;123(4):621-9
16271385
Cell. 2007 Jul 27;130(2):299-308
17662944
Genes Dev. 2004 Jul 15;18(14):1655-66
15231716
Cell. 2004 Mar 19;116(6):831-41
15035985
Nature. 2008 Dec 18;456(7224):921-6
19092929
Methods. 2003 Aug;30(4):330-6
12828947
Genes Dev. 2008 Oct 15;22(20):2773-85
18923076
Mol Cell. 2007 May 25;26(4):523-37
17531811
Genes Dev. 2001 Jan 15;15(2):188-200
11157775
Genes Dev. 2005 Mar 1;19(5):517-29
15741316
Mol Cell. 2004 Jul 23;15(2):185-97
15260970
Cell. 2007 Jul 27;130(2):287-97
17662943
Mol Cell. 2007 Jul 6;27(1):91-105
17612493
EMBO J. 2004 Aug 18;23(16):3356-64
15282547
Science. 2004 Sep 3;305(5689):1434-7
15284453
Genes Dev. 2003 Feb 15;17(4):438-42
12600936
Nat Struct Mol Biol. 2004 Jul;11(7):599-606
15170178
RNA. 2007 Dec;13(12):2324-9
17928574
Cell. 2006 Dec 15;127(6):1193-207
17174894
EMBO J. 2001 Dec 3;20(23):6877-88
11726523
Cell. 2004 Apr 2;117(1):69-81
15066283
RNA. 2008 Dec;14(12):2580-96
18978028
Genes Dev. 2009 Feb 1;23(3):304-17
19174539
Curr Biol. 2005 Dec 6;15(23):2149-55
16289642
Mol Cell. 2008 Nov 21;32(4):592-9
19026789
Genes Dev. 2004 Mar 1;18(5):504-11
15014042
Cell. 2004 Apr 2;117(1):83-94
15066284
Curr Biol. 2008 Jun 3;18(11):795-802
18501606
Cell. 2001 Nov 2;107(3):309-21
11701122
Mol Cell. 2008 Jul 11;31(1):67-78
18571452
PLoS Biol. 2005 Jul;3(7):e236
15918770
Genome Res. 2007 Dec;17(12):1850-64
17989254
Proc Natl Acad Sci U S A. 2008 Jun 10;105(23):7964-9
18524951
Nature. 2008 May 22;453(7194):534-8
18404147
Science. 2008 May 23;320(5879):1077-81
18403677
Curr Biol. 2007 Mar 20;17(6):539-44
17306537
Cell. 2005 Jan 14;120(1):15-20
15652477
Curr Biol. 2008 Jan 22;18(2):147-51
18207740
Cell. 2003 Oct 17;115(2):209-16
14567918
Nat Struct Mol Biol. 2005 Apr;12(4):340-9
15800637
PLoS Biol. 2005 Mar;3(3):e85
15723116
RNA. 2008 Oct;14(10):2104-14
18719242
Cell. 2005 Jul 15;122(1):17-20
16009129
Nature. 2008 Nov 13;456(7219):209-13
18754009
Nature. 2008 Jun 5;453(7196):793-7
18463636
Cell. 2005 Nov 18;123(4):607-20
16271386
EMBO Rep. 2007 Nov;8(11):1052-60
17932509
Genes Dev. 2005 Dec 1;19(23):2837-48
16287716
PLoS Biol. 2005 Jul;3(7):e235
15918769
Science. 2004 Sep 3;305(5689):1437-41
15284456
Mol Cell. 2009 Apr 10;34(1):58-67
19268617
Cell. 2003 Oct 17;115(2):199-208
14567917
Cell. 2004 Jan 23;116(2):281-97
14744438
Proc Natl Acad Sci U S A. 2007 Nov 13;104(46):18097-102
17989215
Nature. 2008 Sep 4;455(7209):64-71
18668037
Nat Genet. 2008 Mar;40(3):351-5
18278047
EMBO Rep. 2006 Mar;7(3):314-20
16439995