Genome-wide identification of Ago2 binding sites from mouse embryonic stem cells with and without mature microRNAs.
MicroRNAs (miRNAs) are 19-22-nucleotide noncoding RNAs that post-transcriptionally regulate mRNA targets. We have identified endogenous miRNA binding sites in mouse embryonic stem cells (mESCs), by performing photo-cross-linking immunoprecipitation using antibodies to Argonaute (Ago2) followed by deep sequencing of RNAs (CLIP-seq). We also performed CLIP-seq in Dicer⁻/⁻ mESCs that lack mature miRNAs, allowing us to define whether the association of Ago2 with the identified sites was miRNA dependent. A significantly enriched motif, GCACUU, was identified only in wild-type mESCs in 3' untranslated and coding regions. This motif matches the seed of a miRNA family that constitutes ~68% of the mESC miRNA population. Unexpectedly, a G-rich motif was enriched in sequences cross-linked to Ago2 in both the presence and absence of miRNAs. Expression analysis and reporter assays confirmed that the seed-related motif confers miRNA-directed regulation on host mRNAs and that the G-rich motif can modulate this regulation.
The Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
Anthony K L
AK
Leung
Amanda G
AG
Young
Arjun
A
Bhutkar
Grace X
GX
Zheng
Andrew D
AD
Bosson
Cydney B
CB
Nielsen
Phillip A
PA
Sharp
D000818:Animals; D060565:Argonaute Proteins; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D053487:DEAD-box RNA Helicases / Q000235:genetics; D053595:Embryonic Stem Cells / Q000378:metabolism*; D004722:Endoribonucleases / Q000235:genetics; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D017353:Gene Deletion; D005786:Gene Expression Regulation; D006147:Guanine / Q000032:analysis; D059014:High-Throughput Nucleotide Sequencing; D051379:Mice; D035683:MicroRNAs / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D043244:Ribonuclease III; D017423:Sequence Analysis, RNA
C526735:Ago2 protein, mouse; D060565:Argonaute Proteins; D015852:Eukaryotic Initiation Factor-2; D035683:MicroRNAs; D006147:Guanine; D004722:Endoribonucleases; C509781:Dicer1 protein, mouse; D043244:Ribonuclease III; D053487:DEAD-box RNA Helicases
Bioinformatics. 1998;14(1):48-54
9520501
Nat Rev Mol Cell Biol. 2008 Mar;9(3):219-30
18270516
Nat Struct Mol Biol. 2008 Mar;15(3):268-79
18311151
Nucleic Acids Res. 2004 Dec 07;32(21):6284-91
15585662
Nature. 2010 Feb 4;463(7281):621-6
20054295
Nature. 2005 Jun 9;435(7043):839-43
15944709
Nat Struct Mol Biol. 2010 Feb;17(2):144-50
20062058
Dev Cell. 2003 Aug;5(2):351-8
12919684
Nat Struct Mol Biol. 2010 Jan;17(1):17-23
19966796
Science. 2007 Oct 12;318(5848):271-4
17761850
RNA. 2007 Nov;13(11):1894-910
17872505
Genes Dev. 2008 Oct 15;22(20):2773-85
18923076
Cold Spring Harb Symp Quant Biol. 2006;71:523-30
17381335
Nature. 2005 Feb 17;433(7027):769-73
15685193
Cell. 2009 Feb 20;136(4):642-55
19239886
Methods. 2008 Jan;44(1):3-12
18158127
Nucleic Acids Res. 2002 Dec 15;30(24):5549-60
12490723
Dev Biol. 2009 Feb 15;326(2):431-43
19073166
Genome Res. 2009 Jan;19(1):92-105
18955434
Mol Cell. 2007 Jul 6;27(1):91-105
17612493
Nature. 2009 Jul 23;460(7254):479-86
19536157
Nature. 2008 Nov 27;456(7221):464-9
18978773
Nat Immunol. 2008 Apr;9(4):405-14
18327259
Nat Genet. 2008 Dec;40(12):1478-83
18978791
Genome Res. 2005 Aug;15(8):1034-50
16024819
Nature. 2008 Oct 23;455(7216):1124-8
18806776
PLoS Genet. 2009 Aug;5(8):e1000620
19714213
Genome Res. 2004 Apr;14(4):708-15
15060014
Dev Cell. 2009 Apr;16(4):517-27
19386261
RNA. 2004 Oct;10(10):1518-25
15337849
Nat Genet. 2007 Oct;39(10):1278-84
17893677
Differentiation. 2009 Sep-Oct;78(2-3):69-78
19628328
Genes Dev. 2004 Mar 1;18(5):504-11
15014042
Proc Int Conf Intell Syst Mol Biol. 1994;2:28-36
7584402
Nat Biotechnol. 2009 May;27(5):459-61
19363475
Proc Natl Acad Sci U S A. 2006 Nov 28;103(48):18125-30
17116888
Mol Cell. 2010 Oct 22;40(2):205-15
20965416
Nat Struct Mol Biol. 2008 Mar;15(3):259-67
18311153
RNA. 2006 Dec;12(12):2092-102
17135486
Neuron. 2010 Feb 11;65(3):373-84
20159450
Cell. 2010 Apr 2;141(1):129-41
20371350
Cell. 2007 Jun 29;129(7):1401-14
17604727
Cell. 2009 Jan 23;136(2):215-33
19167326
Methods. 2005 Dec;37(4):376-86
16314267
Cell. 2005 Jan 14;120(1):15-20
15652477
Nat Struct Mol Biol. 2009 Feb;16(2):130-7
19136955
Genes Dev. 2002 Oct 1;16(19):2491-6
12368260
PLoS Biol. 2005 Mar;3(3):e85
15723116
Nature. 2004 Sep 16;431(7006):350-5
15372042
Cell. 2009 Apr 17;137(2):273-82
19379693
EMBO Rep. 2007 Nov;8(11):1052-60
17932509
Mol Cell. 2007 Jun 8;26(5):753-67
17560377
Nat Struct Mol Biol. 2009 Feb;16(2):144-50
19182800
Proc Natl Acad Sci U S A. 2007 Nov 13;104(46):18097-102
17989215
Nature. 2005 Mar 17;434(7031):338-45
15735639
Cell. 2008 Aug 8;134(3):521-33
18692474
Nature. 2008 Sep 4;455(7209):64-71
18668037
Nat Struct Mol Biol. 2010 Feb;17(2):173-9
20062054
Nucleic Acids Res. 2009 Dec;37(22):7533-45
19808937
Science. 2005 Dec 16;310(5755):1817-21
16308420