Genome-wide identification of targets and function of individual MicroRNAs in mouse embryonic stem cells.
Mouse Embryonic Stem (ES) cells express a unique set of microRNAs (miRNAs), the miR-290-295 cluster. To elucidate the role of these miRNAs and how they integrate into the ES cell regulatory network requires identification of their direct regulatory targets. The difficulty, however, arises from the limited complementarity of metazoan miRNAs to their targets, with the interaction requiring as few as six nucleotides of the miRNA seed sequence. To identify miR-294 targets, we used Dicer1-null ES cells, which lack all endogenous mature miRNAs, and introduced just miR-294 into these ES cells. We then employed two approaches to discover miR-294 targets in mouse ES cells: transcriptome profiling using microarrays and a biochemical approach to isolate mRNA targets associated with the Argonaute2 (Ago2) protein of the RISC (RNA Induced Silencing Complex) effector, followed by RNA-sequencing. In the absence of Dicer1, the RISC complexes are largely devoid of mature miRNAs and should therefore contain only transfected miR-294 and its base-paired targets. Our data suggest that miR-294 may promote pluripotency by regulating a subset of c-Myc target genes and upregulating pluripotency-associated genes such as Lin28.
Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, UK.
Sophie A
SA
Hanina
William
W
Mifsud
Thomas A
TA
Down
Katsuhiko
K
Hayashi
Dónal
D
O'Carroll
Kaiqin
K
Lao
Eric A
EA
Miska
M Azim
MA
Surani
D020413:3' Untranslated Regions / Q000235:genetics; D000818:Animals; D060565:Argonaute Proteins; D002478:Cells, Cultured; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000378:metabolism*; D004722:Endoribonucleases / Q000235:genetics / Q000378:metabolism; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling; D053263:Gene Regulatory Networks; D016678:Genome / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D057507:Mice, 129 Strain; D018345:Mice, Knockout; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D043244:Ribonuclease III; D014162:Transfection
D020413:3' Untranslated Regions; C526735:Ago2 protein, mouse; D060565:Argonaute Proteins; D015852:Eukaryotic Initiation Factor-2; C488352:Lin-28 protein, mouse; C556303:MIRN294 microRNA, mouse; D035683:MicroRNAs; C489428:Myc protein, mouse; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D004722:Endoribonucleases; C509781:Dicer1 protein, mouse; D043244:Ribonuclease III; D053487:DEAD-box RNA Helicases