A biochemical approach to identifying microRNA targets.
Identifying the downstream targets of microRNAs (miRNAs) is essential to understanding cellular regulatory networks. We devised a direct biochemical method for miRNA target discovery that combined RNA-induced silencing complex (RISC) purification with microarray analysis of bound mRNAs. Because targets of miR-124a have been analyzed, we chose it as our model. We honed our approach both by examining the determinants of stable binding between RISC and synthetic target RNAs in vitro and by determining the dependency of both repression and RISC coimmunoprecipitation on miR-124a seed sites in two of its well characterized targets in vivo. Examining the complete spectrum of miR-124 targets in 293 cells yielded both a set that were down-regulated at the mRNA level, as previously observed, and a set whose mRNA levels were unaffected by miR-124a. Reporter assays validated both classes, extending the spectrum of mRNA targets that can be experimentally linked to the miRNA pathway.
Watson School of Biological Sciences, Howard Hughes Medical Institute, Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA.
Fedor V
FV
Karginov
Cecilia
C
Conaco
Zhenyu
Z
Xuan
Bryan H
BH
Schmidt
Joel S
JS
Parker
Gail
G
Mandel
Gregory J
GJ
Hannon
D020413:3' Untranslated Regions / Q000032:analysis / Q000378:metabolism; D060565:Argonaute Proteins; D002460:Cell Line; D015852:Eukaryotic Initiation Factor-2; D005786:Gene Expression Regulation; D006801:Humans; D047468:Immunoprecipitation; D035683:MicroRNAs / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010749:Phosphoprotein Phosphatases / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis* / Q000378:metabolism; D034743:RNA-Induced Silencing Complex / Q000378:metabolism; D014158:Transcription, Genetic; D051040:Vesicle-Associated Membrane Protein 3 / Q000235:genetics
D020413:3' Untranslated Regions; C466422:AGO2 protein, human; D060565:Argonaute Proteins; D015852:Eukaryotic Initiation Factor-2; C509207:MIRN124 microRNA, human; D035683:MicroRNAs; D009687:Nuclear Proteins; D012333:RNA, Messenger; D034743:RNA-Induced Silencing Complex; D051040:Vesicle-Associated Membrane Protein 3; C413483:CTDSP1 protein, human; D010749:Phosphoprotein Phosphatases