Isolation of microRNA targets by miRNP immunopurification.
microRNAs (miRNAs) serve as post-transcriptional regulators of gene expression, by guiding effector complexes (miRNPs) to target RNAs. Although considerable progress has been made in computational methods to identify miRNA targets, only a relatively limited assessment of their ability to function in vivo has been reported. Here we describe an alternative approach to miRNA target identification based on a biochemical method for purifying miRNP complexes with associated miRNAs and bound mRNA targets. Microarray analysis revealed a high degree of enrichment for miRNA complementary sites in the 3'UTRs of the miRNP-associated mRNAs. mRNAs specifically associated with an individual miRNA were identified by comparing the miRNP-associated mRNAs from wild-type flies and mutant flies lacking miR-1, and their regulation by the miRNA was validated. This approach provides a means to identify functional miRNA targets based on their physical interaction in vivo.
Easow
George
G
European Molecular Biology Laboratory, Heidelberg, Germany.
Teleman
Aurelio A
AA
Cohen
Stephen M
SM
D000818:Animals; D060565:Argonaute Proteins; D004330:Drosophila / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D029721:Drosophila Proteins / Q000378:metabolism; D039642:Eukaryotic Initiation Factors; D047468:Immunoprecipitation / Q000379:methods*; D035683:MicroRNAs / Q000378:metabolism*; D009154:Mutation; D012261:Ribonucleoproteins / Q000302:isolation & purification* / Q000378:metabolism
C489654:AGO1 protein, Drosophila; D060565:Argonaute Proteins; D029721:Drosophila Proteins; D039642:Eukaryotic Initiation Factors; D035683:MicroRNAs; D012261:Ribonucleoproteins