Tandem affinity purification of miRNA target mRNAs (TAP-Tar).
MicroRNAs (miRNAs) bind to Argonaute proteins, and together they form the RISC complex and regulate target mRNA translation and/or stability. Identification of mRNA targets is key to deciphering the physiological functions and mode of action of miRNAs. In mammals, miRNAs are generally poorly homologous to their target sequence, and target identification cannot be based solely on bioinformatics. Here, we describe a biochemical approach, based on tandem affinity purification, in which mRNA/miRNA complexes are sequentially pulled down, first via the Argonaute moiety and then via the miRNA. Our 'TAP-Tar' procedure allows the specific pull down of mRNA targets of miRNA. It is useful for validation of targets predicted in silico, and, potentially, for discovery of previously uncharacterized targets.
Nonne
Nora
N
CNRS FRE 2944, Institut André Lwoff, Villejuif F-94801 and Université Paris-Sud, Villejuif F-94801, France.
Ameyar-Zazoua
Maya
M
Souidi
Mouloud
M
Harel-Bellan
Annick
A
D019921:Biotinylation; D050687:E2F1 Transcription Factor / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D035683:MicroRNAs / Q000302:isolation & purification* / Q000378:metabolism; D012333:RNA, Messenger / Q000302:isolation & purification* / Q000378:metabolism
D050687:E2F1 Transcription Factor; C534451:MIRN20a microRNA, human; D035683:MicroRNAs; D012333:RNA, Messenger