A biochemical approach to identifying microRNA targets

Fedor V. Karginov, Cecilia Conaco, Zhenyu Xuan, Bryan H. Schmidt, Joel S. Parker, Gail Mandel, Gregory J. Hannon

Research output: Contribution to journalArticle

294 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)19291-19296
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume104
Issue number49
DOIs
StatePublished - Dec 4 2007

Keywords

  • Gene regulation
  • Immunoprecipitation
  • RISC
  • RNAi

ASJC Scopus subject areas

  • General

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