Affinity purification of microRNA-133a with the cardiac transcription factor, Hand2

Ngan K. Vo, Ryan P. Dalton, Ning Liu, Eric N. Olson, Richard H. Goodman

Research output: Contribution to journalArticle

44 Scopus citations

Abstract

Predictions of microRNA-mRNA interactions typically rely on bioinformatic algorithms, but these algorithms only suggest the possibility of microRNA binding and may miss important interactions as well as falsely predict others. We developed an affinity purification approach to empirically identify microRNAs associated with the 3′ UTR of the mRNA encoding Hand2, a transcription factor essential for cardiac development. In addition to miR-1, a known regulator of Hand2 expression, we determined that the Hand2 3′UTR also associated with miR-133a, a microRNA cotranscribed with miR-1 in cardiac and muscle cells. Using a sequential binding assay, we showed that miR-1 and miR-133a could occupy the Hand2 3′UTR concurrently. miR-133a inhibited Hand2 expression in tissue culture models, and miR-133a double knockout mice had elevated levels of Hand2 mRNA and protein. We conclude that Hand2 is regulated by miR-133a in addition to miR-1. The affinity purification assay should be generally applicable for identifying other microRNAmRNA interactions.

Original languageEnglish (US)
Pages (from-to)19231-19236
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume107
Issue number45
DOIs
StatePublished - Nov 9 2010

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Keywords

  • C2C12
  • Cardiomyocytes
  • Heart
  • MS2

ASJC Scopus subject areas

  • General

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