In vivo processing assay based on a dual-luciferase reporter system to evaluate DROSHA enzymatic activity

Methods Mol Biol. 2014:1095:87-93. doi: 10.1007/978-1-62703-703-7_6.

Abstract

Luciferase reporter assays are widely used to study promoter activity, transcription factors, intracellular signaling, protein interactions (Jia et al., PloS One 6:e26414), miRNA processing (Allegra and Mertens, Biochem Biophys Res Commun 406:501-505), and target recognition (Jin et al., Methods Mol Biol 936:117-127). Here we describe the use of a dual-luciferase reporter system to evaluate the enzymatic activity of a key enzyme involved in RNA maturation-DROSHA. This dual system is a simple and fast method for the quantification of the DROSHA processing activity in live cells.

MeSH terms

  • 3' Untranslated Regions / genetics
  • Cell Adhesion
  • Enzyme Assays / methods*
  • Gene Knockdown Techniques
  • Genes, Reporter / genetics*
  • HEK293 Cells
  • Humans
  • Inverted Repeat Sequences
  • Luciferases / genetics*
  • MicroRNAs / genetics
  • MicroRNAs / metabolism
  • Plasmids / genetics
  • RNA Cleavage
  • RNA Precursors / genetics
  • RNA Precursors / metabolism
  • RNA, Small Interfering / genetics
  • Ribonuclease III / deficiency
  • Ribonuclease III / genetics
  • Ribonuclease III / metabolism*
  • Transfection

Substances

  • 3' Untranslated Regions
  • MIRN16 microRNA, human
  • MicroRNAs
  • RNA Precursors
  • RNA, Small Interfering
  • Luciferases
  • DROSHA protein, human
  • Ribonuclease III